HK1247245B - Methods and compositions for inducing hematopoietic cell differentiation - Google Patents

Description

Methods and compositions for inducing hematopoietic cell differentiation

Related Application

This application claims priority to U.S. Provisional Patent Application No. 62/107,517, filed January 26, 2015, and U.S. Provisional Patent Application No. 62/251,016, filed November 4, 2015, which are hereby incorporated by reference in their entireties.

Technical Field

The present invention generally relates to compositions and methods for producing cells of all hematopoietic lineages from pluripotent stem cells. In particular, the present invention relates to an improved culture platform for producing cells of all hematopoietic lineages from pluripotent stem cells, including human induced pluripotent stem cells.

background

Human induced pluripotent stem cell (hiPSC) technology represents a very promising and potentially unlimited source of therapeutically viable hematopoietic cells for the treatment of many hematological and non-hematological malignancies, including cancer. To advance the promise of hiPSC and genetically engineered hiPSC technology as an allogeneic source of hematopoietic cell therapy, it is imperative to be able to efficiently and reproducibly generate not only hematopoietic stem and progenitor cells (HSCs), but also immune effector populations, including diverse subsets of T, B, NKT and NK lymphoid cells and their progenitors.

Due to the presence of at least two different blood formation stages (waves) in time and space during embryonic development - primitive (primitive) hematopoiesis and permanent (definitive) hematopoiesis - the in vitro derivation of HSC with the potential to produce lymphocytes is complex. Primitive hematopoiesis occurs in the extraembryonic yolk sac and produces transient and restricted hematopoietic cells that mainly include primitive erythrocytes and bone marrow cells rather than HSC. Initial HSC only appears in the final stage of the specific endothelial progenitor cells in the arterial blood vessels called permanent hemogenic endothelium (HE). Permanent HE then undergoes endothelial cell-hematopoietic transition to produce HSC, and then HSC eventually migrates to the bone marrow throughout adulthood, where they maintain multilineage hematopoiesis, including T, B, NKT and NK lymphoid cells. Therefore, the generation of HSC and lymphoid effector cells from pluripotent stem cells depends on the ability to accurately recapitulate (recapitulate) the complex stages of early embryonic hematopoietic development into permanent programs by carefully designed and validated methods and compositions.

A limited number of studies have described the direct differentiation of hiPSC to permanent HE in vitro. The main obstacle to using hiPSC for therapeutic purposes is the need to co-culture these cells with mouse or human stromal cells in the presence of an unclear serum-containing culture medium to maintain pluripotency and induce differentiation. In addition, existing protocols have also adopted strategies including culturing iPSC to form embryoid bodies (EBs), which are heterogeneous aggregates of cells containing various differentiated cells of ectoderm, mesoderm, and endoderm. These methods require aggregating pluripotent cells to form clumps by, for example, rotation, allowing cells to settle and aggregate in wells or allowing passive aggregation and clump formation in suspension cultures. The formed EBs are maintained in a differentiation induction culture system for a certain duration, typically 7 to 10 days, to allow proper differentiation, and then the EBs are transferred to adherent cultures for further maturation, or dissociated into single cells for cell type selection, so as to proceed to subsequent differentiation steps. (Kennedy et al., Cell Reports 2012: 1722-1735; Knorr et al., Stem Cell Translational Medicine 2013(2): 274-283). For example, Kennedy et al. teach the generation of EBs for iPSC differentiation, in which pluripotent cells are treated with collagenase and trypsin to allow scraping of cells to form small aggregates, which are then cultured to form EBs. EB formation has been shown to promote pluripotent stem cell differentiation, however, the need to form aggregates and subsequent EBs is labor intensive, cell numbers are minimally increased in the process, and the cell contents in the three-dimensional EB aggregates are inconsistent and unevenly exposed to culture medium factors, which results in the formation of heterogeneous cell products at different stages of differentiation and greatly hinders the scalability and reproducibility of manufacturing processes that require efficient and streamlined processes.

Therefore, there is a need for methods and compositions for differentiating stem cells into definitive hematopoietic cells that do not rely on co-culture or serum-containing media and do not require the formation of embryoid body aggregates as an intermediate.

SUMMARY OF THE INVENTION

The present invention generally relates to cell culture conditions, media, culture platforms and methods for culturing and differentiating stem cells to a hematopoietic cell fate.

Specifically, the present invention provides methods and compositions for producing hematopoietic cell lineages under serum/no feeder conditions, and in expandable and monolayer culture platforms, without the need for EB formation, by permanent hemogenic endothelium (HE) and permanent hematopoietic stem cells (HSC) from pluripotent stem cells (including hiPSC). The range of cells that can be differentiated according to the method of the present invention ranges from pluripotent stem cells to progenitor cells that are determined as specific terminal differentiated cells and transdifferentiated cells, and the cells of various lineages are not directly converted into hematopoietic fates by pluripotent intermediates. Similarly, the range of cells produced by stem cell differentiation ranges from specialized stem cells or progenitor cells to terminally differentiated stem cells, and all intermediate hematopoietic cell lineages.

The present invention provides methods and compositions for differentiating and expanding hematopoietic lineage cells from pluripotent stem cells in monolayer culture, comprising contacting pluripotent solid cells with a BMP pathway activator and, optionally, bFGF. Thus, mesodermal cells derived from pluripotent stem cells are obtained and expanded without forming embryoid bodies from the pluripotent stem cells. The mesodermal cells are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded mesodermal cells with permanent hemogenic endothelial (HE) potential without forming embryoid bodies from the pluripotent stem cells. Subsequently, the mesodermal cells with permanent HE potential are differentiated into permanent HE cells by contact with bFGF and, optionally, a ROCK inhibitor and/or a WNT pathway activator, which are also expanded during the differentiation process.

The methods provided herein for obtaining cells of the hematopoietic lineage are advantageous over EB-mediated differentiation of pluripotent stem cells because EB formation results in moderate to minimal cell expansion, does not allow monolayer culture and uniform differentiation of cells in a population, which are important for many applications requiring uniform expansion and differentiation, and is laborious and inefficient.

Provided herein is a monolayer differentiation platform that facilitates differentiation into permanent hemogenic endothelium, resulting in the derivation of hematopoietic stem cells and differentiated progeny such as T, B, NKT, and NK cells. The monolayer differentiation strategy shown combines enhanced differentiation efficiency with large-scale expansion to deliver therapeutically relevant quantities of pluripotent stem cell-derived hematopoietic cells for various therapeutic applications. In addition, the present invention discloses that monolayer culture using the methods provided herein results in functional hematopoietic lineage cells that are capable of a full range of in vitro differentiation, ex vivo regulation, and in vivo long-term hematopoietic self-renewal, reconstruction, and transplantation.

One aspect of the present invention provides a culture platform for obtaining a hematopoietic cell line derived from pluripotent stem cells, comprising: Group I: (i) a culture medium comprising a BMP activator, and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, wherein the culture medium is optionally free of Wnt pathway activators and TGFβ receptor/ALK inhibitors, and is suitable for differentiating and expanding permanent HSCs from permanent hemogenic endothelium; (ii) a culture medium comprising a GSK3 inhibitor, a BMP activator and an optional TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for differentiating and expanding permanent hemogenic endothelium from mesoderm cells; and (iii) a culture medium comprising a GSK3 inhibitor, a BMP activator, wherein the culture medium is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells.

Alternatively, the culture platform for obtaining a hematopoietic cell line derived from a pluripotent stem cell comprises Group II: (i) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11, and optionally without a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for differentiating and expanding permanent hemogenic endothelium with permanent hemogenic endothelial potential from mesoderm cells; (ii) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor, and optionally without a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for obtaining mesoderm cells with permanent hemogenic endothelial potential; and (iii) a culture medium comprising a BMP activator, and optionally bFGF, wherein the culture medium is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells. In some embodiments, the pluripotent stem cells of the above-mentioned culture platform are iPSCs. In some embodiments, the iPSCs are initial iPSCs. In some embodiments, group (II) of the above culture platform further comprises: (iv) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and does not contain a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells.

In some embodiments of the above culture platform, each of Group (I) and Group (II) further comprises additional culture medium.

Group (I) may further comprise: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6; and one or more Notch pathway activators; wherein the culture medium does not contain a BMP activator and is suitable for differentiating pluripotent stem cell-derived T cell progenitors into T cells, or (ii) a culture medium comprising a BMP activator; one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6; and one or more Notch pathway activators, wherein the culture medium is suitable for differentiating pluripotent stem cell-derived definitive HSCs into T cell progenitors. These additional culture media are suitable for generating pluripotent stem cell-derived T cell lines.

Group (II) may further comprise: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7, but lacking one or more of VEGF, bFGF, a BMP activator and a ROCK inhibitor, wherein the culture medium is suitable for differentiating pluripotent stem cell-derived pre-T progenitor cells into T progenitor cells or T cells; or (ii) a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7, wherein the culture medium is suitable for differentiating pluripotent stem cell-derived definitive hemogenic endothelium into pre-T progenitor cells; and these additional culture media are suitable for generating pluripotent stem cell-derived T cell lines.

In some embodiments of the above culture platform, each of group (I) and group (II) further comprises an additional culture medium:

Group (I) may further comprise: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15, wherein the culture medium does not contain a BMP activator and is suitable for differentiating NK progenitor cells derived from pluripotent stem cells into NK cells; or (ii) a culture medium comprising a BMP activator; one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15; wherein the culture medium is suitable for differentiating definitive HSCs derived from pluripotent stem cells into NK progenitor cells; and these additional culture media are suitable for generating pluripotent stem cell-derived NK cell lines.

Regarding group (II), in addition to the above-mentioned culture medium, it may further comprise: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors and is suitable for differentiating pre-NK progenitor cells derived from pluripotent stem cells into NK progenitor cells or NK cells; or (ii) a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium is suitable for differentiating definitive hemogenic endothelium derived from pluripotent stem cells into pre-NK progenitor cells; and these additional culture media are suitable for generating pluripotent stem cell-derived NK cell lines.

In another embodiment, the provided culture platform, group (II), further comprises: (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, but without a ROCK inhibitor, wherein the culture medium is suitable for differentiating pre-HSCs derived from pluripotent stem cells into hematopoietic multipotent progenitor cells; (ii) a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, wherein the culture medium is suitable for differentiating definitive hemogenic endothelium derived from pluripotent stem cells into pre-HSCs; and these culture media are provided for generating hematopoietic multipotent progenitor cells derived from pluripotent stem cells.

Another aspect of the present invention provides a composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, comprising one or more of the following group (I) or group (II).

Group (I): (i) a culture medium comprising a BMP activator; one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO; and pluripotent stem cell-derived definitive hemogenic endothelium, wherein the culture medium is optionally free of Wnt pathway activators and TGFβ receptor/ALK inhibitors, and is suitable for differentiating and expanding definitive HSCs from pluripotent stem cell-derived definitive hemogenic endothelium; (ii) a culture medium comprising a GSK3 inhibitor, a BMP activator and optionally a TGFβ receptor/ALK inhibitor; and pluripotent stem cell-derived mesoderm cells, wherein the culture medium is suitable for differentiating and expanding definitive hemogenic endothelium from pluripotent stem cell-derived mesoderm cells; and (iii) a culture medium comprising a GSK3 inhibitor, a BMP activator; and iPSCs, wherein the culture medium is suitable for differentiating and expanding mesoderm cells from pluripotent stem cells.

Group (II): (i) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11; and mesodermal cells derived from pluripotent stem cells with definitive hemogenic endothelial potential, wherein the culture medium optionally does not contain a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for differentiating and expanding definitive hemogenic endothelium from mesodermal cells derived from pluripotent stem cells with hemogenic endothelial potential; (ii) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor, but does not contain a TGFβ receptor/ALK inhibitor; and mesodermal cells derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating and expanding mesodermal cells with definitive hemogenic endothelial potential from mesodermal cells derived from pluripotent stem cells; and (iii) a culture medium comprising a BMP activator, and optionally bFGF; and iPSCs, wherein the culture medium is suitable for differentiating and expanding mesodermal cells from pluripotent stem cells.

In some embodiments of the composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

In some embodiments of the composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, group (II) comprises additional components, such as (vi) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and not containing a TGFβ receptor/ALK inhibitor; and pluripotent stem cells; wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

In some embodiments of the above-mentioned composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, group (I) further comprises: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6; and one or more Notch pathway activators; and pluripotent stem cell-derived T cell progenitors, wherein the culture medium does not contain a BMP activator and is suitable for differentiating the pluripotent stem cell-derived T cell progenitors into T cells, or (ii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6; one or more Notch pathway activators; and pluripotent stem cell-derived definitive HSCs, wherein the culture medium is suitable for differentiating the pluripotent stem cell-derived definitive HSCs into T cell progenitors; and these additional culture media are suitable for generating pluripotent stem cell-derived T cell lines.

Regarding the above-mentioned group (II) of compositions for differentiating and expanding pluripotent stem cell-derived hematopoietic cells, in some embodiments, it further comprises: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, and IL7, but not comprising one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors; and pluripotent stem cell-derived pre-T progenitor cells, wherein the culture medium is suitable for differentiating pluripotent stem cell-derived pre-T progenitor cells into T progenitor cells or T cells; or (ii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, and IL7, and pluripotent stem cell-derived definitive hemogenic endothelium, wherein the culture medium is suitable for differentiating definitive hemogenic endothelium into pre-T progenitor cells. These additional culture media are suitable for generating pluripotent stem cell-derived T cell lines.

In another embodiment of the above-mentioned composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells, group (I) further comprises (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15; and NK progenitor cells derived from pluripotent stem cells, wherein the culture medium does not contain a BMP activator and is suitable for differentiating NK progenitor cells into NK cells; or (ii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15; and permanent HSCs derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating permanent HSCs derived from pluripotent stem cells into NK progenitor cells. These additional culture media are suitable for generating NK cell lines derived from pluripotent stem cells. Alternatively, the above-mentioned group (II) of the composition for differentiating and expanding pluripotent stem cell-derived hematopoietic cells further comprises: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, and not containing one or more of VEGF, bFGF, BMP activators and ROCK inhibitors; and pre-NK progenitor cells derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating pre-NK progenitor cells into NK progenitor cells or NK cells; or (ii) a culture medium comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15 and definitive hemogenic endothelium derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating definitive hemogenic endothelium into pre-NK progenitor cells. These culture media are suitable for generating pluripotent stem cell-derived NK cell lines.

In some other embodiments, the above-mentioned group (II) of the composition for differentiating and expanding hematopoietic cells derived from pluripotent stem cells further comprises one or more culture media for producing hematopoietic multipotent progenitor cells derived from pluripotent stem cells, wherein the culture media comprises: (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, but without a ROCK inhibitor, and pre-HSC derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating pre-HSC into hematopoietic multipotent progenitor cells; and/or (ii) a culture medium comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, and definitive hematopoietic endothelium derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating definitive hematopoietic endothelium into pre-HSC.

One aspect of the present invention provides a culture platform for generating a pluripotent stem cell-derived T cell line, comprising: Group I - (i) a culture medium comprising a GSK3 inhibitor and a BMP activator, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for differentiating and expanding definitive hemogenic endothelium from mesodermal cells; (iii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; wherein the culture medium is optionally free of Wn t pathway activator and TGFβ receptor/ALK inhibitor, and is suitable for differentiating and expanding definitive HSC from definitive hemogenic endothelium; and (iv) a culture medium comprising a BMP activator; one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6; and one or more Notch pathway activators, wherein the culture medium is suitable for differentiating T progenitor cells from definitive HSC; and optionally, (v) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6; and one or more Notch pathway activators; wherein the culture medium does not contain a BMP activator and is suitable for differentiating T cells from T progenitor cells.

Alternatively, the culture platform for generating pluripotent stem cell-derived T cell lines comprises: Group II - (i) a culture medium comprising a BMP activator and, optionally, bFGF, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally, without a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with definitive HE potential from mesodermal cells; (iii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6, and IL11, and optionally, without a TGFβ receptor/ALK inhibitor. wherein the culture medium is suitable for differentiating and expanding definitive hemogenic endothelium from mesodermal cells with definitive hemogenic endothelial potential; (iv) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7, and definitive hemogenic endothelium derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating definitive hemogenic endothelium into pre-T progenitor cells; and (v) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7, but without one or more of VEGF, bFGF, BMP activator and ROCK inhibitor; and pre-T progenitor cells derived from pluripotent stem cells, wherein the culture medium is suitable for differentiating pre-T progenitor cells into T progenitor cells or T cells.

In some embodiments of the above-described culture platform for generating a pluripotent stem cell-derived T cell line, the Group II culture platform further comprises: (vi) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and optionally lacking a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for seeding and expanding the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

Another aspect of the present invention provides a culture platform for generating NK cells derived from pluripotent stem cells, comprising: Group I - (i) a culture medium comprising a GSK3 inhibitor, a BMP activator, wherein the culture medium is suitable for differentiating pluripotent stem cells into mesodermal cells; (ii) a culture medium comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for differentiating mesodermal cells into definitive hemogenic endothelium; (iii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the culture medium can be (iv) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15; wherein the culture medium is suitable for differentiating permanent HSC into NK progenitor cells; and optionally, (v) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15, wherein the culture medium does not contain a BMP activator and is suitable for differentiating NK progenitor cells into NK cells.

Alternatively, the culture platform for generating pluripotent stem cell-derived NK cells comprises: Group II - (i) a culture medium comprising a BMP activator, and optionally bFGF, wherein the culture medium is suitable for differentiating and expanding mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor, and optionally without a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with definitive hemogenic endothelial potential from mesodermal cells; (iii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11, and optionally without a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with definitive hemogenic endothelial potential from mesodermal cells. (iv) a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium is suitable for differentiating definitive hemogenic endothelium from mesodermal cells with definitive hemogenic endothelial potential; (v) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors and is suitable for differentiating pre-NK progenitor cells into NK progenitor cells or NK cells.

In some embodiments of the above-mentioned culture platform for generating pluripotent stem cell-derived NK cells, the Group II culture platform further comprises: (vi) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and lacking a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

Another aspect of the present invention provides a culture platform for generating definitive hemogenic endothelium (iHE) derived from pluripotent stem cells, comprising: (i) a culture medium comprising a BMP activator, and optionally bFGF, wherein the culture medium is suitable for differentiating and expanding mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor, and optionally without a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with definitive hemogenic endothelial potential from pluripotent stem cell-derived mesoderm cells; and (iii) a culture medium comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6, IL11, wherein the culture medium optionally does not contain a TGFβ receptor/ALK inhibitor, and wherein the culture medium is suitable for differentiating and expanding definitive hemogenic endothelium from mesodermal cells with definitive hemogenic endothelial potential.

In some embodiments of the culture platform for generating pluripotent stem cell-derived definitive hemogenic endothelium (iHE), the culture platform further comprises (iv) a culture medium comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, and lacking a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells.

Another aspect of the present invention also provides a culture platform for producing hematopoietic multipotent progenitor cells derived from pluripotent stem cells, which comprises: (i) a culture medium comprising a BMP activator and, optionally, bFGF, wherein the culture medium is suitable for differentiating and expanding pluripotent stem cell-derived mesodermal cells from pluripotent stem cells; (ii) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor, and optionally, no TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for obtaining mesodermal cells with permanent hematopoietic endothelial potential from pluripotent stem cell-derived mesodermal cells; (iii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11; wherein the culture medium optionally does not contain TGFβ receptor /ALK inhibitor, wherein the culture medium is suitable for differentiating and expanding permanent hemogenic endothelium with permanent hemogenic endothelial potential from mesoderm cells; (iv) culture medium comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11; wherein the culture medium is suitable for differentiating permanent hemogenic endothelium into pre-HSC; and (v) culture medium comprising a BMP activator, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, wherein the culture medium does not contain a ROCK inhibitor, wherein the culture medium is suitable for differentiating pre-HSC into hematopoietic multipotent progenitor cells. In some embodiments, the culture platform further comprises (vi) culture medium comprising a MEK inhibitor, a GSK3 inhibitor and a ROCK inhibitor, and does not contain a TGFβ receptor/ALK inhibitor, wherein the culture medium is suitable for seeding and expanding pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs.

Another aspect of the present invention provides a method for directing the differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage, comprising: Group I - (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor and a BMP activator to initiate differentiation and expansion from pluripotent stem cells to mesoderm cells derived from pluripotent stem cells; (ii) contacting pluripotent stem cell-derived mesoderm cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor/ALK inhibitor to initiate differentiation and expansion from pluripotent stem cell-derived mesoderm cells to definitive hemogenic endothelial cells derived from pluripotent stem cells; and (iii) contacting pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the composition optionally does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor to initiate differentiation and expansion from hemogenic endothelial cells to definitive HSCs derived from pluripotent stem cells.

Alternatively, the method for directing the differentiation of pluripotent stem cells into cells of a definitive hematopoietic lineage comprises: Group II - (i) contacting pluripotent stem cells with a composition comprising a BMP activator and optionally bFGF to initiate differentiation and expansion from pluripotent stem cells to mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, wherein the composition optionally does not contain a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion from mesodermal cells to mesodermal cells with definitive HE potential; (iii) contacting mesodermal cells with definitive H E-potential mesodermal cells are contacted with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11; wherein the composition optionally does not contain a TGFβ receptor/ALK inhibitor to initiate differentiation and expansion from pluripotent stem cell-derived mesodermal cells with definitive hemogenic endothelial potential to definitive hemogenic endothelium; and optionally, the pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, mesodermal cells with hemogenic endothelium and/or definitive hemogenic endothelium are placed under a low oxygen tension of about 2% to about 10%.

In some embodiments of the method for guiding the differentiation of pluripotent stem cells into hematopoietic lineage cells, the group (II) method further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, wherein the composition does not contain a TGFβ receptor/ALK inhibitor to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs.

In some embodiments of the method for directing the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the differentiation of pluripotent stem cells into cells of the hematopoietic lineage does not produce embryoid bodies and is in a monolayer culture format.

In some embodiments of the above methods, the obtained pluripotent stem cell-derived definitive hemogenic endothelial cells are CD34+. In some embodiments, the obtained definitive hemogenic endothelial cells are CD34+CD43-. In some embodiments, the definitive hemogenic endothelial cells are CD34+CD43-CXCR4-CD73-.

In some other embodiments of the above methods, Group I further includes (i) contacting the pluripotent stem cell-derived definitive HSCs with a composition comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6, and one or more Notch pathway activators to initiate differentiation of the pluripotent stem cell-derived definitive HSCs into T progenitor cells and optionally contacting the T progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6; and one or more Notch pathway activators to initiate differentiation of the T progenitor cells into T cells. In some embodiments, the method of Group II further comprises (i) contacting the pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7 to initiate differentiation of the definitive hemogenic endothelium into pre-T progenitor cells; and optionally, (ii) contacting the pre-T progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7, but without VEGF, bFGF, BMP activators and ROCK inhibitors to initiate differentiation of the pre-T progenitor cells into T progenitor cells or T cells. In some embodiments of the method, the pluripotent stem cell-derived T progenitor cells are CD34+CD7+.

In some other embodiments of the above-mentioned method for guiding the differentiation of pluripotent stem cells into cells of the hematopoietic lineage, the method of Group I further comprises: (i) contacting the pluripotent stem cell-derived definitive HSC with a composition comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15 to initiate the differentiation of the definitive HSC into NK progenitor cells; and optionally, (ii) contacting the NK progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15, but without a BMP activator, to initiate the differentiation of the NK progenitor cells into NK cells; or the method of Group II further comprises : (i) contacting pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15, and a BMP activator to initiate differentiation of the definitive hemogenic endothelium into pre-NK progenitor cells; and optionally, (ii) contacting pluripotent stem cell-derived pre-NK progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors to initiate differentiation of pre-NK progenitor cells into NK progenitor cells or NK cells. In some embodiments, the method of Group II further comprises: (i) contacting the pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to initiate differentiation of the definitive hemogenic endothelium into pre-HSC; (ii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but without a ROCK inhibitor, wherein the culture medium is suitable for differentiating the pluripotent stem cell-derived pre-HSC into hematopoietic multipotent progenitor cells. In some embodiments, the pluripotent stem cell-derived definitive HSC obtained using the above method is CD34+CD45+ and suitable for long-term transplantation.

Another aspect of the present invention provides a method for generating a T cell line derived from pluripotent stem cells, comprising: Group I - (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor and a BMP activator to initiate differentiation and expansion from pluripotent stem cells to mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a GSK3 inhibitor, a BMP activator, and optionally a TGFβ receptor/ALK inhibitor to initiate differentiation and expansion from mesodermal cells to definitive hemogenic endothelium; (iii) contacting definitive hemogenic endothelium with a composition comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; wherein the composition does not contain Wn t pathway activator and TGFβ receptor/ALK inhibitor to initiate differentiation and expansion from definitive hemogenic endothelium to definitive HSC; and (iv) contacting definitive HSC with a composition comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6, and one or more Notch pathway activators to initiate differentiation of definitive HSC into T progenitor cells; and optionally, (v) contacting T progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6; one or more Notch pathway activators; but without a BMP activator to initiate differentiation of T progenitor cells into T cells.

Alternatively, the method for generating a pluripotent stem cell-derived T cell line comprises: Group II - (i) contacting pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to initiate differentiation and expansion from pluripotent stem cells to mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, but without a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion from mesodermal cells to mesodermal cells with definitive HE potential; (iii) contacting mesodermal cells with definitive HE potential with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6, and IL11; wherein the composition does not contain a TGFβ receptor/ALK inhibitor, to initiate differentiation from mesodermal cells with definitive HE potential. (iv) contacting the definitive hemogenic endothelium with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7 to initiate differentiation of the definitive hemogenic endothelium into pre-T progenitor cells; and (v) contacting the pre-T progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7, wherein the composition does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors; to initiate differentiation of the pre-T progenitor cells into T progenitor cells or T cells; and optionally, the seeded pluripotent stem cells, mesodermal cells, mesodermal cells with definitive HE potential and/or definitive hemogenic endothelium can be placed under a low oxygen tension of about 2% to about 10%. In some embodiments, the above methods of Group II further comprise: contacting iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor/ALK inhibitor, to seed and expand pluripotent stem cells; and/or wherein the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. In some embodiments of the method, the differentiation of pluripotent stem cells into T cell lineages does not produce embryoid bodies and is in a monolayer culture format.

Another aspect of the present invention provides a method for generating a NK cell line derived from pluripotent stem cells, comprising: Group I - (i) contacting pluripotent stem cells with a composition comprising a GSK3 inhibitor and a BMP activator to initiate differentiation of the pluripotent stem cells into mesoderm cells; (ii) contacting the mesoderm cells with a composition comprising a GSK3 inhibitor, a BMP activator and an optional TGFβ receptor/ALK inhibitor to initiate differentiation of the mesoderm cells into definitive hemogenic endothelium; (iii) contacting the definitive hemogenic endothelium with a composition comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, wherein the composition does not contain Wn t pathway activator and TGFβ receptor/ALK inhibitor; to initiate the differentiation of pluripotent stem cell-derived definitive hemogenic endothelium into definitive HSC; and (iv) contacting definitive HSC with a composition comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15 to initiate the differentiation of definitive HSC into NK progenitor cells; and optionally, (v) contacting pluripotent stem cell-derived NK progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15, but without a BMP activator, to initiate the differentiation of NK progenitor cells into NK cells. Alternatively, the method for generating a pluripotent stem cell-derived NK cell line comprises: Group II - (i) contacting pluripotent stem cells with a composition comprising a BMP activator and, optionally, bFGF, to initiate differentiation and expansion from pluripotent stem cells to mesodermal cells; (ii) contacting mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally, without a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion from mesodermal cells to mesodermal cells with definitive HE potential; (iii) contacting mesodermal cells with definitive HE potential with a composition comprising one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6, and IL11, and a ROCK inhibitor, and optionally, without a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion from pluripotent stem cell-derived mesodermal cells with definitive HE potential to definitive hemogenic endothelium derived from pluripotent stem cells ; (iv) contacting the pluripotent stem cell-derived definitive hemogenic endothelium with a composition comprising one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15, a BMP activator and a ROCK inhibitor to initiate the differentiation of the pluripotent stem cell-derived definitive hemogenic endothelium to pre-NK progenitor cells; and (v) contacting the pluripotent stem cell-derived pre-NK progenitor cells with a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, but without one or more of VEGF, bFGF, BMP activator and ROCK inhibitor to initiate the differentiation of the pluripotent stem cell-derived pre-NK progenitor cells to pluripotent stem cell-derived NK progenitor cells or NK cells; and optionally, subjecting the seeded pluripotent stem cells, pluripotent stem cell-derived mesoderm cells and/or definitive hemogenic endothelium to a low oxygen tension of about 2% to about 10%. In some embodiments, the group II method for generating a pluripotent stem cell-derived NK cell line further comprises contacting the iPSC with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor/ALK inhibitor, to seed and expand the iPSC. In some embodiments, the iPSC is an initial iPSC. In some embodiments, the method for generating a pluripotent stem cell-derived NK cell line does not generate embryoid bodies and is a monolayer culture format.

Another aspect of the present invention provides a method for generating definitive hemogenic endothelium derived from pluripotent stem cells, comprising: (i) contacting iPSCs with a composition comprising a BMP activator and, optionally, bFGF, to initiate differentiation and expansion of pluripotent stem cells into mesodermal cells derived from pluripotent stem cells; (ii) contacting pluripotent stem cell-derived mesodermal cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor, and optionally, without a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion of pluripotent stem cell-derived mesodermal cells into pluripotent stem cell-derived mesodermal cells with definitive HE potential. (iii) contacting the pluripotent stem cell-derived mesodermal cells with definitive HE potential with a composition comprising one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6, and IL11; and a ROCK inhibitor, and optionally, a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion of the pluripotent stem cell-derived mesodermal cells with definitive HE potential into pluripotent stem cell-derived definitive hemogenic endothelium; and optionally, subjecting the seeded pluripotent stem cells, pluripotent stem cell-derived mesodermal cells, and/or definitive hemogenic endothelium to a low oxygen tension of about 2% to about 10%. In some embodiments, the above method for generating pluripotent stem cell-derived definitive hemogenic endothelium further comprises: contacting iPSCs with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor, but without a TGFβ receptor/ALK inhibitor, to seed and expand the iPSCs; and/or wherein the iPSCs are naive iPSCs. In some embodiments, the above-described method of differentiating iPSCs into definitive hemogenic endothelial cells does not generate embryoid bodies and is in a monolayer culture format.

Another aspect of the present invention provides a method for generating multipotent hematopoietic lineage progenitor cells derived from pluripotent stem cells, comprising: (i) contacting iPSCs with a composition comprising a BMP activator and, optionally, bFGF, to initiate differentiation and expansion of iPSCs into mesodermal cells derived from pluripotent stem cells; (ii) contacting mesodermal cells derived from pluripotent stem cells with a composition comprising a BMP activator, bFGF, and a GSK3 inhibitor but without a TGFβ receptor/ALK inhibitor to initiate differentiation and expansion of the mesodermal cells into mesodermal cells with definitive HE potential; (iii) contacting mesodermal cells with definitive HE potential with a composition comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6, and IL11, wherein the composition does not contain a TGFβ receptor/ALK inhibitor, to initiate differentiation and expansion of the mesodermal cells into mesodermal cells with definitive HE potential. (iv) contacting the definitive hemogenic endothelium with a composition comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to initiate differentiation of the definitive hemogenic endothelium into pre-HSCs; and (v) contacting the pre-HSCs with a composition comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, but without a ROCK inhibitor, to initiate differentiation of the pre-HSCs into multipotent hematopoietic progenitor cells; and optionally, subjecting the seeded pluripotent stem cells, mesoderm cells, and/or definitive hemogenic endothelium to a low oxygen tension of about 2% to about 10%. In some embodiments, the above-mentioned method for producing hematopoietic multipotent progenitor cells derived from pluripotent stem cells further comprises contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor but without a TGFβ receptor/ALK inhibitor to seed and expand the pluripotent stem cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. In some embodiments, using the above-mentioned method to differentiate pluripotent stem cells into hematopoietic multipotent progenitor cells does not produce embryoid bodies and is in a monolayer culture format.

A further aspect of the present invention provides a composition comprising: one or more cell populations generated from the culture platform disclosed herein: (i) CD34+ definitive hemogenic endothelium (iCD34) derived from pluripotent stem cells, wherein iCD34 cells are capable of differentiating into multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, and NK cells, and wherein iCD34 cells are CD34+CD43-; (ii) definitive hemogenic endothelium (iHE), wherein iHE cells are CD34+; (iii) definitive hemogenic endothelium derived from pluripotent stem cells; (iv) hematopoietic multipotent progenitor cells, wherein iMPP cells are CD34+CD45+; (v) T cell progenitor cells, wherein T cell progenitor cells are CD34+CD7+; (vi) T cells, wherein T cells are CD4+ or CD8+; (vii) NK cell progenitor cells, wherein NK cell progenitor cells are CD56+CD7+CD161+; and (viii) NK cells, wherein NK cells are CD56+CD57+CD16+CD94-.

Yet a further aspect of the present invention provides one or more cell lines, or clonal cells produced using the methods disclosed herein: (i) CD34+ definitive hemogenic endothelium (iCD34) derived from pluripotent stem cells, wherein iCD34 cells are capable of differentiating into multipotent progenitor cells, T cell progenitor cells, NK cell progenitor cells, T cells, and NK cells, and wherein iCD34 cells are CD34+CD43-; (ii) definitive hemogenic endothelium (iHE), wherein the iHE cell line or clonal cell is CD34+; (iii) definitive HSC, wherein iHSCs are CD34+CD45+; (iv) hematopoietic multipotent progenitor cells (iMPPs), wherein iMPP cells are CD34+CD45+; (v) T progenitor cells, wherein T progenitor cells are CD34+CD7+; (vi) T cells, wherein T cells are CD4+ or CD8+; (vii) NK progenitor cells, wherein NK progenitor cells are CD56+CD7+CD161+; and (viii) NK cells, wherein NK cells are CD56+CD57+CD16+CD94-.

Another aspect of the present invention provides a method for promoting hematopoietic self-renewal, reconstitution, or transplantation using one or more cell populations, cell lines, or clones produced by the disclosed methods: (i) CD34+ definitive hemogenic endothelium (iCD34) derived from pluripotent stem cells, wherein the iCD34 cells are capable of differentiating into multipotent progenitor cells, T cell progenitors, NK cell progenitors, T cells, and NK cells, and wherein the iCD34 cells are CD34+CD43-; (ii) definitive hemogenic endothelium (iHE), wherein the iHE cell line or clone is CD34+; (iii) definitive HSCs, wherein iHSCs are CD34+CD45+; (iv) hematopoietic multipotent progenitor cells, wherein iMPP cells are CD34+CD45+; (v) T progenitor cells, wherein T progenitor cells are CD34+CD7+; (vi) T cells, wherein T cells are CD4+ or CD8+; (vii) NK progenitor cells, wherein NK progenitor cells are CD56+CD7+CD161+; and (viii) NK cells, wherein NK cells are CD56+CD57+CD16+CD94-.

In short, the present invention provides methods and compositions that enable the direct differentiation of pluripotent stem cell monolayers without generating embryoid bodies from pluripotent stem cells, thereby enabling the differentiation and expansion of mesodermal cells, definitive HE and definitive HSC, among other hematopoietic cell lineages that can be obtained in a scalable, reliable format with very high efficiency levels.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 shows an exemplary schematic diagram of the multi-stage process of hematopoietic differentiation of human induced pluripotent stem cells (hiPSCs) into fully differentiated T cells.

FIG2 shows an exemplary schematic diagram of the multi-stage process of hematopoietic differentiation of hiPSCs into fully differentiated NK cells.

Figures 3A-3E show morphological changes indicating the transition from hiPSCs to hematopoietic cells over the course of 9 days.

4A-4D show the expression profiles of stem cells as they fully transition from pluripotent stem cells to a hematopoietic fate.

5A-5C show the CD34/CD45 expression profiles of hiPSC-differentiated hematopoietic cells generated by various methods and starting conditions.

6A-6C show the improvement in CD34 differentiation efficiency when cultured as a monolayer in the described differentiation medium.

FIG7 shows the survival and proliferation of hematopoietic differentiated cells under ROCK inhibition after single cell dissociation.

FIG. 8 shows maintenance of CD34 expression in hematopoietic cells differentiated from hiPSCs after 22 days of culture.

Figures 9A-9B show in vivo reconstitution of CD34+ cells transplanted in the presence of cells expressing only the human CD45 marker and reconstitution of CD34+ cells in the presence of T and B cells. The CD34+ cells were derived from naive hiPSCs cultured in monolayer format with a GSK3 inhibitor without forming EBs or aggregated intermediates.

Figure 10 shows CD56-positive cells and single-positive CD4 and CD8 cells, identified from a ^CD56- /CD7 ⁺ /CD3 ⁺ /TCRαβ ⁺ gating strategy, when hiPSC-derived CD34-positive cells were further cultured in differentiation medium without stromal cells and in the presence of soluble DLL1 and DLL4 recombinant peptides (T cells only).

Figure 11 shows that hiPSC-derived CD34-positive cells are responsive to pharmacological modulation as indicated by enhanced PD-L1 surface expression.

Figure 12 shows a simplified diagram of the multi-stage process of hematopoietic differentiation of induced pluripotent stem cells (iPSCs) into definitive hemogenic endothelium (iHE) and multipotent progenitor cells (iMPPs). Note that this culture can be converted to fully defined by replacing vitronectin with Matrigel ^™ .

Figure 13 shows a simplified diagram of the multi-stage process of hematopoietic differentiation of induced pluripotent stem cells into T progenitor cells (iproT) and fully differentiated T (iT) cells. Note that this culture can be converted to fully defined by using Matrigel ^™ instead of vitronectin.

Figure 14 shows a simplified diagram of the multi-stage process of hematopoietic differentiation of induced pluripotent stem cells into NK progenitor cells (iproNK) and fully differentiated NK (iNK) cells. Note that this culture can be converted to fully defined by using Matrigel ^™ instead of vitronectin.

Figures 15A-C show flow cytometric profiles depicting the appearance of iHE after a 10-day process and the generation of iCD34 and iHE cells per iPSC differentiation. Calculations were based on photographs of representative and non-optimized cultures.

Figures 16A-E show protocol modifications, including seeding density and growth factor titration, to improve HE generation on day 10. A) Seeding density on day 0 affects HE population on day 10. B) BMP4 concentration on days 2-6 affects HE population on day 10. C) CHIR concentration on day 3.75 affects HE population on day 10. D) Seeding density on day 6 affects HE population on day 10. E) Addition of IGF1 and EPO on day 8 reduces HE on day 10.

Figure 17A-B shows that HE at day 10 exhibits multipotent and definitive hematopoiesis dependent on the Notch signaling pathway. A) Morphological changes during the 7-day MPP assay as determined by flow cytometry of newly emerged CD45+ hematopoietic cells. B) iPSC-derived CD34+ cells generate Notch-dependent definitive CD45+ cells during the iMPP assay.

Figures 18A-B show the effect of differentiation under hypoxic conditions on the generation of iHE and iMPP hematopoietic progenitor cells. A) Monolayer differentiation under hypoxia increases the percentage of iCD34-positive cells and iHE cells at day 10. B) iCD34+ HE cells generated under hypoxia on day 10 can be further differentiated in the iMPP assay.

Figures 19A-D show the ability of unsorted or sorted day 10 cultures to be cryopreserved and maintain hematopoietic potential. A) Cryopreserved day 10 unsorted differentiated cultures can survive and generate CD45+ hematopoietic cells during the iMPP assay. B), C), and D) Cryopreserved day 10 iCD34+ sorted cells can survive and generate CD45+ hematopoietic cells during the iMPP assay.

Figures 20A-B show that day 10 differentiated cultures can be shipped overnight at ambient temperature without losing HE potential. A) Day 7 cultures were either maintained in an incubator (control) or shipped overnight and then reintroduced into the incubator for an additional two days. Both cultures were then analyzed for the presence of iCD34 and iHE cells on day 10. In the overnight shipped cultures, T-flasks contained either 30% medium containing 70% matrix or 100% medium. B) Cell number enumeration.

Figure 21A-C shows the early CD34+CD7+ T progenitors and mature CD4+ and CD8+ T cell subsets derived from hiPSCs using a CD45+CD56- gating method. A) The early T cell lineage signature marks the presence of iproT cells defined by CD34+/CD7+. B) The mature T cell signature marks the presence of mature T cells defined by CD4+ or CD8+ cells. C) Comparison of the potential of CD34-positive cells and iCD34-positive cells from umbilical cord blood to generate iproT cells after 5 days of T cell differentiation.

Figures 22A-C show the early CD56+CD7+CD161+ NK cell progenitors and mature CD56+CD16+CD8+ NK cell subsets derived from hiPSCs using a CD45+ gating method. A) The early NK lineage signature marks the presence of iproNK cells defined by CD7 and CD56. B) The mature NK lineage signature marks the presence of mature NK cells defined by CD57, CD16, CD94, and CD56. C) Comparison of the potential of CD34-positive cells and iCD34-positive cells from umbilical cord blood to generate iproNK cells after 5 days of NK cell differentiation.

Figures 23A-C demonstrate that the monolayer hiPSC hematopoietic differentiation platform allows for scalable expansion strategies not available during EB formation. A. hiPSCs were aggregated to form embryoid bodies and differentiated for 14 days before analysis of CD34 and 43 expression. B. hiPSC monolayers were seeded and differentiated for 8 days, followed by analysis of CD34, 43, CXCR4, and CD73. C. CD34-positive cells were counted and hematopoietic differentiation in monolayer and EB cultures was plotted over time.

Figure 24 shows a simplified diagram of the scale-up strategy for the monolayer hiPSC hematopoietic differentiation platform to produce finished iNK and iT cells. Calculations were performed based on photographs of representative and non-optimized cultures.

FIG. 25 shows that hiPSC-derived CD34-positive cells have immunomodulatory properties by suppressing CD3+ T cell survival.

FIG26 shows hiPSC-derived mature CD4+ and CD8+ T cell subsets using CD45+CD56- gating.

FIG27 shows that feeder-based suspension culture supports maturation of iCD34-derived NK cells.

FIG28 shows that iCD34-derived iNK cells can secrete proinflammatory cytokines in response to cytokine stimulation in a similar pattern to peripheral blood NK cells.

FIG29 shows that stroma-free differentiation of pro-NK cells from cord blood CD34-positive cells using a CD45+ gating approach is more rapid than conventional stroma-based differentiation platforms.

Figure 30 shows the matrix-free differentiation of iPSC-derived iCD34+ cells into NK cells. Plate-bound DLL4 supports the differentiation of CD56+CD7+CD161+ NK progenitor cells, but not CD11b+ myeloid cells.

FIG31 shows the stroma-free differentiation of UCB CD34+ cells into T cells.

FIG32 shows the matrix-free differentiation of iPSC-derived iCD34+ cells into T cells.

FIG33 shows the transplantation of hiPSC-derived iCD34+ cells.

Detailed Description of the Invention

The present invention generally relates to methods and compositions for differentiating stem cells toward a permanent hematopoietic cell fate. More specifically, the present invention provides a multi-stage differentiation platform in which iPSCs or iPSC-derived cells at different developmental stages can be induced to produce a permanent hematopoietic phenotype, ranging from permanent hematopoietic endothelium to fully differentiated hematopoietic cells, the latter including T cells, B cells, NKT cells, and NK cells. That is, the present invention provides methods and compositions for making cells more likely to form a permanent hematopoietic fate, such as CD34+ permanent hematopoietic stem cells. Alternatively, the methods and compositions of the present invention generate permanent hematopoietic endothelium (HE) in a scaled manner from initial iPSCs by avoiding the formation of EBs or aggregates.

A. Definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention belongs. For purposes of the present invention, the following terms are defined below. The articles "a", "an" and "the" are used herein to refer to one or more than one (i.e., at least one) grammatical object of the article. For example, "element" means one element or more than one element.

The use of alternatives (eg, "or") should be understood to mean either, both, or any combination of the alternatives.

The term "and/or" should be understood to mean either or both of the alternatives.

As used herein, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that varies by up to 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the term "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that ranges by ±15%, ±10%, ±9%, ±8%, ±7%, ±6%, ±5%, ±4%, ±3%, ±2%, or ±1% around a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

As used herein, the terms "substantially" or "generally" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more compared to a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length. In one embodiment, the terms "substantially the same" or "substantially the same" refer to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that is about the same range as a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length.

As used herein, the terms "substantially free" and "substantially free" are used interchangeably and, when used to describe a composition, such as a cell population or culture medium, refer to a composition that is free of a specified substance or its source, e.g., a composition that is 95% free, 96% free, 97% free, 98% free, 99% free of a specified substance or its source, or is undetectable by conventional means. The term "free" or "substantially free" of a component or substance in a composition also means that the component or substance (1) is not included in the composition at any concentration, or (2) has an inert function in the composition but is present at a low concentration. Similar meanings may be applied to the term "absent" when referring to the absence of a particular substance or its source from a composition.

As used herein, the term "assessable" refers to a quantity, level, value, number, frequency, percentage, yardstick, size, amount, weight or length scale that can be easily detected by one or more standard methods. The terms "not-appreciable" and "not appreciable" and equivalents refer to a quantity, level, value, number, frequency, percentage, yardstick, size, amount, weight or length scale that can not be easily detected or can not be detected by standard methods. In one embodiment, an event is not assessable if it occurs at a rate less than 5%, 4%, 3%, 2%, 1%, 0.1%, 0.01%, 0.001% or less.

Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises," and "comprising" should be understood to imply the inclusion of a stated step or element or group of steps or elements, but not the exclusion of any other step or element or group of steps or elements. In particular embodiments, the terms "comprises," "comprising," "having," "containing," and "including" are used synonymously.

“Consisting of is meant to include and be limited to whatever follows the phrase “consisting of.” Thus, the phrase “consisting of indicates that the listed elements are required or mandatory, and other elements may not be present.

"Consisting essentially of is meant to include any elements listed after the phrase and is limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure of the listed elements. Thus, the phrase "consisting essentially of indicates that the listed elements are required or mandatory, but other elements are not optional and may or may not be present depending on whether they affect the activity or action of the listed elements.

Reference throughout this specification to "one embodiment," "an embodiment," "a specific embodiment," "a related embodiment," "an embodiment," "an additional embodiment," or "a further embodiment," or combinations thereof, means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the aforementioned phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The term "ex vivo" generally refers to an activity occurring outside an organism, such as an experiment or measurement completed in or on a living tissue in an artificial environment outside an organism, preferably with minimal change in natural conditions. In a specific embodiment, an "ex vivo" procedure relates to living cells or tissues obtained from an organism, and is cultivated in a laboratory instrument, usually under aseptic conditions, and usually for several hours or up to about 24 hours, but including up to 48 or 72 hours, depending on the environment. In some embodiments, such tissues or cells can be collected and frozen, and subsequently thawed for ex vivo processing. Using living cells or tissues to continue tissue culture experiments or procedures longer than a few days is generally considered "in vitro", although in some embodiments, the term can be used interchangeably with ex vivo.

The term "in vivo" generally refers to activities that occur within an organism.

As used herein, the term "mesoderm" refers to one of the three germ layers that emerge during early embryogenesis and gives rise to a variety of specialized cell types, including blood cells of the circulatory system, muscle, heart, dermis, skeleton, and other supportive and connective tissues.

As used herein, the term "definitive hemogenic endothelium" (HE) or "pluripotent stem cell-derived definitive hemogenic endothelium" (iHE) refers to a subpopulation of endothelial cells that give rise to hematopoietic stem and progenitor cells in a process known as the endothelial-hematopoietic transition. The development of embryonic hematopoietic cells proceeds sequentially from the lateral plate mesoderm through angioblasts to definitive hemogenic endothelium and hematopoietic progenitor cells.

As used herein, the term "hematopoietic stem cell" or "definitive hematopoietic stem cell" refers to a CD34+ stem cell that is capable of giving rise to mature myeloid and lymphoid cell types, including T cells, natural killer cells, and B cells.

As used herein, the term "reprogramming" or "dedifferentiation" or "increasing cell potential" or "increasing developmental potential" refers to a method of increasing cell potential or dedifferentiating a cell into a less differentiated state. For example, a cell with increased cell potential has more developmental plasticity (i.e., it can be differentiated into more cell types) compared to the same cell in a non-reprogrammed state. In other words, a reprogrammed cell is a cell in a less differentiated state than the same cell in a non-reprogrammed state.

As used herein, the term "differentiation" is the process by which a nonspecific ("free") or less specific cell acquires the characteristics of a specific cell (e.g., a blood cell or a muscle cell). A differentiated or differentiation-induced cell is a cell that has taken on a more specific ("committed") position in a cell lineage. The term "committed," when applied to a differentiation process, refers to a cell that has progressed to a point in a differentiation pathway where, under normal circumstances, it would continue to differentiate into a specific cell type or subset of cell types and, under normal circumstances, would not differentiate into a different cell type or would revert to a less differentiated cell type.

As used herein, the term "differentiation marker gene" or "differentiation gene" refers to a gene whose expression indicates cell differentiation occurring in a cell, such as a pluripotent cell. Differentiation marker genes include, but are not limited to, the following genes: FOXA2, FGF5, SOX17, XIST, NODAL, COL3A1, OTX2, DUSP6, EOMES, NR2F2, NR0B1, CXCR4, CYP2B6, GATA3, GATA4, ERBB4, GATA6, HOXC6, INHA, SMAD6, RORA, NIPBL, TNFSF11, CDH11, ZIC4, GAL , SOX3, PITX2, APOA2, CXCL5, CER1, FOXQ1, MLL5, DPP10, GSC, PCDH10, CTCFL, PCDH20, TSHZ1, MEGF10, M YC, DKK1, BMP2, LEFTY2, HES1, CDX2, GNAS, EGR1, COL3A1, TCF4, HEPH, KDR, TOX, FOXA1, LCK, PCDH7, CD1D FOXG1, LEFTY1, TUJ1, T gene (Brachyury), ZIC1, GATA1, GATA2, HDAC4, HDAC5, HDAC7, HDAC9, NOTCH1, NOTCH2, NOTCH4, PAX5, RBPJ, RUNX1, STAT1 and STAT3.

As used herein, the terms "differentiation marker gene profile" or "differentiation gene profile," "differentiation gene expression profile," "differentiation gene expression signature," "differentiation gene expression panel," "differentiation gene set," or "differentiation gene signature" refer to the expression or expression levels of multiple differentiation marker genes.

As used herein, the term "potency" refers to the sum of all developmental options accessible to a cell (i.e., developmental potential). The continuum of cell potential includes, but is not limited to, totipotent cells, pluripotent cells, multipotent cells, oligopotent cells, unipotent cells, and terminally differentiated cells.

As used herein, the term "pluripotent" refers to the ability of cells to form all lineages of a body or soma (i.e., embryonic body). For example, embryonic stem cells are a type of pluripotent stem cells that can form cells from each of the three germ layers: ectoderm, mesoderm, and endoderm. Pluripotency is a continuum of developmental potential, ranging from incomplete or partially pluripotent cells (e.g., ectoderm stem cells or EpiSCs) that cannot produce complete organs to original, more pluripotent cells (e.g., embryonic stem cells) that can produce complete organs.

As used herein, the term "induced pluripotent stem cells," or iPSCs, means stem cells generated from differentiated adult, neonatal, or fetal cells that have been induced or altered, i.e., reprogrammed to be capable of differentiating into tissues of all three germinal or dermal layers: mesoderm, endoderm, and ectoderm. The iPSCs produced do not refer to cells found in nature.

As used herein, the term "embryonic stem cell" refers to the naturally occurring pluripotent stem cells of the inner cell mass of an embryonic blastocyst. Embryonic stem cells are pluripotent and, during development, give rise to all derivatives of the three major germ layers: ectoderm, endoderm, and mesoderm. They do not contribute to the extraembryonic membranes or the placenta, i.e., they are not totipotent.

As used herein, the term "multipotent stem cell" refers to a cell with the developmental potential to differentiate into cells of one or more germ layers (ectoderm, mesoderm and endoderm), but not all three. Therefore, multipotent cells may also be referred to as "partially differentiated cells". Multipotent cells are well known in the art, and examples of multipotent cells include adult stem cells, such as hematopoietic stem cells and neural stem cells. "Multipotent" means that a cell may form many types of cells in a given lineage, but cannot form cells of other lineages. For example, a multipotent hematopoietic cell can form many different types of blood cells (erythrocytes, leukocytes, platelets, etc.), but cannot form neurons. Therefore, the term "multipotency" refers to a cell state in which the degree of developmental potential is lower than that of omnipotence and pluripotency.

The differentiation of pluripotent stem cells requires the change of culture system, such as changing the stimulant in culture medium or the physical state of cell. The most conventional strategy is to utilize the formation of embryoid bodies (EBs) as the common and key intermediate for starting lineage-specific differentiation. EBs are three-dimensional clusters that have been shown to simulate embryonic development because they have produced many lineages in their three-dimensional regions. Through the differentiation process, usually a few hours to a few days, simple EBs (for example, causing the aggregation pluripotent stem cells of differentiation) continue to mature and develop into cystic EBs, and now usually a few days to a few weeks, they are further processed to continue differentiation. By making pluripotent stem cells close to each other in three-dimensional multilayer cell clusters to start EB formation, usually this is achieved by one of several methods, including allowing pluripotent cells to precipitate in droplets, cells are precipitated to " U " bottom well plates or by mechanical agitation. In order to promote EB development, pluripotent stem cell aggregates need further differentiation inducement, because the aggregates maintained in pluripotent culture maintenance medium do not form suitable EBs. Therefore, pluripotent stem cell aggregates need to be transferred to differentiation medium, and this differentiation medium provides inducing factors for selected lineage. The pluripotent stem cell culture based on EB usually results in the differentiated cell group (ectoderm, mesoderm and endoderm germ layer) that produces appropriate proliferation in the EB cell cluster. Although it has been proved that cell differentiation can be promoted, because the cell in the three-dimensional structure is discontinuously exposed to the differentiation factors from environment, EB causes heterogeneous cells under different differentiation states. In addition, EB is difficult to create and maintain. In addition, the cell differentiation by EB is accompanied by appropriate cell amplification, and this also causes low differentiation efficiency.

In contrast, "aggregate formation", which is different from "EB formation", can be used to induce differentiation of pluripotent stem cells and/or expand cell populations derived from pluripotent stem cells. For example, during the expansion of pluripotent stem cells based on aggregates, culture medium is selected to maintain proliferation and pluripotency. Cell proliferation typically increases the size of aggregates, forming larger aggregates, which can be routinely dissociated into smaller aggregates by mechanical or enzymatic means to maintain cell proliferation within the culture and increase cell number. Unlike EB culture, cells cultured in aggregates in maintenance culture maintain pluripotency markers.

As used herein, "monolayer differentiation" refers to a term that is different from differentiation by three-dimensional multilayer cell clusters, i.e., "EB formation." Monolayer differentiation, along with other advantages disclosed herein, avoids the need for EB formation for differentiation initiation. Because monolayer culture cannot mimic embryonic development such as EB formation, differentiation toward specific lineages is minimal compared to differentiation across all three germ layers in EBs.

Pluripotency can be determined in part by evaluating cells for pluripotency characteristics. Pluripotency characteristics include, but are not limited to: (i) pluripotent stem cell morphology; (ii) the potential for unlimited self-renewal, (iii) expression of, but not limited to, the following pluripotent stem cell markers: SSEA1 (mouse only), SSEA3/4, SSEA5, TRA1-60/81, TRA1-85, TRA2-54, GCTM-2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD90, CD105, OCT4, NANOG, SOX2, CD30, and/or CD50; (iv) the ability to differentiate into all three somatic cell lineages (ectoderm, mesoderm, and endoderm), (v) teratoma formation composed of three somatic cell lineages; and (vi) embryoid body formation composed of cells from three somatic cell lineages.

Two types of pluripotency have been previously described: a "primed" or "metastable" state similar to the pluripotency of epiblast stem cells (EpiSCs) of late blastocysts, and a "naive" or "basal" state similar to the pluripotency of the inner cell mass of early/preimplantation blastocysts. While both pluripotent states exhibit the characteristics described above, the naive or basal state also exhibits: (i) pre-inactivation or regeneration of the X chromosome in female cells, (ii) improved clonogenicity and survival during single-cell culture, (iii) an overall reduction in DNA methylation, (iv) a reduction in the deposition of H3K27me3 repressive chromatin marks on developmental regulatory gene promoters, and (v) reduced expression of differentiation markers relative to primed pluripotent cells. Standard methods of cell reprogramming in which exogenous pluripotency genes are introduced into somatic cells, expressed, and subsequently silenced or removed from the resulting pluripotent cells generally exhibit characteristics of a primed pluripotent state. Under standard pluripotent cell culture conditions, such cells remain in a primed state unless exogenous transgene expression is maintained, in which characteristics of a basal state are observed.

As used herein, the term "pluripotent stem cell morphology" refers to the typical morphological characteristics of embryonic stem cells. Normal embryonic stem cell morphology is characterized by a round and small shape, with a high nuclear to cytoplasmic ratio, the prominent presence of nucleoli, and typical intercellular spacing.

As used herein, the term "feeder cell" or "feeder" is a cell used to describe a type of cell co-cultured with a second type of cell to provide an environment in which the second type of cell can grow, because the feeder cell provides growth factors and nutrients for supporting the second cell type. Feeder cells are optionally derived from species different from the cells they support. For example, certain types of human cells, including stem cells, can be supported by primary cultures of mouse embryonic fibroblasts and immortalized mouse embryonic fibroblasts. When co-cultured with other cells, feeder cells can typically be inactivated by irradiation or treatment with an antimitotic agent such as mitomycin to prevent their growth from exceeding the cells they support. Not limited to the foregoing, a specific feeder cell type can be human feeder cells, such as human skin fibroblasts. Another feeder cell type can be mouse embryonic fibroblasts (MEFs). Typically, various feeder cells can be used in part to maintain pluripotency, guide differentiation to specific lineages and promote maturation to specific cell types, such as effector cells.

As used herein, a "feeder-free" (FF) environment refers to an environment, such as culture conditions, cell culture, or culture medium, that is substantially free of feeder or stromal cells and/or has not been preconditioned by cultivation of feeder cells. "Preconditioned" culture medium refers to culture medium that has been harvested after feeder cells have been cultivated in the culture medium for a period of time, such as at least one day. Preconditioned culture medium contains a number of mediator substances, including growth factors and cytokines secreted by the feeder cells cultivated in the culture medium.

As used herein, the term "subject" refers to any animal, preferably a human patient, livestock or other domesticated animal.

"Pluripotency factors" or "reprogramming factors" refer to agents that, alone or in combination with other agents, can increase the developmental potential of a cell. Pluripotency factors include, but are not limited to, polynucleotides, polypeptides, and small molecules that can increase the developmental potential of a cell. Exemplary pluripotency factors include, for example, transcription factors and small molecule reprogramming agents.

"Adherent" refers to the attachment of cells to a container, e.g., to a sterile plastic (or plastic-coated) cell culture dish or flask, in the presence of an appropriate culture medium. Certain types of cells cannot persist or grow in culture unless they adhere to the cell culture container. Certain types of cells ("non-adherent cells") do not require attachment to maintain and/or proliferate in culture.

"Cultivation" or "cell culture" refers to the maintenance, growth, and/or differentiation of cells in an in vitro environment. "Cell culture medium," "culture medium" (in each case the singular "culture medium"), "supplement," and "culture medium supplement" refer to the nutritional composition for cultivating cell cultures.

"Cultivate" or "maintain" refers to the persistence, propagation (growth), and/or differentiation of cells outside a tissue or organism, for example, in a sterile plastic (or plastic-coated) cell culture dish or flask. "Cultivation" or "maintaining" can utilize a culture medium as a source of nutrients, hormones, and/or other factors that help the cells propagate and/or persist.

As used herein, "dissociated" cells refer to cells that have been substantially separated from other cells or surfaces (e.g., culture plate surfaces) or purified away from other cells. For example, cells can be dissociated from animals or tissues by mechanical or enzymatic methods. Alternatively, cells aggregated in vitro can be dissociated from each other enzymatically or mechanically, for example, by dissociating into a suspension of a mixture of clusters, single cells, or single cells and clusters. In another alternative embodiment, adherent cells are dissociated from culture plates or other surfaces. Dissociation can therefore involve destroying the interaction of cells with extracellular matrix (ECM) and matrix (e.g., culture surface), or destroying the ECM between cells.

B. Overview

The present invention generally relates to a multi-stage process of differentiation of initial pluripotent cells into non-pluripotent cells or partially differentiated cells, including mesoderm cells, permanent hemogenic endothelium, permanent hematopoietic stem or progenitor cells, CD34+ cells, multipotent progenitor cells (MPP) (capable of differentiating into bone marrow, including neutrophil progenitor cells), T progenitor cells, NK progenitor cells; or fully differentiated terminal hematopoietic cells, for example, such as T cells, B cells, NKT cells or NK cells. The present invention also relates to compositions used in the disclosed methods; and cell populations, cell lines or clonal cells produced by the disclosed methods.

In contrast to the methods used in the art, the present invention avoids the formation of EBs during iPSC differentiation. As described above, hematopoietic lineage cells derived from iPSCs are obtained by seeding clonal iPSC cells in a TGFβ-free medium to maintain their basal or initial state of pluripotency, differentiating clonal iPSCs into monolayer iPSCs without EB formation, and using a step-by-step strategy to apply small chemicals, growth factors, and appropriate combinations of cytokines at the early and middle stages of differentiation. Therefore, the present invention enables the direct transfer of expanded clonal iPSCs into adherent monolayer culture for immediate differentiation without the need to form EBs from iPSCs.

The present invention thus provides a culture platform capable of efficiently differentiating stem cells into definitive hematopoietic and functional hematopoietic cell lineages without the use of TGFβ receptor/ALK inhibitors, including SB431532. In addition, unlike previous studies, the present invention also provides a culture platform using feeder-free, serum-free conditions that supports direct differentiation of iPSCs in monolayer culture without the need for EBs or aggregates from iPSCs.

C. Training Platform

Existing methods for culturing pluripotent cells primarily rely on feeder cells or culture media preconditioned with feeder cells and containing fetal bovine serum; however, such environments may not be suitable for producing cells for clinical and therapeutic use. For example, cells cultured in such xeno-contaminated environments are generally considered unsuitable for human cell transplantation because exposure to animal components can present a serious risk of immune rejection and transmission of unidentified pathogens to treated patients, and can potentially reactivate animal retroviruses. Culture systems using animal-free culture media, such as the feeder-free environments considered herein, facilitate the manufacture of clinical-grade cell lines, particularly hESCs, hiPSCs, and pluripotent stem cell-derived HSCs, T, B, NKT, or NK cell lines.

In a specific embodiment, no feeder environment is substantially free of human feeder cells, and is not preconditioned by feeder cells, including but not limited to mouse embryonic fibroblasts, human fibroblasts, keratinocytes and embryonic stem cells. No feeder cell culture medium is applicable to cultivating pluripotent cells, reprogrammed cells, single cell culture, the dissociation and passage of pluripotent cells, the cell sorting of pluripotent cells, the generation of ground state pluripotent cells, the maintenance of ground state pluripotency, inducing pluripotent cell differentiation. In a specific embodiment, no feeder environment is used for inducing pluripotency, improves reprogrammed efficiency, increases or maintains potential and/or induced differentiation of cells. In some embodiments, no feeder environment is substantially free of cytokines and growth factors, including bFGF.

In some aspects of the present invention, one or more stages of the above-mentioned iPSC differentiation can be carried out under feeder-free conditions. Such feeder-free conditions can be in the form of monolayer culture and suspension culture. In one embodiment of the present invention, the differentiation of pluripotent cells into mesoderm cells is carried out under monolayer feeder-free conditions. In another embodiment of the present invention, the differentiation of mesoderm cells into permanent hemogenic endothelial cells is carried out under monolayer feeder-free conditions. In another embodiment of the present invention, the differentiation of permanent hemogenic endothelial cells into hematopoietic stem cells is carried out under monolayer feeder-free conditions. In one embodiment of the present invention, the differentiation of permanent hematopoietic stem cells into multipotent progenitor cells, T progenitor cells or NK progenitor cells is carried out under suspension feeder-free conditions, or first under monolayer feeder-free conditions and then under suspension feeder-free conditions. In another embodiment of the present invention, the differentiation of T progenitor cells into fully differentiated T cells or the differentiation of NK progenitor cells into fully differentiated NK cells is carried out under suspension feeder-free conditions, or first under monolayer feeder-free conditions and then under suspension feeder-free conditions.

Any suitable culture flask or cell culture container can be used as the carrier of the cell culture in basal medium and/or cell culture supplement.In some embodiments, the surface of the culture container is coated with adhesion-promoting matrix/substrate (for example, collagen, fibronectin, polypeptide containing RGD, gelatin, etc.) to promote the attachment of cells, and the effect of cell culture medium disclosed herein and supplement can be enhanced in a specific embodiment. Suitable substrates for cultivating and subculturing cells are known in the art, and include but are not limited to vitronectin, gelatin, laminin, fibronectin, collagen, elastin, osteopontin, thrombospondin, the matrix produced by naturally occurring cell lines such as Matrigel ^™ mixture and synthetic or artificial surface such as polyamine monolayer and carboxyl-terminated monolayer.In some embodiments, providing no feeding conditions is included in culturing cells on the surface of matrix coating.In one embodiment, the culture platform considered herein includes the matrix/substrate containing Matrigel ^™ or vitronectin.In some embodiments of cultivating, using Matrigel ^™ , so that culture is fully defined.

In some aspects of the invention, one or more of the above-mentioned differentiation stages can be performed under serum-free conditions. Examples of commercially available serum-free culture media suitable for cell attachment and/or induction include mTeSR ^™ 1 or TeSR ^™ 2 from Stem Cell Technologies (Vancouver, Canada), Primate ES/iPS cell culture medium from ReproCELL (Boston, MA), hESC SFM from Invitrogen (Carlsbad, CA), and X-VIVO ^™ from Lonza (Basel, Switzerland).

In another embodiment, the one or more culture media of the culture platform are feeder-free environments and are optionally substantially free of cytokines and/or growth factors. In other embodiments, the cell culture medium contains supplements, such as serum, extracts, growth factors, hormones, cytokines, etc. Typically, the culture platform comprises one or more stage-specific feeder-free, serum-free culture media, each culture medium also comprising one or more of the following: nutrients/extracts, growth factors, hormones, cytokines, and culture medium additives. Suitable nutrients/extracts may include, for example, DMEM/F-12 (Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12), which is a widely used basal culture medium for supporting the growth of many different mammalian cells; KOSR (eliminating serum replacement); L-glut; NEAA (non-essential amino acids). Other culture medium additives may include, but are not limited to, MTG, ITS, βME, antioxidants (e.g., ascorbic acid). In some embodiments, the culture medium of the present invention comprises one or more of the following cytokines or growth factors: epidermal growth factor (EGF), acidic fibroblast growth factor (aFGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), insulin-like growth factor 2 (IGF-2), keratinocyte growth factor (KGF), nerve growth factor (NGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), bone morphogenetic protein (BMP4), vascular endothelial growth factor (VEGF) transferrin, various interleukins (such as IL-1 to IL-18), various colony stimulating factors (such as granulocyte/macrophage colony stimulating factor (GM-CSF)), various interferons (such as IFN-γ) and other cytokines that have an effect on stem cells, such as stem cell factor (SCF) and erythropoietin (EPO). These cytokines can be purchased commercially, for example, from R&D Systems (Minneapolis, Minn.), and can be natural or recombinant. In some other embodiments, the culture medium of the present invention comprises one or more bone morphogenetic protein (BMP4), insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), hematopoietic growth factors (e.g., SCF, GMCSF, GCSF, EPO, IL3, TPO, EPO), Fms-related tyrosine kinase 3 ligand (Flt3L); and one or more cytokines from leukemia inhibitory factor (LIF), IL3, IL6, IL7, IL11, IL15. In some embodiments, the growth factors/mitogens and cytokines are stage and/or cell type specific in concentration, determined empirically or guided by established cytokine technology.

Generally, the technology for differentiating inducible pluripotent cells involves using a method based on polynucleotides, polypeptides and/or small molecules to directly or indirectly regulate specific cell pathways. The developmental potential of a cell can be regulated, for example, by contacting the cell with one or more regulators. "Contact" used herein can relate to culturing cells in the presence of one or more factors (such as small molecules, proteins, peptides, etc.). In some embodiments, the cell is contacted with one or more reagents to induce cell differentiation. This contact can be carried out, for example, by introducing one or more reagents into the cell during in vitro culture. Therefore, contact can be carried out by introducing one or more reagents into the cell in the nutrient cell culture medium. The cell can remain in the culture medium comprising one or more reagents for a time sufficient to allow the cell to reach the desired differentiation phenotype. In some other embodiments, when one or more factors are introduced into the cell by a vector, "contact" occurs. In some embodiments, one or more vectors are introduced by retrovirus, Sendai virus, adenovirus, episome, microring, a vector system or mRNA with an expression cassette.

In other embodiments, one or more stage-specific feeder-free, serum-free culture media of the culture platforms disclosed herein further comprise one or more small molecules. In some embodiments, the culture platform comprises a cell culture medium comprising a GSK-3 inhibitor, a MEK inhibitor, a Rho kinase (ROCK) inhibitor, and does not comprise or is free of small molecule inhibitors of the TGFβ/activin signaling pathway, including but not limited to TGFβ receptor or ALK5 inhibitors.

The culture platform contemplated herein also provides many advantages by utilizing a uniform population of industrial or clinical grade pluripotent cells with reduced spontaneous differentiation and/or reaching basal state pluripotency. In one embodiment, homogeneous iPSCs are maintained in a composition comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor; and the composition does not contain a TGFβ receptor/ALK inhibitor. As used herein, the term "homogeneous" refers to a cell population in which each cell is identical or substantially identical to other cells in the population. In one embodiment, if each cell expresses one or more identical pluripotency markers contemplated herein, such as SSEA4 and TRA1-81, the cell is identical to other cells in the population. In one embodiment, if at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more of the cells are identical or substantially identical to other cells in the population, the population is homogeneous.

In a number of embodiments, the cell culture medium for the culture platform for producing hematopoietic cell lineages by the permanent hemogenic endothelium of the present invention does not include or is substantially free of inhibitors of TGFβ/activin signaling pathways, including TGFβ receptor (TGFβR) inhibitors and ALK5 inhibitors. In one embodiment, the culture platform includes a seeding culture medium for maintaining initial hiPSC, the culture medium comprising a GSK-3 inhibitor, a MEK inhibitor, and a Rho kinase (ROCK) inhibitor. The inventors do not wish to be bound by any specific theory and have found that although TGFβR/ALK5 inhibitors improve the efficiency of reprogramming, these inhibitors offset the long-term maintenance, quality, and uniformity of the pluripotent cell population. That is, although the inhibition of TGFβ pathway signaling improves the efficiency of cell reprogramming, the alleviation of this inhibition contributes to the subsequent maintenance of pluripotent cell populations in an in vitro culture system, particularly in a system using no feeder cells and single cells, enzymatic digestion passage, wherein preferably there is reduced spontaneous differentiation and a homogeneous pluripotent population maintained in a "basal" or "initial" pluripotency state. As used herein, the term "long-term" is measured based on, but not limited to, the number of passages, and generally means at least 10, 15, 20, 25, 30, 35, 40, 45, 50 or more generations. As defined, "passaging" refers to the act of separating and spreading cells onto a multi-cell culture surface or culture flask when the cells have proliferated to the desired extent. Additionally, metastable pluripotent cells are cultured in a culture medium disclosed herein comprising a GSK3 inhibitor and a MEK inhibitor and optionally a ROCK inhibitor but without a TGFβR/ALK5 inhibitor, transforming pluripotent cells to achieve reduced spontaneous differentiation, and/or achieving basal pluripotency.

Achieving the basic or initial pluripotency of iPSCs is also important for obtaining hematopoietic lineage cells by differentiating iPSCs without forming EB intermediates. In addition, the efficiency of initial iPSC differentiation into permanent HE is also severely affected by the use of monolayer culture without forming EBs and their aggregates. In some embodiments, the culture platform includes a culture medium containing a ROCK inhibitor and free of or substantially free of TGFβR/ALK5 inhibitors. In some other embodiments, the culture platform includes a culture medium containing a GSK3 inhibitor but free of TGFβR/ALK5 inhibitors, which promotes the generation of permanent HE and/or permanent HSC cells using the culture platform provided herein.

1. TGFβ receptor/ALK inhibitors

TGFβ receptor (e.g., ALK5) inhibitors may include antibodies to TGFβ receptor (e.g., ALK5), dominant negative (negative) variants of TGFβ receptor, and antisense nucleic acids that inhibit TGFβ receptor expression. Exemplary TGFβ receptor/ALK inhibitors include, but are not limited to, SB431542 (see, e.g., Inman et al., Molecular Pharmacology 62(1):65-74 (2002)); A-83-01, also known as 3-(6-methyl-2-pyridine)-N-phenyl-4-(4-quinoline)-1H-pyrazole-1-thioamide (see, e.g., Tojo et al., Cancer Science 96(11):791-800 (2005) and commercially available from, e.g., Toicris Bioscience); 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine; Wnt3a/BIO (see, e.g., Dalton et al., WO2008/094597, incorporated herein by reference); GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide) (see, e.g., Gellibert et al., Journal of Medicinal Chemistry 49(7):2210-2221 (2006)); SM16 (see, e.g., Suzuki et al., Cancer Research 67(5):2351-2359 (2007)); IN-1130 (3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide) (see, e.g., Kim et al., Xenobiotica 38(3):325-339 (2008)); GW6604 (2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., deGouville et al., Drug News Perspective 19(2):85-90 (2006)); SB-505124 (2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride) (see, e.g., DaCosta et al., Molecular Pharmacology 65(3):744-752 (2004)); and pyrimidine derivatives (see, e.g., those listed in Stiefl et al., WO2008/006583, incorporated herein by reference). In addition, although "ALK5 inhibitor" is not intended to encompass non-specific kinase inhibitors, "ALK5 inhibitor" should be understood to encompass inhibitors that inhibit ALK4 and/or ALK7 in addition to ALK5, such as SB-431542 (see, e.g., Inman et al., J Mol. Pharmacol. 62(1):65-74 (2002)). Without intending to limit the scope of the present invention, we believe that ALK5 inhibitors affect the mesenchymal to epithelial transition/transition (MET) process. The TGFβ/activin pathway is a driver of epithelial to mesenchymal transition (EMT). Therefore, inhibiting the TGFβ/activin pathway may contribute to the MET (i.e., reprogramming) process.

Taking into account the data showing the effect of inhibiting ALK5 herein, we believe that inhibition of the TGFβ/activin pathway will have a similar effect of inhibiting ALK5. Therefore, any inhibitor of the TGFβ/activin pathway (e.g., upstream or downstream) can be used in combination with or instead of an ALK5 inhibitor, as described in each paragraph herein. Exemplary TGFβ/activin pathway inhibitors include, but are not limited to, TGFβ receptor inhibitors, SMAD 2/3 phosphorylation inhibitors, SMAD 2/3 and SMAD 4 interaction inhibitors, and SMAD 6 and SMAD 7 activators/agonists. In addition, the classification described below is only for organizational purposes, and those skilled in the art will know that the compound can affect one or more points within the pathway, and therefore the compound can work in more than one limited category.

TGFβ receptor (TGFβR) inhibitors may include antibodies against TGFβ receptor, dominant negative variants of TGFβ receptor, and siRNA or antisense nucleic acids targeting TGFβ receptor. Specific examples of TGFβ receptor inhibitors include, but are not limited to, SU5416; 2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridine hydrochloride (SB-505124); lerdelimumb (CAT-152); metilimumab (CAT-192); GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580 276; LY364947; LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894; SB-203580; SD-093; Gleevec; 3,5,7,2',4'-pentahydroxyflavone (Morin); activin-M108A; P144; soluble TBR2-Fc; and antisense-transfected tumor cells targeting the TGFβ receptor. (See, e.g., Wrzesinski et al., Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska et al., Acta Biochimica Polonica 52(2):329-337 (2005); and Chang et al., Frontiers in Bioscience 12:4393-4401 (2007).)

SMAD 2/3 phosphorylation inhibitors may include antibodies to SMAD2 or SMAD3, dominant negative variants of SMAD2 or SMAD3, and antisense nucleic acids targeting SMAD2 or SMAD3. Specific examples of inhibitors include PD169316; SB203580; SB-431542; LY364947; A77-01; and 3,5,7,2',4'-pentahydroxyflavone (Morin). (See, for example, Wrzesinski, supra, incorporated herein by reference; Kaminska, supra; Shimanuki et al., Oncogene 26:3311-3320 (2007); and Kataoka et al., EP1992360).

SMAD 2/3 and SMAD4 interaction inhibitors may include antibodies against SMAD2, SMAD3 and/or SMAD 4, dominant negative variants of SMAD2, SMAD3 and/or SMAD 4, and antisense nucleic acids targeting SMAD2, SMAD3 and/or SMAD 4. Specific examples of SMAD 2/3 and SMAD4 interaction inhibitors include, but are not limited to, Trx-SARA, Trx-xFoxH1b, and Trx-Lef1. (See, e.g., Cui et al., Oncogene 24:3864-3874 (2005) and Zhao et al., Molecular Biology of the Cell, 17:3819-3831 (2006)).

SMAD 6 and SMAD 7 activators/agonists include, but are not limited to, antibodies against SMAD 6 or SMAD 7, dominant negative variants of SMAD 6 or SMAD 7, and antisense nucleic acids targeting SMAD 6 or SMAD 7. Specific examples of inhibitors include, but are not limited to, SMAD 7-as PTO-oligonucleotides. (See, for example, Miyazono et al., US6534476, both of which are incorporated herein by reference, and Steinbrecher et al., US2005119203).

2. WNT pathway agonists

As used herein, the terms "Wnt signaling promoting agent," "Wnt pathway activating agent," or "Wnt pathway agonist" refer to agonists of the Wnt signaling pathway, including but not limited to agonists of one or more of Wnt1, Wnt2, Wnt2b/13, Wnt3, Wnt3a, Wnt4, Wnt5a, Wnt5b, Wnt6, Wnt7a, Wnt7b, Wnt7c, Wnt8, Wnt8a, Wnt8b, Wnt8c, Wnt10a, Wnt10b, Wnt11, Wnt14, Wnt15, and Wnt16. Wnt pathway agonists also include but are not limited to one or more of the following polypeptides or fragments thereof: a Dkk polypeptide, a crescent polypeptide, a cerberus polypeptide, an axin polypeptide, a Frzb polypeptide, a T cell factor polypeptide, or a dominant negative dishevelled polypeptide.

Non-limiting examples of Wnt pathway agonists also include one or more of the following: a nucleic acid comprising a nucleotide sequence encoding a Wnt polypeptide, a polypeptide comprising an amino acid sequence of a Wnt polypeptide, a nucleic acid comprising a nucleotide sequence encoding an activated Wnt receptor, a polypeptide comprising an amino acid sequence that activates a Wnt receptor, a small organic molecule that promotes Wnt/β-catenin signaling, a small organic molecule that inhibits the expression or activity of a Wnt antagonist, an antisense oligonucleotide that inhibits the expression of a Wnt antagonist, a ribozyme that inhibits the expression of a Wnt antagonist, an RNAi construct, siRNA, or shRNA that inhibits the expression of a Wnt antagonist, an antibody that binds to a Wnt antagonist and inhibits the activity of the Wnt antagonist, a nucleic acid comprising a nucleotide sequence encoding a β-catenin polypeptide, a polypeptide comprising an amino acid sequence of a β-catenin polypeptide, a nucleic acid comprising a nucleotide sequence encoding a Lef-1 polypeptide, a polypeptide comprising an amino acid sequence of a Lef-1 polypeptide.

Wnt pathway agonists also include GSK3 inhibitors, such as nucleic acids comprising a nucleotide sequence encoding a dominant negative GSK-3, GSK3α or GSK3β polypeptide, polypeptides comprising an amino acid sequence of a dominant negative GSK-3, GSK3α or GSK3β polypeptide, small organic molecules that bind to GSK-3, GSK3α or GSK3β and inhibit the expression or activity of GSK-3, GSK3α or GSK3β, RNAi constructs that bind to GSK-3, GSK3α or GSK3β and inhibit the expression and/or activity of GSK-3, GSK3α or GSK3β, siRNA or shRNA, antisense oligonucleotides that bind to GSK-3, GSK3α or GSK3β and inhibit the expression of GSK-3, GSK3α or GSK3β, antibodies that bind to GSK-3, GSK3α or GSK3β and inhibit the expression and/or activity of GSK-3, GSK3α or GSK3β, ribozymes that bind to GSK-3, GSK3α or GSK3β and inhibit the expression of GSK-3, GSK3α or GSK3β, and any GSK-3-independent agent that activates β-catenin target genes with effects similar to those of GSK3 inhibition.

3. GSK3 inhibitors

GSK-3β inhibitors are specific exemplary Wnt pathway agonists suitable for use in the compositions contemplated herein and may include, but are not limited to, polynucleotides, polypeptides, and small molecules. GSK-3β inhibitors contemplated herein may reduce GSK-3β expression and/or GSK-3β activity. Illustrative examples of GSK-3β inhibitors contemplated herein include, but are not limited to, anti-GSK-3β antibodies, dominant negative GSK-3β variants, siRNA, shRNA, miRNA, and antisense nucleic acids targeting GSK-3β.

Other illustrative GSK-3β inhibitors include, but are not limited to, kemperolone, 1-azacamperolone, CHIR99021, CHIR98014, AR-A014418, CT 99021, CT 20026, SB216763, AR-A014418, lithium, SB415286, TDZD-8, BIO, BIO-acetone oxime, (5-methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine, pyridinecarbazol-cyclopentadienylruthenium complex, TDZD-8 4-Benzyl-2-methyl-1,2,4-thiadiazolidine-3-,5-dione, 2-thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole, OTDZT, α-4-dibromoacetophenone, AR-AO 144-18, 3-(1-(3-hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione; TWS1 19 pyrrolopyrimidine compound, L803H-KEAPPAPPQSpP-NH2 or its myristoylated form; 2-chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone; GF109203X; RO318220; TDZD-8; TIBPO; and OTDZT.

In certain illustrative embodiments, the GSK-3β inhibitor is CHIR99021, BIO, or kemperolone.

In a preferred embodiment, the GSK-3β inhibitor is CHIR99021.

In another embodiment, the GSK3 inhibitor is BRD0705.

4. ERK/MEK inhibitors

ERK/MEK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides, and small molecules. ERK/MEK inhibitors contemplated herein can reduce MEK or ERK expression and/or MEK or ERK activity. Illustrative examples of ERK/MEK inhibitors contemplated herein include, but are not limited to, anti-MEK or anti-ERK antibodies, dominant negative MEK or ERK variants, siRNA, shRNA, miRNA, and antisense nucleic acids targeting MEK or ERK.

Other illustrative ERK/MEK inhibitors include, but are not limited to, PD0325901, PD98059, U0126, SL327, ARRY-162, PD184161, PD184352, sunitinib, sorafenib, vandetanib, pazopanib, axitinib, GSK1120212, ARRY-438162, RO5126766, XL518, AZD8330, RDE119, AZD6244, FR180204, and PTK787.

Additional illustrative MEK/ERK inhibitors include those compounds disclosed in International Published Patent Applications WO 99/01426, WO 02/06213, WO 03/077914, WO 05/051301 and WO 2007/044084.

Further illustrative examples of MEK/ERK inhibitors include the following compounds: 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2,3-dihydroxy-propoxy)-amide; 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-pyran-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide; 1-[6-(4-bromo- -2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-yl]-2-hydroxyethanone, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-(tetrahydro-furan-2-ylmethyl)-3H-benzimidazole-5-carboxylic acid (2-hydroxy-1,1-dimethyl-ethoxy)-amide -amide, 6-(4-bromo-2-fluoro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(2,4-dichloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide, 6-(4-bromo-2-chloro-phenylamino)-7-fluoro-3-methyl-3H-benzimidazole-5-carboxylic acid (2-hydroxy-ethoxy)-amide -amide, hereinafter referred to as MEK inhibitor 1; 2-[(2-fluoro-4-iodophenyl)amino]-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridine-3-carboxamide (hereinafter referred to as MEK inhibitor 2); and 4-(4-bromo-2-fluorophenylamino)-N-(2-hydroxyethoxy)-1,5-dimethyl-6-oxo-1,6-dihydropyridazine-3-carboxamide and pharmaceutically acceptable salts thereof.

In a preferred embodiment, the MEK/ERK inhibitor is PD98059.

5.ROCK inhibitors

Rho-associated kinase (ROCK) is a serine/threonine kinase that acts as a downstream effector of Rho kinase (of which there are three isoforms - RhoA, RhoB and RhoC). ROCK inhibitors suitable for use in the compositions contemplated herein include, but are not limited to, polynucleotides, polypeptides and small molecules. ROCK inhibitors contemplated herein can reduce ROCK expression and/or ROCK activity. Illustrative examples of ROCK inhibitors contemplated herein include, but are not limited to, anti-ROCK antibodies, dominant negative ROCK variants, siRNA, shRNA, miRNA and antisense nucleic acids targeting ROCK.

Illustrative ROCK inhibitors contemplated herein include, but are not limited to, thiazovivin, Y27632, Fasudil, AR122-86, Y27632H-1152, Y-30141, Wf-536, HA-1077, hydroxy-HA-1077, GSK269962A, SB-772077-B, N-(4-pyridyl)-N'-(2,4,6-trichlorophenyl)urea, 3-(4-pyridyl)-1H-indole, and (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide, as well as the ROCK inhibitors disclosed in U.S. Pat. No. 8,044,201, which is incorporated herein by reference in its entirety.

In one embodiment, the ROCK inhibitor is thiazovivin, Y27632, or pyrintegrin.

In a preferred embodiment, the ROCK inhibitor is thiazovivin.

The amount of small molecules in the compositions and cell culture media contemplated herein can vary and can be optimized based on specific culture conditions, including the specific molecules and combinations used, the cell types cultured in the culture medium, and the specific application. In one embodiment, the small molecule is present in the composition at a concentration sufficient to induce pluripotency, improve reprogramming efficiency, increase or maintain the potency of the cell, or induce or maintain basal pluripotency.

Another aspect of the present invention relates to Notch activators for use in the present invention. Notch includes all members of the Notch receptor family, including but not limited to Notch1. Notch activators include but are not limited to agonists of Notch receptors. Notch agonists will bind to Notch receptors and also trigger or mediate signaling events associated with Notch receptors, such as causing the intracellular domain of Notch to be cleaved and translocated to the nucleus. Notch activators include but are not limited to Jag1, Jag2, DLL-1, DLL-3 and DLL-4. Notch activators include but are not limited to those disclosed in EP 2606884, US 6689744 and US 5780300, the disclosures of which are incorporated herein by reference. In some embodiments, one or more Notch ligands can be introduced as soluble peptides or immobilized on a solid material. Solid materials can include but are not limited to polystyrene plates or beads. The beads used for Notch ligand immobilization can be agarose beads, magnetic beads and latex beads. In one embodiment, the Notch ligand peptide is conjugated/immobilized to the beads. In another embodiment, the Notch ligand peptide is conjugated/immobilized to the surface of the polystyrene plate. In some embodiments, the immobilization of the Notch ligand is non-covalent. In some embodiments, the Notch ligand peptide is presented by the cell.

Another aspect of the present invention relates to BMP pathway activators, including those disclosed in the following publications, the disclosures of which are incorporated herein by reference: WO 2014011540, WO 2014062138, and WO 2005117994. BMP pathway activators for use in the present invention include, but are not limited to, BMP-5, BMP-6, BMP-7, BMP-8, BMP-2, and BMP-4. In one non-limiting embodiment of the present invention, the BMP pathway activator is BMP-4. BMPs are multifunctional cytokines that are members of the transforming growth factor-β superfamily. Bone morphogenetic protein (BMP) receptors mediate BMP signaling by activating Smads. BBMP ligands bind to the BMP receptors BMPRI and BMPRII. Upon phosphorylation, BMPRII activates BMPRI. Phosphorylated BMPRI then phosphorylates receptor-activated Smad proteins (R-Smads), which bind to the common mediator SMad (co-Madad) and enter the cell nucleus, where they regulate gene expression. In one embodiment, the BMP pathway activator is BMP4.

The present invention provides compositions for obtaining hematopoietic lineage cells from iPSCs, either by definitive HSCs differentiated from iPSCs or by definitive hemogenic endothelium differentiated from iPSCs, each without EB formation from iPSCs for desired cell differentiation.

6. hiPSC differentiation platform

I.iHSC Platform

One aspect of the present invention provides a culture medium for obtaining mesodermal cells from pluripotent stem cells, including iPSC. In some embodiments, iPSC is an initial iPSC. In one embodiment, the culture medium comprises a Wnt pathway activator and a BMP activator. In one embodiment, the culture medium comprises a Wnt pathway activator, and the iHSC basal culture medium comprises a BMP activator. In one embodiment, the Wnt pathway activator in the culture medium is a GSK3 inhibitor. In one embodiment, the GSK3 inhibitor is CHIR99021. In one embodiment, the BMP activator is BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines in concentration ranges as shown in Table 1. In some embodiments, the culture medium is fully defined when Matrigel ^™ is used instead of vitronectin.

One aspect of the present invention provides a culture medium for obtaining definitive hemogenic endothelium from mesodermal cells. In one embodiment, the culture medium comprises a Wnt pathway activator, a BMP activator, and an optional TGFβ receptor/ALK inhibitor. In one embodiment, the culture medium comprises a Wnt pathway activator, a BMP activator, and the culture medium does not contain or is substantially free of a TGFβ receptor/ALK inhibitor. In one embodiment, the culture medium comprises a Wnt pathway activator and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, the latter comprising a BMP activator. In one embodiment, the Wnt pathway activator in the culture medium is a GSK3 inhibitor. In one embodiment, the GSK3 inhibitor is CHIR99021. In one embodiment, the BMP activator is BMP4. In one embodiment, the optional TGFβ receptor/ALK inhibitor is SB431542. In some embodiments, the culture medium herein comprises an extracellular matrix protein. In other embodiments, the culture medium comprises small molecules, growth factors, and/or cytokines in concentration ranges as shown in Table 2. In some embodiments, Matrigel ^™ is used instead of vitronectin to fully define the culture medium.

One aspect of the present invention provides a culture medium for obtaining permanent HSCs from the hemogenic endothelium. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, and does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor. In one embodiment, the culture medium comprises iHSC basal medium, which comprises a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. In one embodiment, the BMP activator is BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines in the concentration ranges shown in Table 3. In some embodiments, the culture medium is fully defined using Matrigel ^™ instead of vitronectin.

One aspect of the present invention provides a culture medium for obtaining T progenitor cells from permanent HSCs. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, and IL7. In one embodiment, the culture medium comprises SCF, Flt3L, IL7, a BMP activator, and an iTC basal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3, and IL6, and one or more Notch pathway activators. In one embodiment, the iTC basal medium comprises a combination of IL2, IL3, IL6, and one or more Notch pathway activators. In one embodiment, the iTC basal medium does not contain fibronectin. In one embodiment, the BMP activator is BMP4. In one embodiment, the Notch pathway activator is a Notch ligand, including but not limited to Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides. In some embodiments, the culture medium comprises extracellular matrix proteins. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines in the concentration ranges shown in Table 4. In some embodiments, the culture medium is fully defined using Matrigel ^™ instead of vitronectin.

One aspect of the present invention provides a culture medium for obtaining T cells from T progenitor cells. In one embodiment, the culture medium comprises one or more growth factors and cytokines selected from SCF, Flt3L, IL7, and IGF. In one embodiment, the culture medium comprises one or more growth factors and cytokines selected from SCF, Flt3L, IL7, and IGF, and does not contain a BMP activator. In one embodiment, the culture medium comprises a combination of growth factors and cytokines selected from SCF, Flt3L, IL7, and IGF, and an iTC basal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3, and IL6, and one or more Notch pathway activators. In one embodiment, the iTC basal medium comprises a combination of IL2, IL3, IL6, and one or more Notch pathway activators. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and/or DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides. In some embodiments, the BMP activator comprises BMP4. In some embodiments, the culture medium comprises extracellular matrix proteins. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines at concentrations in the ranges shown in Table 5. In some embodiments, the culture medium is fully defined using Matrigel ^™ in place of vitronectin.

One aspect of the present invention provides a culture medium for obtaining NK progenitor cells from definitive HSCs. In one embodiment, the culture medium comprises a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, and VEGF. In one embodiment, the culture medium comprises SCF, Flt3L, VEGF, and a BMP activator and an iNK basal medium comprising one or more growth factors and cytokines selected from IL2, IL3, IL6, and IL15. In one embodiment, the iNK basal medium comprises a combination of IL2, IL3, IL6, and IL15. In one embodiment, the BMP activator is BMP4. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines in the concentration ranges shown in Table 6. In some embodiments, the culture medium is fully defined using Matrigel ^™ instead of vitronectin.

One aspect of the present invention provides a culture medium for obtaining NK cells from NK progenitor cells. In one embodiment, the culture medium comprises one or more growth factors and cytokines selected from SCF, Flt3L, IGF and IL7. In one embodiment, the culture medium comprises SCF, Flt3L, IGF, IL7 and iNK minimal culture medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3, IL6 and IL15, wherein the culture medium does not contain a BMP activator. In one embodiment, the iNK minimal culture medium comprises a combination of IL2, IL3, IL6 and IL15. In some embodiments, the culture medium comprises extracellular matrix proteins. In other embodiments, the culture medium herein comprises small molecules, growth factors and/or cytokines in a concentration range as shown in Table 7. In some embodiments, Matrigel ^™ is used to replace vitronectin to fully define the culture medium. In some embodiments, artificial antigens used to stimulate NK growth, development and maturation are introduced in the form of microbead conjugation, plasma membrane particles or antigen presenting cells.

Another aspect of the present invention provides a culture platform for obtaining T cells, comprising one or more of (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7 and IGF and an iTC basal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators, wherein the culture medium does not contain a BMP activator and is suitable for generating T cells from T progenitor cells; (ii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and an iTC basal medium, wherein the culture medium is suitable for generating T progenitor cells from definitive HSCs; (iii) a culture medium comprising one or more A plurality of growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO and iHSC basal medium, the latter comprising a BMP activator, wherein the medium does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for generating definitive HSCs from hemogenic endothelium; (iv) a medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, the latter comprising a BMP activator, wherein the medium is suitable for generating hemogenic endothelium from mesodermal cells; and (v) a medium comprising a GSK3 inhibitor and a basic iHSC basal medium, the latter comprising a BMP activator, wherein the medium is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

In one embodiment, the culture platform for obtaining T cells comprises (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF and IL7 and an iTC basal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3 and IL6, and one or more Notch pathway activators, wherein the culture medium does not contain a BMP activator and is suitable for generating T cells from T progenitor cells. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and an iTC basal medium, wherein culture medium (ii) is suitable for generating T progenitor cells from definitive HSCs. In one embodiment, the culture platform comprising culture medium (i) and (ii) further comprises (iii) culture medium, which comprises one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO and iHSC basic culture medium, the latter comprising BMP activator, wherein culture medium (iii) does not contain Wnt pathway activator and TGFβ receptor/ALK inhibitor and is suitable for producing permanent HSC from hemogenic endothelium. In one embodiment, the culture platform comprising culture medium (i), (ii) and (iii) further comprises (iv) culture medium, which comprises GSK3 inhibitor and optional TGFβ receptor/ALK inhibitor and iHSC basic culture medium, wherein culture medium (iv) is suitable for producing permanent hemogenic endothelium from mesoderm cells. In another embodiment, the culture platform comprising culture medium (i), (ii), (iii) and (iv) further comprises (v) culture medium, which comprises GSK3 inhibitor and basic iHSC basic culture medium, wherein culture medium (v) is suitable for producing mesoderm cells from iPSC. In some embodiments, iPSC is initial iPSC.

One aspect of the present invention provides a culture platform for obtaining T progenitor cells, comprising one or more (i) culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and IL7, and iTC basal medium, wherein the culture medium is suitable for generating T progenitor cells from definitive HSCs; (ii) culture medium comprising one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, and iHSC basal medium comprising a BMP activator, wherein the culture medium does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for generating HSCs from definitive hemogenic endothelium; (iii) culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, wherein the culture medium is suitable for generating definitive hemogenic endothelium from mesoderm cells; and (iv) culture medium comprising a GSK3 inhibitor and basic iHSC basal medium, wherein the culture medium is suitable for generating mesoderm cells from iPSCs. In some embodiments, the iPSCs are initial iPSCs.

In one embodiment, the culture platform for obtaining T progenitor cells comprises (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and IL7, and an iTC basic culture medium, wherein the culture medium (i) is suitable for producing T progenitor cells from permanent HSC. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, and an iHSC basic culture medium, the latter comprising a BMP activator, wherein the culture medium (ii) does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for producing HSC from permanent hemogenic endothelium. In one embodiment, the culture platform comprising culture medium (i) and (ii) further comprises (iii) a culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and an iHSC basic culture medium, wherein the culture medium (iii) is suitable for producing permanent hemogenic endothelium from mesoderm cells. In another embodiment, the culture platform comprising culture medium (i), (ii) and (iii) further comprises (iv) culture medium comprising a GSK3 inhibitor and basic iHSC basic medium, wherein culture medium (iv) is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

One aspect of the present invention provides a culture platform for obtaining permanent HSCs, comprising one or more (i) culture media comprising one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO and iHSC basal medium, the latter comprising a BMP activator, wherein the culture media does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for generating HSCs from permanent hemogenic endothelium; (ii) culture media comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, wherein the culture media is suitable for generating permanent hemogenic endothelium from mesoderm cells; (iii) culture media comprising a GSK3 inhibitor and a basic iHSC basal medium, wherein the culture media is suitable for generating mesoderm cells from iPSCs. In some embodiments, the iPSCs are initial iPSCs.

In one embodiment, the culture platform for obtaining permanent HSC comprises (i) culture medium, which comprises one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO and iHSC basic culture medium, the latter comprising a BMP activator, wherein the culture medium (i) does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for producing HSC from permanent hemogenic endothelium. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) culture medium, which comprises a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basic culture medium, wherein culture medium (ii) is suitable for producing permanent hemogenic endothelium from mesoderm cells. In another embodiment, the culture platform comprising culture medium (i) and (ii) further comprises (iii) culture medium, which comprises a GSK3 inhibitor and basic iHSC basic culture medium, wherein culture medium (iii) is suitable for producing mesoderm cells from iPSC. In some embodiments, the permanent HSC obtained is CD34+HSC. In some embodiments, iPSC is initial iPSC.

Yet another aspect of the present invention provides a culture platform for obtaining hemogenic endothelium, comprising one or more of (i) a culture medium comprising a GSK3 inhibitor and optionally a TGFβ receptor/ALK inhibitor and an iHSC basal medium comprising a BMP activator, wherein the culture medium is suitable for generating definitive hemogenic endothelium from mesodermal cells; and (ii) a culture medium comprising a GSK3 inhibitor and a basal iHSC basal medium, wherein the culture medium is suitable for generating mesodermal cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

In one embodiment, the culture platform for obtaining hemogenic endothelium comprises (i) a culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and an iHSC basal culture medium comprising a BMP activator, wherein the culture medium (i) is suitable for producing permanent hemogenic endothelium from mesoderm cells. In another embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising a GSK3 inhibitor and a basic iHSC basal culture medium, wherein the culture medium (ii) is suitable for producing mesoderm cells from iPSC. In some embodiments, iPSC is an initial iPSC.

A further aspect of the present invention provides a culture platform for obtaining NK cells, comprising one or more of (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IGF and IL7 and an iNK basal medium, the latter comprising one or more growth factors and cytokines selected from IL2, IL3, IL6 and IL15, wherein the culture medium does not contain a BMP activator and is suitable for generating NK cells from NK progenitor cells; (ii) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and VEGF, a BMP activator and an iNK basal medium, wherein the culture medium is suitable for generating NK progenitor cells from definitive HSCs; (iii) a culture medium comprising One or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO and iHSC basal medium, the latter comprising a BMP activator, wherein the medium does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for generating HSCs from definitive hemogenic endothelium; (iv) a culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, the latter comprising a BMP activator, wherein the culture medium is suitable for generating definitive hemogenic endothelium from mesoderm cells; and (v) a culture medium comprising a GSK3 inhibitor and basic iHSC basal medium, wherein the culture medium is suitable for generating mesoderm cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

One embodiment of a culture platform for obtaining NK cells comprises (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and an iNK basal medium comprising one or more growth factors and cytokines selected from IL2, IL3, IL6 and IL15, wherein the culture medium (i) does not contain a BMP activator and is suitable for generating NK cells from NK progenitor cells. In one embodiment, the culture platform comprising the culture medium (i) further comprises (ii) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and VEGF, a BMP activator and an iNK basal medium comprising one or more growth factors and cytokines selected from IL2, IL3, IL6 and IL15, wherein the culture medium (ii) is suitable for generating NK progenitor cells from definitive HSCs. In one embodiment, the culture platform comprising culture medium (i) and (ii) further comprises (iii) culture medium, which comprises one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO and iHSC basic culture medium, the latter comprising a BMP activator, wherein culture medium (iii) does not contain Wnt pathway activators and TGFβ receptor/ALK inhibitors and is suitable for producing HSC from permanent hemogenic endothelium. In one embodiment, the culture platform comprising culture medium (i), (ii) and (iii) further comprises (iv) culture medium, which comprises a GSK3 inhibitor and optional TGFβ receptor/ALK inhibitor and iHSC basic culture medium, wherein culture medium (iv) is suitable for producing permanent hemogenic endothelium from mesoderm cells. In another embodiment, the culture platform comprising culture medium (i), (ii), (iii) and (iv) further comprises (v) culture medium, which comprises a GSK3 inhibitor and basic iHSC basic culture medium, wherein culture medium (v) is suitable for producing mesoderm cells from iPSC. In some embodiments, iPSC is initial iPSC.

Another aspect of the present invention provides a culture platform for obtaining NK progenitor cells, comprising one or more of (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, and iNK basal medium comprising one or more growth factors and cytokines selected from the group consisting of IL2, IL3, IL6, and IL15, wherein the culture medium is suitable for generating NK progenitor cells from definitive HSCs; (ii) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and (iii) a culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, wherein the culture medium is suitable for generating definitive hemogenic endothelium from mesoderm cells; and (iv) a culture medium comprising a GSK3 inhibitor and basal iHSC basal medium, wherein the culture medium is suitable for generating mesoderm cells from iPSCs. In some embodiments, the iPSCs are naive iPSCs.

One embodiment of a culture platform for obtaining NK progenitor cells comprises (i) a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L and VEGF, and an iNK basal medium comprising one or more growth factors and cytokines selected from IL2, IL3, IL6 and IL15, wherein the culture medium (i) is suitable for generating NK progenitor cells from definitive HSCs. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) a culture medium comprising one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, and an iHSC basal medium comprising a BMP activator, wherein the culture medium (ii) does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for generating HSCs from definitive hemogenic endothelium. In one embodiment, the culture platform comprising culture medium (i) and (ii) further comprises (iii) culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, wherein culture medium (iii) is suitable for producing permanent hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform comprising culture medium (i), (ii) and (iii) further comprises (iv) culture medium comprising a GSK3 inhibitor and basic iHSC basal medium, wherein culture medium (v) is suitable for producing mesodermal cells from iPSC. In some embodiments, iPSC is an initial iPSC.

One embodiment of a culture platform for obtaining NK progenitor cells comprises one or more artificial antigens in the form of microbead conjugates, plasma membrane particles, and/or antigen presenting cells to stimulate NK growth, development, and maturation.

Yet another aspect of the present invention provides a culture platform for generating permanent CD34+ cells, comprising one or more (i) culture medium comprising one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO and iHSC basal medium, the latter comprising a BMP activator, wherein the culture medium does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor and is suitable for generating HSCs from permanent hemogenic endothelium; (ii) culture medium comprising a GSK3 inhibitor and an optional TGFβ receptor/ALK inhibitor and iHSC basal medium, wherein the culture medium is suitable for generating permanent hemogenic endothelium from mesoderm cells; and (iii) culture medium comprising a GSK3 inhibitor and basic iHSC basal medium, wherein the culture medium is suitable for generating mesoderm cells from pluripotent stem cells including iPSCs. In some embodiments, the iPSCs are initial iPSCs.

II. iCD34 Platform

Another aspect of the present invention provides a culture platform for obtaining permanent hemogenic endothelium using pluripotent stem cells. As used herein, permanent hemogenic endothelium refers to a hematopoietic cell population that is directed to permanent hematopoiesis and has the ability to generate all hematopoietic cells, including but not limited to permanent HSCs, hematopoietic multipotent progenitor cells (MPPs), T progenitor cells, NK progenitor cells, mature T cells and/or NK cells.

In one embodiment, the culture platform for obtaining permanent hemogenic endothelium using pluripotent stem cells including iPSC includes an inoculation culture medium containing MEKi, GSKi and ROCKi. In some embodiments, the inoculation culture medium does not contain or is substantially free of TGFβ receptor/ALK inhibitors. In one embodiment, the combination of small molecules in the inoculation culture medium of the present invention is shown as fate maintenance culture medium (FMM) in Table 9. The components of the culture medium can be present in the culture medium in an amount within the concentration range shown in Table 9. In one embodiment, the iPSC for obtaining permanent hemogenic endothelium is a cell line produced using fate reprogramming culture medium (FRM), and is further maintained in FMM to establish and maintain the basis or initial state of the iPSC cell line suitable for stage-specific differentiation disclosed herein. The basis or initial iPSC thus obtained is suitable for cryopreservation. In the present invention, the iPSC cell line preserved or the iPSC cloned can be seeded in FMM for subsequent differentiation into permanent hemogenic endothelium.

Table 9: Initial iPSC seeding culture to obtain CD34+ definitive hemogenic endothelium, multipotent progenitor cells, T progenitor cells and NK progenitor cells:

One aspect of the present invention provides a culture medium for differentiating and expanding mesoderm from pluripotent stem cells including iPSC. In some embodiments, iPSC is an initial iPSC. In one embodiment, the culture medium comprises a BMP activator, and optional bFGF and CD34 basic culture medium, the latter comprising a small molecule combination as shown in Table 10. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors and/or cytokines in a concentration range as shown in Table 10. In some embodiments, Matrigel ^™ is used to replace vitronectin to fully define the culture medium.

In one embodiment, the culture medium for differentiating and expanding mesoderm from pluripotent stem cells further comprises 0.2-50 ng of bFGF.

One aspect of the present invention provides a culture medium for obtaining mesodermal cells with definitive hemogenic endothelial potential from pluripotent stem cells, including iPSCs. In some embodiments, the iPSCs are naive iPSCs. In one embodiment, the culture medium comprises a BMP activator, a GSK3 inhibitor, and bFGF. In one embodiment, the culture medium comprising the GSK3 inhibitor is applied only after the mesodermal cells have specialized to achieve definitive HE potential. In one embodiment, the culture medium comprising the BMP activator, the GSK3 inhibitor, and bFGF further comprises CD34 minimal medium comprising a combination of small molecules as shown in Table 11. In one embodiment, the culture medium does not contain a TGFβ receptor/ALK inhibitor. In some embodiments, the culture medium comprises an extracellular matrix protein. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines in the concentration ranges shown in Table 11. In some embodiments, Matrigel ^™ is used instead of vitronectin to fully define the culture medium.

One aspect of the present invention provides a culture medium for obtaining definitive hemogenic endothelium from mesodermal cells. In one embodiment, the culture medium comprises a ROCK inhibitor and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11. In one embodiment, the culture medium comprises VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor and a CD34 minimal medium, the latter comprising a combination of small molecules as shown in Table 12. In one embodiment, the culture medium comprising VEGF, bFGF, SCF, IL6, IL11, and a ROCK inhibitor does not contain IGF1 and/or EPO. In other embodiments, the culture medium herein comprises small molecules, growth factors, and/or cytokines in concentrations ranging as shown in Table 12.

One aspect of the present invention provides a culture platform for obtaining multipotent progenitor (MPP) cells from permanent hemogenic endothelium.MPP can further differentiate into bone marrow cells, including neutrophil progenitor cells. In one embodiment, the culture platform comprises (i) culture medium, which comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, wherein the culture medium is suitable for differentiating permanent hemogenic endothelium into pre-HSC (Table 13). In another embodiment, the culture platform comprising a culture medium for differentiating permanent hemogenic endothelium into pre-HSC further comprises (ii) culture medium, which comprises a BMP activator, TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11, wherein the culture medium does not contain a ROCK inhibitor and is suitable for differentiating pre-HSC into multipotent progenitor cells. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium herein comprises small molecules, growth factors and/or cytokines in concentrations ranging as shown in Table 13.

One aspect of the present invention provides a culture platform for producing T progenitor cells or T-cells from permanent hemogenic endothelium. In one embodiment, the culture platform comprises (i) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7, wherein the culture medium is suitable for differentiating permanent hemogenic endothelium into pre-T progenitor cells (pre-proT); and (ii) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7, wherein the culture medium does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors, and is suitable for differentiating pre-T progenitor cells into T progenitor cells or T cells (Table 14). In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium herein comprises small molecules, growth factors and/or cytokines in a concentration range as shown in Table 14.

One aspect of the present invention provides a culture platform for producing NK progenitor cells or NK cells from permanent hemogenic endothelium. In one embodiment, the culture platform comprises (i) culture medium, which comprises a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium is suitable for differentiating permanent hemogenic endothelium into pre-NK progenitor cells (pre-proNK); and (ii) culture medium, which comprises one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors and is suitable for differentiating pre-NK progenitor cells into NK progenitor cells or NK cells. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In other embodiments, the culture medium herein comprises small molecules, growth factors and/or cytokines in concentration ranges as shown in Table 8.

Another aspect of the present invention provides a culture platform for obtaining T progenitor cells or T cells, comprising one or more of (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L and IL7 and being free of or substantially free of one or more of VEGF, bFGF, BMP activators and ROCK inhibitors, the culture medium being suitable for differentiating pre-T progenitor cells into T progenitor cells or T cells; (ii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7, and being suitable for differentiating definitive hemogenic endothelium into pre-T progenitor cells; and (iii) a culture medium, It comprises a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11 and is suitable for differentiating and expanding permanent hemogenic endothelium from mesodermal cells; (iv) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor and is suitable for obtaining permanent hemogenic endothelial potential in mesodermal cells; (v) a culture medium comprising a BMP activator, and optionally bFGF and suitable for generating and expanding mesodermal cells from iPSCs; and (vi) a culture medium comprising MEKi, GSKi and ROCKi, free of or substantially free of TGFβ receptor/ALK inhibitors and suitable for seeding and expanding initial iPSCs. In some embodiments, all of the above culture media are free of or substantially free of TGFβ receptor/ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

In one embodiment, the culture platform for producing T progenitor cells or T cells comprises (i) a culture medium comprising SCF, Flt3L and IL7, free of or substantially free of one or more of VEGF, bFGF, BMP activators and ROCK inhibitors, and suitable for converting pre-T progenitor cells into T progenitor cells or T cells. In another embodiment, the culture platform for producing T progenitor cells or T cells comprising culture medium (i) further comprises (ii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L and IL7, and suitable for differentiating permanent hemogenic endothelium into pre-T progenitor cells. In another embodiment, the culture platform for producing T progenitor cells or T cells comprising culture medium (i) and (ii) further comprises (iii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11, and suitable for differentiating and expanding permanent hemogenic endothelium from mesoderm cells. In another embodiment, the culture platform for producing T progenitor cells or T cells comprising culture medium (i), (ii) and (iii) further comprises (iv) culture medium, which comprises BMP activator, bFGF and GSK3 inhibitor and is suitable for obtaining permanent hemogenic endothelial potential in mesodermal cells. In another embodiment, the culture platform for producing T progenitor cells or T cells comprising culture medium (i), (ii), (iii) and (iv) further comprises (v) culture medium, which comprises BMP activator, and optional bFGF and is suitable for producing and amplifying mesodermal cells from iPSC. In another embodiment, the culture platform for producing T progenitor cells or T cells comprising culture medium (i), (ii), (iii), (iv) and (v) further comprises (vi) culture medium, which comprises MEKi, GSKi and ROCKi, is free of or substantially free of TGFβ receptor/ALK inhibitors and is suitable for inoculating and amplifying initial iPSC. In some embodiments, all of the above culture media are free of or substantially free of TGFβ receptor/ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, the culture platform for generating T progenitor cells or T cells uses Notch factors. In some embodiments, Notch factors include Jag1, Jag2, DLL-1, DLL-3 and DLL-4, which can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides or cell-presented peptides.

Another aspect of the present invention provides a culture platform for obtaining NK progenitor cells or NK cells, comprising one or more of: (i) a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor and is suitable for differentiating pre-NK progenitor cells into NK progenitor cells or NK cells; (ii) a culture medium comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15, wherein the culture medium is suitable for differentiating definitive hemogenic endothelial cells into definitive hemogenic endothelial cells. Differentiation into pre-NK progenitor cells; (iii) a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11 and suitable for differentiating and expanding permanent hemogenic endothelium from mesoderm cells; (iv) a culture medium comprising a BMP activator, bFGF and a GSK3 inhibitor and suitable for obtaining permanent hemogenic endothelial potential in mesoderm cells; (v) a culture medium comprising a BMP activator, and optional bFGF and suitable for generating and expanding mesoderm cells from iPSCs; (vi) a culture medium comprising MEKi, GSKi and ROCKi, free of or substantially free of TGFβ receptor/ALK inhibitors and suitable for seeding and expanding initial iPSCs. In some embodiments, all of the above culture media are free of or substantially free of TGFβ receptor/ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, NK maturation is induced using one or more artificial antigens designed to stimulate NK growth, development, and maturation, wherein the antigens are introduced in the form of microbead conjugates, plasma membrane particles, and/or antigen-presenting cells.

In one embodiment, the culture platform for producing NK progenitor cells or NK cells comprises: (i) a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium does not contain or substantially does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors and is suitable for differentiating pre-NK progenitor cells into NK progenitor cells or NK cells. In some embodiments, NK maturation is induced using one or more artificial antigens for stimulating NK growth, development and maturation, and the antigens are introduced in the form of microbead conjugation, plasma membrane particles and/or antigen presenting cells. In another embodiment, the culture platform for producing NK progenitor cells or NK cells comprising culture medium (i) further comprises (ii) a culture medium comprising a BMP activator, a ROCK inhibitor, one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7 and IL15, wherein the culture medium is suitable for differentiating permanent hemogenic endothelium into pre-NK progenitor cells. In another embodiment, the culture platform for producing NK progenitor cells or NK cells comprising culture medium (i) and (ii), further comprises (iii) culture medium, which comprises ROCK inhibitor, one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11 and is suitable for differentiating and expanding permanent hemogenic endothelium from mesodermal cells. In another embodiment, the culture platform for producing NK progenitor cells or NK cells comprising culture medium (i), (ii) and (iii), further comprises (iv) culture medium, which comprises BMP activator, bFGF and GSK3 inhibitor and is suitable for obtaining permanent hemogenic endothelial potential in mesodermal cells. In another embodiment, the culture platform for producing NK progenitor cells or NK cells comprising culture medium (i), (ii), (iii) and (iv), further comprises (v) culture medium, which comprises BMP activator, and optional bFGF and is suitable for producing and expanding mesodermal cells from iPSC. In another embodiment, the culture platform for producing NK progenitor cells or NK cells comprising culture media (i), (ii), (iii), (iv) and (v) further comprises (vi) a culture medium comprising MEKi, GSKi and ROCKi, which is free of or substantially free of TGFβ receptor/ALK inhibitors and is suitable for seeding and expanding initial iPSCs. In some embodiments, all of the above culture media are free of or substantially free of TGFβ receptor/ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

One aspect of the present invention provides a culture platform for generating permanent hemogenic endothelium, comprising one or more (i) a culture medium for differentiating and expanding permanent hemogenic endothelium from mesoderm cells, comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11; (ii) a culture medium for obtaining permanent hematopoietic potential in mesoderm cells, comprising a BMP activator, bFGF and a GSK3 inhibitor; (iii) a culture medium for differentiating and expanding mesoderm cells from initial iPSCs, comprising a BMP activator, and optional bFGF; and (iv) initial iPSC seeding and expansion culture medium, comprising MEKi, GSKi and ROCKi, and the seeding culture does not contain TGFβ receptor/ALK inhibitors. In some embodiments, the permanent hemogenic endothelium is CD34+. In some embodiments, all of the above culture media do not contain or are substantially free of TGFβ receptor/ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

In one embodiment, the culture platform for obtaining permanent hemogenic endothelium comprises (i) culture medium, which comprises a ROCK inhibitor, and one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11, wherein the culture medium is suitable for differentiating and expanding permanent hemogenic endothelium from mesoderm cells. In one embodiment, the culture platform comprising culture medium (i) further comprises (ii) culture medium, which comprises a BMP activator, bFGF and a GSK3 inhibitor, wherein culture medium (ii) is suitable for obtaining permanent hematopoietic potential in mesoderm cells. In another embodiment, the culture platform comprising culture medium (i) and (ii) further comprises (iii) culture medium, which comprises a BMP activator, and optional bFGF, wherein culture medium (iii) is suitable for differentiating and expanding mesoderm cells from initial iPSC. In another embodiment, the culture platform comprising culture medium (i), (ii) and (iii) further comprises (iv) culture medium comprising MEKi, GSKi and ROCKi, and culture medium (v) does not contain or is substantially free of TGFβ receptor/ALK inhibitors, wherein culture medium (v) is suitable for inoculating and expanding initial iPSCs. In some embodiments, all of the above culture media do not contain or are substantially free of TGFβ receptor/ALK inhibitors. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

One aspect of the present invention provides a culture platform for generating CD34+ permanent hemogenic endothelium, comprising one or more (i) a culture medium for differentiating and expanding permanent hemogenic endothelium from mesoderm cells, comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11, wherein the permanent hemogenic endothelium comprises CD34+ permanent hemogenic endothelium; (ii) a culture medium for obtaining permanent hematopoietic potential in mesoderm cells, comprising a BMP activator, bFGF and a GSK3 inhibitor; (iii) a culture medium for differentiating and expanding mesoderm cells from initial iPSCs, comprising a BMP activator, and optional bFGF; and (iv) an initial iPSC seeding or expansion culture medium comprising MEKi, GSKi and ROCKi, and the seeding culture medium does not contain or substantially does not contain a TGFβ receptor/ALK inhibitor. In some embodiments, all of the above culture media do not contain or substantially do not contain a TGFβ receptor/ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4.

One aspect of the present invention provides a culture platform for producing mesodermal cells, which comprises one or more (i) a culture medium for differentiating and expanding mesodermal cells from initial iPSCs, which comprises a BMP activator, and optionally bFGF; and (ii) an initial iPSC seeding or expansion culture medium, which comprises MEKi, GSKi, and ROCKi, and the seeding culture medium does not contain or substantially does not contain a TGFβ receptor/ALK inhibitor. In some embodiments, all of the above culture media do not contain or substantially do not contain a TGFβ receptor/ALK inhibitor. In some embodiments, the GSK3 inhibitor is CHIR99012 or BIO. In some embodiments, the GSK3 inhibitor is CHIR99012. In some embodiments, the ROCK inhibitor is thiazovivin or Y27632. In some embodiments, the ROCK inhibitor is Y27632. In some embodiments, the BMP activator is BMP4. In some embodiments, the culture platform for generating mesodermal cells may further comprise (iii) a culture medium comprising a BMP activator, bFGF, and a GSK3 inhibitor, wherein the culture medium is used to obtain permanent hematopoietic potential in the mesodermal cells. In some embodiments, the culture medium comprises a BMP activator, bFGF, and a GSK3 inhibitor, but does not contain a TGFβ receptor/ALK inhibitor.

C. Methods for Obtaining CD34+ Cells, Definitive Hemogenic Endothelium, Multipotent Progenitor Cells, T Cell or NK Cell Progenitor Cells, T Cells and/or NK Cells

The present invention provides a method for producing permanent hematopoietic cells derived from pluripotent stem cells using a multi-stage culture platform comprising one or more culture media. The method is suitable for feeder-free conditions. The method is also suitable for monolayer culture, and thus compared with methods known in the art, pluripotent stem cell differentiation can be carried out without the need for EB formation or aggregate intermediates. The provided method produces and simultaneously amplifies permanent hematopoietic endothelium (iHE), permanent HSC (iHSC), CD34+HE (iCD34) derived from pluripotent stem cells, which can be further differentiated into multipotent progenitor cells (iMPP), natural killer progenitor cells (ipro-NK), T progenitor cells (ipro-T), mature NK cells (iNK) and T cells (iT). Another aspect of the present invention also provides a method for producing bone marrow cells from CD34+, HE, HSC and/or MPP differentiation derived from pluripotent stem cells.

In one embodiment, the present invention provides a method for differentiating and expanding hematopoietic lineage cells from pluripotent cells in monolayer culture, comprising contacting pluripotent cells with a BMP pathway activator and, optionally, bFGF, wherein pluripotent stem cell-derived mesoderm cells are obtained and expanded from the pluripotent stem cells without forming embryoid bodies, which are then contacted with a BMP pathway activator, bFGF, and a WNT pathway activator to obtain expanded pluripotent stem cell-derived mesoderm cells with definitive hemogenic endothelial (HE) potential from the pluripotent stem cells without forming embryoid bodies. By subsequently contacting the cells with bFGF and, optionally, a ROCK inhibitor and/or a WNT pathway activator, the mesoderm cells with definitive HE potential are differentiated into definitive HE cells, which are also expanded during the differentiation process.

The provided methods for obtaining hematopoietic lineage cells are advantageous over EB-cultured pluripotent stem cell differentiation, as EB formation does not result in cell expansion, does not allow monolayer culture, and is laborious and inefficient. Furthermore, the present invention discloses that monolayer culture using the methods provided herein can result in functional hematopoietic cell lineages that are capable of long-term hematopoietic self-renewal, reconstitution, and engraftment in vivo.

As described in detail below, the present invention provides methods for deriving hematopoietic cell lineages from pluripotent cells by obtaining definitive HSCs or definitive hemogenic endothelial cells. In particular, the present invention provides methods for directing the differentiation of pluripotent cells toward cells of the hematopoietic lineage without the goal of differentiation to form EBs.

I.iHSC Platform

1. Differentiation and expansion from pluripotent stem cells, pluripotent stem cell-derived mesoderm, or HE

iHSC—iHSC Platform

One aspect of the present invention provides a method for generating and expanding permanent HSCs (iHSCs) using a multi-stage process. Generally, the method begins in the first stage, in which pluripotent stem cells are differentiated into mesodermal cells, and then in the second stage, the mesodermal cells are differentiated and expanded into hemogenic endothelium (HE). In the third stage, HE cells are differentiated into permanent HSCs (iHSCs), which are also expanded. The present invention also provides a method for generating permanent HSCs (iHSCs), comprising differentiating and expanding mesodermal cells derived from pluripotent stem cells into HE and differentiating HE into iHSCs. Alternatively, the present invention provides a method for generating and expanding permanent HSCs (iHSCs), comprising differentiating HE derived from pluripotent stem cells into iHSCs. In some embodiments of the above methods, the pluripotent stem cells include iPSCs. In some embodiments, the iPSCs are initial iPSCs.

In one embodiment of a method for generating definitive HSCs (iHSCs) from initial pluripotent stem cells, the method comprises: (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein, wherein the differentiated mesodermal cells expand; (2) differentiating the mesodermal cells into definitive hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein the definitive HE cells expand; and (3) differentiating the definitive HE cells into definitive HSCs by contacting the definitive HE cells with a third culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the definitive HSCs expand. In some embodiments, the pluripotent stem cells comprise iPSCs. In some embodiments, the iPSCs are initial iPSCs. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSC (iHSC) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, CD34 is positive and CD43 is negative. In some embodiments, CD34 is positive, CD43 is negative and CD73 is negative. In some other embodiments, CD34 is positive, CD43 is negative, CD73 is negative and CXCR4 is negative. In some embodiments, the above method comprises differentiating the mesodermal cells into definitive hemogenic endothelium by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator, a Wnt pathway activator and a TGFβ receptor inhibitor.

In one embodiment of a method for generating and expanding permanent HSCs (iHSCs) from mesodermal cells derived from pluripotent stem cells, the method comprises differentiating the mesodermal cells into permanent HE cells by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein the permanent HE cells expand; and (2) differentiating the obtained HE cells into iHSCs by contacting the HE cells with a second culture medium comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, wherein the iHSCs expand. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

In one embodiment of a method for generating and expanding definitive HSCs (iHSCs) from pluripotent stem cell-derived HE, the method comprises contacting the HE cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the iHSCs are differentiated and expanded from pluripotent stem cell-derived HE. In some embodiments, the iHSCs obtained by the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4.

2. HE-iHSC platform for differentiation and expansion from pluripotent stem cells or pluripotent stem cell-derived mesoderm

One aspect of the present invention provides a method for producing and amplifying permanent hemogenic endothelium using a multi-stage process. Generally, the method begins with a first stage, wherein pluripotent stem cells are differentiated into mesodermal cells, which are then amplified and differentiated into hemogenic endothelium in a second stage. Initial pluripotent cells include, but are not limited to, inductive basis or initial pluripotent stem cells and embryonic stem cells. In some embodiments, the hemogenic endothelium produced and amplified is permanent. In some embodiments, the permanent hemogenic endothelium produced is CD34 positive. Alternatively, the present invention provides a method for producing and amplifying hemogenic endothelium by differentiating mesodermal cells derived from pluripotent stem cells into permanent hemogenic endothelium.

In one embodiment of a method for differentiating and expanding definitive hemogenic endothelium from pluripotent stem cells, the method comprises (1) differentiating pluripotent stem cells into mesodermal cells by contacting the cells with a first culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein; and (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and, optionally, a TGFβ receptor inhibitor, wherein definitive HE cells are expanded. In some embodiments, the HE cells obtained from the above method express CD34. In some embodiments, the above method comprises a GSK3 inhibitor as a Wnt pathway activator. In some embodiments, the above method comprises CHIR99021 or BIO as a GSK3 inhibitor. In some embodiments, the above method comprises CHIR99021 as a GSK3 inhibitor. In some embodiments, the above method comprises SB431542 or A83-01 as a TGFβ receptor inhibitor. In some embodiments, the above method comprises SB431542 as a TGFβ receptor inhibitor.

In one embodiment of a method for differentiating and expanding hemogenic endothelium from pluripotent stem cell-derived mesodermal cells, the method comprises differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and, optionally, a TGFβ receptor inhibitor. In some embodiments, the definitive HE cells obtained from the above method express CD34.

3. Differentiation and Expansion of Mesodermal Cells from iPSCs—iHSC Platform

One aspect of the present invention provides a method for generating mesodermal cells from pluripotent stem cells. Starting pluripotent cells include, but are not limited to, induced basal or initial pluripotent stem cells and embryonic stem cells.

In one embodiment of the method for producing and expanding mesodermal cells from pluripotent stem cells, the method includes differentiating pluripotent stem cells into mesodermal cells by contacting pluripotent stem cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein. In some embodiments, the above method includes a GSK3 inhibitor as a Wnt pathway activator. In some embodiments, the above method includes CHIR99021 or BIO as a GSK3 inhibitor. In some embodiments, the above method includes CHIR99021 as a GSK3 inhibitor.

4. Deriving T cell progenitors from pluripotent stem cells, pluripotent stem cell-derived mesoderm, HE, or definitive HSC – iHSC and iTC platforms

One aspect of the present invention provides a method for producing T progenitor cells from pluripotent stem cells using a multi-stage process. Generally, the method begins in the first stage, in which pluripotent stem cells are differentiated into mesodermal cells, wherein the mesodermal cells expand, and then in the second stage, hemogenic endothelial cells differentiate and expand. In the third stage, HE cells are differentiated into permanent HSCs (iHSCs), wherein the iHSCs expand. In the fourth stage, iHSCs are differentiated into T progenitor cells. The present invention also provides a method for producing T progenitor cells, comprising differentiating pluripotent stem cell-derived mesodermal cells into HE, then differentiating HE into iHSC, and then differentiating iHSC into T progenitor cells. The present invention further provides a method for producing T progenitor cells, comprising differentiating pluripotent stem cell-derived HE into iHSC, and then differentiating iHSC into T progenitor cells. Alternatively, the present invention provides a method for producing T progenitor cells, comprising differentiating pluripotent stem cell-derived iHSC into T progenitor cells.

In one embodiment of the method for generating T progenitor cells from pluripotent stem cells, the method comprises (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein, wherein the mesodermal cells expand; (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE cells expand; (3) contacting the HE cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE cells expand; The HE cells are differentiated into definitive HSCs by a third culture medium comprising a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein iHSCs expand; and (4) the iHSCs are differentiated into T progenitor cells by contacting the iHSCs with a fourth culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the BMP activator is BMP4. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides.

In one embodiment of a method for generating T progenitor cells from pluripotent stem cell-derived mesodermal cells, the method comprises differentiating the mesodermal cells into HE cells by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE expansion occurs; (2) differentiating the obtained HE cells into iHSCs by contacting the HE cells with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein iHSC expansion occurs; and (3) differentiating the iHSCs into T progenitor cells by contacting the iHSCs with a third culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, CD34 is positive and CD43 is negative for sorting. In some embodiments, CD34 is positive, CD43 is negative and CD73 is negative for sorting. In some other embodiments, CD34 is positive, CD43 is negative, CD73 is negative and CXCR4 is negative for sorting. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3 and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides or cell-presented peptides.

In one embodiment of the method for generating T progenitor cells from HE derived from pluripotent stem cells, the method comprises (1) differentiating the HE into iHSCs by contacting the HE cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the iHSCs expand; and (2) differentiating the iHSCs into T progenitor cells by contacting the iHSCs with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathways. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting is CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting is CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

In one embodiment of the method for generating T progenitor cells from iHSCs derived from pluripotent stem cells, the method comprises differentiating iHSCs into T progenitor cells by contacting the iHSCs with a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the above method further comprises sorting and obtaining pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides.

5. T cells derived from pluripotent stem cells, pluripotent stem cell-derived mesoderm, HE, iHSC, or T cell progenitors – iHSC and iTC platforms

One aspect of the present invention provides a method for producing T cells from pluripotent stem cells using a multi-stage process. Generally, the method begins in the first stage, wherein the pluripotent stem cells are differentiated into mesodermal cells, which expand during differentiation, and then in the second stage, the mesodermal cells are differentiated into hemogenic endothelium, wherein HE cells expand. In the third stage, HE cells are differentiated into permanent HSCs (iHSCs), wherein iHSCs expand. In the fourth stage, iHSCs are differentiated into T progenitor cells (ipro-T). In the fifth stage, iHSCs are differentiated into T cells. The present invention also provides a method for producing T cells, comprising differentiating pluripotent stem cell-derived mesodermal cells into HE, differentiating HE into iHSCs, differentiating iHSCs into T progenitor cells, and then differentiating T progenitor cells into T cells. The present invention further provides a method for producing T cells, comprising differentiating pluripotent stem cell-derived HE into iHSCs, differentiating iHSCs into T progenitor cells, and differentiating T progenitor cells into T cells. Alternatively, the present invention provides a method for producing T cells, comprising differentiating pluripotent stem cell-derived iHSCs into T progenitor cells and differentiating the T progenitor cells into T cells. Further, the present invention provides a method for producing T cells, comprising differentiating pluripotent stem cell-derived T progenitor cells into T cells.

In one embodiment of the method for generating T cells from pluripotent stem cells, the method comprises (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein, wherein the mesodermal cells expand; (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE cells expand; (3) differentiating the HE cells into iHSCs by contacting the HE cells with a third culture medium comprising a BMP activator and one or more selected from VEGF, SCF, F Flt3L, IL15, IL3, IL6, IGF and TPO growth factors and cytokines, wherein iHSCs expand; (4) differentiating the iHSCs into T progenitor cells by contacting the iHSCs with a fourth culture medium, the fourth culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6, and one or more Notch pathway activators; and (5) differentiating the T progenitor cells into T cells by contacting the T progenitor cells with a fifth culture medium, the fifth culture medium comprising one or more growth factors and cytokines selected from the following: SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6, and one or more Notch pathway activators. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the BMP activator is BMP4. In one embodiment, the Notch pathway activator is Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides.

In one embodiment of the method for generating T cell-derived mesodermal cells from pluripotent stem cells, the method comprises differentiating the mesodermal cells into HE cells by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and optionally a TGFβ receptor inhibitor, wherein expansion of the definitive HE cells occurs; (2) differentiating the obtained HE cells into iHSCs by contacting the HE cells with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein (3) differentiating the iHSCs into T progenitor cells by contacting the iHSCs with a third culture medium, the third culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6, and one or more Notch pathway activators; and (4) differentiating the T progenitor cells into T cells by contacting the T progenitor cells with a fourth culture medium, the fourth culture medium comprising one or more growth factors and cytokines selected from the following: SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6, and one or more Notch pathway activators. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positive. In some embodiments, sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the Notch pathway activator is Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides.

In one embodiment of the method for generating T cell-derived HE from pluripotent stem cells, the method comprises (1) differentiating the pluripotent stem cell-derived HE into iHSC by contacting the HE cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the iHSC expands; (2) differentiating the iHSC into T progenitor cells by contacting the iHSC with a second culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators; and (3) differentiating the T progenitor cells into T cells by contacting the T progenitor cells with a third culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHSCs using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, sorting uses CD34 positivity and CD43 negativity. In some embodiments, sorting uses CD34 positivity, CD43 negativity and CD73 negativity. In some other embodiments, sorting uses CD34 positivity, CD43 negativity, CD73 negativity and CXCR4 negativity. In one embodiment, the Notch pathway activators are Jag1, Jag2, DLL-1, DLL-3 and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides or cell-presented peptides.

In one embodiment of the method for generating iHSCs of T cell origin from pluripotent stem cells, the method comprises (1) differentiating iHSCs into T progenitor cells by contacting iHSCs with a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3 and IL6, and one or more Notch pathway activators, wherein iHSCs expand; and (2) differentiating T progenitor cells into T cells by contacting T progenitor cells with a culture medium comprising one or more growth factors and cytokines selected from the following: SCF, Flt3L, IL7, IGF, IL2, IL3 and IL6, and one or more Notch pathway activators. In some embodiments, the above method further comprises sorting and obtaining pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positive. In some embodiments, sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In one embodiment, the Notch pathway activator is Jag1, Jag2, DLL-1, DLL-3, and DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides.

In one embodiment of the method of generating T cell-derived T progenitor cells from pluripotent stem cells, the method comprises differentiating the T progenitor cells into T cells by contacting the T progenitor cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators.

6. Deriving NK progenitors from pluripotent stem cell-derived mesoderm, HE, or iHSC – iHSC and iNK platforms

One aspect of the present invention provides a method for generating NK progenitor cells from pluripotent stem cells using a multi-stage process. Generally, the method begins in the first stage, in which pluripotent stem cells are differentiated into mesodermal cells, wherein the mesodermal cells expand; then in the second stage, the mesodermal cells are differentiated into hemogenic endothelium, wherein HE cells expand. In the third stage, HE cells are differentiated into permanent HSCs, wherein permanent HSCs expand. In the fourth stage, iHSCs are differentiated into NK progenitor cells. The present invention also provides a method for generating NK progenitor cells, comprising differentiating pluripotent stem cell-derived mesoderm cells into HE, then differentiating HE into iHSC, and then differentiating iHSC into NK progenitor cells. The present invention further provides a method for generating NK progenitor cells, comprising differentiating pluripotent stem cell-derived HE into iHSC and then differentiating iHSC into NK progenitor cells. Alternatively, the present invention provides a method for generating NK progenitor cells, comprising differentiating pluripotent stem cell-derived iHSC into NK progenitor cells.

In one embodiment of the method for generating NK progenitor cells from pluripotent stem cells, the method comprises (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein, wherein the mesodermal cells expand; (2) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE cells expand; (3) differentiating the mesodermal cells into hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE cells expand; HE cells are contacted with a third culture medium to differentiate the HE cells into iHSCs, wherein the third culture medium comprises a BMP activator, one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and TPO, wherein the iHSCs expand; and (4) the iHSCs are differentiated into NK progenitor cells by contacting the iHSCs with a fourth culture medium, wherein the fourth culture medium comprises a BMP activator, and one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity and CD73 negativity. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

In one embodiment of the method for generating NK progenitor-derived mesodermal cells from pluripotent stem cells, the method comprises differentiating the pluripotent stem cell-derived mesodermal cells into HE cells by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and optionally a TGFβ receptor inhibitor, wherein the HE cells expand; (2) differentiating the obtained HE cells into iHSCs by contacting the HE cells with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the iHSCs expand; and (3) differentiating the iHSCs into NK progenitor cells by contacting the iHSCs with a third culture medium comprising a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity and CD73 negativity. In some other embodiments, the sorting uses CD34 positivity, CD43 negativity, CD73 negativity and CXCR4 negativity.

In one embodiment of the method for generating NK progenitor cells from HE derived from pluripotent stem cells, the method comprises (1) differentiating HE derived from pluripotent stem cells into iHSCs by contacting the HE cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the iHSCs expand; and (2) differentiating the iHSCs into NK progenitor cells by contacting the iHSCs with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting is CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting is CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

In one embodiment of the method for producing iHSC derived from NK progenitor cells from pluripotent stem cells, the method includes differentiating iHSC into NK progenitor cells by contacting iHSC with a culture medium comprising a BMP activator, and one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15. In some embodiments, the above method further includes sorting and obtaining HSC (iHSC) derived from pluripotent stem cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the above method further includes sorting using CD34 positive. In some embodiments, sorting uses CD34 positive and CD43 negative. In some embodiments, sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative.

7. Derivation of NK cells from pluripotent stem cells, pluripotent stem cell-derived mesoderm, HE, iHSC, or NK progenitor cells – iHSC and iNK platforms

One aspect of the present invention provides a method for producing NK cells from pluripotent stem cells using a multi-stage process. Generally, the method begins in the first stage, wherein pluripotent stem cells are differentiated into mesodermal cells, wherein the mesodermal cells are expanded, and then in the second stage the mesodermal cells are differentiated into hemogenic endothelium, wherein HE cells are expanded. In the third stage, HE cells are differentiated into permanent HSCs (iHSCs); wherein HSCs are expanded. In the fourth stage, iHSCs are differentiated into NK progenitor cells (ipro-NK). In the fifth stage, iHSCs are differentiated into NK cells. The present invention also provides a method for producing NK cells, comprising differentiating pluripotent stem cell-derived mesodermal cells into HE, differentiating HE into iHSCs, differentiating iHSCs into NK progenitor cells and then differentiating NK progenitor cells into NK cells. The present invention further provides a method for producing NK cells, comprising differentiating pluripotent stem cell-derived HE into iHSCs, differentiating iHSCs into NK progenitor cells and differentiating NK progenitor cells into NK cells. Alternatively, the present invention provides a method for producing NK cells, comprising differentiating pluripotent stem cell-derived iHSCs into NK progenitor cells and differentiating pluripotent stem cell-derived NK progenitor cells into NK cells. Further, the present invention provides a method for producing NK cells, comprising differentiating pluripotent stem cell-derived NK progenitor cells into NK cells.

In one embodiment of the method for generating NK cells from pluripotent stem cells, the method comprises (1) differentiating the pluripotent stem cells into mesodermal cells by contacting the pluripotent stem cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an extracellular matrix protein, wherein the mesodermal cells expand; (2) differentiating the mesodermal cells into definitive hemogenic endothelium by contacting the mesodermal cells with a second culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and an optional TGFβ receptor inhibitor, wherein HE cells expand; (3) differentiating the HE cells into iHSCs by contacting the HE cells with a third culture medium comprising a BMP activator and one or more (4) differentiating the iHSCs into NK progenitor cells by contacting the iHSCs with a fourth culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO; and (5) differentiating the NK progenitor cells into NK cells by contacting the NK progenitor cells with a fifth culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the method comprises differentiating the mesodermal cells into definitive hemogenic endothelium by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator, a Wnt pathway activator, and a TGFβ receptor inhibitor. In one embodiment, the BMP activator is BMP4.

In one embodiment of the method for generating NK cells from pluripotent stem cell-derived mesodermal cells, the method comprises differentiating the pluripotent stem cell-derived mesodermal cells into HE cells by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and optionally a TGFβ receptor inhibitor, wherein the mesodermal cells expand; (2) differentiating the obtained HE cells into iHSCs by contacting the HE cells with a second culture medium comprising a BMP activator and one or more selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF and (1) differentiating the iHSCs into NK progenitor cells by contacting the iHSCs with a third culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and (2) differentiating the NK progenitor cells into NK cells by contacting the NK progenitor cells with a fourth culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In some embodiments, the above method comprises differentiating the mesodermal cells into definitive hemogenic endothelium by contacting the mesodermal cells with a culture medium comprising at least one BMP pathway activator and a Wnt pathway activator and a TGFβ receptor inhibitor.

In one embodiment of the method for generating NK cells from pluripotent stem cell-derived HE, the method comprises (1) differentiating the pluripotent stem cell-derived definitive HE into iHSC by contacting the HE cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO, wherein the iHSC expands; (2) differentiating the iHSC into NK progenitor cells by contacting the iHSC with a second culture medium comprising a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and (3) differentiating the NK progenitor cells into NK cells by contacting the NK progenitor cells with a third culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the iHSC cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained HSCs (iHSCs) using CD34, CD43, CD73 and/or CXCR4. In some embodiments, the above method further comprises sorting using CD34 positivity. In some embodiments, the sorting uses CD34 positivity and CD43 negativity. In some embodiments, the sorting uses CD34 positivity, CD43 negativity and CD73 negativity. In some other embodiments, the sorting uses CD34 positivity, CD43 negativity, CD73 negativity and CXCR4 negativity.

In one embodiment of the method for generating NK cells from iHSCs derived from pluripotent stem cells, the method comprises (1) differentiating iHSCs into NK progenitor cells by contacting the iHSCs with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6, and IL15; and (2) differentiating the NK progenitor cells into NK cells by contacting the NK progenitor cells with a second culture medium comprising one or more growth factors and cytokines selected from the following: SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15. In some embodiments, the above method further comprises sorting and obtaining pluripotent stem cell-derived HSCs (iHSCs) using CD34, CD43, CD73, and/or CXCR4.

In one embodiment of the method of generating NK cells from pluripotent stem cell-derived NK progenitor cells, the method comprises differentiating the NK progenitor cells into NK cells by contacting the NK progenitor cells with a fifth culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, IL2, IL3, IL6, and IL15.

II. iCD34 Platform

1. Obtain and expand permanent iHE-iCD34 platform

One aspect of the present invention provides a method for producing permanent hemogenic endothelium (iHE) using an optimized multi-stage process. Generally, the method begins in the first stage, in which pluripotent stem cells are seeded and amplified. The pluripotent stem cells are then differentiated into mesodermal cells, which are amplified in this stage. The amplified mesodermal population is then differentiated into a mesodermal population with permanent hemogenic endothelium potential, and then the permanent hemogenic endothelium is differentiated and amplified from the mesodermal cells with permanent hemogenic endothelium potential. Alternatively, the present invention provides a method for producing permanent hemogenic endothelium (iHE), comprising differentiating and amplifying mesodermal cells from pluripotent stem cells; then differentiating and amplifying permanent hemogenic endothelium (iHE) from the mesodermal cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. The present invention further provides a method for producing and amplifying permanent hemogenic endothelium (iHE), comprising differentiating and amplifying mesodermal cells derived from pluripotent stem cells and obtaining mesodermal cells with permanent iHE potential, which are then differentiated into iHE. Alternatively, the present invention provides a method for generating and expanding definitive hemogenic endothelium, comprising differentiating pluripotent stem cell-derived mesodermal cells into iHE. The method disclosed herein utilizes an optimized monolayer iCD34 culture platform, without EB formation and without or substantially without TGFβ receptor/ALK inhibitors.

In one embodiment of a method for generating definitive hemogenic endothelium (iHE) from pluripotent stem cells, the method comprises (1) differentiating and expanding a mesodermal population from the pluripotent stem cells by contacting the cells with a culture medium comprising a BMP activator and, optionally, bFGF; (2) differentiating and expanding the mesodermal population to obtain definitive HE potential in the mesodermal cells by contacting the cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (3) differentiating and expanding the mesodermal cells having definitive HE potential into definitive HE cells by contacting the cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In some embodiments, the culture medium in the above method does not contain or is substantially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the cells are contacted with a culture medium containing a GSK3 inhibitor only after the mesodermal cells are specified to achieve permanent HE potential. In some embodiments, the above method further comprises subjecting the seeded iPSCs and/or mesodermal cells to a low oxygen tension of about 2% to about 10%. In some embodiments, the above method further comprises seeding pluripotent stem cells by contacting the pluripotent cells with a culture medium containing a MEKi, a GSKi, and a ROCKi, wherein the pluripotent stem cells expand.

In one embodiment of a method for generating definitive hemogenic endothelium (iHE) from seeded pluripotent stem cells, the method comprises (1) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the pluripotent stem cells with a culture medium comprising a BMP activator and, optionally, bFGF; (2) obtaining mesodermal cells with definitive iHE potential by contacting the mesodermal cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (3) differentiating and expanding definitive HE cells from the mesodermal cells with iHE potential by contacting the mesodermal cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the above method further comprises using CD34 positive sorting. In some embodiments, CD34 positive and CD43 negative are used for sorting. In some embodiments, CD34 positive, CD43 negative and CD73 negative are used for sorting. In some other embodiments, CD34 positive, CD43 negative, CD73 negative and CXCR4 negative are used for sorting. In some embodiments, the culture medium in the above method does not contain or is substantially free of TGFβ receptor inhibitors. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the seeded iPSCs and/or mesoderm cells to a low oxygen tension of about 2% to about 10%.

In one embodiment of a method for generating definitive hemogenic endothelium (iHE) from pluripotent stem cell-derived mesodermal cells, the method comprises (1) obtaining mesodermal cells with definitive HE potential by contacting the mesodermal cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; and (2) differentiating and expanding definitive HE cells from the mesodermal cells with definitive HE potential by contacting the mesodermal cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative and CXCR4 negative. In some embodiments, the culture medium in the above method does not contain or substantially does not contain a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the mesodermal cells to a low oxygen tension of about 2% to about 10%.

In one embodiment of a method for obtaining definitive hemogenic endothelial (iHE) potential in pluripotent stem cell-derived mesodermal cells, the method comprises contacting the mesodermal cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11, wherein the mesodermal cells expand. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the mesodermal cells to a low oxygen tension of about 2% to about 10%.

2. Identify and expand pluripotent stem cell-derived mesoderm cells with permanent hemogenic endothelial potential—the iCD34 platform

One aspect of the present invention provides a method for producing mesodermal cells derived from pluripotent stem cells using an optimized multi-stage process. Generally, the method begins in the first stage, in which pluripotent stem cells are seeded. The seeded pluripotent stem cells then develop into mesoderm. In the third stage, the mesoderm is further differentiated into mesodermal cells with permanent hematopoietic endothelial potential. Alternatively, the present invention provides a method for producing mesodermal cells derived from pluripotent stem cells, comprising differentiating the seeded pluripotent stem cells into mesoderm and differentiating the mesoderm into mesodermal cells with permanent hematopoietic potential. The present invention further provides a method for producing mesodermal cells with permanent hematopoietic potential derived from pluripotent stem cells, comprising differentiating the pluripotent stem cell-derived mesoderm into mesodermal cells with permanent hematopoietic endothelial potential. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that does not contain or is substantially free of TGFβ receptor/ALK inhibitors.

In one embodiment of a method for obtaining definitive hemogenic endothelial potential in mesodermal cells derived from pluripotent stem cells, the method comprises (1) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the pluripotent stem cells with a culture medium comprising a BMP activator and, optionally, bFGF; and (2) obtaining definitive hemogenic endothelial potential in the mesodermal cells by contacting the cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the culture medium in the above method does not contain or is substantially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the pluripotent stem cells to a low oxygen tension of about 2% to about 10%. In some embodiments, the above method further comprises seeding the pluripotent stem cells by contacting the cells with a culture medium comprising a MEKi, a GSKi, and a ROCKi.

In one embodiment of a method for producing pluripotent stem cell-derived mesodermal cells with permanent hemogenic endothelial potential from pluripotent stem cell-derived mesoderm, the method comprises differentiating the mesoderm into mesodermal cells with permanent hemogenic endothelial potential by contacting the cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. In some embodiments, the culture medium in the above method does not contain or substantially does not contain a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the above method further comprises subjecting the mesoderm cells to a low oxygen tension of about 2% to about 10%.

3. Deriving and Expanding Mesoderm from Pluripotent Stem Cells

One aspect of the present invention provides a method for generating pluripotent stem cell-derived mesoderm using an optimized multi-stage process. Generally, the method begins in a first stage, in which pluripotent stem cells are seeded, and then the seeded cells are differentiated into mesoderm in a second stage. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that is free of or substantially free of TGFβ receptor/ALK inhibitors.

In one embodiment of the method for producing a pluripotent stem cell-derived mesoderm from pluripotent cells, the method includes differentiating and amplifying mesoderm cells from seeded pluripotent stem cells by contacting the cells with a culture medium comprising a BMP activator and, optionally, bFGF. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. In some embodiments, the culture medium in the above method does not contain or is substantially free of a TGFβ receptor inhibitor. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the above method further includes subjecting the seeded iPSC to a low oxygen tension of about 2% to about 10%. In some embodiments, the above method further includes seeding and amplifying iPSCs by contacting the pluripotent cells with a culture medium comprising MEKi, GSKi, and ROCK.

4. Obtaining hematopoietic multipotent progenitor cells (iMPP)—iCD34 platform and iMPP platform

One aspect of the present invention provides a method for producing multipotent progenitor cells (iMPP) derived from pluripotent stem cells using an optimized multi-stage process. Generally, the method begins in the first stage, in which pluripotent stem cells are inoculated. The inoculated cells are amplified and differentiated into mesodermal cells. The mesoderm is amplified and differentiated into mesodermal cells with permanent hemogenic endothelial potential, and then the mesodermal cells are differentiated into permanent hemogenic endothelial cells. HE cells are amplified and differentiated into pre-HSC, followed by multipotent progenitor cells, which can differentiate into bone marrow cells, including neutrophil progenitor cells. Alternatively, the present invention provides a method for producing multipotent progenitor cells (iMPP) derived from pluripotent stem cells, comprising differentiating the inoculated multipotent cells into mesoderm, differentiating the mesoderm into mesoderm cells with permanent hemogenic endothelial potential, and then differentiating the mesoderm cells into permanent iHE, which is then differentiated into iMPP. The present invention further provides a method for producing iMPPs derived from pluripotent stem cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesodermal cells with permanent hemogenic endothelial potential, and then differentiating the mesodermal cells into permanent iHE, which are then differentiated into iMPPs. Alternatively, the present invention provides a method for producing iMPPs derived from pluripotent stem cells, comprising differentiating pluripotent stem cell-derived mesoderm cells into permanent iHE, which are then differentiated into iMPPs. Further, the present invention provides a method for producing iMPPs derived from pluripotent stem cells, comprising differentiating iHE derived from pluripotent stem cells into iMPPs. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. The methods disclosed herein utilize an optimized monolayer iCD34 culture platform without EB formation, which does not contain or substantially does not contain TGFβ receptor/ALK inhibitors.

In one embodiment of a method for generating hematopoietic multipotent progenitors (iMPPs) from pluripotent stem cells, the method comprises (1) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the pluripotent cells with a culture medium comprising a BMP activator and, optionally, bFGF; (2) obtaining definitive hematopoietic endothelial potential in the mesodermal cells by contacting the mesodermal cells with a culture medium comprising a BMP activator, a Wnt pathway activator and bFGF; (3) differentiating and expanding definitive HE cells from the mesodermal cells having definitive hematopoietic endothelial potential by contacting the cells with a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6 and IL11; and (4) differentiating the definitive HE cells into iMPPs by contacting the HE cells with a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11 and, optionally, a ROCK inhibitor. In some embodiments, the method further comprises seeding and expanding the pluripotent stem cells by contacting the cells with a culture medium comprising a MEKi, a GSKi, and a ROCKi. In some embodiments, the method further comprises differentiating the definitive HE cells into pre-HSCs by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11. In other embodiments, the method comprises differentiating the definitive HE cells into pre-HSCs, further comprising differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the culture medium in the above methods is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the above methods further comprise subjecting the seeded pluripotent stem cells, mesoderm, and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above methods express CD34. In some embodiments, the above methods further comprise sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting is performed using CD34-positive and CD43-negative cells. In some embodiments, the sorting is performed using CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting is performed using CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the BMP activator in the above methods is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin.

In one embodiment of a method for generating multipotent stem cell-derived multipotent progenitors (iMPPs) from multipotent stem cell-derived mesoderm, the method comprises (1) obtaining definitive hemogenic endothelial potential in the multipotent stem cell-derived mesoderm cells by contacting the mesoderm cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (2) differentiating and expanding definitive HE cells from the mesoderm cells having definitive hemogenic endothelial potential by contacting the cells with a culture medium comprising a ROCK inhibitor, and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11; and (3) differentiating the definitive HE cells into iMPPs by contacting the HE cells with a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and optionally a ROCK inhibitor. In some embodiments, the multipotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the definitive HE cells into pre-HSCs by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11. In other embodiments, the method comprising differentiating the definitive HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the above method further comprises subjecting the mesoderm and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%.

In one embodiment of a method for generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs) from pluripotent stem cell-derived mesodermal cells with definitive hemogenic endothelial potential, the method comprises (1) differentiating and expanding definitive HE cells from pluripotent stem cell-derived mesodermal cells with definitive hemogenic endothelial potential by contacting the cells with a culture medium comprising one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11, and a ROCK inhibitor; and (2) differentiating the definitive HE cells into iMPPs by contacting the HE cells with a culture medium comprising a BMP activator, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and an optional ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the definitive HE cells into pre-HSCs by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11. In other embodiments, the method comprising differentiating the definitive HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the above method further comprises subjecting the mesodermal cells having definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%.

In one embodiment of a method for generating pluripotent stem cell-derived multipotent progenitor cells (iMPPs) from pluripotent stem cell-derived permanent HE cells, the method comprises differentiating the permanent HE cells into iMPPs by contacting the HE cells with a culture medium comprising a BMP activator, one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, and an optional ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the permanent HE cells into pre-HSCs by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11. In other embodiments, the method comprising differentiating definitive HE cells into pre-HSCs further comprises differentiating the pre-HSCs into iMPPs by contacting the pre-HSC cells with a culture medium comprising a BMP activator and one or more growth factors and cytokines selected from the group consisting of TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11, wherein the culture medium is free of or substantially free of a ROCK inhibitor. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor. In some embodiments, the above method further comprises subjecting the mesodermal cells having definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%.

5. Obtaining pluripotent stem cell-derived T progenitor cells (ipro-T) or T cells—iCD34 platform and iT platform

One aspect of the present invention provides a method for producing pluripotent stem cell-derived T progenitor cells (ipro-T) or pluripotent stem cell-derived T cells using an optimized multi-stage process. Generally, the method starts with pluripotent stem cells, from which mesodermal cells are differentiated and amplified. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. The mesoderm is then differentiated into mesodermal cells with permanent hemogenic endothelial potential. The mesodermal cells with permanent hemogenic endothelial potential are subsequently differentiated into permanent hemogenic endothelium, which is simultaneously amplified in culture medium. The permanent HE cells are then differentiated into pre-proT, which is then differentiated into T progenitor cells (pro-T), which can be continuously differentiated into T cells in the same culture medium. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived T progenitor cells (ipro-T) or T cells, comprising differentiating seeded pluripotent stem cells into mesoderm, differentiating the mesoderm into mesodermal cells with permanent hemogenic endothelial potential, and then differentiating the mesodermal cells with permanent hemogenic endothelial potential into iHE, which are then differentiated into T progenitor cells or T cells. The present invention further provides a method for producing pluripotent stem cell-derived T progenitor cells (ipro-T) or T cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesodermal cells with permanent hemogenic endothelial potential, which are then differentiated into iHE, which are then differentiated into ipro-T or T cells. Alternatively, the present invention provides a method for producing pluripotent stem cell-derived T progenitor cells or T cells, comprising differentiating pluripotent stem cell-derived mesoderm cells with permanent hemogenic endothelial potential into iHE, which are then differentiated into ipro-T or T cells. Further, the present invention provides a method for producing pluripotent stem cell-derived T progenitor cells (ipro-T) or T cells, comprising differentiating pluripotent stem cell-derived iHE into ipro-T or T cells. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. The methods disclosed herein utilize an optimized iCD34 culture platform that does not contain or substantially does not contain TGFβ receptor/ALK inhibitors. In some embodiments, Notch factors including but not limited to Jag1, Jag2, DLL-1, DLL-3, and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides.

In one embodiment of the method for generating pluripotent stem cell-derived T progenitors (ipro-T) or T cells (iT) from pluripotent stem cells, the method comprises (1) differentiating the seeded pluripotent stem cells into mesoderm by contacting the cells with a culture medium comprising a BMP activator and, optionally, bFGF; (2) differentiating the mesoderm into mesodermal cells with definitive hemogenic endothelial potential by contacting the cells with a culture medium comprising a BMP activator, a Wnt pathway activator and bFGF; (3) differentiating the mesodermal cells with definitive hemogenic endothelial potential into definitive HE cells by contacting the cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6 and IL11 and (4) differentiating the definitive HE cells into ipro-T or iT by contacting the HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L and IL7 and, optionally, one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator and a ROCK inhibitor. In some embodiments, the above method further comprises seeding and expanding pluripotent stem cells by contacting the cells with a culture medium comprising a MEKi, a GSKi, and a ROCKi. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the definitive HE cells into pre-proT cells by contacting the HE cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating the definitive HE cells into pre-proT cells further comprises differentiating the pre-proT cells into ipro-T cells or iT cells by contacting the pre-proT cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and the culture medium being free of or substantially free of one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the above method comprises pluripotent stem cell-derived pro-T cells with one or more Notch factors. In some embodiments, the Notch factor is Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides. In some embodiments, the above method further comprises subjecting the seeded pluripotent stem cells, mesoderm, and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method is free of, or substantially free of, a TGFβ receptor inhibitor.

In one embodiment of a method for generating pluripotent stem cell-derived T progenitors (ipro-T) or T cells from pluripotent stem cell-derived mesoderm, the method comprises (1) differentiating the mesoderm into mesodermal cells with definitive hemogenic endothelial potential by contacting the cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (2) differentiating the mesodermal cells with definitive hemogenic endothelial potential into definitive HE cells by contacting the cells with a culture medium comprising a ROCK inhibitor, one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (3) differentiating the definitive HE cells into ipro-T or iT by contacting the HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7 and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the definitive HE cells into pre-proT cells by contacting the HE cells with a medium containing a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating the definitive HE cells into pre-proT cells further comprises differentiating the pre-proT cells into iproT cells or iT cells by contacting the pre-proT cells with a medium containing one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and the medium being free or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method comprises introducing pluripotent stem cell-derived proT cells into one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides. In some embodiments, the above method further comprises subjecting the mesoderm and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor.

In one embodiment of a method for generating pluripotent stem cell-derived T progenitors (ipro-T) or T cells (iT) from pluripotent stem cell-derived mesodermal cells with definitive hemogenic endothelial potential, the method comprises (1) differentiating the mesodermal cells with definitive hemogenic endothelial potential into definitive HE cells by contacting the cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, IL6, and IL11; and (2) differentiating the definitive HE cells into ipro-T or iT by contacting the HE cells with a culture medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, and IL7 and optionally one or more factors selected from VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the definitive HE cells into pre-proT cells by contacting the HE cells with a medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating the definitive HE cells into pre-proT cells further comprises differentiating the pre-iproT cells into iproT cells or iT cells by contacting the pre-proT cells with a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and the medium being free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method comprises introducing pluripotent stem cell-derived proT cells into one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides. In some embodiments, the above method further comprises subjecting the mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor.

In one embodiment of a method for generating pluripotent stem cell-derived T cell progenitors (ipro-T) or T cells (iT) from pluripotent stem cell-derived HE cells, the method comprises differentiating the definitive HE cells into ipro-T or T cells by contacting the definitive HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, and IL7, and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the definitive HE cells into pre-pro-T cells by contacting the HE cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7, VEGF, and bFGF. In other embodiments, the method comprising differentiating definitive HE cells into pre-pro-T further comprises differentiating the pre-ipro-T into pro-T or iT by contacting the pre-pro-T cells with a medium comprising one or more growth factors and cytokines selected from SCF, Flt3L, and IL7, and wherein the medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the above method comprises pluripotent stem cell-derived pro-T cells with one or more Notch factors. In some embodiments, the Notch factors are Jag1, Jag2, DLL-1, DLL-3, or DLL-4. In some embodiments, DLL-1 and DLL-4 can be introduced as soluble peptides, microbead-conjugated peptides, surface-conjugated peptides, or cell-presented peptides. In some embodiments, the above method further comprises subjecting the pluripotent stem cell-derived HE cells to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor.

6. Obtaining pluripotent stem cell-derived NK progenitor cells (ipro-NK) or NK cells—iCD34 platform and iNK platform

One aspect of the present invention provides a method for producing NK progenitor cells (ipro-NK) or NK cells (iNK) derived from pluripotent stem cells using an optimized multi-stage process. Generally, the method begins with pluripotent stem cells, which are inoculated in some embodiments. Pluripotent stem cells develop into mesodermal cells, which are amplified and subsequently differentiated into mesodermal cells with permanent hemogenic endothelial potential. Permanent hemogenic endothelium is then differentiated and amplified from the mesodermal cells with permanent hemogenic endothelial potential. HE cells can be differentiated into pro-NK, which are then differentiated into NK progenitor cells (pro-NK), which can continue to be differentiated into NK cells in the same culture medium. Alternatively, the present invention provides a method for producing NK progenitor cells (ipro-NK) or NK cells derived from pluripotent stem cells, comprising differentiating the inoculated pluripotent stem cells into mesoderm, differentiating the mesoderm into mesoderm cells with permanent hemogenic endothelial potential, and then differentiating the mesoderm cells with permanent hemogenic endothelial potential into iHE, which then differentiate into NK progenitor cells. The present invention further provides a method for producing NK progenitor cells (ipro-NK) or NK cells derived from pluripotent stem cells, comprising differentiating pluripotent stem cell-derived mesoderm into mesodermal cells with permanent hemogenic endothelial potential, and then differentiating the mesodermal cells with permanent hemogenic endothelial potential into iHE, which are then differentiated into ipro-NK or iNK. Alternatively, the present invention provides a method for producing NK progenitor cells or iNK derived from pluripotent stem cells, comprising differentiating pluripotent stem cell-derived mesoderm cells with permanent hemogenic endothelial potential into iHE, which are then differentiated into ipro-NK or iNK. Further, the present invention provides a method for producing NK progenitor cells (ipro-NK) or NK cells derived from pluripotent stem cells, comprising differentiating pluripotent stem cell-derived iHE into ipro-NK or iNK. In some embodiments, the pluripotent stem cell is an iPSC. In some embodiments, the iPSC is an initial iPSC. In some embodiments, the culture platform for obtaining NK progenitor cells comprises obtaining NK cells by contacting pro-NK cells with one or more artificial antigens for stimulating NK growth, development, and maturation, wherein the artificial antigens are introduced in the form of microbead conjugates, plasma membrane particles, and/or antigen-presenting cells. The methods disclosed herein utilize an optimized iCD34 culture platform that is free of or substantially free of TGFβ receptor/ALK inhibitors.

In one embodiment of a method for generating pluripotent stem cell-derived NK progenitors (ipro-NK) or NK cells (iNK) from seeded iPSCs, the method comprises (1) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the cells with a culture medium comprising a BMP activator and, optionally, bFGF; (2) obtaining definitive hemogenic endothelial potential in the mesodermal cells by contacting the mesodermal cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (3) differentiating and expanding mesodermal cells from pluripotent stem cells by contacting the cells with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF; (4) differentiating the definitive HE cells into ipro-NK or iNK by contacting the HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the definitive HE cells into pre-iproNK by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating definitive HE cells into pre-proNK cells further comprises differentiating the pre-proNK cells into pro-iNK cells or iNK cells by contacting the pre-proNK cells with a medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and wherein the medium is free of or substantially free of one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK progenitor cells comprises obtaining NK cells by contacting the pro-NK cells with one or more artificial antigens for stimulating NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of microbead conjugates, plasma membrane particles, and/or antigen-presenting cells. In one embodiment, the method further comprises seeding and expanding the primary pluripotent cells by contacting the pluripotent cells with a medium comprising a MEKi, a GSKi, and a ROCKi. In some embodiments, the method further comprises subjecting the seeded iPSCs, mesoderm, and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above methods express CD34. In some embodiments, the above methods further comprise sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In some embodiments, the BMP activator in the above methods is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above methods does not contain or is substantially free of a TGFβ receptor inhibitor.

In one embodiment of a method for generating pluripotent stem cell-derived NK progenitors (ipro-NK) or NK cells (iNK) from pluripotent stem cell-derived mesoderm, the method comprises (1) differentiating the mesoderm by contacting the mesoderm with a culture medium comprising a BMP activator, a Wnt pathway activator, and bFGF to obtain mesodermal cells with definitive hemogenic endothelial potential; (2) differentiating the mesodermal cells with definitive hemogenic endothelial potential by contacting the cells with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11 to obtain definitive HE cells; and (3) differentiating the definitive HE cells by contacting the HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor to obtain ipro-NK or iNK. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSC is a naive iPSC. In some embodiments, the above method further comprises differentiating the definitive HE cells into pro-proNK cells by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating the definitive HE cells into pro-proNK cells further comprises differentiating the pro-proNK cells into ipro-NK cells or iNK cells by contacting the pro-proNK cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the culture platform for obtaining NK progenitor cells comprises obtaining NK cells by contacting the pro-NK cells with one or more artificial antigens for stimulating NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of microbead conjugates, plasma membrane particles, and/or antigen-presenting cells. In some embodiments, the above method further comprises subjecting the mesoderm and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting is performed using CD34-positive and CD43-negative cells. In some embodiments, the sorting is performed using CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting is performed using CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor.

In one embodiment of a method for generating pluripotent stem cell-derived NK progenitors (ipro-NK) or NK cells (iNK) from pluripotent stem cell-derived mesodermal cells with definitive hemogenic endothelial potential, the method comprises: (1) differentiating and expanding definitive HE cells by contacting the mesodermal cells with definitive hemogenic endothelial potential with a culture medium comprising a ROCK inhibitor and one or more growth factors and cytokines selected from the group consisting of VEGF, bFGF, SCF, IL6, and IL11; and (2) differentiating the definitive HE cells into ipro-NK or iNK by contacting the HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15 and optionally one or more factors selected from the group consisting of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the above method further comprises differentiating the definitive HE cells into pro-ipro-NK cells by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating the definitive HE cells into pro-pro-NK cells further comprises differentiating the pro-proNK cells into ipro-NK cells or iNK cells by contacting the pro-proNK cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and wherein the culture medium is free of or substantially free of one or more of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the culture platform for obtaining NK progenitor cells comprises obtaining NK cells by contacting the pro-NK cells with one or more artificial antigens for stimulating NK growth, development, and maturation, wherein the artificial antigens are introduced in the form of microbead conjugates, plasma membrane particles, and/or antigen-presenting cells. In some embodiments, the above method further comprises subjecting the seeded pluripotent stem cells, mesoderm, and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting is performed using CD34-positive and CD43-negative cells. In some embodiments, the sorting is performed using CD34-positive, CD43-negative, and CD73-negative cells. In some other embodiments, the sorting is performed using CD34-positive, CD43-negative, CD73-negative, and CXCR4-negative cells. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method is free of or substantially free of a TGFβ receptor inhibitor.

In one embodiment of a method for generating pluripotent stem cell-derived NK progenitors (ipro-NK) or NK cells (iNK) from pluripotent stem cell-derived HE cells, the method comprises differentiating the definitive HE cells into ipro-NK or iNK by contacting the definitive HE cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and optionally one or more factors selected from the group consisting of VEGF, bFGF, a BMP activator, and a ROCK inhibitor. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are naive iPSCs. In some embodiments, the method further comprises differentiating the definitive HE cells into pro-ipro-NK by contacting the HE cells with a culture medium comprising a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, IL15, VEGF, and bFGF. In other embodiments, the method comprising differentiating definitive HE cells into pre-ipro-NK cells further comprises differentiating the pre-proNK cells into ipro-NK cells or iNK cells by contacting the pre-proNK cells with a culture medium comprising one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL3, IL7, and IL15, and wherein the culture medium is free of, or substantially free of, one or more of VEGF, bFGF, BMP activators, and ROCK inhibitors. In some embodiments, the culture platform for obtaining NK progenitor cells comprises obtaining NK cells by contacting ipro-NK cells with one or more artificial antigens for stimulating NK cell growth, development, and maturation, wherein the artificial antigens are introduced in the form of microbead conjugates, plasma membrane particles, and/or antigen-presenting cells. In some embodiments, the method further comprises subjecting the seeded pluripotent stem cells, mesoderm, and/or mesodermal cells with definitive hemogenic endothelial potential to a low oxygen tension of about 2% to about 10%. In some embodiments, the iHE cells obtained from the above method express CD34. In some embodiments, the above method further comprises sorting the obtained iHE cells using CD34, CD43, CD73, and/or CXCR4. In some embodiments, the sorting uses CD34 positive and CD43 negative. In some embodiments, the sorting uses CD34 positive, CD43 negative, and CD73 negative. In some other embodiments, the sorting uses CD34 positive, CD43 negative, CD73 negative, and CXCR4 negative. In some embodiments, the BMP activator in the above method is BMP4. In some embodiments, the Wnt pathway activator is a GSK3 inhibitor. In some embodiments, the ROCK inhibitor is Y27632 or thiazovivin. In some embodiments, the culture medium in the above method does not contain or is substantially free of a TGFβ receptor inhibitor.

Under the above teachings, one of the advantages provided by the culture platform contemplated herein is the enhanced viability and survival rate of culturing, passage, and dissociating single pluripotent cells without the formation of EBs used for pluripotent stem cell differentiation. In some embodiments, the pluripotent stem cells are iPSCs. In some embodiments, the iPSCs are initial iPSCs. Dissociating the cells into single cells, for example, into a single cell suspension, can be accomplished enzymatically or mechanically. Any enzyme known in the art that allows cells to be dissociated into single cells can be used in the methods of the present invention. In one embodiment, the dissociating agent is selected from trypsin/EDTA, TrypLE-Select, collagenase IV, and dispase. According to the methods contemplated herein, chelating agents such as EDTA, Accutase, or AccuMax can also be used alone or in combination with enzymes to dissociate cells. The dissociating agent can be dissolved in PBS without calcium and magnesium to promote dissociation into single cells. In order to enhance the survival of cells during and after dissociation, in some embodiments, a survival-promoting substance is added, such as one or more growth factors, inhibitors of cellular pathways involved in cell death and apoptosis, or conditioned medium. In one embodiment, the survival-promoting substance is a ROCK inhibitor, including but not limited to thiazovivin.

Cell culture and culture medium collection techniques are summarized in Hu et al., Curr. Opin. Biotechnol. 8:148, 1997; K. Kitano, Biotechnology 17:73, 1991; Curr. Opin. Biotechnol. 2:375, 1991; Birch et al., Bioprocess Technol. 19:251, 1990; "Teratocarcinomas and embryonic stem cells: A practical approach" (E. J. Robertson, ed., IRL Press Ltd. 1987); "Guide to Techniques in Mouse Development" (P. M. Wasserman et al., eds., Academic Press 1993); "Embryonic Stem Cell Differentiation in vitro" (M. V. Wiles, Meth. Enzymol. 225:900, 1993); "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Genetics" (eds., 1992); and "Properties and uses of Embryonic Stem Cells: Prospects for Application to Human Biology and Genetics." Therapy" (P.D. Rathjen et al., 1993). Stem cell differentiation is summarized in Robertson, Meth. Cell Biol. 75:173, 1997; and Pedersen, Reprod. Fertil. Dev. 10:31, 1998.

In the present invention, strategies for enriching cell populations with specific characteristics are provided at various stages of the method. In one embodiment, the method for enriching pluripotent stem cells from a cell population includes preparing a single cell suspension by dissociating cells in the population and resuspending the cells. The dissociated cells can be resuspended in any suitable solution or culture medium for maintaining the cells or performing cell sorting. In a specific embodiment, the pluripotent single cell suspension contains a GSK3 inhibitor, a MEK inhibitor, and a Rock inhibitor, and lacks a TFGβ inhibitor. In certain embodiments, the GSK3 inhibitor is CHIR99021, the MEK inhibitor is PD0325901, and/or the Rock inhibitor is thiazovivin.

In a specific embodiment, the cell population is sorted into positive selection pluripotent cells, and/or non-reprogrammed or non-pluripotent cells are removed from the group to obtain a cell population rich in pluripotent cells. In one embodiment, a single cell suspension is prepared and then a single cell is prepared for sorting, such as by staining a marker of pluripotency using, for example, an appropriate antibody. Cells can be sorted by any suitable method of sorting cells, such as by magnetic beads or flow cytometry (FACS) sorting.

Cells can be sorted based on the expression of one or more markers of pluripotency or differentiation, including but not limited to, SSEA3/4, TRA1-60/81, TRA1-85, TRA2-54, ^GCTM -2, TG343, TG30, CD9, CD29, CD133/prominin, CD140a, CD56, CD73, CD105, OCT4, NANOG, SOX2, KLF4, SSEA1 (mouse), CD30, SSEA5, CD90, and/or CD50. In various embodiments, cells are sorted based on at least one, at least two, at least three, or at least four markers of pluripotency or differentiation. In certain embodiments, cells are sorted based on the expression of SSEA4, and in certain specific embodiments, based on the expression of SSEA4 in combination with TRA1-81 and/or TRA1-60. In certain embodiments, cells are sorted based on the expression of SSEA4, TRA1-81, or TRA1-60, and/or CD30. In one embodiment, cells are sorted based on SSEA4, TRA1-81, and CD30. In another embodiment, cells are sorted based on SSEA4, TRA1-60, and CD30. In certain embodiments, cells are sorted using one or more surface markers of differentiation, including but not limited to CD13, CD26, CD34, CD45, CD31, CD46, and CD7, and pluripotency markers such as SSEA4, TRA1-81, and/or CD30.

In some embodiments, the cell population or pluripotent cell population being reprogrammed is depleted of differentiated cells. In one embodiment, cells having one or more cell surface markers of differentiated cells in the pluripotent cells or cell population induced to reprogram can be removed. Illustrative examples of cell surface markers of differentiated cells include but are not limited to CD13, CD26, CD34, CD45, CD31, CD46, and CD7. In a specific embodiment, CD13 is used as a surface marker of differentiated cells.

In other embodiments, the cell group is induced to differentiate into the desired pedigree, and pluripotent cells are removed to obtain the differentiated cells or differentiated cell colony of enrichment. In some embodiments, the differentiated cell colony comprises a cell group that has been induced to differentiate into a specific pedigree, such as ESCs or iPSC. In some embodiments, the above-mentioned negative cell sorting technique (" panning ") can be used to remove pluripotent cells in a cell group, such as according to the cells in the magnetic beads or FAC sorting colony based on pluripotency markers. In some embodiments, the cell group comprising differentiated cells is sorted by FAC using pluripotency markers, and the cell fraction expressing pluripotency markers is removed. In other embodiments, based on differentiation markers, such as lineage-specific markers (including but not limited to CD13, CD26, CD34, CD45, CD31, CD46 and CD7), cell groups are sorted by FAC to obtain the fraction removing pluripotency markers. In some embodiments of the present invention, CD13 is used as a surface marker of differentiated cells.

D. Cell Populations and Cell Lines Generated by the Methods and Platforms Provided Herein

In some embodiments, the cells cultured after reprogramming are induced to differentiate for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 20, 22, 24, 26, 28, 30, 32, 35, 40, 42 or 45 days, or any number of days during. In some embodiments, the cells cultured after reprogramming are induced to differentiate for at least 1-42 days, 2-40 days, 2-35 days, 2-20 days, 2-10 days, 4-30 days, about 4-24 days, about 6-22 days or about 8-about 12 days. In some embodiments, the cell is a pluripotent stem cell, including iPSC. In some embodiments, iPSC is initial iPSC. In one embodiment, enrichment provides a method for obtaining cloned pluripotent stem cell-derived differentiated cell colonies in a relatively short time, thereby improving the efficiency of producing differentiated cells derived from pluripotent stem cells in each stage. In one embodiment, enrichment provides a method for obtaining HE cells expressing CD34, HSC cells expressing CD34, T or NK progenitor cells, and T or NK cells, thereby improving the efficiency of generating each cell population. Enrichment can include sorting the cell population to identify and obtain cells expressing specific characteristic markers that can indicate the differentiation stage/cell type. In some embodiments, sorting uses CD34, CD43, CD73 and/or CXCR4. In some embodiments, sorting uses CD34 positivity. In some embodiments, sorting uses CD34 positivity and CD43 negativity. In some embodiments, sorting uses CD34 positivity, CD43 negativity, and CD73 negativity. In some other embodiments, sorting uses CD34 positivity, CD43 negativity, CD73 negativity, and CXCR4 negativity. Additional enrichment methods include removing cells expressing markers representing undesirable cell types to obtain an enriched population of desired cell types.

Thus, one aspect of the present invention provides a composition comprising one or more of the following cell populations, cell lines or clones: (i) pluripotent stem cell-derived CD34+ HE cells (iCD34), wherein the iCD34 cells are capable of differentiating into multipotent progenitor cells and wherein the iCD34 cells are CD34+CD43-; (ii) pluripotent stem cell-derived definitive hemogenic endothelium (iHE), wherein the iHE cell line or clone is CD34+; (iii) pluripotent stem cell-derived definitive HSCs (iHSCs), wherein the iHSCs are CD34+CD45+ and suitable for long-term transplantation; (iv) pluripotent stem cell-derived definitive HSCs (iHSCs), wherein the iHSCs are CD34+CD45+ and suitable for long-term transplantation; Stem cell-derived multipotent progenitor cells (iMPP), wherein iMPP cells are CD34+CD45+; (v) pluripotent stem cell-derived T progenitor cells (iproT), wherein T progenitor cells are CD34+CD7+; (vi) pluripotent stem cell-derived T cells (iTC), wherein T cells are CD4+ or CD8+; (vii) pluripotent stem cell-derived NK progenitor cells (iproNK), wherein NK progenitor cells are CD56+CD3-; and (viii) pluripotent stem cell-derived NK cells (iNK), wherein NK cells are CD56+CD57+CD16+. In some embodiments, the above-mentioned composition, cell population, cell line or cloned cell is suitable for low-temperature storage. In some embodiments, the composition, cell population, cell line or cloned cell is suitable for storage under room temperature conditions for more than 12 hours, 24 hours, 36 hours, 48 hours, but not longer than 3 days, 4 days, 5 days, 6 days or a week.

Another aspect of the present invention provides a mixture comprising one or more pluripotent stem cell-derived (i) CD34+ HE cells (iCD34), and one or more culture media selected from the group consisting of iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-A1, iNK-A2, iNK-B1, and iNK-B2; (ii) definitive hemogenic endothelium (iHE), and one or more culture media selected from the group consisting of iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-A1, iNK-A2, iNK-B1, and iNK-B2; (iii) definitive HSC, and one or more culture media selected from the group consisting of iMPP-A, iTC-A1, iTC-A2, iTC-B1, iTC-B2, iNK-A1, iNK-A2, iNK-B1, and iNK-B2. 1, iTC-B2, iNK-A1, iNK-A2, iNK-B1 and iNK-B2; (iv) multipotent progenitor cells (iMPP) and iMPP-A; (v) T progenitor cells (iproT), and one or more culture media selected from the following: iTC-A1, iTC-A2, iTC-B1 and iTC-B2; (vi) T cells (iTC) and iTC-B1 or iTC-B2; (vii) NK progenitor cells (iproNK), and one or more culture media selected from the following: iNK-A1, iNK-A2, iNK-B1 and iNK-B2; and/or (viii) NK cells (iNK) and iNK-B1 or iNK-B2; (ix) HSC (iHSC) and iHSC-A, iHSC-B and iHSC-C; wherein

a. iHSC-A contains Wnt pathway activators and BMP activators;

b. iHSC-B contains Wnt pathway activators, BMP activators, and optional TGFβ receptor/ALK inhibitors;

c. iHSC-Cs contain a BMP activator and one or more growth factors and cytokines selected from VEGF, SCF, Flt3L, IL15, IL3, IL6, IGF, and TPO. In some embodiments, the composition does not contain a Wnt pathway activator and a TGFβ receptor/ALK inhibitor;

d. iTC-A1 comprises a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IL2, IL3, and IL6, and one or more Notch pathway activators selected from Jag1, Jag2, DLL-1, DLL-3, and DLL-4; in some embodiments, the composition does not contain VEGF and/or IL15;

e. iTC-B1 comprises one or more growth factors and cytokines selected from SCF, Flt3L, IL7, IGF, IL2, IL3, and IL6, and one or more Notch pathway activators selected from Jag1, Jag2, DLL-1, DLL-3, and DLL-4; (iHSC platform); in some embodiments, the composition does not contain a BMP activator;

f. iNK-A1 comprises a BMP activator, one or more growth factors and cytokines selected from SCF, Flt3L, VEGF, IL2, IL3, IL6 and IL15;

g. iNK-B1 comprises one or more growth factors and cytokines selected from SCF, Flt3L, IGF, IL7, IL2, IL3, IL6 and IL15;

h.iCD34-C contains a ROCK inhibitor and one or more growth factors and cytokines selected from bFGF, VEGF, SCF, IL6 and IL11; and does not contain a TGFβ receptor/ALK inhibitor;

i.iMPP-A comprises a BMP activator, a ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11;

j.iTC-A2 contains a ROCK inhibitor; and one or more growth factors and cytokines selected from SCF, Flt3L and IL7;

k.iTC-B2 comprises one or more growth factors and cytokines selected from SCF, Flt3L and IL7; wherein the composition does not contain one or more of VEGF, bFGF, BMP activator and ROCK inhibitor;

1. iNK-A2 contains a BMP activator, a ROCK inhibitor, VEGF and bFGF, and one or more growth factors and cytokines selected from SCF, Flt3L, IL7, and IL15; and

m.iNK-B2 comprises one or more growth factors and cytokines selected from the group consisting of SCF, Flt3L, IL7 and IL15.

Example

The following examples are offered by way of illustration and not by way of limitation.

Example 1 - hiPSC Generation and Maintenance

In the presence of reprogramming medium containing ROCK, MEK, GSK3 pathway and TGFβ receptor inhibitors (Valamehr et al., Sci Rep. 2002; 2: 213), various factor combinations including OCT4/SOX2/LargeT, OCT4/SOX2 or OCT4/SOX2/NANOG/LargeT were used to induce somatic cells including fibroblasts and blood cells to reprogram to a pluripotent state. 14 days after induction, the reprogrammed population was transferred to maintenance medium containing ROCK, GSK3 and MEK pathway inhibitors, basic fibroblast growth factor (bFGF) and leukemia inhibitory factor (LIF) (Valamehr et al., Stem Cell Reports, 2014, 2(3): 366-381). The cells were kept in the maintenance medium indefinitely.

Approximately three weeks after induction, the reprogrammed population was sorted into individual wells of a 96-well plate. Selected clones were characterized, and fully reprogrammed clones representing the initial hiPSCs were selected for differentiation studies (Valamehr et al., Stem Cell Reports 2014, 2(3): 366-381). To determine the percentage of undifferentiated cells during maintenance and after differentiation, flow cytometry analysis was performed to analyze the co-surface expression of SSEA4, TRA181, and CD30.

Example 2 - Hematopoietic differentiation using iHSC culture platform

To initiate differentiation into the hematopoietic lineage, the initial hiPSCs were seeded as a monolayer in maintenance medium and allowed to expand until approximately 25% confluence was reached. At this point, hematopoietic differentiation was initiated by switching the culture medium to iHSC-A (see Figure 1). As shown in Figure 1, the culture was then switched to iHSC-B 48 hours after the start of differentiation, and then to iHSC-C on days 4-5 after the start of differentiation. The attached cultures remained adherent and undisturbed during the culture medium change.

In the early stages of the differentiation process, direct differentiation was monitored by lineage markers CD57, NESTIN, SOX17, and BRACHYURY. Figure 3 shows direct differentiation changes towards the mesoderm lineage and away from the ectoderm lineage. Through the subsequent differentiation stage, the hiPSC morphology was given a differentiation colony consisting of round cells, which was a morphology similar to that of hematopoietic cell clusters (Fig. 4A), lacking hiPSC-related surface markers (Fig. 4B), and surface markers such as Brachyury, CD34, CXCR4, and CD45 (Fig. 4C and 4D). On the 9th day (this time point can be extended, optimal to the 14th day), the cells were dissociated into single cells and the surface expression of CD34 and CD45 was analyzed (Fig. 5A-5C). Single cell dissociation was usually assisted by Accutase and filtered through a 40 μm sieve to collect single cells. Using FACS Aria or MACS enrichment, CD34 positive or CD34 and CD45 positive cells were collected for further processing and testing.

Example 3 - In vitro characterization and testing after differentiation using the iHSC culture platform

In order to determine the scalability and maintenance of the permanent hematopoietic stem cells of differentiation, the CD34 sorting or enriched colony was transferred to suspension culture and supplemented with Stemspan hematopoietic stem cell culture medium (StemCell Technologies, Vancouver, Canada), 1x CC110 supplement (StemCell Technologies), 10ng/mL bFGF and 5μM Thiazovivin or 10μM 27632ROCK inhibitor (for the first few days of culture to improve survival). Fresh culture medium was added to the culture every other day and pipetted to destroy the aggregates produced by the CD34 positive sorting cells of division. After culturing for several weeks, the suspension culture was evaluated and scaled by surface marker expression and viable cell count. As shown in Figure 8, the CD34 sorting colony remained in culture for 22 days, and the loss of the CD34 colony was minimal. In order to improve the viability of suspension culture, a small molecule inhibitor of the ROCK pathway was added. Figure 7 illustrates the survival and proliferation of cells after single cell dissociation and passage in the presence of ROCK inhibition. Figures 6A-6C show a modification of the differentiation method described in Figure 1 applied to monolayer and EB cultures, with a higher percentage of CD34 cells shown to be present in the monolayer format compared to EB-mediated differentiation.

Example 4 - In vivo reconstruction after differentiation using the iHSC culture platform

In order to show the in vivo functionality and transplantation potential of the permanent hematopoietic stem cells obtained, CD34 positive cells from the hiPSC differentiation process described above were enriched using FACS Aria sorting or MACS microbeads. The collected cells were counted by determining activity using trypan blue staining. Approximately 30,000 active CD34 positive cells were resuspended in HBSS and introduced into NSG mice via retroorbital injection. NSG mice were irradiated with 300 rad semi-lethal radiation for 24 hours before transplantation. In addition, unsorted differentiated groups (up to 250,000) and human peripheral blood were also introduced into NSG mice as controls. Mice were bled every two weeks and human-specific markers including CD45, CD11b, CD19 and CD3 were used to evaluate human blood contribution. Figure 9A shows the 12-week reconstruction of transplanted CD34 positive cells, as shown by the presence of cells that exclusively express the human CD45 marker. The data also show the multi-lineage ability of transplanted cells to produce bone marrow and lymphoid populations. Figure 9B shows the 18-week reconstruction of CD34+ cells in the presence of T and B cells.

Example 5 - Small molecule modulation after differentiation using the iHSC culture platform

In order to evaluate the ability of hiPSC-derived CD34-positive permanent hematopoietic stem cells to respond to pharmacological regulation, CD34 sorted or enriched cells were treated with known regulators. After incubation overnight at 37°C, the pharmacological response of CD34-positive cells, including upregulation of surface PDL1 expression, was evaluated by flow cytometry. Compared to the vehicle control, significantly more treated CD34 cells showed upregulation of PDL1 surface protein and increased expression in the total population (see Figure 11), which is an indicator of the immunomodulatory potential of CD34-positive cells obtained from initial hiPSC using the disclosed method.

Example 6 - Continued differentiation into specific hematopoietic lineages after differentiation using the iHSC culture platform

The sorted or enriched CD34-positive cells are further differentiated into various specific cell types along the hematopoietic lineage, including T cells and NK cells. Specific to T cells, after enrichment, the CD34-positive cells are transferred to a suspension culture without feeder cells or an adherent culture containing OP9 stromal cells or a matrigel-coated surface. Regardless of the environment (setting), the culture is supplemented with iTC-A1 (Figure 1) containing soluble DLL1 and DLL4. After about 10 days, the culture environment is replaced with iTC-B1 to complete T cell maturation. About 30-40 days (after the original induction of differentiation), the composition of T cells is assessed, including the surface expression of CD3, CD7, TCRαβ, CD4 and CD8. Figure 10 shows the in vitro differentiation ability of the obtained CD34-positive cells to produce different T cell populations defined by the expression of CD4 and CD8 from the CD7 population.

Specifically for NK cells, CD34-positive cells were treated with differentiation medium containing IL15 and iNK-A1 medium for approximately 10 days (Figure 2), and switched to iNK-B1 medium (Figure 2) for an additional 10-20 days (see the CD56-positive population in Figure 10). Culture was performed in suspension.

Multi-stage differentiation platform described herein shows the process of obtaining permanent hematopoietic stem cells from a variety of stem, progenitor cells or transdifferentiation cells (including pluripotent stem cells) using sequential differentiation methods. The CD34 positive hematopoietic stem cells obtained can be retained in suspension culture for amplification (scaling), and produce multi-lineage hematopoietic cell types, including hematopoietic stem cells, T cells and NK cells. In addition, the CD34 positive permanent hematopoietic stem cells obtained show that pharmacological regulation (Figure 11) is responded to by raising immunomodulatory surface protein PDL1. In addition, when implanted, the CD34 positive cells obtained can rebuild in vivo including bone marrow and lymphoid colonies (Fig. 9). Permanent hematopoietic stem cells from various colonies (including pluripotent stem cells) are ideal candidates for patient-specific treatment and regenerative medicine applications.

Example 7 - Hematopoietic differentiation and identification of HE populations with transplantation potential using the iCD34 culture platform

The above-mentioned iHSC platform is further optimized for hematopoietic lineage cell differentiation. In order to start differentiation to the hematopoietic lineage, hiPSC is seeded as a monolayer in maintenance medium at day 0 (D0), and is adhered and expanded for about 24 hours. At this time, the maintenance medium is removed and replaced with a basal medium without maintenance factors at D1. By switching the culture medium to iCD34-A (see Figure 12), hematopoietic differentiation begins around D2. As shown in Figure 12, the culture medium is supplemented with growth factor bFGF at D3, and then switched to iCD34-B culture medium for differentiation. The monolayer is maintained until around D5-D6, at which point it is dissociated into single cells and seeded in a low-density monolayer in iCD34-C culture medium until differentiation around D10. Maintain low oxygen tension (2-10% O ₂ ) from the start of hematopoietic differentiation around D2 until around D10 of differentiation.

During the culture process, direct differentiation into the hematopoietic lineage was monitored by dissociating the monolayer into single cells and analyzing the expression of surface markers such as CD34 and optionally CD43, CD45, CXCR4 and CD73 (Figure 15A). At about D8 of differentiation, the appearance of a cell population representing HE was observed by the cell surface expression characteristic CD34+. CD43-CXCR4-CD73- was also observed in CD34+ cells. The CD34+ population remained until around D10 (Figure 15A). At D10 (the time point can be shortened to about D9 or extended to about D12), the cells were dissociated into single cells and the CD34+HE population was sorted by FACS using a BD FACS Aria for further analysis and functional assessment. As an example of hematopoietic output capacity, 463 total CD34+ cells (Figure 15A) and 41 CD34+CD43-CXCR4-CD73- cells (Figure 15A) were generated for each input of a single iPSC, with a conversion rate of at least 2.5% to permanent HE cells.

To demonstrate the transplantation potential of hiPSC-derived iHE, day 10 CD34+ cells were sorted and cultured for 7 days in the iMPP assay as described above. After a total of 17 days in culture (10 days for iCD34 plus 7 days for iMPP), approximately 400,000 cells were injected into the NSG via retroorbital injection. 200,000 cord blood CD34+ cells were injected into separate mice as a control. Figure 33 shows the 5-week reconstitution of transplanted CD34-positive cells, as indicated by the presence of cells expressing the human CD45 marker in the peripheral blood of the mice.

Example 8 - Optimization of HE production by small molecules, cytokines, and plating density modulation

To optimize the efficient generation of HE from hiPSC after about 10 days of differentiation, several parameters were examined. The optimal plating density of the monolayer at D0 of differentiation was assessed by increasing the amount of hiPSC from 7.5x10 ⁴ / well to 1.5x10 ⁵ / well on a matrigel ^TM coated 6-well culture dish, and then analyzing the generation of CD34+HE groups at about D10. Figure 16A shows that increasing the cell plating density increased the total percentage of CD34+ cells, but reduced the CXCR4-CD73-HE subpopulation. Despite this reduction, the highest conversion rate of hiPSC to HE after 10 days of monolayer differentiation was the highest plating density tested at 1.5×10 5 / well (Figure 15C).

The effect of BMP4 modulation on HE production during the initial stages of hematopoietic differentiation was evaluated by treating cultures with increasing BMP4 concentrations from 0 ng/ml to 30 ng/ml from approximately D2 to approximately D6 of differentiation. HE production on D10 was assessed by measuring the CD34+ HE population. Figure 16B shows that increasing BMP4 concentrations from D2 to D6 resulted in a HE population below the threshold BMP4 concentration on D10, indicating an optimal concentration of approximately 3 ng/mL.

The Wnt pathway activator CHIR99021 is responsible for inducing the definitive hematopoietic program in hiPSCs. The effects of CHIR regulation during the induction phase of the hematopoietic differentiation protocol were assessed by treating cultures with increasing concentrations of CHIR from approximately D3.75 to approximately D6. Figure 16C shows that increasing concentrations of CHIR99021 increased the total percentage of CD34+ cells while decreasing the percentage of the HE subset, with the optimal CHIR99021 concentration being approximately 1 μM.

At D6 of the direct differentiation protocol, the monolayer cultures were dissociated into single cells and replated as a monolayer for further differentiation into HE. As shown in Figure 16D, the plating density at D6 was shown to affect the generation of HE, where decreasing the cell concentration from 1.5 x 10 ⁵ to 7.4 x 10 ⁴ increased the percentage of the HE population.

Previous protocols for direct differentiation required the addition of IGF1 and EPO cytokines during the generation of HE. Figure 16E shows that the addition of IGF1 and EPO reduced the percentage of HE observed around D10, and that removal of these factors resulted in an improvement in the differentiation process.

Example 9 - Determination of the hematopoietic potential of HE by Notch-dependent hematopoiesis and MPP differentiation

To demonstrate the hematopoietic potential of the hiPSC-derived definitive population, HE cells were sorted using FACS and assessed for their ability to undergo an endothelial-to-hematopoietic transition to generate CD45+ hematopoietic progenitors, as described in the multipotent progenitor assay (iMPP) in Figure 12. Approximately 30,000 CD34+ HE cells were cultured overnight in suspension as aggregates in iMPP-A medium, then plated as monolayers and further cultured for 6-8 days. Figure 17A shows the phenotypic changes in monolayer culture with the appearance of rounded hematopoietic cells, and flow cytometric staining identified the presence of CD45+ during 6-8 days of culture.

To assess whether the HE population generated at D10 of the direct differentiation protocol represents permanent HE, CD34+ HE sorted cells were treated with the Notch pathway inhibitor γ-secretase inhibitor (γSI) during the 7-9 day iMPP experiment. Fresh γSI was added to the iMPP-A culture medium every 2 days. After approximately 8 days, the CD45+ hematopoietic cells present in the monolayer culture were assessed by flow cytometry. Compared to the vehicle control, significantly fewer CD45+ cells were observed in the γSI-treated cultures, indicating dependence on the Notch signaling pathway and the presence of permanent HE (Figure 17B).

Example 10 - Optimization of HE and iMPP Production by Manipulating Oxygen Conditions

To evaluate whether the oxygen environment affects the generation of permanent HE and iMPP hematopoietic progenitor cells, hiPSCs were differentiated under normal oxygen (21% O ₂ ) or hypoxic (5% O ₂ ) conditions as shown in FIG12 . Around D10 of differentiation, the CD34+HE population present in the monolayer was counted and evaluated by flow cytometry. As shown in FIG18A , differentiation under hypoxic conditions resulted in an increase in the amount of CD34+HE produced. To confirm that the HE produced under hypoxic conditions retains the potential to generate CD45+ hematopoietic progenitor cells, CD34+ cells were isolated from normal oxygen and hypoxic differentiation conditions by FACS and evaluated by iMPP assay. The HE generated under both conditions had comparable iMPP potential, indicating that hematopoietic differentiation under hypoxic conditions increased the output of permanent HE ( FIG18B ).

Example 11 - Cryopreservation of Day 8 Differentiation Cultures and HE

At approximately day 10 of the direct differentiation protocol, all cultures were dissociated to assess their ability to maintain hematopoietic potential after cryopreservation in day 8 culture medium supplemented with 10% DMSO. Thawed cells were resuspended and then cultured for an additional 7 days in iMPP-A medium as described in Figure 12 before flow cytometric analysis. As shown in Figure 19A, cryopreserved D10 cells survived the freeze/thaw process and had hematopoietic potential comparable to that of unfrozen controls as evidenced by the presence of CD45+ cells.

The ability of the sorted CD34+ cells to maintain hematopoietic potential after cryopreservation was assessed. The D10 iCD34 produced under hypoxic conditions was separated and directly plated in the iMPP test or in iMPP-A culture medium for 7 days as described in Figure 12, then thawed and plated in the iMPP test. As shown in Figure 19B, the cryopreserved HE survived the freezing/thawing process and was able to maintain the hematopoietic potential observed by the presence of CD45+ cells, although the efficiency was reduced compared to the unfrozen control (Figure 19B top figure). The cryopreserved iCD34 was also plated at a higher density (200,000 cells/well), which seemed to increase the yield of CD45+ cells (Figure 19D).

Example 12 - Recovery of Differentiation Cultures on Day 8 After Overnight Shipment

Day 6 differentiation cultures from 6-well plates were passaged into T25 flasks at a seeding density of 200,000 cells/flask, then completely filled with medium on day 8 and stored overnight in polystyrene foam boxes to assess the feasibility of direct delivery of fresh cells without the need for cryopreservation. Cold packs, initially stored in a 37°C water bath, were also added to the foam boxes to maintain a temperature of 37°C as long as possible. Two media compositions were tested alongside control flasks in a 37°C incubator: flasks containing 30% of the cytokines and morphogens used in the day 8 step ( FIG. 12 ) and flasks containing 100% of the cytokines and morphogens used in the day 8 step ( FIG. 12 ). On day 9, the flasks were removed from the boxes, the medium was replaced with 10 mL of day 8 medium, and new caps were placed on the flasks, and they were allowed to recover before being processed for flow cytometry analysis on day 10 for the presence of a CD34+ HE population. As shown in FIG. 20 , the overall HE output was comparable between all conditions, demonstrating the feasibility of fresh overnight delivery of day 8 cultures as an effective means of delivering HE cells.

Example 13 - Further differentiation of HE cells into mature T and NK lymphoid lineages using DLL4-expressing stromal cells

The sorted CD34+HE cells were further differentiated into T and NK lymphoid lineages. Specifically for T cells, after sorting, HE cells were cultured as aggregates on low-attachment tissue culture plates in iTC-A serum-free differentiation medium containing BMP4, SCF, Flt3L and IL7 for 16 hours (Figure 13). After 16 hours, the aggregated cells were transferred to adherent iTC-B differentiation medium containing stromal cells expressing DLL4, and the iTC-B differentiation medium contained SCF, Flt3L and IL7. After 5 days, iTC-B medium was maintained to complete T cell differentiation. After about 10 days of culture (after HE isolation), the T progenitor cells produced by the culture were evaluated by co-expression of cell surface markers CD34 and CD7. After further differentiation for about 15-20 days, these CD34+CD7+ T progenitor cells produced different mature T cell populations, as shown by the expression of CD4 and CD8. Figure 21 depicts the in vitro differentiation capacity of day 10 HE populations to give rise to early T cell progenitors and mature T cell subsets by analyzing CD45+CD56- populations generated in co-culture. Figure 26 depicts the in vitro differentiation capacity of day 10 HE populations to give rise to mature T cell subsets by analyzing CD45+CD56- populations generated in co-culture approximately 30 days in culture (after HE isolation).

Specific to NK cells, when sorting, HE cells were cultured as aggregates on low-attachment tissue culture plates in iNK-A serum-free differentiation medium containing BMP4, SCF, IL3, IL15, Flt3L and IL7 for 16 hours (Figure 14). After 16 hours, the aggregated cells were transferred to adherent cultures containing stromal cells expressing DLL4 in iNK-B differentiation medium containing SCF, IL3, IL15, Flt3L and IL7. After 5 days, iNK-B culture medium was maintained to complete NK cell differentiation. After approximately 10-15 days of culture (after HE separation), the NK progenitor cells produced by the culture were evaluated, followed by mature NK subpopulations after another 10-15 days of culture. CD56 and CD161 (NKR-P1A) are the first cell surface markers expressed in early NK cell development, followed by CD16, KIR, CD8 and NKG2D (CD314) on later mature NK cell subpopulations. Figure 22 depicts the in vitro differentiation capacity of day 10 HE populations to generate early NK cell progenitors and mature NK cell subsets obtained by analyzing CD45+ populations generated in co-culture. Another approach to enhance NK progenitor maturation is to co-culture with feeder cells in suspension culture. Day 20 iNK cells were transferred from DLL4 stromal cell cultures to feeder-based suspension cultures in iNK-B medium containing SCF, IL15, FLT3L, and IL7 and cultured for another 12 days. Figure 27 depicts the in vitro differentiation capacity of day 10 HE populations to generate mature NK cell subsets using feeder suspension cultures by analyzing CD45+ populations generated in stromal and feeder-based co-cultures compared to peripheral blood-derived ones. HiPSC-derived CD34+ cells were differentiated toward the NK cell lineage for 20 days and then placed in suspension culture for further maturation. Mature NK lineage markers identify the presence of mature NK cells as defined by CD56, CD122, NKp30, CD94, CD16, NKG2D, and KIR.

Example 14 - Monolayer hiPSC hematopoietic differentiation platform allows highly scalable expansion strategies

HiPSCs were seeded as a monolayer and differentiated into hematopoietic cells in a defined serum-free and feeder-free culture, as described in Figures 12-14, and compared with hiPSCs that form embryoid bodies in aggregates before the start of hematopoietic differentiation (Kennedy et al., Cell Reports 2012: 1722-1735). Both culture environments used 100,000 hiPSCs as the initial starting number. During the hematopoietic differentiation process, cell counting and phenotypic assessment were routinely performed to show the expansion potential of each system. As shown in Figure 23, on day 6 of differentiation, a significant number of CD34-positive cells—more than 2 million CD34+ cells—were detected in the monolayer culture, while no CD34-positive cells were detected in the EB format. On day 8 of differentiation, the monolayer format produced approximately 2.4 million cells, while in the EB format, although roughly the same number of iPSCs were used as starting material, only about 100,000 CD34-positive cells were detected, a difference of about 24 times. In addition, as evaluated at the time of evaluation, the monolayer format only produced CD34+CD43- cells, indicating permanent hematopoiesis, and the EB format produced a majority of CD34+CD43+ cells, indicating primitive hematopoiesis (Kennedy et al., 2012). Figure 24 further illustrates the scale-up of the monolayer hiPSC hematopoietic differentiation platform disclosed herein for producing finished iNK and iT cells. The pluripotent cell cloning expansion platform provided herein ensures, for example, the scale-up of finished products from single pluripotent cell clones to therapeutic doses of approximately 1 million vials, each of which has no less than 10 ⁶ NK or T cells with therapeutic function. The disclosed clonal expansion further provides extensive homogeneity, thereby ensuring product consistency, quality control and quality assurance. In some embodiments, single pluripotent cell clones are genetically engineered.

Example 15 - Immunomodulatory properties of iCD34+ cells

To determine the immunomodulatory capacity of hiPSC-derived CD34-positive cells (iCD34), CD34-sorted cells were co-cultured with activated peripheral blood-derived CD3-expressing T cells in iMPP-A medium. After incubation at 37°C for 5 days, the co-cultures were mixed with counting beads, and the absolute cell number in each sample was determined by flow cytometry. Figure 25 describes the immunomodulatory potential of hiPSC-derived CD34+ cells observed by comparison with cultures containing only CD3+ T cells. CD3+ T cells co-cultured with hiPSC-derived CD34+ cells had reduced cell survival, while the total amount of CD34+ cells in the culture was not affected.

Example 16 - Characterization of cytokine-induced activation by cytokine release and degranulation to determine iNK cell function

In order to show the function of mature iNK cells derived from hiPSC, iNK cells at day 20 (after HE separation) were transferred to iNK-B culture medium containing SCF, IL15, IL7 and Flt3L for another 10 days of suspension culture based on feeding. After 10 days of additional culture, iNK cells were stimulated with IL12 and IL18 to induce iNK cell activation. iNK derived from iCD34 responds to cytokine stimulation and secreted proinflammatory cytokines in a manner similar to peripheral blood NK cells. Figure 28 shows the expression of CD107A (a cell surface marker representing degranulation) and the activation of iNK cells shown by interferon γ intracellular staining based on CD45+CD56+ gating method compared with umbilical cord blood-derived NK cells and peripheral blood-derived NK cells produced using the same matrix and feeding suspension co-culture.

Example 17 - Establishment of stromal-free differentiation cultures for the generation of T and NK cells

The above T and NK lymphoid differentiation platforms were further optimized for the generation of cord blood-derived and hiPSC-derived iT and iNK progenitor cells using a stromal-free differentiation platform.

Specifically for NK cells, enriched umbilical cord blood CD34+ cells were seeded in iNK-A serum-free differentiation medium containing SCF, Flt3L, IL3, IL15 and IL7 on culture plates containing DLL4 protein or control protein. After 5 days, iNK-B medium was maintained to complete NK cell differentiation. After about 10-15 days of culture, the NK progenitor cells produced by the culture were evaluated for the absence of bone marrow cells. CD56, CD7 and CD161 are the first cell surface markers expressed during NK cell development. CD11b and CD14 are cell surface markers expressed on bone marrow cell subsets. Figure 29 describes the matrix-free differentiation of umbilical cord blood CD34+ cells into NK cells. The results showed that plate-bound DLL4 supported more rapid and efficient differentiation of CD56+CD7+CD161+ NK cell progenitors compared to both matrix-based and matrix-free control cultures, and that cord blood CD34+ cells had the in vitro differentiation capacity to generate early NK progenitors (pro-NK) in a matrix-free differentiation platform expressing DLL4 in a manner (phenotypically) similar to that using a CD45+ gating approach in a conventional matrix-based differentiation platform. Early NK lineage markers identified proNK cells as defined by the presence of CD56, CD7, and CD161 and the absence of the myeloid markers CD11b and CD14.

To demonstrate the ability of hiPSC-derived HE cells to generate iNK progenitors in a matrix-free differentiation platform, day 10 CD34+ iHE sorted cells were cultured in iNK-A2 medium in a culture medium containing DLL4 protein or a control protein. The hiPSC-derived CD34+ cells were then differentiated toward the NK cell lineage for 20 days and then placed in suspension culture for further maturation. Figure 30 shows the ability of hiPSC-derived iHE to generate iNK progenitors, as shown by the expression of CD56, CD161, and CD94 using a CD45+ gating method. Plate-bound DLL4 supports the differentiation of CD56+CD7+CD161+ NK progenitors but not CD11b+ bone marrow cells. After 5 days, iNK-B2 medium was maintained to complete NK cell differentiation. Maturation of hiPSC-derived iNK cells in feeder-based suspension culture resulted in mature NK cells with a phenotype similar to peripheral blood NK cells using a CD45+ gating method. Mature NK lineage markers identify the presence of mature NK cells as defined by CD56, CD122, NKp30, CD94, CD16, NKG2D, and KIR.

Specifically for T cells, CD34+ cells enriched from umbilical cord blood were inoculated in iT-A2 culture medium containing DLL4 protein or control protein. After 5 days, iT-B2 culture medium was maintained for the production of T progenitor cells (proT). After about 10-15 days in culture, the production of T progenitor cells in the culture was evaluated by co-expression of cell surface markers CD34 and CD7. Figure 31 depicts the ability of CD34+ umbilical cord blood cells using a CD45+ gating method to produce T progenitor cells in a matrix-free differentiation platform expressing DLL4. The matrix-free differentiation platform of proT cells from umbilical cord blood CD34-positive cells showed faster differentiation than the conventional matrix-based differentiation platform using a CD45+CD56 gating method. Early T lineage markers identified the presence of proT cells defined by CD34, CD5, and CD7.

To demonstrate the ability of hiPSC-derived HE cells to generate iT cells in a matrix-free platform, day 10 CD34+ iHE sorted cells were cultured in iT-A2 medium containing DLL4 protein or control protein. Figure 32 shows the ability of hiPSC-derived iHE to generate iT progenitor cells as indicated by the expression of CD45 and CD7.

Those skilled in the art will readily appreciate that the methods, compositions, and products described herein are exemplary embodiments and are not intended to limit the scope of the invention. It will be apparent to those skilled in the art that various replacements and modifications may be made to the disclosure herein disclosed without departing from the scope and spirit of the invention.

All patents and publications mentioned in this specification are indicative of the levels of those skilled in the art to which the disclosure pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

The present disclosure illustratively described herein may suitably be practiced in the absence of any elements or limitations not specifically disclosed herein. Thus, for example, herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions that have been used are used as terms of illustration rather than limitation, and it is not intended that such terms and expressions be used to exclude any equivalents of the features shown and described, or portions thereof, but it will be appreciated that various modifications are possible within the scope of the disclosure claimed. Therefore, it will be understood that although the present disclosure has been specifically disclosed by preferred embodiments and optional features, modifications and variations of the concepts disclosed herein may be adopted by those skilled in the art and that such modifications and variations are considered to be within the scope of the invention as defined in the appended claims.

Claims (13)

1. A method for guiding pluripotent stem cells to differentiate into hematopoietic cell lines, comprising: (i) Expose pluripotent stem cells to a first composition comprising a BMP pathway activator and optionally bFGF to obtain mesodermal cells; (ii) Exposing mesodermal cells to a second composition comprising a BMP pathway activator, bFGF, and a WNT pathway activator to obtain mesodermal cells with permanent hematopoietic endothelial (HE) potential; and (iii) Contacting mesodermal cells with permanent HE potential with a third composition containing bFGF and ROCK inhibitor to obtain permanent HE cells; The permanent HE cells described therein can provide hematopoietic cell lines, including hematopoietic stem and progenitor cells (HSCs); Mesodermal cells and mesodermal cells with permanent HE potential are obtained in steps (i) and (ii) without forming embryoid bodies; and The differentiation of the pluripotent stem cells into hematopoietic cell lines does not involve TGFβ receptor/ALK inhibitors. 2. The method of claim 1, wherein the hematopoietic cell line comprises hematopoietic stem and progenitor cells (HSC), hematopoietic progenitor cells (MPP), pre-T progenitor cells, pre-NK progenitor cells, T progenitor cells, NK progenitor cells, T cells, NK cells, NKT cells, or B cells. 3. The method of claim 1, further comprising: (i) Expose pluripotent stem cells to a composition containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the cells; or (ii) Exposing pluripotent stem cells, mesodermal cells and/or mesodermal cells with permanent hematopoietic endothelial potential to a low oxygen stress of 2% to 10%. 4. The method of claim 1, further comprising: (i) Expose permanently hematopoietic progenitor cells (MPPs) to a composition comprising a BMP activator, an optional ROCK inhibitor, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11; or (ii) Expose permanently HE cells to a composition containing one or more growth factors and cytokines selected from SCF, Flt3L, and IL7; and optionally a ROCK inhibitor, to obtain pre-T progenitor cells, T progenitor cells, and/or T cells; or (iii) Expose permanent HE cells to a composition comprising one or more growth factors and cytokines selected from SCF, Flt3L, IL7 and IL15, and optionally one or more BMP activators, ROCK inhibitors, VEGF and bFGF, to obtain pre-NK progenitor cells, NK progenitor cells and/or NK cells. 5. The method of claim 1, wherein (i) Differentiation of pluripotent stem cells into hematopoietic cell lines occurs under feeder-free conditions; or (ii) Differentiation of pluripotent stem cells into hematopoietic cell lines occurs under matrix-free conditions; or (iii) Pluripotent stem cells include induced pluripotent stem cells (iPSCs), optionally wherein the iPSCs are naïve iPSCs; or (iv) The WNT pathway activator is a GSK3 inhibitor, optionally wherein the GSK3 inhibitor is CHIR99021; or (v) The BMP pathway activator is BMP4. 6. The method of claim 1, wherein the ROCK inhibitor is: (i) Thiazovin, or (ii) Y-27632. 7. The method of claim 1, comprising: Contacting pre-T progenitor cells derived from pluripotent stem cells with a composition containing one or more growth factors and cytokines selected from SCF, Flt3L and IL7 to obtain T progenitor cells or T cells derived from pluripotent stem cells, wherein the composition does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors. The pluripotent stem cell-derived progenitor cells are generated by exposing the permanent hematopoietic endothelium to a composition containing a ROCK inhibitor and one or more growth factors and cytokines selected from SCF, Flt3L and IL7, to allow cell differentiation and expansion. 8. The method of claim 7, wherein: (a) The method further includes Expose the pluripotent stem cells to a composition containing a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor for seeding and expansion of the pluripotent stem cells, wherein the composition does not contain a TGFβ receptor/ALK inhibitor; or (b) Where the T cell lines derived from pluripotent stem cells (i) do not produce embryoid bodies; (ii) are cultured in a monolayer; (iii) are cultured under feeder-free conditions; or (iv) are cultured under substrate-free conditions; or (c) Where the pluripotent stem cells are iPSCs or naïve iPSCs. 9. The method of claim 1, further comprising: Contacting pre-NK progenitor cells derived from pluripotent stem cells with a composition containing one or more growth factors and cytokines selected from SCF, Flt3L, IL3, IL7 and IL15 to obtain NK progenitor cells or NK cells derived from pluripotent stem cells, wherein the composition does not contain one or more of VEGF, bFGF, BMP activators and ROCK inhibitors. The pluripotent stem cell-derived pre-NK progenitor cells are made to permanently contact hematopoietic endothelial cells with a combination of BMP activators, ROCK inhibitors, and one or more growth factors and cytokines selected from VEGF, bFGF, SCF, Flt3L, IL3, IL7, and IL15 to allow cell differentiation and expansion. 10. The method of claim 9, wherein (a) The method further comprises: (i) subjecting the pluripotent stem cells, the mesodermal cells, the mesodermal cells with permanent hematopoietic endothelial potential, and/or the permanent hematopoietic endothelium to a hypoxic stress of 2% to 10%; or (ii) contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the cells, wherein the composition does not contain a TGFβ receptor/ALK inhibitor; or (b) NK cell lines that produce pluripotent stem cells (i) do not produce embryoid bodies, (ii) are cultured in a monolayer, (iii) are cultured under feeder-free conditions, or (iv) are cultured under substrate-free conditions; or (c) Where the pluripotent stem cells are iPSCs or naïve iPSCs. 11. The method of claim 1, further comprising: Contacting pre-HSCs derived from pluripotent stem cells with a composition comprising a BMP activator and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6 and IL11 to obtain pluripotent stem cell-derived progenitor cells, wherein the composition does not contain ROCK inhibitors. The pre-HSCs derived from pluripotent stem cells are made to achieve permanent hematopoietic endothelial contact with a combination of BMP activators, ROCK inhibitors, and one or more growth factors and cytokines selected from TPO, IL3, GMCSF, EPO, bFGF, VEGF, SCF, IL6, and IL11 to allow cell differentiation and expansion. 12. The method of claim 11, wherein (a) The method further comprises one or both of the following: (i) contacting the pluripotent stem cells with a composition comprising a MEK inhibitor, a GSK3 inhibitor, and a ROCK inhibitor to seed and expand the pluripotent stem cells, wherein the composition does not contain a TGFβ receptor/ALK inhibitor; (ii) subjecting the pluripotent stem cells, the mesodermal cells, the mesodermal cells with permanent hematopoietic endothelial potential, and/or the permanent hematopoietic endothelium to a hypoxic stress of 2% to 10%; or (b) Among them, progenitor cells of pluripotent stem cell lineages (i) do not produce embryoid bodies. (ii) monolayer culture, (iii) under no-feeding conditions, or (iv) under substrate-free conditions; or (c) Where the pluripotent stem cells are iPSCs or naïve iPSCs. 13. The method according to any one of claims 1-12, wherein: (i) The permanent HE cells comprise CD34+ permanent hematopoietic endothelial cells (iCD34 HE), wherein the iCD34 cells are capable of differentiating into specialized progenitor cells, T progenitor cells, NK progenitor cells, T cells and NK cells and wherein the iCD34 cells are CD34+CD43-. (ii) The permanent HE cells are CD34+; (iii) The HSC is CD34+CD45+; (iv) Integrative progenitor cells (iMPPs) are CD34+CD45+; (v) T progenitor cells are CD34+CD7+; (vi) T cells are CD4+ or CD8+; (vii) NK progenitor cells are CD56+CD7+CD161+; and (viii)NK cells are CD56+CD57+CD16+CD94-.