JP2014023519A - Generation of lung and airway epithelium from human pluripotent stem cells and use thereof - Google Patents

Abstract

The present invention provides a method for inducing differentiation of epithelial cells of lung and airways from human pluripotent stem cells (hPSC).
An isolated cell population enriched in epithelial cells of the lungs and airways. An isolated cell population enriched in lineage cells in the lung field. The cells are NKX2.1, FOXP2, GATA6, P63, mucin 5ac, mucin 2, mucin 5b, FOXJ1, acetylated tubulin +, CC-10, pro-SPC, SPB, mucin 1, lysozyme, lectin DBA, podoplanin +, A cell population expressing aquaporin 1+, lectin RCA120 +, or a combination thereof.
[Selection] Figure 1

Description

CROSS REFERENCE TO RELATED APPLICATIONS This application is filed on April 24, 2012, US Provisional Application No. 61 / 675,069, entitled “Production and Use of Lung and Airway Epithelia from Human Pluripotent Stem Cells”. In contrast, the entire application is incorporated herein by reference and claims.

BACKGROUND OF THE INVENTION Lung disease is a major cause of death. For some lung diseases, transplantation is an effective treatment option, however, due to the low availability of donor organs, surgical and medical complications associated with transplant procedures, and immunological rejection. Be disturbed. Therefore, the possibility of regenerating lung tissue from autologous cells would be an important medical advance.

  According to the first series of embodiments, the platform is for diseases such as, for example, cystic fibrosis, tracheoesophagal fistula, surfactant deficiency syndrome, infant respiratory distress syndrome, respiratory infections, etc. Drug screening using patient-specific iPS cells is provided.

  According to a second series of embodiments, the method comprises seeding autologous iPSC-derived respiratory cells into a human decellularized lung matrix for transplantation to replace a terminally ill lung, and therefore Avoid donor organ shortages and immunological rejection.

  According to a third series of embodiments, the isolated cell population is enriched with lung and airway epithelial cells and cells lineaged in the lung field. In some embodiments, the cells are NKX2.1, FOXP2, GATA6, P63, mucin 5ac, mucin 2, mucin 5b, FOXJ1, acetylated tubulin +, CC-10, pro -Express SPC, SPB, mucin 1, lysozyme, lectin DBA, podoplanin +, aquaporin 1+, lectin RCA120 +, or combinations thereof. In some embodiments, the cell population comprises up to 90% lung and airway epithelial specific cells. In some embodiments, the cell population comprises up to 80% lineaged cells in the lung field. The cell population has cells including tracheal cells, bronchial cells, alveolar cells, or combinations thereof. In some embodiments, the purified preparation of cells lineaged in the lung field express NKX2.1, GATA6, SOX2, p63, FOXP2, FOXJ1, or combinations thereof.

  According to a fourth series of embodiments, the method of enhancing the induction of cells lineaged in the lung field from anterior foregut endoderm cells comprises: (a) at least anterior foregut endoderm (AFE) cells, Culturing with a BMP inhibitor or TGF-β signaling inhibitor for 1 day; and (b) said cells for at least 5 days, Wnt protein or a pharmacological agonist thereof (eg, CHIR99021), BMP factor, FGF protein, Culturing in the presence of EGF protein, retinoic acid, or a combination thereof.

  In some embodiments, the cell culture occurs in the presence of a Wnt inhibitor and a TGF-β signaling inhibitor. In some embodiments, the cell culture occurs in the presence of matrigel and / or maturation medium. The maturation medium includes dexamethasone, methylbutyryl cAMP, hypoxanthine, or a combination thereof. In some embodiments, the culturing of step (b) occurs in the presence or absence of BMP4. In some embodiments, the culture of step (b) occurs in the presence or absence of retinoic acid, dexamethasone, methylbutyryl cAMP, hypoxanthine, or a notch inhibitor. In various embodiments, the notch inhibitor includes DAPT (gamma-secretase inhibitor).

  In some embodiments, the method further comprises adding a SHH inhibitor. The SHH inhibitor is cyclopamine. In some embodiments, the Wnt inhibitor includes a Wnt3a inhibitor or IWP2. BMP inhibitors are noggin or dorsomorphin, or other selective pharmacological BMP4 inhibitors. The TGF-β signaling inhibitor is SB341543.

  According to a fifth series of embodiments, the cells express NKX2.1, GATA6, SOX2, p63, FOXP2, FOXJ1, or combinations thereof. Cells that have been lineaged in the lung field are new uncharacterized p63-expressing epithelium, goblet cells, submucosal gland epithelium, Clara cells, basal cells, ciliated cells, type I alveolar cells, type II alveolar cells Or a group of combinations thereof. In some embodiments, the lung lineage cells are Muc5a, Muc2, or combinations thereof, Muc5b, Muc2, or combinations thereof, CC10, p63, acetylated tubulin, FOXJ1, or combinations thereof , Muc1, SP-B, pro-SP-C, lysozyme, lectin DBA, or combinations thereof, podoplanin, aquaporin 1, aquaporin 5, T1α, lectin RCA120, or combinations thereof are also expressed. In some embodiments, the BMP factor is BMP4. The FGF protein is FGF10 or FGF7.

  According to a sixth series of embodiments, purified preparations of cells lined in the lung field are NKX2.1, GATA6, FOXP2, CGRP, CCSP, FOXJ1, SP-B, SP-C, p63, CC10. , Cells expressing MUC5a, MUC1, MUC2, or combinations thereof.

  In the following figures, various embodiments are described for illustrative purposes, but embodiments of the invention are not limited to the details shown in these figures. This patent application contains at least one drawing executed in color.

Potential of AFE produced in vitro in vivo (a) H / E staining of teratoma induced 5 weeks after HES2 transplanted under the kidney capsule of NSG mice. The three right panels show the neuroectoderm (neurorosette), endoderm (intestinal epithelium) and mesoderm (cartilage), respectively. (B) H / E staining of proliferation that occurred 5 weeks after transplantation of NS-induced AFE cells (derived from HES2 cells) under the kidney capsule of immunodeficient mice. (C) Immunofluorescence analysis of the tissue of (b) stained for FOXA2, PAX9, AIRE and Surfactant protein C (SP-C). Overview of AFE induction from hPSC. Expression of NKX2.5, NKX2.1, FOXA2 and EPCAM in cells differentiated according to the scheme at the top of the figure. Induction of SP-C in ventralized AFE. Expression of NKX2.1, p63, FOXA2 and Muc5a in hPSCs cultured according to the scheme at the top of the figure. Colony morphology and expression of NKX2.1, p63 and FOXA2 in spherical colonies obtained after seeding ventilated AFE in the presence of WFKBE + RA (see top of figure) in DCI and Matrigel. Relative expression of NKX2.1 (blue) and PAX1 (red) after exposure to NS, NS> SI or SI> NS followed by WFKBE + RA and induction of AFE (analysis on day 13). Expression of NKX2.1 and FOXA2 after ventralization of AFE produced by either NOGGIN / SB or NOGGIN / SB followed by IWP2 / SB, as schematically shown at the top of the figure. Ventralization of AFE: (a) Expression of SOX2, NKX2.1, NKX2.5, PAX1 and P63 in HES2-derived cells produced under the two conditions schematically shown at the top of the panel (n = 6 culture wells) (From two independent experiments), * significantly different from NOGGIN / SB-431542) (b) HDF2 (top) and HFD9 (bottom) hiPS differentiated to AFE under the conditions schematically shown at the top of the panel Expression of SOX1, NKX2.1 and PAX1 mRNA in cells (n = 4-6 culture wells (from two independent experiments) * significantly different from NS) (c) Treated sequentially with Activin A, NS and WFKBE Later, immunofluorescence of NKX2.1 in differentiated HDF9 hiPS cells. NKX2.1, NKX2.5 and PAX1 expression (n = 4-6 culture wells (from 3 independent experiments)) in HES2-derived cells produced under the three conditions shown schematically at the top of the panel, * Significantly different from other conditions). Potential of ventral AFE as lung. (A) Expression of PAX1, NKX2.1, FOXP2, GATA6 and FOXJ1 (n = 4-6 culture wells (from multiple experiments)) in cells derived from HES2 produced under the two conditions shown at the top of the panel, * WFKBE Significantly different). (B) Induction of SP-C in ventralized AFE in the presence of the indicated factors. (C) Expression of NKX2.1 (green) on day 13 (blue = DAPI) in AFE cultured with WFKBE + ATRA (d11) followed by Wnt3a + FGF10 + FGF7 (d13). Effect on GATA6 expression when increasing concentrations of ATRA added to WFKBE ventralization cocktail (n = 3). Spherical colonies at 11 and 17 days in culture from Ngfr + cells isolated from mouse trachea according to the protocol of Rock et al. Day 4 endoderm (A) has a higher capacity for lineage determination of lung field cells (Nkx2.1 + FOXA2 +) compared to day 0.5 (B) and day 5 (C) endoderm. Have Day 4 endoderm (A) has a higher capacity for lineage determination of lung field cells (Nkx2.1 + FOXA2 +) compared to day 0.5 (B) and day 5 (C) endoderm. Have On day 15 of culture, a group of p63 + FOXA2-SOX2-Nkx2.1- cells was also produced. These cells exist as small populations adjacent to lineaged cells (NKX2.1 +) in the lung field or, if the culture is of low density, surround the NKX2.1 + cells It exists as a linear array of cells with nuclei. On day 55 of culture, the following cells were produced: (A) cc-10 + cells; (B) mucin 2+ cells; (C) SPB + NKX2.1 + cells; (D) acetylated alpha-tubulin expressing cells. On day 48 of culture, the following cells were produced: (A) mucin 1+ cells; (B) lysozyme + cells; (C) and (D) lectin-DBA expressing cells. On day 48 of culture, enrichment of CC10 + pro-SP-C + SP-B + cell population was observed. (A)> 40% of cells were CC10 and pro-SPC double positive; (B)> 40% of cells were CC10 and mature SPB double positive. Effect on Nkx2.1 and FOXA2 expression when increasing concentrations of retinoic acid (ATRA) added to WFKBE ventralization cocktail. The dose of RA during ventralization was critical and 50 μM was determined to be optimal (indicated by the amount of Nkx2.1 + FOXA2 + cells produced on day 15 of culture).

DETAILED DESCRIPTION OF THE INVENTION Each year as many as 400,000 people die from lung disease in the United States. However, the lung is a very complex organ containing many different cell types, and regenerative medicine for lung diseases is immature. For many end-stage lung diseases, transplantation is an effective treatment option, but is hampered by the low availability of donor organs and surgical, medical, and immunological complications. Therefore, there is a need for new approaches to cell replacement therapy for lung disease. Lung and airway epithelial cells have never been derived from human pluripotent stem cells. The first report of an efficient protocol for producing AFE and its derivatives was by Green et al. Longmir et al. Used the mouse NKX2: GFP reporter (which is not applicable to human hPSCs) and the efficiency was up to 20%. In contrast, Mou et al. Achieved an efficiency of 10-15% in lung field epithelial production and less than 5% for further differentiation into proximal airway cells in contrast to that in humans. In the system of the present invention, it is 90%.

  Treatment using a cell-based approach is very promising, but also difficult to implement. With the exception of one study (though the results are controversial), any type of stem cell or its progeny has not yet become possible to engraft in the lung of a lung injury animal model.

  Respiratory stem cells have a variety of applications: (1) Tissue repair: seeding autologous iPSC-derived lung and airway cells into decellularized lung matrix for organ transplantation to replace end-stage disease lungs (2) Disease modeling: Lung and airway epithelium produced in vitro from hPSC is an excellent research platform for congenital and acquired human lung disease; (3) Drug screening: Producing a sufficient number of biologically relevant patient-specific cells for high-throughput drug screening by utilizing the unlimited self-renewal ability of hPSC used to produce lung epithelium And (4) elucidation of the mechanism of lung development: studies that give valuable insight into the characteristic properties and functions of postnatal tracheobronchial and lung stem cells An in vitro model is provided that specifically examines.

  The first step in this field is to develop strategies to produce various lung cell types from stem cells and to understand the underlying mechanisms. A possible alternative approach is to use tissue reconstituted in a decellularized lung matrix to achieve replacement of affected lungs and airways with either adult stem cells or hPSC-derived cells. is there. Rat lungs can be decellularized by perfusion with a mild detergent and can be recellularized using fetal or neonatal airway-side lung cell suspension and vascular-side endothelial cells Was recently shown.

ES cell

Embryonic stem (ES) cells are derived from the inner cell mass of blastocysts and can be maintained in a pluripotent state under certain conditions. Without being bound by theory, ES cells can differentiate into all somatic and germ cell types. Thus, the development of suitable conditions that differentiate ES into various cell types and tissues is highly promising for future cell replacement therapies ¹⁰¹ . Development begins in the gastrulation process (during this time, undifferentiated cells of the inner cell mass of the blastocyst differentiate into three germ layers, from which all the body's tissues develop). For example, ES cells can ultimately differentiate into endoderm that develops the intestine, liver, pancreas, lungs, esophagus, pharynx, thyroid, parathyroid, thymus, and the like. Alternatively, ES cells can eventually differentiate into mesoderm that develops muscle, bone, adipose tissue, connective tissue, urogenital system, cardiovascular system, and the like. Finally, ES cells can further differentiate into ectoderm, which ultimately develops the skin, nervous system, neural crest, and the like. Recent discoveries ^102-106 that adult somatic cells can be reprogrammed to a pluripotent state (induced pluripotent cells, or iPS cells) using a relatively simple procedure are patient specific pluripotent cells (which are , Which will overcome the problem of rejection, and ethical issues regarding the use of hES cell-derived tissue).

Lung development

Lung development from the endoderm is driven primarily by a combination of activation and inhibition of six signaling pathways tightly controlled in time and space: BMP / TGF-β, FGF, Wnt, retinoic acid (RA), Hedgehog (HH) and Notch ^98,99,116 . These signals originate in part from the surrounding mesenchyme and vasculature and partly from the developing lung tissue itself. Gata6, FOXA2, SOX2 and NKX2.1 are transcriptional regulators for lung and tracheobronchial development ^98,99,116 . The lung develops from the ventral side of the anterior foregut endoderm (FOXA2 + SOX2 +) (region with NKX2.1 expression as a marker). In E9.5 in mice, trachea primordia and two HaiHara group separates from the ventral side of the endodermal primitive gut ^98,99. An airway develops through a complex process of branching morphogenesis ¹⁰⁸ . This is followed by alveolar morphogenesis (partially progressing after birth and undergoing the ^{prescribed pseudogonadic} , saccular and ^{vesicular stages} ) ^98,99,116 . During early postnatal development, lung and tracheobronchial stem cells that provide extensive regenerative capacity are also defined ^100,107 . Further relevant details of pulmonary development and postnatal lung stem cells are discussed further in this application as needed. In accordance with the developmental paradigm, it can be inferred that in order to produce tracheobronchial cells and alveolar cells, hPSCs should first differentiate into endoderm and then to AFE. Next, the lung field must be induced and subsequently differentiated into late embryonic and postnatal respiratory cell types and structures.

lung

The respiratory system has significant implications for gas exchange and consists of a complex branching system of alveoli (also comparable to a branched vasculature). In humans, the trachea and bronchi are lined by pseudostratified epithelium (including ciliated cells, mucus cells, secretory (Clara) cells, neuroendocrine cells and basal cells). The alveoli are lined by two types of cells: type I alveolar epithelial cells and type II alveolar epithelial cells (ATI and ATII cells). ATII cells produce surfactant ^{98 and 99.}

The lung consists of a complex branching system of bronchi and bronchioles that terminate in the alveoli where gas exchange occurs ^98,99 . Thus, if lung tissue can be regenerated from autologous cells, it would be a major medical advance ¹⁰⁰ . One way to achieve this may be the differentiation of pluripotent stem cells into a variety of respiratory epithelial cells and / or putative postnatal stem cells of the respiratory system ^100,101 . There are two types of pluripotent stem cells. As previously mentioned, ES cells are derived from the inner cell mass of blastocysts and can be maintained in a pluripotent state in certain conditions in both humans and mice ¹⁰¹ . Artificial pluripotent state cells (iPS cells) are cells in which somatic cells are initialized to a pluripotent state by expressing 3-4 genes with lentivirus or retrovirus ^102-106 . iPS technology opens the way to the generation of patient-specific pluripotent cells that will overcome the problem of rejection and the ethical issues associated with the use of ES cell-derived tissue ¹⁰² .

AFE

  AFE is the most anterior part of the endoderm. During embryonic development, the formation of the anterior and pharyngeal endoderm is the establishment of a body plan and the development of various organ systems such as the ears, palatine tonsils, thymus, parathyroid, thyroid, lungs, esophagus, and trachea Is a critical stage. Following its formation, the definitive endoderm undergoes differentiation into the endoderm lower lineage. The more posterior endoderm gives rise to the midgut and hindgut. The pharyngeal endoderm (the front side of the lung field) forms four exposed parts called the pharyngeal sac. Each sac develops into a specific organ (eg, inner leaflet of the ear canal and tympanic membrane (first sac), tonsil (second sac), thymus (front third), parathyroid gland (dorsal) Lateral third and fourth sac), and parathyroid follicle C cells of the thyroid (fourth sac)). The thyroid gland develops from the bottom of the pharynx. The lungs, esophagus, and trachea originate from the AFE distal to the sac.

  The pharyngeal endoderm (including the thymus) and lung primordium are both derived from AFE. Directed differentiation into lung tissue should proceed by first differentiating ES cells into definitive endoderm and then patterning to their most anterior fate. Induction of midgut and hindgut tissues (pancreas, liver, intestinal tract) has been more successful, whereas induction of AFE organ fate has been delayed because of difficulty rather than lack of interest. AFE is an embryonic germ layer that also produces the intestine, liver, pancreas, stomach, esophagus, lung, pharynx and thyroid gland. Several important organs (including lung and thymus) are derived from AFE, so AFE is of great importance.

  First, definitive endoderm is derived from hPSC using high concentration of Activin A (100 ng / ml), followed by BMP inhibitors (such as Noggin and Dorsomorphin) (NS or DMS) and TGF-β signaling inhibitors AFE was produced by culturing the cells in the presence of (SB341543 or the like). After culturing for 2 days in NS, the cells were subjected to ventralization conditions consisting of Wnt3a or small molecule GSK inhibitor, BMP4, FGF10, FGF7 and EGF. Under these conditions, ventral AFE is obtained, containing approximately 40% NKX2.1 + cells (an index of lineage determination to the lung field). Lung and airway epithelia were produced from AFEs derived from human pluripotent stem cells (embryonic and induced pluripotent state stem cells).

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Exemplary methods and materials are described below, but methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention.

  All patents, patent applications and publications and other references mentioned herein are referenced as if each individual publication or reference was specifically and individually indicated to be incorporated by reference. Is incorporated in its entirety. The publications and references cited herein are not admitted to be prior art.

  In order to facilitate a more complete understanding of the invention, the following examples are provided. The following examples and virtual examples illustrate exemplary aspects of making and implementing the present invention. However, the scope of the present invention is not limited to the specific embodiments disclosed in these examples, but it is for illustration purposes only, since similar methods can be obtained using alternative methods. Absent.

  Certain embodiments of the invention are directed to methods of inducing differentiation of lung and airway epithelial cells from human pluripotent stem cells (hPSCs). Embodiments of the present invention further provide cell populations enriched in lung field lineaged cells (NKX2.1 +).

  In certain embodiments, the following culture conditions were developed to induce lung and airway epithelium from hPSC-derived AFE.

(1) In order to increase the production of cells lined up in the lung field (NKX2.1 +) in early and late endoderm (ie, medium containing Activin A, 3, 3.5 and 4 days, respectively) Cultured) were tested for their ability to be specific to the lung. The initial endoderm, compared with late endoderm (with the efficiency of only 10% of the Nkx2.1 ⁺ cells), at 50-60% higher efficiency, and produce Nkx2.1 ⁺ cells.

  (2) In order to increase the production of cells lined in the lung field (NKX2.1 +), the anteriorization condition (NS) was improved, NS was first applied for 1 day, and then TGF-β and Wnt Anteriorization was performed by applying a combination of inhibition (using SB and IWP2). This results in NKX2.1 + production with 80% efficiency on day 15 without evidence of neural or thyroid differentiation.

  (3) On the 15th day of culture, a p63 + FOXA2-SOX2-Nkx2.1- cell group was also produced using the above conditions. These cells exist as small populations adjacent to lineaged cells (NKX2.1 +) in the lung field or, if the culture is of low density, surround the NKX2.1 + cells It existed as a linear array of cells with nuclei.

  (4) On the 15th day of culture, the cells were gently trypsinized and replated under culture conditions containing Wnt3a or GSK inhibitor, FGF10, FGF7, with or without BMP4 and retinoic acid. After further culture for 10-40 days, the following cell types were observed: goblet cells (Muc5a +, Muc2 +); submucosal gland epithelium (Muc5b +, mucin2 +); Clara cells (CC10 +); basal cells (p63 +); Ciliated cells (acetylated tubulin +, Foxj1 +); type II alveolar cells (Muc1 +, pro-SP-C +, SP-B +, lysozyme +, lectin DBA +); and type I alveolar cells (podoplanin +, aquaporin 1+, Lectin RCA120 +).

  The data showed that the addition of BMP4 and retinoic acid helped the initial determination of airway basal cell fate. Overall,> 90% of cells were determined to be airway or lung epithelial cell types.

  (5) On the 25th day of culture, the gamma-secretase inhibitor DAPT (inhibits notch signaling) is added to the condition containing Wnt3a or GSK inhibitor, FGF10, FGF7, with or without BMP4 and retinoic acid, After further culturing for 20-25 days, enrichment of the CC10 + pro-SP-C + SP-B + cell population was observed and in conditions containing notch inhibitor DAPT, Wnt3a or GSK inhibitors, FGF10 and FGF7> 40% of the cells were triple positive.

  (6) By adding maturation components (dexamethasone, methylbutyryl cAMP and hypoxanthine) to the condition containing Wnt3a or GSK inhibitor, FGF10, FGF7 with or without BMP4 and retinoic acid on the 25th day of culture. After further culture for 20-25 days, the SPB + Nkx2.1 + cell population is enriched.

  (7) Culture of ventral AFE in the presence of matrigel and maturation medium (dexamethasone, methylbutyryl cAMP and hypoxanthine) resulted in a globular structure. The globular structure expressed various proximal airway markers and was probably derived from developmental airway progenitor cells.

Example 1 : Induction of cells lineaged in the lung field

  This example describes the production of lung and respiratory cell types from hPSCs, which are either ESCs of iPSCs. The ability to produce various cell types of the respiratory system makes it possible to test drugs against developmental lung abnormalities and to test the teratogenic effects of drugs on lung development.

  Differentiating pluripotent cells into the earliest stages of the developing lung has never been possible before. With high efficiency and high purity, these cells were successfully differentiated into early cells belonging to the lungs and airways. These cells further differentiated into cells expressing at least some mature lung and airway cell markers. This model presents new avenues for regenerative treatment of lung disease and to gain a better understanding of the mechanisms underlying human lung development and human lung disease.

  The ability to produce respiratory cells from human pluripotent stem cells opens a new avenue to explore the human developmental biology of the respiratory system, drug trials for the treatment of developmental lung abnormalities, and drugs in lung development It will be possible to test the teratogenic effect of. Appropriate late embryonic, postnatal cell type differentiation suggests that successive stages in development are reproduced in vitro (a strategy called directed differentiation). The lung is derived from the endoderm (more specifically, the ventral frontal foregut endoderm). This example shows that highly enriched AFE was produced from human pluripotent stem cells. In addition, this tissue was serialized to express a marker corresponding to the lung field producing the lung primordium. Subsequently, conditions were established that aided cell fate in the proximal (airway) or more distal (alveolar). Importantly, it was also observed that the conditions that produced AFE subsequently determined the potential to express lung markers, suggesting early prespecification during endoderm development. . The conditions were further optimized to functionally examine the resulting cells and tissues and to gain an understanding of the mechanism of human lung development.

In this example, we explored a strategy to produce lung endoderm compartments (ie, tracheal, bronchial and alveolar epithelial cells) and putative postnatal stem cells associated with these structures from hPSCs. In addition, this model was used to discover the mechanisms involved in early human lung development. Directional differentiation was used for appropriate late embryonic or postnatal cell differentiation. The lungs originate from the ventral aspect of the AFE, from which the tracheal primordium and two pulmonary primaries separate ^1,2,98,99 . Airway and alveoli develop through a complex branching process ^3,108 and subsequent alveolar morphogenesis ^1,2,98,99 . Efficient induction of definitive ^endoderm and differentiation to various degrees to the posterior foregut, midgut and hindgut derived organs (liver, pancreas, and intestinal tract) ^4-9,109-113 , directed differentiation However, the art has not been able to produce endoderm tissue proximal to the liver ^100,114 . A plausible reason is that it was impossible to efficiently determine AFE from pluripotent stem cells. In addition, AFE could be lineaged to cells corresponding to the lung field of ventral AFE, and conditions were identified such that the culture consisted of respiratory cells substantially uniformly. Therefore, it was possible to further optimize these conditions and use this model to gain a better understanding of the mechanisms underlying the AFE lung fate decision.

  According to the developmental paradigm, tracheobronchial and alveolar cells, and hPSCs should first differentiate into endoderm and then lineaged to AFE. The lung field must then be induced and then differentiated into late embryonic and postnatal respiratory cell types and structures.

Production of ventral frontal foregut endoderm

The published strategy of inducing endoderm, consisting of exposing differentiated ES cells to high concentrations of Activin ^25-27 , resulted in posterior biased results (probably in contrast to foregut tissue). Explains why the induction of midgut and hindgut tissues (pancreas, liver, intestine) ^25-27 is more successful). However, the definitive endoderm is a combination of NOGGIN (physiological inhibitor of BMP signaling) and SB-431452 (pharmacological inhibitor of Activin A / nodal and TGF-β signaling) ( N OGGIN / S B-431542 or NS). to, followed by exposure, before the onset of intestinal markers SOX2, rear inhibition of marker CDX2, and to result in maintenance of endodermal markers FOXA2, revealed by morphogen sorting ^31,32. More than 90% of cells expressed FOXA2 and SOX2 at this stage.

  An overview of the ventral AFE production protocol from hPSC is shown in FIG. The in vitro differentiation scheme (shown in FIGS. 2 and 3) reproduces the hierarchical and sequential induction of lung progenitor cells that occurs in vivo. Blocking the other fate of AFE increases the production of lung cells, further matures lung progenitor cells, major types of lung (ATI and ATII) and airway epithelial cells (basal cells, Clara cells, The aim was to lineage cilia cells and goblet cells) from hPSCs.

Preferential decision to lung field for pharyngeal endoderm ¹⁰

NS-induced AFE exposure to WFKBE resulted in increased expression of both pharyngeal and lung markers. Therefore, conditions were determined that favored the lung over the pharyngeal fate. WFKBE + RA biases the lung field, while WFKBE aids pharyngeal fate. The role of retinoic acid in lineage determination to the lung indicates posteriorization of ventral AFE and prevention of pharyngeal sac formation by inhibiting TBX1 signaling. Under these conditions, substantially 100% of the cells are NKX. 2.5 was expressed, most of which also expressed FOXA2 and EPCAM (FIG. 3) (a group of typical markers of ventral AFE). Insular cells expressed NKX2.1. Flat NKX2.5 ⁺ FOXA2 ⁻ cells were generated interspersed between dense colonies of cells expressing NKX2.1 (FIG. 3). They expressed vascular smooth muscle actin and were therefore mesodermal (FIG. 3). Continued treatment with WFKBE + RA up to day 19 resulted in low levels of mature ATII marker (SP-C) (FIG. 4).

Induction of proximal vs distal lung fate

When the fate of the cell continuously cultured with WFKBE + RA was examined, SP-C was not induced. After re-seeding (13th to 15th day) and continuous treatment under these conditions until 23rd to 33rd day, all mesodermal cells disappeared, and cells including tubular structures were filled with high density The resulting culture consisted of colonies (Fig. 5). These colonies were entirely FOXA2 ⁺ , while most cells expressed NKX2.1 except those at the outer edge. The tubular structure expressed mucins (MUC2 (not shown) and MUC5a (FIG. 5)). The airway basal marker p63 was expressed mainly at the outer edge (FIG. 5). Toward the more central part of the structure, p63 and NKX2.1 are coexpressed (middle panel in FIG. 5), suggesting differentiation of p63 ⁺ cells into NKX2.1 ⁺ cells.

Thereafter, the cells were cultured for 3 days in a maturation medium ⁵⁰ consisting of dexamethasone, butyryl cAMP and isobutylmethylxanthine (DCI). Under these conditions, large globular structures expressing FOXA2, NKX2.1 and p63 were formed (FIG. 6). These structures are very similar to tracheospheres ⁵¹ obtained from the adult trachea. Cells at this stage are fully destined for the respiratory tract, but are still in a relatively early developmental stage. Lung field cells that were successfully produced were seeded onto decellularized human lung scaffolds. Preliminary data show that seeded cells align on the scaffold and express Nkx2.1 and p63.

AFE prior series determination

Although the observation that AFE is determined by adding NS after Activin A induction was made according to a relatively unbiased selection, this protocol is reasonably reasonable. Most AFEs originate from cells that first pass through the primitive streak and then leave the nodule ⁵⁴ . Therefore, this part of the endoderm is furthest away from the nodal signaling region of the upper blastoderm for the longest time ⁵⁵ . This explains why blockade of TGF-β signaling after a period of exposure to Activin A is involved in determining this part of the endoderm. During its anterior migration, cells destined to become AFE pass through a region that expresses Noggin ⁵⁵ , which provides a compelling explanation for the desire to block BMP signaling. . Interestingly, the most anterior fate cells were then exposed to a Wnt inhibitor (Dkk1) ⁵⁵ . These cells then fold ventrally when the intestinal tract is formed, and the foremost endoderm eventually reaches the ventral, where the pulmonary primordium ^54,55 occurs.

We tested whether inhibition of Wnt signaling could lead to a more detailed lineage determination of the lung field. NS (day 6), followed by SB + IWP2 (Wnt inhibitor) (SI, day 7) in sequence, so that NKX2.1 mRNA of PAX1 mRNA (pharyngeal endoderm) on day 13 after ventralization The ratio to has increased. Consistent with these findings, the number of NKX2.1 ⁺ cells was greater after NS and subsequent AFE induction with SI than with NS alone (FIG. 8). In addition, after induction of AFE by using NOGGIN / SB followed by SB + IWP2 (NS> SI), more in 19 in the presence of DCI than when AFE was induced by using NS alone on day 5 A spherical structure was obtained. Although more detailed research and quantification is desired, these data indicate that the history of cell differentiation determines their future potential, perhaps making it easier to later lineage the lungs By fixing cells in an epigenetic state. These observations have important implications for the development of directed differentiation strategies into the respiratory lineage and pose fundamental questions regarding epigenetic control and pre-sequencing decisions during endoderm development.

  Optimizing conditions for the induction of respiratory cells

  Use qPCR and, if possible, fluorescence and confocal microscopy. In order to achieve maximum potential in subsequent lung lineage determination, an optimization factor for AFE induction was determined. Factors involved in far-proximal determination were analyzed. Once these are established, the lineage produced in culture can be manipulated.

Since there is currently no recognized lung injury model that has been demonstrated convincingly to engraft stem cell populations, transplantation of immunodeficient mice under the kidney capsule was used as an in vivo assay. After transplantation of NS-induced AFE under the kidney capsule of immunodeficient mice, many SP-C ⁺ tube structures and structures with pseudostratified epithelium were observed (FIG. 1b, c). These data suggest that AFE produced in vitro has significant lung potential in vivo, and conversely, this analysis is relatively efficient as a lung of transplanted cells. It was suggested to clarify the potential. This finding also transplanted embryonic lung E11.5 mouse to under the kidney capsule is consistent with the observation that allowed the differentiation through false glandular phase and canalicular stage lung胞発raw ^56.

During this process, the grafted blood vessels joined ⁵⁶ with the host blood vessels extending inward. Furthermore, recently, postnatal lung stem cell populations are mixed with cell suspensions produced from E14 embryonic lungs to include traces of alveolar morphogenesis derived from adult stem cells after transplantation of the kidney capsule. ⁵⁷ has been shown to yield structure.

It can be determined whether the potential of the cell in vivo occurs. Accordingly, in certain embodiments, the following is performed. 10 ⁶ cells are dissolved in Matrigel (4 μl) at 4 ° C., allowed to clot at 37 ° C., and transplanted under the kidney capsule of immunodeficient NSG mice. Groups of 5 mice can be analyzed for each condition and differentiation stage 4 and 8 weeks after transplantation, respectively. The analysis consists of staining of airway epithelial cells (FOXJ1, CCSP, CGRP, MUC5a, p63) and alveolar cells (SP-C, T1α, AQ5). In addition, other fate can be examined by staining for GCM2 (parathyroid), PAX9 and TBX1 (pharyngeal sac), Calcitonin (C cells), and AIRE (thymus). If growth is not observed, support from other cells may be involved. In that case, it is possible to employ Chapman et al. Analysis ⁵⁷ in which the cell population to be tested is mixed with an E14 embryonic lung cell suspension.

Factors that induce lung fields from AFE

  Prior series determination. Data show that better induction of NKX2.1 is achieved after 1 week and ventralization when AFE is induced by continuous exposure of cells to NS followed by SI (NS> SI) Indicated. These data further showed that lung primordium fate may already be determined when AFE is formed. In some embodiments, the following occurs:

1. Further quantification of these data regarding the absolute number and enrichment of NKX2.1 ⁺ FOXA2 ⁺ cells can be performed.

  2. It was determined that the NS> SI mode combined with inhibition of Wnt, BMP and TGF-β was optimal. And

  3. It is possible to determine how the prior lineage determination will affect subsequent differentiation into proximal and distal lung cell types. This is because we already have evidence that DCI-induced globular structures are more efficiently produced when AFE is induced in the presence of NS> SI than in the presence of NS.

In other embodiments, the following occurs. AFE can be induced in the presence of NS, SI, NS> SI, NSI, or SI> NS. Subsequently, cells can be ventralized using WFKBE + RA, and lineage determined to lung primordium equivalents. On days 13-15, the culture can be divided into WFKBE + RA continuation (putative proximal lung) or WFK (putative distal lung). On day 23, DCI conditions can be applied for final maturation. Replicates of these cultures can be analyzed at various stages of culture. At each stage, cytospin preparations can be used to quantify the cell number, NKX2.1 ⁺ , FOXA2 ⁺ and p63 ⁺ cell fractions. Furthermore, the number of DCI-derived spheres per unit of input cells can be evaluated. Finally, qPCR and staining can be performed on the markers described herein.

Factors involved in near-distal differentiation

Continued culture in WFKBE + RA leads to a more proximal fate, while switching to a condition in which BMP4 and RA were removed between days 13 and 19 leads to a more distal fate. Induced (proven by induction of SP-C). However, fate decisions during pulmonary development are quite complex and not well understood. During branching morphogenesis, pluripotent progenitor cells regenerate at the tip ^1,2,63 . The cells left in the stalk differentiated into the tracheobronchial epithelium. When the tubular / saccular phase begins, the distal cells begin to differentiate into alveolar cells ^1,2 . Application of Wnt3a, FGF7 and FGF10 after ventralization of AFE induces large amounts of SP-C mRNA on day 19. Proteins are expected to be expressed by longer cultures. Although these conditions have been identified in unbiased selection, FGF10 and Wnt signaling are developmentally reasonable ^45-49 because they establish distal fate. In some embodiments:

1. ^Make a determination of whether all the factors in our culture are desirable, and whether their concentrations can be optimized (since many morphogens have a concentration-dependent effect) ^1,63 .

2. Since Wnt signaling is developmentally important in establishing distal fate ^45-47 , in proximal conditions, removing Wnt may enhance proximal fate.

3. Non-standard Wnt (Wnt5a) also plays a role in lung development, possibly through control of SHH and FGF10 signaling, and can be validated ^65,66 .

4). Blocking Notch in early developmental signaling also promotes more distal fate ⁶⁷ . This was done by using the gamma-secretase inhibitor DAPT.

5. DAPT increased pro-SPC (SPC precursor) expressing cells. Down-regulation of RA signaling is involved in terminal differentiation of AT cells ⁵⁷ . However, the role of RA is complex. RA affects the expression of Wnt, FGF10 and BMP4 in vivo and thus has an indirect effect ⁴⁴ . Furthermore, RA appears to enhance postnatal alveolar development ⁶⁸ . Finally, corticosteroids, butyryl-cAMP and isobutylmethyl hypoxanthine (DCI) enhance lung maturation ^50,69 . This condition induced the development of a structure similar to trachiosphere from our “proximal” condition.

6). Once the conditions that determine the more proximal fate were refined, it was determined whether additional factors would affect the differentiation of these proximal regions. Notch signaling determines the fate selection among the three mature cell types of the bronchial tree by helping Clara cell fate ⁷⁰ , and controls basal cell differentiation ⁷¹ . Therefore, the inhibitory effect of Notch signaling by DAPT was interesting. We have found that DAPT helps enrich the CC10 ⁺ spb ⁺ pro-SPC ⁺ cells. This is consistent with the literature that blocking Notch at the endoderm part helps distal (as opposed to proximal) fate, while blocking Notch signaling in the mesenchyme promotes cilia cell fate To do. SHH affects neuroendocrine cell differentiation through direct effects that would not be mediated by mesenchyme ⁶⁰ . Thus, both blocking or enhancing SHH signaling can affect in vitro differentiation.

7). Factors that control branching morphogenesis can be investigated. The establishment of conditions that can model the branching morphogenesis of hPSC-derived cells represents a major advance in the field. In addition, since progenitor cells regenerate at the tip of the growing branch, stimulation of branching morphogenesis, followed by application of appropriate stimulation for alveolar differentiation, increases the production of mature cells. It can open the way. Both EGF and HGF induce branching morphogenesis by mammary epithelial progenitor cells in collagen gel cultures ^72,73 . Furthermore, TGF-β1 produced in the stalk portion of branching mammary epithelial cells inhibits branching, whereas inhibition of TGF-β promotes branching ⁷² . In some embodiments: It can be tested whether the addition of EGF, HGF, SB-431542, or combinations thereof enhances branching (if any) in extracellular matrix (ECM) embedded culture.

  Putative postnatal lung stem cell detection

Postnatal lung stem cells

Although postnatal lung progenitor cells are established as development proceeds, the characteristic properties of putative lung stem cells (which seem to function primarily after injury ⁷⁸ ) are unclear ^{11,12,75,79-87} . ⁸¹ type of stem cell that is defined by the localization at the junction of the bronchoalveolar (bronchopulmonary胞幹cells (BASC)) have been identified. This cell expresses both SP-C (ATII marker) and CCSP (Clara cell marker) ⁸¹ . However, lineage tracking suggests that alveoli are not regenerated from CCSP ⁺ or SP-C ⁺ cells ^57,84 . can result in airway and alveolar cells ^{in vitro, CD49f + (integrinβ6)} CD104 + Epcam hi CD24 evidence for pulmonary stem cells with a ^lo phenotype have been published ^86. Similarly in other papers, rare α4β6 (CD49f ⁺ ) cells were shown as lung stem cells, which were located in terminal bronchioles and alveoli ⁵⁷ . These putative postnatal lung stem cells have at least CD49f expression in common with basal cells ⁵¹ . The cells can form sac-like and cyst-like colonies in vitro. Interestingly, FGF10 was involved in these but was inhibited by BMP-4 ⁸⁶ . ⁸⁷ Without being bound by theory, p63 ⁺ cells bronchioles, migrating the mesenchyme of the lung to at least after influenza infection, repair alveoli. An NKX2.1 ⁺ FOXA2 ⁺ structure surrounded by p63 ⁺ cells was produced. However, in mouse bronchioles, most cilia cells are derived from Clara ^cells84 . In the trachea, Clara-like cells appear to function as conditionally permeable amplified cells, while most regenerative capacity is derived from basal cells (it forms 30% of the epithelium, and Ngfr in at least some cells). And can express CD49f and form so-called trachiospheres in vitro) ⁵¹ . Another type of basal cell has been identified by the CD49f ⁺ Sca1 ⁺ ALDH ⁺ phenotype, which forms so-called “bordered” colonies in vitro ⁸⁵ .

  We have identified cells in our culture that are very similar to bronchoalveolar stem cells (BASC). It is determined whether any cells produced that are similar to other postnatal lung stem cells, or embryonic stem cells, can regenerate, proliferate and differentiate. Production of such cells from patient-specific iPSCs may be key to lung regenerative medicine (eg, by seeding on decellularized lung matrix).

approach

Spherical colonies expressing post-differentiation NKX2.1, FOXA2 and p63 were obtained using DCI for 3 days. Interestingly, the application of the published “trachiosphere” condition ⁵¹ at this stage does not induce the formation of spheroids, whereas we use these conditions to Lachiospher could be produced. Furthermore, more mature cellular markers (except mucin 5a) were not yet present in these structures. These data indicate that they originate from relatively early progenitor cells and have not been cultured for a sufficient period of time to achieve full maturation. This has been confirmed simply by culturing the cells for a longer period. The potential for reseeding and differentiation of the cells is further investigated.

The colonies were isolated and reseeded to either similar conditions (DCI) or published conditions ⁵¹ for trachiosphere production from adult trachea. Colonies in 2D were p63, Muc5a, FOXJ1, acetylated tubulin (cilia cells), CCSP (Clara cells), and CRPP (neuroendocrine cells), SP-C (ATII cells), T1α, AQ5 (ATI cells) The expression of was analyzed. In some embodiments: Analysis can be performed in 3D spherical culture. Subsequent reseeding may reveal more mature marker expression. Alternatively, differentiation potential may only be acquired when further progenitor cells of these colonies develop. Differentiating cells were exposed to (proximal) WFKBE + RA or (distal) WFK conditions for extended periods on 2D cultures before seeding in DCI or trachiosphere conditions. In some embodiments, the analysis can be performed in 3D spherical culture.

  We have successfully established a published trachiosphere culture system that maintains adult mouse basal stem cells. Cultivation of the lung progenitor cells on day 16 in published trachiosphere conditions does not induce the formation of spheroids, while the lung field cells on day 16 in 3D Matrigel in the presence of the mature component DCT. Spheroids were produced by culturing with After differentiation using DCI for 3 days, the spherical colonies expressed NKX2.1, FOXA2 and p63.

After exposure of AFE to WFKBE + RA and reseeding on days 15-19, colonies containing tubular structures, Muc5a positive, surrounded by p63 ⁺ cells were obtained (FIG. 5). Sub-fractions of these cells generated spheroids after seeding in Matrigel and DCI media (FIG. 6). The marginal p63 ⁺ NKX2.1 ⁺ cells were more primitive cells in these colonies. In some embodiments: Analyzing cell populations for surface markers associated with lung stem cells (NGFR, CD49f, EPCAM, CD24, ALDH (detected using colorimetric ALDEFLUOR)) ^{51, 85, 86} which can be ^separated . This can be done either in culture prior to spheroid formation or in the spheroid itself, and can be done by flow cytometry and immunofluorescence. Without being bound by theory, colony-bound p63 ⁺ cells and cells lining the spheroids express stem cell markers (especially given the recently published data on lung stem cell identification) , Α6β4 integrin (CD49f)). Interestingly, several putative lung stem cell markers are also used to isolate human mammary stem cells. These, EPCAM (however, human mammary stem cells EPCAM ^- a is) include CD49f, CD24 and ALDH1 ^88. The developing lung and adult mammary gland have the common feature that both are epithelial tissues that undergo branching morphogenesis.

In some embodiments: Expression of other reported markers of mammary stem cells may be monitored in the models described herein. These include c-KIT, CD10, CD133 and CD90 ^89. Markers that are also present on hematopoietic stem cells (such as CD38, CD34 and endoglin) are tested. SLAM markers are also used in hematopoietic stem cells, but their expression tends to be restricted to the hematopoietic ^system89 . Cell populations expressing any of these markers are isolated by cell sorting and seeded under conditions that produce spheroids. Limit dilution analysis reveals the frequency of spheroid-inducing cells and determines whether spheroid formation depends on one cell type (single hit kinetics). Greater enrichment is achieved by combining markers of cell populations rich in colony-inducing ability.

In other embodiments, the following is performed. Application of published conditions for postnatal putative lung stem cell growth to cells produced from hPSCs in the system described herein produces cells that are consistent with postnatal lung stem cells To investigate. This is done by sorting cells that ultimately express a putative stem cell marker and then reseeding to the reported conditions for trachiosphere and lung stem cells ^51,85,86,90 .

  Mechanisms involved in lung field lineage determination

  Rationale. In other embodiments, the following is performed. By leveraging the developed high efficiency and standardized differentiation protocols, this model can be used to understand the mechanisms involved in human early lung development. This approach can change conditions to obtain selective cell fate, thus providing a good basis for comparison in microarray and ChIP studies, and the ability of cells determined to a particular lineage The advantage is that the number is not theoretically limited. This is in stark contrast to the mouse genetic approach and mouse embryo dissection. Both types of approaches are highly complementary.

NKX2.5 ⁺ Role of mesoderm

After AFE production, more than 90% of the cells were FOXA2 ⁺ SOX2 ⁺ , but after ventralization in the presence of WFKBE + RA and determination of lung primordia lineage, a variable percentage (5 to 40%) of cells were NKX2 .5 ⁺ EPCAM ^- VSMA ⁺ (FIG. 3). A genealogical follow-up study showed that the mesenchyme surrounding the ventral AFE was also NKX2.5 ^{+ 91} . Thus, these data indicate that the contaminating mesoderm in the culture is equivalent to the mesoderm surrounding the ventral AFE. This raises the question of whether this mesoderm is advantageous for lung lineage determination in vitro. After reseeding, virtually no mesoderm cells are present, but the cells are capable of differentiation (although minimal contamination of mesoderm cells may still play a role) The germ layer may be unnecessary for further differentiation.

In some embodiments: It may be possible to establish a role for the mesoderm. Cells are sorted after induction of AFE and after ventralization of AFE into EPCAM ⁺ (endoderm) and EPCAM ⁻ (mesoderm) populations. These cells are then seeded separately or together at various ratios during subsequent differentiation. Quantification and analysis proceeds as previously described in this example.

If the presence of mesoderm is found to affect lung differentiation, then gene expression analysis is performed on this mesoderm to detect soluble factors that may be involved. This is determined by analyzing cells “present” in the Affymetrix microarray and comparing expression to EPCAM ⁺ AFE cells. Without being bound by theory, ligand expression (membrane-bound or soluble) is increased in the mesoderm fraction, while putative receptor expression is high or at least present in the AFE fraction.

Genome-wide expression analysis during lung lineage determination

  The goal of these experiments is to genome-wide chart the differentiation pathway from ES cells to cells destined to the distal or proximal lung. This is important because no such data is currently available.

In some embodiments: CDNA is prepared at the following stages of culture: ES cells, definitive endoderm, NS, or AFE produced using any combination of N, S and I optimal for lung lineage determination, WFKBE (pharynx ) Or after ventralization in the presence of WFKBE + RA (lung field). In the WFKBE + RA group, cDNA is prepared from cells cultured in the presence of continued WFKBE + RA (putatively proximal) and WFK (putatively distal). In all populations (except ES cells), to compare only the endoderm cells and to ensure that the data is not biased by a fluctuating mix of mesoderm cells, cell-sorting EPCAM ⁺ cells Isolate. Furthermore, at each stage, the culture undergoes extensive quality control (consisting of IF and qPCR for the appropriate markers at each stage of differentiation).

  In other embodiments, the following is performed. After general standardization, the data is subjected to clustering without objective variables. Without being bound by theory, successive stages of differentiation are continuously clustered. These studies can yield several results.

  In certain embodiments, the following is performed. Surface markers may be identified that allow for the probable isolation of subpopulations that occur during differentiation. In addition to its obvious scientific importance, this will be a major advance in developing protocols for directed differentiation.

  In some embodiments: Data may indicate specific signaling pathways that are activated or inhibited during successive stages of differentiation. Morphogen is added to the culture, but nothing is known about the intrinsic signaling activity.

  In some embodiments: The data may reveal novel stage-specific markers and transcription factors that are useful for endoderm and lung development studies. Such markers are tested by in situ hybridization in developmental stage mouse embryos.

  In certain embodiments, the following is performed. Of primary interest are genes that are differentially expressed after induction of AFE by use of NS or a combination of N, S and I. This is the first step in understanding how and how pre-sequence decisions occur at this stage of development.

Pre-sequencing: staying balanced with essential lung-derived genes? Or don't keep it?

Without being bound by theory, AFE lineage determination through the use of either NS or a combination of N, S, and I (N / S / I) has shown that liver and pancreas during early definitive endoderm development. similar to the observations recently published for the previous patterned to impart different histone modifications or CpG methylation in genes important in the development of the pharynx and HaiHara group ^92.

This study could be approached in two ways. The first approach would be to compare NS to N / S / I and perform a Chip-Seq on the AFEs derived from them. Costs due which will be able to make only a series of histone modifications very limited ^93. Thus, a hypothesis-based approach can give more information. Accordingly, in some embodiments: After normal endoderm induction in the presence of NS or N / S / I (day 7 of the protocol in FIG. 9), normal histone modifications at the promoter regions of the two genes (PAX9 and NKX2.1) ChIP and subsequent PCR, and CpG methylation can be examined by bisulfite sequencing. NKX2.1 is used for lung establishment ^1,2,20 , while ^PAX9 (although not essential for pharyngeal endoderm production) is expressed in the pharyngeal endoderm ³² and is high after induction of AFE using NS Express ¹⁰ . Furthermore, PAX9 is immediately downstream of human chromosome 14 NKX2.1, the only gene in between is NKX2.1 homolog NKX2.8 (recently shown to play a role in lung cancer) ⁹³ . In other embodiments, the following is performed. In some embodiments, highly differentially regulated regulatory sites may be found within this narrow region. Histone modification being investigated is, H3K4me3, H4K4me2, and H3K27ac (activated promoter), a H3K9me3, H3K27me3 and H4K20me3 (inhibition of promoter) ^{95, 96.} NKX2.1 While detailed analysis of the promoter have been published some ^97, in some embodiments, PCR primers (up to 100-200Bp), to focus on the promoter region having a conserved transcription binding sites Is possible (dcode.org). For NKX2.1, these are between the two selective transcription start sites and (interestingly) in the 3 ′ UTR of the 5 ′ gene (SFTA3) important for surfactant production. Both sites contain several binding sites for NKX2.1 itself.

  In other embodiments, the following is performed. NKX2.1 may be transcribed in the antisense orientation, with a dense cluster of conserved transcriptional binding sites present at the 5 'end of the gene (which is also included). Similarly, in other embodiments, the region in PAX9 that contains a conserved transcription factor binding site is evaluated. As “positive” controls, cells differentiated mainly into pharyngeal endoderm (AFE induction using NS, ventralization in the presence of WFKBE), and lung field (AFE induction using N / S / I and WFKBE + RA presence) Use cells that have differentiated into the lower ventral).

In yet another embodiment, the following is performed. Differential histone modifications and / or CpG methylation depending on whether AFE was induced in NS of N / S / I are identified in the control region of PAX9 and NKX2.1. If this is not the case, the experiment is repeated using another series of genes derived from the microarray data collected as described herein. For example, during pharyngeal pre-sequencing, the putative promoter of the antisense NKX2.1 transcript may assume an “open” conformation (a finding that would be of great interest). For example, if extensive H3K4me3 is found, proteins with the SET domain (Trisolax group, histone methylase) and Jumonji family protein (histone demethylase) gather ⁹⁶ .

Example 2 : Induction of lung airway epithelial cell differentiation from hPSC

This example describes a strategy for producing, from iPS cells, lung endoderm compartments (ie, tracheal, bronchial and alveolar epithelial cells) and putative postnatal stem cells associated with these structures. In addition to the potential of this approach to regenerative medicine, the ability to produce a diverse array of cell types in the respiratory system is the result of drug testing for developmental lung abnormalities and drug teratogenicity for lung development. Allows testing of effects. Furthermore, this study provides useful insights into the characteristic properties and functions of postnatal tracheobronchial and pulmonary stem cells and provides an in vitro model to investigate human development in detail ¹⁰⁷ .

We have shown that highly enriched AFE can be produced, which removes a major obstacle in the field of anterior endoderm differentiation ¹¹⁵ . In addition, we were able to lineage AFE into cells corresponding to the lung field of ventral AFE, and we were able to identify conditions under which cells expressing alveolar markers were obtained through moderate throughput screening. ¹¹⁵ .

Strategies for directed differentiation of ES cells

IPS cells were used as discussed herein. hES cells were also used as a control. Some iPS strains fall within the normal variability range of ES strains for differentiation potential ¹²⁰ . Currently used iPS cells are produced by transduction with a lentivirus of a cassette containing excisable SOX2, OCT4, KLF4 and MYC ¹²¹ . The recently described PiggyBac system ¹²² works with a mouse. Among the new technologies for producing iPS cells, repeated mRNA transfection and hiPS produced by non-integrated Sendai virus are currently used. This approach yields safe iPS cells ¹²² because no exogenous genetic material should be incorporated.

10 ⁶ cells were transplanted under the kidney capsule of NOD / SCIDI12rg − / − (NSG) mice. Undifferentiated HES2 cells produced teratomas containing cells from all three germ layers (FIG. 1a), whereas NS-treated cells lacked identifiable ectoderm or mesodermal component growth. Produced (FIG. 1b). Numerous luminal structures were observed, lined by either pseudostratified epithelium (typical of upper airway epithelium) or less organized epithelium containing 1 to 3 layers of nuclei (FIG. 1b). The latter was consistently stained with Surfactant Protein-C (SP-C), a marker ^98,99 specific for lung type II alveolar cells (FIG. 1c). The remaining cells were almost uniformly stained with FOXA2. However, except for the luminal structure where FOXA2 was expressed in the nucleus and cytoplasm, FOXA2 was localized in the cytoplasm (FIG. 1c). Islands of cells expressing PAX9 (pharyngeal ^{sac) 31,} and a region showing a ³⁴ expression (specific medullary thymic epithelial ^{cells) (parathyroid) 33} or AIRE separate nuclear speckles of GCMB was also detected (FIG. 1c; FIG. 2). Like FOXA2, GCMB staining is predominantly cytoplasmic, indicating that the parathyroid tissue will not be functional, although very specific for the parathyroid gland. These findings indicate that the potential of definitive endoderm induced by NS is fairly limited to those derived from AFE.

Ventralization of AFE in vitro. AFE undergoes dorsal ventral patterning and responds to WNT, BMP, and FGF signals from ventral mesoderm, resulting in ventral lineage determination of lung primordia and trachea ^{1,2,35-37,98,99 143-146} . This stage produces a dorsal-ventral SOX2 gradient, while ventral-specific markers NKX2.1 (lung and thyroid fields 1, 2, ³⁸ , ⁹⁸ , ⁹⁹ , ¹⁴⁷ ), NKX2.5 (ventral pharynx) ^39,148 ) transiently expressed in the endoderm, and PAX1 ( ^40,149 specifically expressed in the pharyngeal sac within the endoderm) were induced. Treatment with NS extended to day 13 resulted in continued expression of SOX2, suggesting dorsal fate (FIG. 9a), while BMP4 is expressed ventral, whereas Noggin is AFE. ^1,37,98,146 consistent with the fact that it is expressed on the dorsal side of In contrast, replacing NS with WNT3a, KGF, FGF10, BMP4, and EGF (WKFBE) on day 7 of culture resulted in low expression of SOX2, and on day 13 ventral markers NKX2.1, PAX1 and NKX2.5 was induced (Figure 9a for HES cells, Figure 9b for HDF2 and HDF9 hiPS cells). Furthermore, p63 (airway progenitor cell marker ⁹⁸ ) was strongly induced (FIG. 9a). The addition of individual factors was not sufficient for this transcription induction. Expression of the early thyroid markers (PAX8 ⁵³⁾ is not observed, this is, Nkx2.1 induction, rather than the thyroid, is rather suggested that shows the fate of the lungs. Immunofluorescence revealed NKX2.1 expression in 37 + 6% cells (sometimes occurring in densely populated cell colonies) (FIG. 9c).

  The timing of WFKBE ventralization stimulation is significant because it had the ability to express NKX2.1, PAX1, and NKX2.5 only in cultures that were treated on day 7 instead of day 9. > 90% of the cells were SOX2 + FOXA2 +. Next, it was tested whether NS exposure was advantageous to achieve ventral AFE fate in response to WFKBE. Cultures treated with NS on day 7 were compared to cultures on day 7 in conditions that aid in medium alone or liver (posterior foregut) fate. Only exposure to NS allowed subsequent up-regulation of PAX1, NKX2.1 and NKX2.5 by WFKBE (FIG. 10), indicating that NS treatment of definitive endoderm induced with Activin A is a ventral AFE. It is shown to be involved in the differentiation into.

Potential of ES / iPS-derived AFE as lung in vitro. NS-induced exposure of AFE to WFKBE did not result in expression of lung terminal differentiation markers on day 13 or 19 of culture. Lineage tracing of the reporter gene shows that the lung field, rather than the pharyngeal sac, experiences retinoic acid (RA) signaling ^{42-44, 151,152} . Furthermore, RA is involved during lung primordium formation ^45,153 . Indeed, the addition of RA to the WFKBE cocktail decreased the expression of the anterior sac marker PAX1, but increased FOXP2, NKX2.1, GATA6, and the cilia cell marker FOXJ1 (depletion of lung field fate and pharyngeal sac fate) 1), 1, 2, ^{98, 99} (FIG. 11a). No expression of SP-C was observed. However, continued treatment with WFKBE + RA up to day 19 produced low levels of SP-C (FIG. 11b).

In order to optimize SP-C expression, ventral AFE was produced in the presence of WKFBE and RA. On day 11, in a 48-well plate, 400 binary combinations of selected factors (FGF10 / 7, Wnt5a, Wnt3a, BMP4, Noggin, Sonic Hedgehog (SHH), SB-431542, TGF-β1, Notch Cells were seeded in the inhibitors DAPT, RA inhibitor DEAB, FGF inhibitor SU-, SHH inhibitor cyclopamine, and RA). On day 19, wells were pooled to consist of 4 wells per group for SPC qPCR. SPC was induced in 6 groups of wells. Next, the same conditions as those included in the pool of positive wells were set up, and qPCR was performed on cells cultured under individual conditions. Among these combinations, Wnt3a + FGF10 / 7 only has consistently induce high levels of SP-C (Fig. ^{11b) (Wnt 154,156} and ^{FGF10 147,157,158} signaling, important distal lung generation This is consistent with the idea that

  Under these conditions, large cystic and sac-like structures were formed in the culture. Staining on day 13 of culture revealed that most of these were NKX2.1 (FIG. 11c). However, no expression of SP-C at the protein level was detected, indicating that the maturation of SP-C mRNA expressing cells is still sufficiently advanced to the extent that SP-C protein expression is detectable. Suggests not.

Establishment of conditions for determination of AFE lung field series

Factor that induces presumed lung fields from AFE. Treatment of AFE with WFKBE and RA resulted in preferential induction of AFE posterior aspect markers (including GATA6 and NKX2.1) producing trachea and lung primordia. These conditions were refined to achieve more efficient induction with up to 80% lung cell efficiency compared to the original protocol (only 40% cells expressed NKX2.1). . More efficient guidance of the AFE lung field at the expense of more anterior regions allows more efficient subsequent guidance of the lung and bronchial tree.

The individual factors of the WFKBE combination had no effect on the induction of NKX2.1, but it was not determined which combination, which concentration was optimal, and which factors were essential. For example, GATA6 expression is strongly dependent on ATRA dose (FIG. 12). Within AFE, GATA6 is a marker for the most posterior region where lung primordia occur ^98,99,116 . Although SHH is used for foregut development and branching morphogenesis via signaling to the surrounding mesenchyme, ^{58,59,159-161} , in later developments, SHH signaling is at least some epithelial (neural Endocrine) cells are also targeted ^60,162 . SHH also plays a role in the formation of front and back patterns in AFE ^61,162 . Deletion of Shh in mice reduced the expression of the pharyngeal sac marker Pax1 ^61,163 . Further Shh - / - in mice, it has dictated to form the parathyroid, the later portion of the third pharyngeal pouch expands at the expense of the front portion that is destined to become a thymus ¹⁶⁴ . Thus, inhibition of SHH using cyclopamine can help the later fate of lung lineage within the AFE. Any effect of cyclopamine will facilitate the study of the role of contaminating FOXF1 mesenchyme in culture ¹¹⁶ .

After induction of AFE by using NS for 2 days, the effects of several combinations of BMP-4, Wnt3a, KGF, FGF10 (binding to FGFR2b), EGF, and multiple concentrations of ATRA were tested. The effect of FGF9 (which binds to FGFR2c and also affects branching morphogenesis) ^98,99 , and in some embodiments, cyclopamine is tested. In the first stage, wells are pooled for GATA6 and NKX2.1 qPCR. At two time points, the following are analyzed: In vitro, analysis is performed 3 and 5 days after the start of the culture stage, since the initial stage of development proceeds fairly quickly. In addition, some manipulations (such as blocking SHH signaling) may be useful in establishing lung fields in vitro at the expense of the pharyngeal sac region, but since SHH is involved in lung development, ^{98, 99, 116, 158-161} may be detrimental to the initial development of and lung field enlargement.

  In certain embodiments, the following is performed. The group with the highest conditions for both GATA6 and NKX2.1 is then set up and analyzed separately for expression of GATA6 and NKX2.1 to identify and confirm hits.

In other embodiments, the following is performed. Analyze hits further. If possible, qPCR data is confirmed with a fluorescence microscope (eg using NKX2.1 antibody). Importantly, a corresponding culture of “liver” condition ¹¹⁰ is used as one of the controls for staining of AFE and lung specific markers. Thyroid (PAX8, TG (thyroglobulin), TSHR (TSH receptor)), pharyngeal sac (PAX1, PAX9, TBX1, SIX, EYA and HOXA3), pharyngeal sac derivatives (FOXN1 (thymus), GCMB (parathyroid), Calcitonin Follicular C cells)), and of course, markers for lung and trachea (p63, NKX2.1, GATA6, FOXP2, SP-C, CCSP, FOXJ1, MUC5a) are tested. In particular, with respect to other endoderm derivatives such as neuroectodermal (TUBIII, TUJ1 and PAX6), mesoderm (FOXF1), stomach (ODD1), liver (CEBPA, EVX1, FOXA3) and pancreas (PDX1), etc. Confirm the loss and contamination of the genealogy.

  Finally, the ability to differentiate into more mature cells is evaluated.

Cell proliferation

  It is highly noteworthy that the differentiation of human pluripotent stem cells in vitro proceeds very quickly compared to the rate of human development in vivo. One possible reason is that in vivo, more developmental time is spent on cell growth, but in vitro favors more rapid differentiation than “lateral” growth at each developmental stage. The conditions for working are selected empirically.

G-CSF increases the production of differentiated cultures of cardiomyocytes ^74,164 . In some embodiments: When the lung field induction conditions are optimized, the effect of G-CSF is examined. A useful hormone system for growth during development is the insulin-like growth factor (IGF) ^-growth hormone (GH) axis ^75,164 . In other embodiments, it can be explored whether the addition of IGF1, GH, or both increases the production of cells. This approach is supported by the observation that lungs are significantly hypoplastic in mutants lacking Igf signaling ^75,165 .

The effect of timing

The possibility of expressing ventral AFE markers in the presence of WFKBE exists only in a narrow time frame after NS induction ¹⁰ . Thus, the length of exposure to WKFBE or any optimized derivative of this cocktail may determine the efficiency of subsequent lung or bronchial differentiation induction. In some embodiments, after subsequent culture in either FGF7 / 10 + Wnt3a (SP-C) or FGF7 / 10 + Noggin (tubular structure), the possibility that the ventralized AFE expresses SP-C or the tubular structure. Read the possibilities to form.

Role of extracellular matrix

In other embodiments, the following is performed. These conditions are further optimized by culturing in the presence of extracellular matrix (ECM) ⁶¹ . ^141,167,168 to investigate fibronectin, cultures embedded in Matrigel, and collagen I cultures. It can be determined whether the induction of SP-C can be efficiently reproduced in the presence of FGF7, FGF10 and Wnt3a, as well as in the presence of ECM. Collagen allows branching morphogenesis from mammary epithelial cells ⁷² . Fibronectin plays a role in branch morphogenesis in in vivo, is a target for Wnt signaling ^77. It is also commonly used to embed with Matrigel containing multiple ECM components, as used to obtain DCI-inducing spheres from “proximal” conditions. If good results are obtained with ECM embedded culture, then in some embodiments, all subsequent experiments can be performed in the presence of ECM. In addition, the occurrence of three-dimensional structures (possibly representing branching morphogenesis) is evaluated.

  Optimization of lineage determination of tracheobronchial and alveolar epithelial cells from ventralized AFE

Further optimization of the conditions for obtaining mature respiratory cells is desirable. The reading of these experiments is the expression of lung markers at different stages of development (reviewed in references 98, 99, and 116). FOXA2, NKX2.1 and GATA6 serve as markers for all lung regions and are precursors of the development of respiratory epithelium. In mice, SOX2, p63 and Nkx2.5 (we have already identified after ventralization of AFE) are early tracheal markers, while Id2, Sox9, Nmyc and Irx are distal tip markers. The latter is down-regulated when generation is complete. Thus, the transient expression pattern of human orthologs of these markers provides information regarding the stage of differentiation. Since differentiation proceeds in the perspective direction, the Clara cell marker CCSP, the cilia cell marker FOXJ1, the mucus cell marker MUC5a, and the neuroendocrine marker calcitonin gene-related peptide product (CGRP) appear in the handle. ATII markers SFTPC (SP-C), SFTPA (SP-A) and SFTPB (SP-B), and ATI markers Aq5 and T1α indicate terminal alveolar differentiation. In humans, the pseudostratified columnar epithelium reaches the distal much more ^{98, 99} may find markers associated with the trachea. qPCR and (if possible) fluorescence and confocal microscopy are used. In some embodiments: To determine the cell's potential in vivo, the cells are transplanted under the kidney capsule.

Extracellular matrix

In other embodiments, the following is performed. The establishment of the correct tissue structure uses ECM support ¹⁶⁸ . In some embodiments, it is determined whether SP-C induction can be efficiently reproduced in the presence of FGF7, FGF10 and Wnt3a in the presence of ECM. Fibronectin, collagen I gel, and matrigel are used. Collagen allows branching morphogenesis from mammary epithelial cells ¹⁶⁶ . Fibronectin plays a role in branching morphogenesis in vivo and is a target for Wnt signaling ¹⁶⁸ . Embedding in matrigel containing multiple ECM components is also commonly used ¹⁴¹ . If good results are obtained in ECM embedded culture, then in some embodiments, all subsequent experiments are performed in the presence of ECM. Furthermore, in other embodiments, it is determined whether the occurrence of a three-dimensional structure representing the branching morphogenesis is observed.

Optimal factor concentration

During branching morphogenesis, pluripotent progenitor cells regenerate at the tip ^{98,99,108,170} . Cells left in the stalk differentiate into tracheobronchial epithelium. When the tubular / saccular phase begins, the distal cells begin to differentiate into alveolar cells ^98,99 . Application of Wnt3a, FGF7 and FGF10 after ventralization of AFE was shown to induce large amounts of SP-C mRNA but not protein. Although these conditions were identified in unbiased selection, FGF10 and Wnt signaling establish a distal fate and thus make developmental sense ^154-158 .

In some embodiments: Determines whether all three factors are involved in these cultures and whether their concentrations can be optimized (since many morphogens show concentration-dependent effects) ^98,171,172 .

In some embodiments: To what extent in vitro perspective lung differentiation can be affected. In addition to providing a method for manipulating the characteristic properties of mature cells that can ultimately be produced in vitro for cell replacement therapy, the observations made in the lung development of mice have been observed in our in vitro human Reproduction in the system will prove the validity of this system. BMP-4 is expressed in the developing lung disc, controls FGF signaling, and promotes more distant fate ^98,173-175 . Thus, a more proximal fate characterized by tube formation and budding in the presence of ECM and in the absence of SP-C by removing Wnt3a and blocking endogenous BMP-4 by adding Noggin A series can be determined. This should become clear by further analysis of the expression of proximal and distal airway markers. Furthermore, inhibition of endogenous Wnt using sFRP3 or DKK may further aid proximal fate. Non-standard Wnt (Wnt5a) also plays a role in lung development, possibly through control of SHH and FGF10 signaling ^176-177 . Blocking Notch in early developmental signaling also promotes more distal fate ¹⁷⁷ . This can be achieved using the gamma-secretase inhibitor DAPT.

  Conditions that induce more proximal fate have been identified, but at a later time, the effect of switching the culture from conditions that induce proximal fate to conditions that follow SP-C induction was investigated. Conditions that favored pro-SPC induction (addition of DAPT to medium containing WFK) were at least identified. This would mimic the developmental sequence of branching morphogenesis followed by alveolar differentiation.

  In some embodiments: Once the optimal conditions for SP-C guidance are identified, the optimal timing is determined. Not only mRNA expression but also SP-C protein expression may simply involve long-term culture. A series of determinations of markers for differentiated respiratory cells also reveal the degree of terminal differentiation that has been achieved in these cultures.

In other embodiments, the following is performed. We investigate whether terminal maturation into SP-C expressing cells is a different condition that has not yet been explored. There are several signaling pathways investigated in this context: glucocorticoids, hedgehog and RA. SHH expression stops ¹⁶² during terminal alveolar differentiation. Thus, although SHH is believed to primarily affect the surrounding lung mesenchyme, ^159,160 , inhibition of SHH may promote terminal alveolar differentiation. Similarly, downregulation of RA signaling is desired for terminal differentiation of AT cells ¹⁷⁹ . However, the role of RA is complex. RA affects the expression of Wnt, FGF10 and BMP4 in vivo and thus has an indirect effect ¹⁵³ . Furthermore, RA appears to enhance postnatal alveolar development ¹⁸⁰ . Therefore, blocking SHH signaling by cyclopamine and blocking RA signaling by BMS493 may be advantageous to achieve terminal alveolar differentiation. Finally, corticosteroids promote and investigate lung maturation through effects on mesenchyme ⁹⁹ .

In some embodiments: If conditions can be defined that determine more proximal fate, then it is determined whether additional factors affect the differentiation of these proximal regions. Notch signaling determines fate selection among the three mature cell types of the bronchial tree by helping Clara cell fate ¹⁸¹ . Therefore, the inhibitory effect of Notch signaling by DAPT was investigated. SHH affects neuroendocrine cell differentiation through direct effects, probably not mediated by mesenchyme ¹⁶¹ . Thus, in some embodiments, either blocking or enhancing SHH signaling can affect in vitro differentiation.

In some embodiments: Investigate factors controlling branching morphogenesis. Establishing conditions that can model the branching morphogenesis of iPS-derived cells is a major advance in the field. In addition, since progenitor cells regenerate at the tips of growing branches, application of stimulating branching morphogenesis followed by appropriate stimulation for alveolar differentiation will provide a path to increased production of mature cells. Can be opened. Both EGF and HGF induce branching morphogenesis by mammary epithelial progenitor cells in collagen gel cultures ^167,168 . Furthermore, TGF-β1 produced in the stem of branching mammary epithelial cells inhibits branching, whereas inhibition of TGF-β promotes branching ¹⁶⁷ . Test whether the addition of EGF, HGH, SB-431542 or combinations thereof enhances branching (if any) in ECM-embedded cultures.

Functional analysis of tracheobronchial and alveolar cell populations produced in vitro

Detection of potential postnatal lung stem cells

The postnatal lung has considerable regenerative capacity after injury. However, the characteristic properties of putative lung stem cells ¹⁸² that are thought to function primarily after injury are unclear ^{100,107,183,184} . A type of stem cell (bronchoalveolar stem cell (BASC)), defined by its localization at the bronchoalveolar junction, has been identified ¹⁸⁵ . This cell expresses both SP-C (ATII marker) and CCSP (Clara cell marker) ¹⁸⁵ . Several recent reports suggest that BASCs may be cells of bronchoalveolar carcinoma origin, but they may not function as alveolar stem cells. Initial reports claimed that BASC could be promisingly isolated by the CD45-CD31-Sca1 + CD34 + phenotype, but more recent observations questioned that putative BASC is positive for CD34 and Sca1 ^{186, 187} . The strongest argument against SP-C + CCSP + cells as lung stem cells is from careful lineage tracing experiments showing that only very rare cells (if any) in the alveoli are derived from CCSP-expressing progenitor cells. come. However, in mouse bronchioles, most cilia cells are derived from Clara cells ¹⁸⁸ . In the trachea, Clara-like cells appear to function as conditionally permeable amplified cells, while most regenerative capacity is derived from basal cells (it accounts for 30% of the epithelium, at least some of which are Ngfr) And expresses CD49f and can form so-called trachiospheres in vitro) ¹⁸⁹ . Another type of basal cell, identified by the CD49f + Sca1 + ALDH + phenotype and forming so-called “marginal” colonies in vitro, ¹⁹⁰ was identified. In the alveoli, ATII cells or a subset thereof can differentiate into ATI cells ^100,107,183 . In humans, pseudostratified epithelium reaches the bronchiole level ⁹⁸ . Thus, in humans, there can be only two types of stem cells, tracheobronchial and alveolar, and the corresponding postnatal stem cell niche.

Since the cells at the tip of the budding branch have been shown to give rise to all epithelial cells of the respiratory system, this bisection method may not exist during development. Only in late development these cells are destined to the alveoli ¹⁷⁰ . However, most available data suggest that as development progresses, different progenitor cells are defined ^{100,107,183-187} . Nonetheless, evidence for pulmonary stem cells with the CD49f + CD104 + EpcamhiCD24lo phenotype that can generate airway and alveolar cells in vitro has been published ¹⁹¹ . ^{189 and 190} The postnatal lung stem cells putative (its localization is unknown) is thus having in common with basal cells the expression of at least CD49f. They can form sac-like and cyst-like colonies in vitro. Interestingly, these accompanied FGF10 but were inhibited by BMP-4. The production of these colonies is also accompanied by HGF (a factor proposed to be tested in the above model) ¹⁹¹ .

In the model described herein, two approaches are used to detect putative lung stem cells. In the first approach, the cultures were replated after formation of NKX2.1 + sac colonies (FIG. 11c). Regeneration of NKX2.1 + or SP-C + colonies (see FIG. 11c) is observed (observing NKX2.1 after day 25 and pro-SPC after day 45). Since reseeding ability was observed, in some embodiments, the following is performed. A limiting dilution method is performed to determine the frequency of cells eliciting these structures. A series of replating then gives an indication of the regenerative capacity of these cells. In the next step, these putative stem cells are analyzed for cell populations for surface markers associated with lung stem cells (Ngfr, CD49f, Epcam, CD24, ALDH (detected using colorimetric staining ALDEFLUOR)). ^189-191 with probable isolation by analysis. Interestingly, several putative lung stem cell markers are also used to isolate human mammary stem cells. These include (but human mammary stem cells are EpCAM-) Epcam, include CD49f, CD24 and ALDH1 ^192. The developing lung and adult mammary gland have the common feature that they are both epithelial tissues that undergo branching morphogenesis.

Accordingly, in some embodiments: Monitor the expression of other markers reported for mammary stem cells. As these include c-KIT, CD10, CD133 and CD90 ^192. Test markers present in hematopoietic stem cells, such as CD38, CD34 and endoglin. SLAM markers are also used in hematopoietic stem cells, but their expression tends to be restricted to the hematopoietic system ¹⁹³ . Cell populations expressing any of these markers are isolated by cell sorting and seeded under conditions optimal for production of NKX2.1 and SP-C expressing colonies. Higher enrichment is achieved by combining markers of cell populations rich in colony-inducing ability. Purified putative stem cells are analyzed for expression of lung / tracheal markers such as p63, SOX2, GATA6, FOXA2 and NKX2.1, FOXJ1, SFTPC (SP-C), MUC5A, CGRP, CCSP.

In some embodiments: By applying known conditions for probable lung stem cell growth after birth to cells produced from pluripotent cells in the system described herein, cells consistent with postnatal lung stem cells are produced. Investigate. This re-seeding is finally performed in the conditions reported for trachiospheres and lung stem cells after sorting cells expressing putative stem cell markers ^189-191,194 . As a preliminary experiment demonstrating the feasibility of such an approach, the protocol of Rock et al. Was used to successfully produce trachiosphere from the trachea of adult mice (FIG. 13) ¹⁸⁹ .

Xenotransplantation under the kidney capsule

After transplantation of NS-induced AFE under the kidney capsule of immunodeficient mice, multiple SP-C + tubular structures and structures with pseudostratified epithelium were observed (FIG. 1c). These data show that AFE produced in vitro has significant lung potential in vivo, and conversely, this assay reveals the lung potential of transplanted cells relatively efficiently It shows that. This finding is also consistent with the observation that transplantation of embryonic lungs of E11.5 mice under the kidney capsule allowed differentiation through the pseudoglandular and tubular stages of alveolar development ¹⁹⁵ . During this process, the grafted blood vessel joined with the host blood vessel extending in ¹⁹⁵ .

In some embodiments: It is determined whether the in vivo potential of cells produced according to the methods described herein is narrower or limited by lung production. The time points to be tested are AFE after ventralization using WKFBE + RA or any improved protocol identified, and successive stages of differentiation. 10 ⁶ cells were dissolved in Matrigel (4 μl) at 4 ° C., allowed to clot at 37 ° C., and transplanted under the kidney capsule of immunodeficient NSG mice. Groups of 3-5 mice are analyzed for each condition and differentiation stage at 4 and 8 weeks after transplantation, respectively. The analysis consists of staining of airway epithelial cells (FOXJ1, CCSP, CGRP, MUC5a, p63) and alveolar cells (SP-C, T1a, AQ5). In addition, other fate is investigated by staining for GCMB (parathyroid) PAX9 and TBX1 (pharyngeal sac), Calcitonin (C cells), AIRE (thymus).

A cell population rich in progenitor / stem cell activity in vitro has been identified. In some embodiments: Those after suspension in either collagen gel or Matrigel are also transplanted. This approach is justified by the fact that human mammary stem cells can be quantified using this approach ^196,197 . Furthermore, if in vivo stem cell activity is observed, it is possible to evaluate the self-renewal ability by collecting the growth and transplanting it to a secondary recipient.

Lung injury model

The lung injury model ¹⁹⁷ is typically used to assess regeneration from endogenous progenitor cells ^188-190,199 . There are few treatment examples of lung injury using lung cells. Intratracheal administration of purified ATII cells was able to prevent bleomycin-induced inflammation and subsequent fibrosis ²⁰⁰ . Consistent with these findings, hES-derived ATII cells (obtained using selection) improved survival from bleomycin toxicity ²⁰¹ . However, in both cases, there is a lack of reliable engraftment of transplanted cells in the alveoli, suggesting an indirect effect. In lung injury models, attempts to test the potential of any mature cell population obtained with optimized protocols are useful. If obtained with sufficiently high purity, the cells of interest are ATII cells, Clara cells, and any cell type with pulmonary stem cell activity.

In some embodiments: In principle, injuries to the lungs of NSG mice by inhalation of bleomycin (injury of alveoli) or ip administration of naphthalene (injury of tracheobronchial Clara cells), the cells were intubated in the oral trachea, ²⁰² , ²⁰³ , 3, Inject on days 7 or 14. Survival of mice, weight loss and subsequent increase, lung mass, and a series of histological analyses.

Without being bound by theory, the best functional assay would be ^{recellularization of} decellularized lung matrix ^204-206 . This approach involves not only respiratory epithelium, which includes all mature and stem cell compartments, but possibly also mesenchymal cells as well as blood vessels.

Example 3 Optimization of NKX2.1 + FOXA2 + cell enrichment

In the above culture protocol (FIGS. 2 and 3), the enrichment of lung field cells (NKX2.1 + FOXA2 + Pax8−Pax6-) did not exceed 40%. Therefore, a strategy was developed to achieve better enrichment of NKX2.1 ⁺ FOXA2 ⁺ cells. During mouse embryonic development, endoderm cells that initially migrate away from nodal / activin signaling become AFE. Therefore, AFE was exposed to Activin A signaling for the shortest time compared to endoderm cells in the midgut and hindgut. Early and late endoderm (days 4, 4.5 and 5) were tested for their ability to determine lung cell lineage. Although endoderm production on days 4, 4.5 and 5 was all> 90% (FIG. 14, left panel), the enrichment of lung field cells from these endoderm cells was very different from each other. It was.

Early endoderm produced NKX2.1 + cells with 40-60% higher efficiency than late endoderm (contrast with FIG. 14 (A) and FIGS. 14 (B) and (C)). Day 5 endoderm produced only -10% NKX2.1 + cells (FIG. 14C). These results indicated that the timing of separation of embryoid bodies exposed to Activin A was critical. When DE was induced for longer than 5 days, the potential to produce NKX2.1 ⁺ FOXA2 ⁺ cells was completely lost. Second, during AFE formation in mice, cells destined to become AFE pass through the area where the nodal / activin inhibitor Lefty and the BMP4 inhibitor Noggin are expressed, which is probably why ActivinA It explains how blocking TGF-β and BMP signaling after a period of exposure to can be used to determine this part of the endoderm. Thereafter, the most fateful cells are exposed to the Wnt inhibitor Dkk1.

The hypothesis that inhibition of Wnt signaling at a specific time could lead to a more detailed lineage determination of the lung field was tested. ActivinA-induced DE was exposed to NS from day 4 to day 5 and then from day 5 to day 6 to SB and the Wnt inhibitor IWP-2 (SI). Cells were then cultured in the presence of ventralization cocktail, Wnt3a, BMP-4, FGF10, FGF7 and EGF, and RA (WKFBE + RA) until day 15 as described above. Compared to exposure to NS alone, NS followed by SI increased the fraction of NKX2.1 ⁺ FOXA2 ⁺ cells and NKX2.1 mRNA on day 15 (FIG. 8). Reversing the NS / SI order or using only SI was detrimental to the expression of NKX2.1. Furthermore, it was determined that the dose of RA during ventralization was critical and 50 μM was optimal (FIG. 19).

Furthermore, the importance and contribution of FGF7 and EGF to lung cell lineage determination was investigated. The results showed that the amount of NKX2.1 ⁺ FOXA2 ⁺ cells produced did not decrease by removing each factor or both factors from the culture medium (data not shown). These manipulations (along with the optimal dose of RA) resulted in a culture in which the majority (80%) of the cells were NKX2.1 ⁺ FOXA2 ⁺ . Importantly, the optimal timing of separation of DE and the optimal dose of RA may vary depending on the specific strain of hPSC, and therefore it is advantageous to test against different hPSCs It is. In these cultures, no evidence with pPCR or IF was observed for neural or thyroid differentiation. This is important because NKX2.1 is also expressed in the thyroid and forebrain. No marker of mature lung epithelial cells was detected by IF on day 15 of culture. However, island-like cells that were negative for FOXA2, SOX2, and NKX2.1 but expressed p63 were observed (FIG. 15). If the cultures are of lower density, they exist as linearly aligned cells with spindle-shaped nuclei surrounding NKX2.1 + cells (FIG. 15). The characteristic properties of these cells are currently unknown.

Optimal conditions for inducing lung fields that do not express more mature lung markers at the protein level were established, the cultures were replated to WFKB + RA or WFK conditions on day 15 and cultures were observed until day 35. From day 23 onwards, the entire culture consisted of colonies that were> 90% FOXA2 ⁺ SOX2 ⁺ , most of which expressed NKX2.1 (although NKX2.1 was more patchy) (Lower left panel, FIG. 5, NKX2.1). Colonies surrounded by an edge of FOXA2 ⁺ p63 ⁺ cells, which are stem cells of the respiratory tract, suggesting basal cells (Figure 5, left and middle panels). These expressed FOXA2, but NKX2.1 was thin at the very outer edge. Towards the center of the structure, p63 and NKX2.1 were co-expressed (upper right, FIG. 5). Within the colony there was a tubular structure expressing mucins (MUC2 (not shown) and MUC5a (bottom right, FIG. 5)). Distal markers (SP-A, SP-B, SP-C and SP-D), as well as Clara cell markers (CC-10) and ciliated cell markers (acetylated tubulin) are expressed as IF (sprayed CC-10 Or SP-B positive cells can be found) or not detected by qPCR. When branching morphogenesis begins in the mouse embryo, RA signaling decreases in the most distal compartment of the developing lung.

Furthermore, constitutively active RA signaling prevents the development of the distal lung and helps the development of the proximal airway. We have also shown that to achieve mRNA expression of the distal marker SP-C, BMP4 should be removed from the growth factor cocktail (although no SP-C protein was detected). However, no evidence of differentiation into cells expressing the distal marker was observed on day 35 in these cultures. Therefore, we further cultured with or without the addition of dexamethasone, butyryl cAMP and isobutylmethylxanthine (DCI), which induces alveolar maturation in embryonic mouse lung explants. MRNA expression of all epithelial lung and airway markers tested (CC-10 (Clara cells), FoxJ1 (ciliary cells), MUC2, MUC5C and MUC5AC (goblet cells), SP-A, SP-B, SP- D (ATII) cells, Acq5 and podoplanin (ATI cells)) increased rapidly by day 55 (data not shown), and this increase was more pronounced when BMP4 and RA were absent. A large amount of SP-B ⁺ cells and a population of CC-10 ⁺ or Muc2 ⁺ , lysozyme ⁺ , or lectin DBA ⁺ cells were observed by IF (FIGS. 16 (A)-(C) and 17 (B) − D) These accounted for the majority of cells in culture as a whole. These were interspersed with a population of mucin 5AC ⁺ (not shown) or Muc1 ⁺ cells (FIG. 18 (A)).

Consistent with qPCR data, SP-C ⁺ cells were rare. Acetylated tubulin was mainly detected at the edge of the colony and in the tubular structure within the colony (FIG. 16 (D)). All these markers were detected both in the condition with and without DCI. The addition of DCI enhanced the lineage determination of the population of SPB and Nkx2.1 co-expressing cells (FIG. 16 (C)). Consistently, expression of SPB mRNA was higher in the conditions where DCI was added. The only marker consistently higher expressed in the presence of BMP4 and RA was the basal cell marker p63. In fact, in the absence of BMP4 and RA, NKX2.1 ⁺ p63 ⁺ cells were rarely observed on day 23, but after day 25 some NKX2.1 ⁺ p63 ⁺ cells were observed. It was done.

  These data indicate that either BMP4 or RA, or both, are detrimental to maturation of lung progenitor cells in vitro, but of putative basal cells that would be defined as another lineage Suggested to help outbreak. The role of BMP4 and RA was further tested separately in differentiation cultures. The results show that RA at the dose used (50 nM) is not detrimental to lineage determination, but BMP4 is detrimental to maturation of lung progenitor cells in vitro.

  Finally, we tested the inhibition of Notch signaling and whether it helps maturation of certain lineages. The Notch inhibitor DAPT was added on day 25 of culture in conditions containing WFKB + RA or WFK. After further culture for 20-25 days, enrichment of the CC10 + pro-SP-C + SP-B + cell population was observed and> 40% of the cells were triple positive in conditions containing DAPT + WFK (FIG. 18). Under these conditions, enrichment of mucin 2+ cells and lectin DBA + cells was also observed.

  Differentiation of hPSC-derived lung progenitor cells has been achieved with very low efficiency, yielding only a few percent of cells expressing several proximal markers. In mice, the NKX2.1: GFP reporter had to be used to isolate cells destined for the lung and thyroid. This report is the first to demonstrate complete differentiation of hPSCs into various lung and airway cells. However, it is clear that terminal maturation of alveolar cells has not yet been achieved. Nevertheless, the above protocol provides a model for further studying lineage determination.

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Claims (35)

  An isolated population of cells enriched in lung and airway epithelial cells.   An isolated cell population enriched in lineage cells in the lung field.   The cell population according to claim 1 or 2, wherein the cells express NKX2.1.   The cells are NKX2.1, FOXP2, GATA6, P63, mucin 5ac, mucin 2, mucin 5b, FOXJ1, acetylated tubulin +, CC-10, pro-SPC, SPB, mucin 1, lysozyme, lectin DBA, podoplanin The cell population according to claim 1 or 2, which expresses +, aquaporin 1+, lectin RCA120 +, or a combination thereof.   2. The cell population of claim 1 comprising up to 90% lung and airway epithelial specific cells.   The cell population of claim 2, comprising cells lined up to 80% of the lung field.   The cell population according to claim 1 or 2, wherein the cells include tracheal cells, bronchial cells, alveolar cells, or a combination thereof.   A purified preparation of cells lined in the lung field, wherein the cells express NKX2.1, GATA6, SOX2, p63, FOXP2, FOXJ1, or combinations thereof. A method for enhancing the induction of lineage-derived cells from the anterior foregut endoderm cells into the lung field,
(A) culturing anterior foregut endoderm cells using a BMP inhibitor or TGF-β signaling inhibitor for at least one day; and (b) culturing the cells with a Wnt protein or a pharmacological thereof such as CHIR99021. Culturing in the presence of an agonist, BMP factor, FGF protein, EGF protein, retinoic acid, or combinations thereof for at least 5 days.   The method of claim 9, further comprising culturing the cell in the presence of a Wnt inhibitor and a TGF-β signaling inhibitor.   10. The method of claim 9, further comprising culturing the cells in the presence of matrigel and / or maturation medium.   The method of claim 11, wherein the maturation medium comprises dexamethasone, methylbutyryl cAMP, hypoxanthine, or a combination thereof.   The method according to claim 9, wherein the culture in the step (b) is performed in the presence or absence of BMP4.   The method according to claim 9, wherein the culture in the step (b) is performed in the presence or absence of retinoic acid.   The method according to claim 9, wherein the culture in the step (b) is performed in the presence or absence of dexamethasone, methylbutyryl cAMP, and hypoxanthine.   The method according to claim 9, wherein the culture in the step (b) is performed in the presence or absence of a notch inhibitor.   17. The method of claim 16, wherein the notch inhibitor is the gamma-secretase inhibitor DAPT.   10. The method of claim 9, further comprising adding a SHH inhibitor.   The method of claim 18, wherein the SHH inhibitor is cyclopamine.   11. The method of claim 10, wherein the Wnt inhibitor is a Wnt3a inhibitor.   11. The method of claim 10, wherein the Wnt inhibitor is IWP2.   10. The method of claim 9, wherein the BMP inhibitor is noggin or dorsomorphin, or other pharmacologically selective BMP inhibitor.   The method according to claim 9, wherein the TGF-β signaling inhibitor is SB341543.   10. The method of claim 9, wherein the cell expresses NKX2.1, GATA6, SOX2, p63, FOXP2, FOXJ1, or a combination thereof.   Cells that have been lineaged in the lung field are uncharacterized novel p63-expressing epithelium, goblet cells, submucosal gland epithelium, Clara cells, basal cells, cilia cells, type I alveolar cells, type II alveoli 10. The method of claim 9, comprising a population of cells, or combinations thereof.   10. The method of claim 9, wherein the lung lineage cells express Muc5a, Muc2, or a combination thereof.   10. The method of claim 9, wherein the lineaged cells in the lung field express Muc5b, Muc2, or a combination thereof.   10. The method of claim 9, wherein the lineaged cell line expresses CC10.   10. The method of claim 9, wherein the cells lineaged in the lung field express p63.   10. The method of claim 9, wherein the lineaged cells in the lung field express acetylated tubulin, foxj1, or a combination thereof.   10. The method of claim 9, wherein the lung lineage cells express Mucl, SP-B, pro-SP-C, lysozyme, lectin DBA, or combinations thereof.   10. The method of claim 9, wherein the lung lineage cells express podoplanin, aquaporin 1, aquaporin 5, T1α, lectin RCA120, or combinations thereof.   The method according to claim 9, wherein the BMP factor is BMP4.   The method according to claim 9, wherein the FGF protein is FGF10 or FGF7.   Cells were lineaged in the lung field expressing NKX2.1, GATA6, FOXP2, CGRP, CCSP, FOXJ1, SP-B, SP-C, p63, CC10, MUC5a, MUC1, MUC2, or combinations thereof A purified preparation of cells.