HK1242369A1 - Suspension culture of human embryonic stem cells - Google Patents

Description

Suspension culture of human embryonic stem cells

Other patent applications

This application claims priority from USSN 60/693,266 filed on 22/6/2005.

Background

Regenerative medicine is conducive to recent research advances regarding the isolation, culture, and use of various types of progenitor cells. The present disclosure further enhances the level of commercial development of human pluripotent stem cells and derivatives thereof.

Embryonic stem cells have two very specific characteristics: first, unlike other normal mammalian cell types, they can be cultured and propagated indefinitely with essentially unlimited supply. Second, they can be used to generate a variety of tissue types of interest for use as a source of replacement cells and tissues for tissue therapy or for drug screening.

Thomson et al (U.S. Pat. No. 5,843,780; Proc. Natl. Acad. Sci. USA 92:7844, 1995) first succeeded in isolating and proliferating primate pluripotent stem cells. They subsequently generated a human embryonic stem (hES) cell line from human blastocysts (Science 282:114, 1998). Gearhart and colleagues generated human embryonic germ (hEG) cell lines from fetal gonad tissue (Shamblott et al, Proc. Natl. Acad. Sci. USA 95:13726, 1998; and U.S. Pat. No. 6,090,622). Both hES and hEG cells have long sought characteristics of pluripotent stem cells: they can be cultured indefinitely without differentiation, they have a normal karyotype, and they can produce a wide variety of important cell types.

The main challenge in using pluripotent stem cells for therapy is that they are usually cultured on a layer of feeder cells to prevent differentiation (U.S. Pat. No. 5,843,780; U.S. Pat. No. 6,090,622). hPS cells cultured in the absence of feeder cells either die very quickly, or differentiate into heterogeneous populations of committed cells, according to Thomson et al (Science 282:114, 1998). Leukemia Inhibitory Factor (LIF) inhibits the differentiation of mouse ES cells, but it does not replace the role of feeder cells in preventing differentiation of human ES cells.

U.S. Pat. No. 6,800,480 (Geron Corp.) entitled "Methods and materials for culturing primate-derived primary stem cells" (Methods and materials for the growth of the parent-derived primordial stem cells). International patent publication WO 01/51616 (Jelon) entitled "technique for culturing and differentiating human pluripotent stem cells" (Techniques for growing and differentiation of human pluripotent cells). An article by Xu et al (Nature Biotechnology 19:971, 2001) is entitled "culture of undifferentiated human embryonic stem cells without Feeder cells" (Feeder-free growth of undiffiented dhumman embryo cells). An article by Lebkowski et al (Cancer J.7 supplement 2: S83, 2001) is entitled "human embryonic stem cells: culture, differentiation and genetic modification for regenerative medical applications "(human anatomical stem cells). International patent publication WO 03/020920 entitled "Culture System for Rapid Expansion of human embryonic Stem cells" (Culture System for Rapid Expansion of human embryonic Stem cells). An article by Li et al (Biotechnology and Bioengineering, on-line publication date: 21/6/2005) is entitled "Expansion of human embryonic stem cells" (Expansion of human embryonic stem cells). These publications report exemplary culture reagents and techniques for propagating embryonic stem cells in an undifferentiated state, and their use in preparing cells for human therapy.

The information provided in the following sections further develops the context of hES cell culture, will facilitate the culture and manipulation of undifferentiated pluripotent stem cells, and will help realize the full commercial potential of embryonic cell therapy.

Summary of The Invention

The present invention provides an improved system for culturing and propagating primate pluripotent stem (hES) cells. The suspension culture system of the present invention enables users to produce high quality embryonic stem cells for therapy and drug discovery in a rapid and volume-efficient manner.

One aspect of the invention is suspension culture of human embryonic stem (hES) cells, wherein the hES cells are substantially undifferentiated. The culture may contain one or more of the following: fibroblast growth factor at high concentrations, other media additives such as TGF β, Stem Cell Factor (SCF) or Flt3 ligand (Flt3L), one or more soluble or suspended extracellular matrix components such as laminin and/or fibronectin, or various types of solid microparticles optionally porous or coated with extracellular matrix.

Another aspect of the invention is a method of culturing hES cells, comprising: suspending the cells in a nutrient medium; maintaining said cells in suspension during culturing with said system; replacing the culture medium periodically; optionally, the culture is flask-cultured from time to reduce cell density; finally, the cells in the culture are harvested.

Another aspect of the invention is a system or kit for suspension culture of hES cells comprising one or more of the components already mentioned or described below.

These and other aspects of the invention will be apparent from the following description.

Drawings

FIG. 1 shows hES cell colonies after six passages on a solid surface with unconditioned medium supplemented with growth factors. (A) mEF conditioned ES medium (control) + bFGF (8 ng/mL); (B) X-VIVO^TM10+bFGF(40ng/mL)；(C)X-VIVO^TM10+ bFGF (40ng/mL) + stem cell factor (SCF, graying factor) (15 ng/mL); (D) X-VIVO^TM10+ bFGF (40ng/mL) + Flt3 ligand (75 ng/mL); (E) QBSF^TM-60+ bFGF (40 ng/mL). All three basic media (ES medium, X-VIVO) can be used^TM10 and QBSF^TM-60) expansion of hES cells in feeder cells free culture. In this case, the cells cultured with the combination of conditions shown in (C) were expanded 8.2-fold per passage, while the cells cultured with the conditioned medium were expanded 2.2-fold.

FIG. 2 shows the gene expression profiles of hTERT and Oct3/4 as determined by real-time RT-PCR, as described in example 1.

FIG. 3 demonstrates that cells cultured in unconditioned medium retain their pluripotency. Using mEF conditioned medium or unconditional X-VIVO containing bFGF and SCF^TMhES cells were passaged 7 times in 10 medium, then differentiated into embryoid bodies, plated, and analyzed by immunocytochemistry for phenotypic markers representing three germ layers, respectively.

FIG. 4 shows the cell count of hES cells cultured in suspension in spinner flasks (example 3). After the culture was established, the cells continued to grow at the same density (upper panel). When re-inoculated onto a standard surface for culture, they revert to a morphology typical of the undifferentiated phenotype: i.e., an independent cell colony with a classical hES cell morphology (lower panel).

Fig. 5 was obtained from the following experiment: the suspension cultured cells (fig. 4) were differentiated into embryoid bodies, plated, and then analyzed for specific cell types by immunocytochemistry (top column). Cells maintained entirely by standard surface culture methods are also shown (column below). The cells in suspension culture retain full pluripotency, demonstrating the effectiveness of the suspension culture system in maintaining the important properties of undifferentiated hES cells.

FIG. 6 shows the cell count of another suspension culture grown in a spinner flask.

FIG. 7 shows cells of different hES cell lines cultured in suspension on a shaking device. After 4 weeks, the cells were re-seeded onto a solid surface, showing a classical undifferentiated hES cell morphology, as shown. Continuing the culture in this manner for more than 3 months showed that the cells in suspension culture proliferated in large quantities.

Detailed description of the invention

The prior art for culturing primate pluripotent stem (hES) cells includes on a solid surface: i.e., fibroblast feeder cells (U.S. Pat. No. 6,200,806) or extracellular matrix (U.S. Pat. No. 6,800,480). Feeder cells-free technology can be optimized for rapid expansion (WO 03/020920), greatly reducing the cost of producing hES cells for commercial purposes.

The following information provides a novel system for further development of hES cell culture technology. In particular, the productivity of such a culture is no longer limited by the two-dimensional size of the culture surface, enabling a more complete utilization of the three-dimensional space of the entire culture vessel. Culture conditions have been determined that enable suspension culture of hES cells for more than three months (example 4). Suspension cultured hES cells maintain the phenotypic characteristics of undifferentiated cells and maintain the full potential to differentiate into tissue types representing any of the three germ layers (example 3).

The ability to culture hES cells in three-dimensional space makes the accumulation of hES cells (bulking up) a more cost-effective process and provides more opportunities to optimize the production capacity and growth rate of hES cell cultures. The use of suspension cultures also helps to adapt the hES cell culture process to a closed system, where cells and media are introduced and recovered from the system in a sterile manner, but the system can also be run in a somewhat loose environment.

Other advantages of the present invention will be appreciated from the following sections.

Definition of

The prototype "primate pluripotent stem cells" (pPS cells) are pluripotent cells derived from pre-embryonic, embryonic or fetal tissue at any time after fertilization, and have the characteristic of being able to produce progeny of several different cell types under the correct conditions. In accordance with standard techniques-accepted monitoring, pPS cells are capable of producing progeny that are derivatives of each of the three germ layers (endoderm, mesoderm, and ectoderm), such as cells capable of forming teratomas in a suitable host, or capable of differentiating into markers of tissue types having all three germ layers in culture.

The definition of pPS cells includes various types of embryonic cells such as hES cells (defined below); embryonic stem cells from other primates such as cynomolgus or marmoset stem cells (Thomson et al, Proc. Natl. Acad. Sci. USA 92:7844, 1995; Developmental Biology 38:133, 1998); and human embryonic germ (hEG) cells (Shamblott et al, Proc. Natl. Acad. Sci. USA 95:13726, 1998). The term also includes other types of pluripotent cells. Also included are any cells of primate origin that are capable of producing progeny that are derivatives of all three germ layers. It is preferred to use pPS cells of normal karyotype and not of malignant origin.

"human embryonic stem cells" (hES cells), unless otherwise specifically indicated, the term includes established lineages that have phenotypic characteristics of hES cells, and derivatives of such lineages that are still capable of producing progeny of each of the three germ layers.

hES cell cultures are said to be "undifferentiated" when the majority of stem cells and their derivatives in the population exhibit morphological characteristics of undifferentiated cells, which can clearly distinguish them from differentiated cells of embryonic or adult origin. One skilled in the art will readily recognize undifferentiated hES cells, which typically exhibit a high nucleus to mass ratio and distinct nucleoli in a two-dimensional microscopic field. It will be appreciated that colonies of undifferentiated cells in a population are often surrounded by differentiated adjacent cells. However, undifferentiated colonies persist when cultured or passaged under appropriate conditions, with the majority of the cell population being single undifferentiated cells. From a developmental standpoint, substantially undifferentiated cultures contain at least 20% undifferentiated hES cells, and may contain at least 40%, 60%, or 80% to improve preference (as a percentage of cells of the same undifferentiated genotype).

When a culture or cell population is said to proliferate "undifferentiated" herein, it is meant that after proliferation, its composition is substantially undifferentiated as defined above. At least four passages (20 fold turnover) were propagated while the undifferentiated population contained substantially the same proportion of undifferentiated cells (or possibly a higher proportion of undifferentiated cells) when evaluated at the same degree of confluence as the starting culture.

"nutrient medium" is a cell culture medium containing nutrients that promote proliferation. The nutrient medium typically contains isotonic saline, buffers, protein sources (in the form of one or more added proteins or amino acids), and (possibly) other externally added nutrients and growth factors.

"conditioned media" is prepared by culturing a first population of cells in a culture medium and then harvesting the medium. The conditioned medium (and any substances secreted by the cells into the medium) can then be used to support the growth of the second population of cells. Description a particular component or agent has been added to a culture medium means that the agent (or cells or particles engineered to secrete the agent) has been intentionally incorporated into the culture medium.

"fresh medium" is medium that has not been purposefully conditioned for use before the final cell type designed to support is cultured with a different cell type. In addition, no limitation should be imposed on the manner of preparation, storage or use. It is added fresh (by exchange or infusion) to the final culture where it is consumed or processed by the cell type present.

General technique

Common methods in molecular genetics and genetic engineering are described in the current edition "molecular cloning: a Laboratory Manual (Molecular Cloning: A Laboratory Manual) (Sambrook et al, Cold spring harbor (Cold spring harbor)); gene Transfer Vectors for mammalian cells (Gene Transfer Vectors for Mammaliancells) (eds. Miller and Calos); and "Current Protocols in molecular Biology", edited by F.M. Ausubel et al, Wiley & Sons. Cell biology, protein chemistry, and antibody technology can be found in Current Protocols in protein science, Inc. (J.E. Colligan et al, Inc.); the "Current Protocols in Cell Biology" protocol (J.S. Bonifacino et al, Willi) and the "Current Protocols in Immunology" protocol (J.E. Colligan et al, Willi). The reagents, cloning vectors and kits for genetic manipulation referred to in the present invention are available from manufacturers such as berle (BioRad), scatchard (Stratagene), Invitrogen (Invitrogen), ClonTech (ClonTech) and Sigma-Aldrich Co.

Cell culture methods are generally described in the current version of animal cell culture: a Manual of Basic technology (manufactured by R.I. Freshney, Willi); general Cell Culture Techniques (General Techniques of Cell Culture) (m.a. harrison and i.f. rae, Cambridge university press) and embryonic stem cells: methods and Protocols (Embryonic StemCells: Methods and Protocols) (K.Turksen, Human Press). Other references of interest include "Culture of Our do (Culture Is ours Business) (m.mcluhan, barnacle office (Ballantine Books), 1970); and Understanding Media (m.mcluhan, Signet, 1970). Tissue culture supplies and reagents are commercially available from manufacturers such as Gibbs/BRL (Gibco/BRL), Nalgene-Sack International, Sigma Chemicals (Sigma Chemical Co.), and ICN biopharmaceuticals (ICN Biomedicals).

Stem cell source

The contents of the examples section were generated with hES cells. However, other cells that meet the definition of a primate pluripotent stem cell can be used to practice the invention unless otherwise required.

In no way is it necessary to practice the invention to break down human blastocysts to produce hES or embryonic stem cells for use in practicing the invention. hES cells can be obtained from established lineages available from public depositories such as the WiCell research Institute (WiCellResearch Institute) in madison, wisconsin or the american type culture collection in manassas, virginia. Human embryonic germ (hEG) cells can be prepared from primordial germ cells as described by Shamblott et al, Proc.Natl.Acad.Sci.U.S.A.95:13726, 1998, and U.S. patent 6,090,622. U.S. patent publication 2003/0113910A1 reports pluripotent stem cells obtained without embryonic or fetal tissue. Other progenitor cells can also be reprogrammed to hES cells using factors that induce a pluripotent phenotype (Chambers et al, Cell113: 643, 2003; Mitsui et al, Cell113:631, 2003). Under appropriate conditions, any cell having suitable proliferation and differentiation capabilities can be used to produce differentiated tissues for use in the present invention.

Proliferation of hES cells

Initially, most scientists in the field prefer to culture hES cells on a layer of feeder cells to prevent differentiation, as described by Thomson (U.S. Pat. No. 5,843,780; U.S. Pat. No. 6,090,622).

Early, the scientists of Jelon discovered that culture systems that provided components produced by feeder cells that promoted the proliferation of undifferentiated phenotypes could be provided in an alternative format. U.S. Pat. No. 6,800,480, WO 01/51616 (Jelon) and Xu et al (Nature Biotechnology 19:971, 2001) describe feeder cell-free culture environments that can support proliferation without differentiation.

Feeder cells-free culture methods are, on the one hand, by culturing support hES cells on an extracellular matrix. The matrix can be deposited by pre-culturing and lysing a matrix-forming cell line (U.S. patent 6,800,480), such as an STO mouse fibroblast cell line (ATCC accession number CRL-1503) or human placental fibroblasts. The matrix may also be coated directly into a culture vessel containing the separation matrix components.Is a soluble formulation prepared from Engelbreth-Holm-Swarm tumor cells that forms a gel at room temperature to form a reconstituted basement membrane. Other suitable extracellular matrix components may include laminin, fibronectin, proteoglycans, vitronectin, entactin, heparan sulfate, and the like, alone or in various combinations. The matrix composition may be human and/or produced by recombinant expression. Substrates that can be tested using the assays described herein include not only other extracellular matrix components, but also polyamines, hydrogels, and other commercially available coatings.

Another aspect of feeder cells-free culture systems is the nutrient medium. The medium typically contains common components that enhance cell viability, including isotonic buffers (i.e., buffers that are isotonic when adjusted to working concentrations), essential minerals and serum or some serum replacement.

The direct introduction of hES support factors is accomplished by preconditioning the culture medium with primary mouse embryonic fibroblasts (mEF), which can be prepared as described in U.S. Pat. No. 6,200,806 or WO 01/51616. Also suitable for use as feeder cells are telomerized cell lines, as well as human cell lines obtained from differentiating ES cells (U.S. Pat. No. 6,642,048) or other primitive cell types. hES cell culture media can be conditioned by culturing feeder cells (typically inactivated by radiation or other means). The medium conditioned by incubation at 37 ℃ for 1-2 hours contains factors at concentrations that support hES cell cultures for about 1-2 days. However, the adjustment time may be adjusted up or down, with sufficient adjustment time being empirically determined.

As an alternative to conditioned media, hES cells are cultured in fresh (unconditioned) media containing added factors that initiate appropriate signaling pathways in undifferentiated cells. Suitable minimal media that can be used without adjustment can be empirically identified. The medium typically contains a neutral buffer (e.g., phosphate and/or bicarbonate at high concentrations) in the form of an isotonic solution; protein nutrients (e.g., serum such as FBS, serum replacement, albumin, or essential and non-essential amino acids such as glutamine). It typically also contains lipids (fatty acids, cholesterol, HDL or LDL extracts of serum) and other components found in most such medium stocks (e.g. insulin or transferrin, nucleosides or nucleotides, pyruvic acid, a sugar source such as glucose, selenium in any ionized or salt form, a glucocorticoid such as hydrocortisone and/or a reducing agent such as beta-mercaptoethanol).

Many commercially available basal media have been developed that are suitable for culturing proliferative cell types, such as hematopoietic cells. Such as X-VIVO^TM10 amplification Medium (Biowhittaker) and QBSF^TM-60 (Quality biology Inc.) (example 1). See also WO 98/30679 (life technologies Inc.) and U.S. patent 5,405,772 (Amgen)). X-VIVO^TM10 formulations contain pharmaceutical grade human albumin, recombinant human insulin and pasteurized human transferrin. X-VIVO^TMThe medium 10 does not contain exogenous growth factors, artificial stimulators of cell proliferation or undefined supplements. They also do not contain any protein kinase C stimulators. QBSF^TM-60 is a serum-free formulation containing recombinant or pasteurized human proteins. Other possible alternatives are Ex-C manufactured by JRH Biosciences (JRH Biosciences)ell VPRO^TMMedium and HyQ CDM4 from Hyclone^TM。

The basal medium is supplemented with an additive that promotes the proliferation of the undifferentiated phenotype and inhibits differentiation. High concentrations of fibroblast growth factor are particularly effective in promoting proliferation of hES cells without differentiation. Such as basic FGF (FGF-2), and FGF-4, but other members of the family may also be employed. Equivalent forms are species homologues, artificial analogues, antibodies to the respective FGF receptor and other receptor activating molecules. Undifferentiated hES cells have been shown by gene expression analysis to express a receptor for acidic FGF (FGF-1). At high concentrations, FGF alone is sufficient to promote hES cell growth in an undifferentiated state (examples 1 and 2). The lower limit of the concentration of FGF effective to promote growth of undifferentiated hES cells is typically about 20, 30, or 40ng/mL, with practical lower limits of about 200, 500, or 1000 ng/mL. A concentration of at least 60, 80 or 100ng/mL bFGF is both reliable and cost effective. Equivalent concentrations of other forms of FGF and analogs can be determined empirically by replacing bFGF in culture with a proposed surrogate and monitoring the culture for differentiation according to the marker system described below.

Suspension culture of hES cells

It has now been found that hES cells can be cultured in suspension without having to grow the cells on a solid substrate.

hES cells expanded by another culture method (or obtained from a primary source) are seeded into a vessel suitable for maintaining the cells in suspension. The container wall is generally inert or resistant to adhesion of undifferentiated hES cells. There are also devices that prevent the cells from settling down, for example, stirring mechanisms such as magnetically or mechanically driven stirring plates or paddles, oscillating mechanisms (typically attached to the outside of the vessel), or inverting mechanisms (i.e., devices that rotate the vessel to change the direction of gravity acting on the cells).

Suitable vessels for suspension culture in process development include a common range of commercially available spinner flasks or shaker flasks. Suitable for commercial studentsThe fermentation tank produced was Celligen Plus^TM(New Brunswick scientific Co.) and Stirred-Tank Reactor^TM(Elapricothrix (Applikon Inc.)). These bioreactors may be continuously perfused with culture medium or run in batch feed mode, and may vary in size.

Nutrient media that helps maintain the undifferentiated phenotype and support growth is changed as needed (e.g., cells are pelleted, media changed, and cells are then resuspended). Growth was monitored and the cultures were split into flasks as needed to make room for further growth. After a suitable incubation time, the cells are harvested and used for the desired purpose.

Media and other components designed to culture hES cells without feeder cells on a solid surface can also be used in suspension culture. Conditioned medium or fresh medium (example 4) may be used. However, the kinetics of suspension culture provide the user with another opportunity to optimize the individual components of the culture system. Without being bound by theory, the present invention assumes: suspension cultures are augmented if the hES cells are allowed to form small clusters of undifferentiated cells (three-dimensional equivalents of undifferentiated colonies on solid surfaces) that may be surrounded by cells that partially differentiate into stromal cells, or if the hES cells are dispersed, but dynamic fluid forces that may cause differentiation are avoided.

The suspension culture system can be optimized by empirical testing. Undifferentiated cells from previous surface or suspension cultures were passaged to test conditions for 1 week or more. The hES cell characteristics of the cells can be periodically measured using, for example, the marker system described in the next section and described in example 1. The cells can also be passaged back into a well established culture system to evaluate the classical morphological characteristics of undifferentiated cells (example 3). If it is desired to have hES cells ultimately differentiate into a particular tissue type, the ultimate test goal may not be a marker profile for undifferentiated cultures, but rather the ability of the cells to differentiate as needed. Pluripotency of hES suspension cultures can be verified by sampling cells and then generating teratomas in SCID mice or staining markers representing all three germ layers on EB-derived cells (example 3). The user can thus optimize the system to obtain a high growth rate and maintain the full potential of the cell (or at least maintain the ability of the cell to differentiate into the desired tissue of interest).

Aspects of the culture system that may benefit from further optimization include nutrient media. Alternative basal media and alternative FGF additives are listed in the previous section. It may also be advantageous to sample one or more additional additives as described below:

stem cell factor (SCF, steel factor), other ligands or antibodies that dimerize c-kit, and other activators of the same signal transduction pathway

Other tyrosine kinase-associated receptors, such as ligands for receptors for platelet-derived growth factor (PDGF), macrophage colony stimulating factor, Flt-3 ligand and Vascular Endothelial Growth Factor (VEGF)

Factors which increase cyclic AMP levels, e.g. forskolin

Gp130 inducing factors, such as LIF or oncostatin-M;

hematopoietic growth factors, such as Thrombopoietin (TPO)

Transforming growth factors, e.g. TGF beta 1

Other growth factors, e.g. Epidermal Growth Factor (EGF)

Neurotrophins, e.g. CNTF

In view of preventing the cells from adhering to each other, to the walls of the container, or forming excessively large clusters of cells, it may be advantageous to include an anti-aggregation agent such as the reagent sold by Invitrogen (Cat. No. 0010057 AE).

Although cells are capable of forming a limited degree of their own extracellular matrix, it may also be advantageous to include one or more extracellular matrix components dissolved or suspended in the culture medium. A suitable working range for maintaining laminin in suspension is about 10-33 μ g/mL. Other candidate matrix components for suspension cultures include some of the foregoing, specifically fibronectin, proteoglycans, vitronectin, and artificial equivalents thereof. The extracellular matrix may help the cells to form small aggregates of suitable size.

Alternatively or in addition, the suspension culture may contain particulate carriers that establish a surface in suspension, but still provide the benefit of culturing cells in three-dimensional space. Cells were cultured and passaged in the same manner except that the particles were retained in the vessel when the medium was changed and more particles were added when the flask was divided.

One type of microcarrier is a solid spherical or hemispherical particle made of glass, plastic, dextran (Cytodex) that has a positive charge to enhance cell adhesion, and the like. Another type is a cultured plastic in the form of a disc, such as the fibre-cel Disks sold by New Bronsted science^TM.1 gram of this disk base provides a surface area of 1200cm². The solid support is optionally coated with a hES cell-friendly extracellular matrix, such as laminin, to make the adherent cells the same microenvironment as the cells seeded onto the solid surface.

Another type of microcarrier is a macroporous particle with different pore sizes that can retain cells inside and outside, potentially increasing the protective effect. To recover hES cells with minimal disruption, it is desirable to use particles made of materials (e.g., agarose) that can be readily lysed or dispersed by mild mechanical or enzymatic action, thereby releasing the cells for harvesting or further culture.

Characterization of undifferentiated hES cells

Human ES cells cultured according to the present invention have the characteristic morphological characteristics of undifferentiated stem cells. In standard two-dimensional micrographs, hES cells have a high nucleus/mass ratio in the image plane, distinct nucleoli and form dense colonies of cell junctions that are difficult to distinguish. Cell lines can be karyotyped using standard G-banding techniques (which can be performed by many clinical diagnostic laboratories that provide conventional karyotyping services, such as the cytogenetics laboratory (cytogenetics Lab) in Oakland, Calif.) and compared to published human karyotypes. Advantageously, cells are obtained with a "normal karyotype" (meaning that the cells are euploid), in which all human chromosomes are present and not significantly altered.

hES cells can be characterized by using cell expression markers that can be detected by antibodies (flow cytometry or immunocytochemistry) or by reverse transcriptase PCR. hES cells typically have antibody-detectable SSEA-4, Tra-1-60, and Tra-1-81, but rarely SSEA-1, and have alkaline phosphatase activity. Suitable markers that can be detected at the mRNA level are described in U.S. application 2003/0224411A1 (Jalon). Examples are teratocarcinoma growth factor (Cripto), gastrin-releasing peptide (GRP) receptor, podocalyxin-like Protein (PODXL), human telomerase reverse transcriptase (hTERT) and POU transcription factor Oct 3/4.

As previously mentioned, an important feature of proliferating hES cells is the potential to differentiate into cells of the three germ layers endoderm, mesoderm and ectoderm. The potential of hES cells can be verified by forming teratomas in SCID mice and testing their representative tissue to form three germ layers. Alternatively, hES cells are non-specifically differentiated (e.g., by embryoid body formation) and then immunocytochemically assayed for the cell type represented in the culture to determine potency (example 3). The potential of hES cells to differentiate into specific cell lines can be determined as described in the next section.

Use of proliferating hES cells

The present invention provides methods for producing large quantities of pluripotent cells on a commercial scale. The cells can be used in undifferentiated form for a variety of research and commercial purposes, or can be directed to differentiate into specific cell types.

Undifferentiated hES cells can be used to screen for factors (e.g., small molecule drugs, peptides, polynucleotides, etc.) or conditions (e.g., culture conditions or operating conditions) that affect the characteristics of the cultured hES cells. hES cultures can also be used in drug studies to detect drug compounds. Evaluation of the activity of a candidate pharmaceutical compound generally involves mixing the differentiated cells of the invention with the candidate compound, determining the resulting change, and then correlating the effect of the compound with the observed change. Cytotoxicity or metabolic effects can be determined by cell viability, morphology, expression or release of certain markers, receptors or enzymes, DNA synthesis or repair, and the like.

hES cells cultured in accordance with the present invention can be used to prepare differentiated cells of a variety of commercially and therapeutically important tissue types.

Liver cell

Hepatocytes may be differentiated from hES cells using histone deacetylase inhibitors, as described in U.S. patent 6,458,589 and PCT publication WO 01/81549 (jerong corporation). Undifferentiated hES cells were cultured in the presence of histone deacetylase inhibitors.

A staged approach to differentiating hES cells into hepatocytes is seen in US 2005/0037493a1 (jeron). Cells are cultured sequentially with several combinations of differentiating and maturation agents, such that hES cells differentiate first into early endoderm or hepatocyte precursors and then into mature hepatocyte-like cells. Briefly, differentiation into endodermal-like cells can be initiated using butyrate, DMSO, or fetal bovine serum (optionally in conjunction with fibroblast growth factor). Differentiation can then be continued using commercially available hepatocyte culture media containing various combinations of factors such as Hepatocyte Growth Factor (HGF), Epidermal Growth Factor (EGF) and/or bone morphogenic proteins (e.g., MP-2, 4 or 7). The final maturity can be increased by the presence of drugs such as dexamethasone or oncostatin M. Obtaining a protein having asialoglycoprotein expression, glycogen storage, cytochrome p450 enzyme expression; glucose-6-phosphatase activity and morphological characteristics of liver cells.

Nerve cell

As per U.S. patent 6,833,269; carpenter et al, Exp neurol.2001; 172(2) 383-97; and the method described in WO 03/000868 (jeron) to generate nerve cells from hES cells. Culturing undifferentiated hES cells or embryoid body cells in a medium containing one or more neurotrophins and one or more mitogens produces a cell population in which at least-60% of the cells express A2B5, polysialylated NCAM or dry protein (Nestin) and are capable of at least 20-fold turnover during culture. Exemplary mitogens are EGF, basic FGF, PDGF and IGF-1. Exemplary neurotrophins are NT-3 and BDNF. TGF- β superfamily antagonists or a combination of cAMP and ascorbic acid can be used to increase the proportion of neuronal cells that are positive for tyrosine hydroxylase (a characteristic of dopaminergic neurons). The proliferating cells can then undergo terminal differentiation by culture with neurotrophins in the absence of mitogens.

hES cells are cultured as cell aggregates and suspended in a medium containing a mitogen such as FGF and an oligodendrocyte differentiation factor such as triiodothyronine, selenium and retinoic acid, thereby producing oligodendrocytes from the hES cells. Cells are then seeded onto a solid surface, eliminating retinoic acid, and expanding the population. Terminal differentiation can be achieved by seeding on poly-L-lysine and removing all growth factors. More than 80% of the cells in the obtained cell population were positive for the oligodendrocyte markers NG2 proteoglycan, A2B5 and PDGFR α, while the neuronal marker NeuN was negative. See PCT publication WO 04/007696 and Keirstead et al, J Neurosci.2005; 25(19):4694-705.

Heart cells

Cardiomyocytes or cardiomyocyte precursors can be generated from hES cells according to the method provided in WO 03/006950. The cells are cultured in a suspension containing fetal bovine serum or serum replacement and optionally cardiotrophic factors affecting DNA-methylation, such as 5-azacytidine. Alternatively, a combination of activin a and bone morphogenic protein 4 can be used to directly differentiate cardiomyocyte clusters on a solid substrate: the spontaneously contracting cells can then be separated from other cells in the cell population by density centrifugation.

A further process step may include culturing the cells to form what is known as a heart body^TM(cardiacbodies^TM) Removing individual cells, then dispersing and reforming into the heart body^TMThis process is repeated continuously. The cell population obtained has a high proportion of cells staining positive for cTnI, cTnT, heart-specific Myosin Heavy Chain (MHC) and transcription factor nkx 2.5. See WO 03/006950, Xu et al, Circ Res.2002; 91, (6) 501-8; and PCT/US2005/009081 (Jalon).

Other cell types

hES cells initiate differentiation of hES cells by culturing in a medium containing a combination of several factors selected from the group consisting of: activin a, histone deacetylase inhibitors (e.g., butyrate), mitogens (e.g., bFGF); and antagonists of the TGF-beta superfamily (e.g., noggin). The cells can then be matured by culturing with nicotinamide, resulting in a population of cells in which at least 5% of the cells express Pdx1, insulin, glucagon, somatostatin, and pancreatic polypeptide. See WO 03/050249 (jeron).

Hematopoietic cells can be prepared by co-culturing hES cells with murine bone marrow cells or yolk sac endothelial cells to produce cells with hematopoietic cell markers (us patent 6,280,718). Hematopoietic cells can also be prepared by culturing hES cells with hematopoietic cytokines and bone morphogenic proteins, as described in US 2003/0153082 a1 and WO 03/050251 (roberts Institute).

Mesenchymal progenitor cells can be generated from hES cells according to the method described in WO 03/004605. Then, the hES-derived mesenchymal Cells are further differentiated into osteoblast lineage Cells in a medium containing an osteogenic factor such as a bone morphogenic protein (specifically BMP-4), a ligand for a human TGF-beta receptor, and a ligand for a human vitamin D receptor (WO 03/004605; Sotile et al, Cloning Stem Cells 2003; 5(2): 149-55). hES cells in the microaggregate state can be cultured using an effective combination of differentiation factors listed in WO 03/050250 (Jelon) to produce chondrocytes or their progenitors.

Other differentiation methods known in the art and subsequently developed may be applied to hES cells cultured according to the present invention. hES-derived cells can be used for drug screening, preparation of pharmaceutical compositions, research and many other worthwhile purposes.

Commercial circulation

The components of the culture system of the present invention may be offered for sale, or other distribution by the source to another entity for any purpose. The components may also be sold or distributed in various useful combinations, such as two or more of the following:

media suitable for culturing hES cells with suspension factor

Extracellular matrix components or thickeners present in the culture medium or to be added to the medium

Microcarriers present in the culture medium or ready to be added to the medium

Vessels adapted for suspension culture

hES cells themselves, cultured in a culture system, or stored in another form, but ready for use in the culture system

The products and product combinations are packaged in suitable containers, optionally in kit form, and may be accompanied by written information regarding the materials used in accordance with the invention, such as maintenance or expansion of hES cells. The information may be written in any language on any communication medium that is available to and understood by the intended user. It may be in the form of a label on the container or kit, or a product insert that is packaged and distributed with the container. Equivalent forms have written instructions, specifications or interpretations in hard copy or electronic form available to the user or intended user as references or resources related to commercially distributed products.

The following examples are illustrative and do not limit the scope of the claims of the present invention.

Examples

Example 1: by usingRapid amplification culture medium for culturing pluripotent stem cells

The hES cell line was obtained initially grown on mouse embryonic fibroblast feeder Cells and then grown as per us patent 6,800,480 and Xu et al, Stem Cells 2005; 23(3) 315-23 said inThe extracellular matrix was expanded for 20 passages in feeder cells-free environment and conditioned medium.

The hES cells were then transferred to X-VIVO from Bykint^TM10 amplification Medium or QBSF from Quality biology Inc^TM-60. For these experiments, X-VIVO^TMThe medium 10 was supplemented with the usual substances (goodiis), i.e. 2mM L-glutamine, 1% non-essential amino acids (Gibbo), 0.1mM β -mercaptoethanol and 8ng/mL bFGF. the medium was also supplemented with 8ng/mL or 40ng/mL bFGF (Gibbo), 40ng/mL bFGF and 15ng/mL SCF (R)&D systems Co Ltd (R)&D System)); or 40ng/mL bFGF and 75ng/mLFlt3 ligand (R)&D systems corporation). QBSF^TMhES cells cultured in mEF conditioned medium were used as controls for these experiments, with medium 60 supplemented with 0.1mM β -mercaptoethanol, 1% non-essential amino acids (Gibbco) and 40ng/mL bFGF.

hES cells were first passaged with collagenase IV toThe coated plates were incubated with conditioned medium for 2 days. On day 2, the conditioned medium was replaced with 80% unconditioned ES medium plus 20% expansion medium. Fresh medium was changed daily and passaged once a week. The proportion of the amplification culture medium is increased by about 20% every 2 days until the cells are completely adapted to the amplification culture medium, and then the cells are cultured until 6 generations.

FIG. 1 shows hES cell colonies at the end of 6 passages (sufficient for complete adaptation) with the following media: (A) mEF conditioned medium + bFGF (8 ng/mL); (B) X-VIVO^TM10+bFGF(40ng/mL)；(C)X-VIVO^TM10+ bFGF (40ng/mL) + stem cell factor (SCF, graying factor) (15 ng/mL); (D) X-VIVO^TM10+ bFGF (40ng/mL) + Flt3 ligand (75 ng/mL); (E) QBSF^TM-60+bFGF(40ng/mL)。

The following table shows the results of 4 generations of cultures grown in mEF conditioned medium or with X-VIVO^TM10 or QBSF^TM60 Total fold of expanded cells per generation in 7 generations of undifferentiated hES cells.

In X-VIVO^TM10 and QBSF^TMThe mean fold expanded cells per passage in-60 was greater than cells cultured with mEF conditioned medium. mEF cells cultured in conditioned medium were passaged on average once every 7 days and then treated with X-VIVO^TM10 and QBSF^TM60 cultured cells were passaged on average every 5 days. Therefore, unconditional X-VIVO^TM10 or QBSF^TMThe amplification rate in-60 was 3.2-5.2 times higher than that in mEF conditioned ES medium.

FIG. 2 shows the gene expression profiles of hTERT and Oct 3/4. Cell RNA was isolated using a High purity RNA Isolation Kit (High pure RNA Isolation Kit), Roche Diagnostics, and Taqman^TMThe experiments (real-time RT-PCR) were evaluated. Gene expression in each test condition was plotted against expression in control cultures. Taking into account instrumental error and experimental variability, a difference in expression between test and control samples can only be considered significant if it is more than two-fold. The analysis showed adaptation to unconditional X-VIVO^TM10 or QBSF^TMAfter-60 media, expression of hTERT and Oct-3/4 was slightly reduced (first four columns of each group), but returned to standard levels when the cells were returned to mEF conditioned media (last three columns of each group).

To verify that cells cultured in unconditioned medium retain their potential to form embryoid bodies, each phenotypic marker representing three germ layers was analyzed by immunocytochemistry. After 7 passages with the amplification medium, the cells were dissociated into small pieces by incubating with 200U/mL collagenase IV at 37 ℃ for 10 minutes, placed in a differentiation medium (DMEM + 10% FBS) for differentiation culture for 4 days, and then transferred to a poly-L-ornithine hydrobromide-coated plate for incubation for 10 days. Fixed with 4% paraformaldehyde, permeabilized, and labeled with immunocytochemistry.

Fig. 3 shows the results. For using unconditional X-VIVO^TMAlpha fetoprotein (representing endoderm), actin (representing mesoderm), and β -tubulin III (representing ectoderm) were stained on hES cells at 7 passages in 10 medium.

Thus, hES cells can be rapidly expanded in a feeder cells-free environment with fresh (unconditioned) media at rates suitable for commercial production, which are 2-5 times or more faster than the growth rate in conditioned media or on feeder cells. This cell retains the morphology of undifferentiated hES cells and can differentiate into derivative cells representing all three germ layers.

Example 2: culturing hES cells in a defined system without animal-based products

Will be provided withhES cells cultured in MEF-CM were passaged toFresh (non-conditioned) serum-free Medium X-VIVO supplemented with Glutamine, non-essential amino acids and β -mercaptoethanol and 80ng/mL human basic FGF^TM10, and then adapted to a surface coated with human laminin. Alternatively, cryopreserved cells were thawed directly into the same medium containing 80ng/mL hbFGF. The cells were passaged with collagenase IV every 5-6 days.

Cultures grown under these conditions were similar to or superior toThe culture as described above. (A) Morphology of cells cultured with mEF conditioned medium; (B) determining morphology in the culture medium on laminin; (C) expression of the surface marker SSEA-4 in mEF-CM (H1p62) or defined medium (H1p34+ 28); (D) expression of the surface marker Tra-1-60 in mEF-CM or defined medium. The culture performance of the medium determined on laminin is excellent: very large colonies of ES cells were observed, representing approximately 80% of the culture. Marker expression levels were as follows:

mean. + -. SD of 3 RT-PCR assays

The expression of other markers that are characteristic of undifferentiated hES cells is also comparable: levels of hTERT and teratocarcinoma growth factors in the media were determined to be the same or greater than those in mEF-CM, compared to mEF-CM, while expression of Oct3/4 was approximately 28% lower (average of three experiments) by real-time PCR assay. TRAP analysis showed that the cells retained telomerase activity.

Cells cultured with p34+11 (day 75) in a well-defined culture system were harvested and used to generate teratomas in SCID mice to assess potential. Teratomas exhibit pigment epithelium (endoderm); kidney tissue (mesoderm); mesenchymal tissue (mesoderm); and evidence of the neural tube (ectoderm). This demonstrates that the cells retain their potential.

Example 3: suspension culture of hES cells

To increase the production of hES cells per culture volume, the cells were cultured in suspension and then evaluated for morphology and ability to form differentiated cells representing all three germ layers.

Harvesting from 6-well platesH9hES cells cultured above were seeded into spinner flasks under the following conditions:

a container: 100mL rotary culture bottle

Inoculation density: 3.6X 10⁵Individual cells/mL;

volume of culture medium: 50mL per rotating flask

The culture medium employed: mEF conditioned Medium containing bFGF (8ng/mL)

Stirring rate: 20rpm (Bellco carrier magnetic stirrer)

Atmosphere: CO at 37 deg.C₂Culture box

Replacement of medium: every other day (replacement was done by precipitating cells and replacing supernatant)

The H9hES cells were cultured in spinner flasks for 6 days under these conditions.

Fig. 4 (upper panel) shows the results. After an initial decline during which cultures were established, the cell number began to increase from day 2-6.

At this point, the cells are seeded for reuse6-well plates were coated to determine if they still had the phenotype of undifferentiated cells. The culture was continued with mEF-CM medium containing bFGF (8 ng/mL).

Fig. 4 (lower panel) shows the results. After 1 passage, the cells grew and showed the morphology of undifferentiated cells.

The potential of the cells was assessed by the formation of embryoid bodies. Cells in confluent cultures were harvested with collagenase IV and transferred to low adhesion 6-well plates containing DMEM + 20% FBS. EBs were formed and maintained for 4 days. Then, EBs were again inoculated onto polyornithine-coated culture slides (chamber slides). After 11 more days of culture, the EBs were stained for alpha fetoprotein (endoderm), actin (mesoderm) and β -tubulin (ectoderm) with neuronal morphology in the outgrowth.

Fig. 5 shows the results. The upper row shows cells differentiated from hES cells seeded back on laminin under standard conditions after suspension culture. The lower row shows cells differentiated from the same hES cell line, completely cultured as plated cells. As shown, suspension cultured hES cells completely maintained their ability to differentiate into derivatives of all three germ layers.

In another experiment, H9hES cells were cultured under the following conditions:

a container: 100mL rotary culture bottle

Inoculation density: 3.5X 10⁵Individual cell/mL

Volume of culture medium: 50ml in each rotary culture flask

The culture medium employed: mEF-CM + bFGF (8ng/mL)

Stirring rate: 20rpm (same as before)

Replacement of medium: is replaced every 3 days

Fig. 6 shows the results. Once the culture is established, the cells are properly maintained for the entire 12 day period.

Example 4: long term suspension culture

This experiment was done with another hES cell line. The shake flask was used instead of a spinner flask to culture cells under several different culture conditions, the culture process lasted more than two months.

Harvest of H1hES cells from 6-well plates (inUpper mEF conditioned medium) were inoculated into shake flasks under the following conditions:

a container: 100mL shake flask

Inoculation density: 5.0x 10⁵Individual cell/mL

Volume of culture medium: 15mL in each flask

Stirring rate: 80rpm (Labline) rotor/shaker at 37 ℃ CO₂In incubator)

Replacement of medium: the replacement is performed every other day, and every 2-3 days

The media and incubation times used were as follows:

a: mEF-CM + bFGF (8 ng/mL). The maintenance period is 98 days.

B: mEF-CM + bFGF (8ng/mL) + laminin (33. mu.g/mL at the beginning, followed by 10. mu.g/mL for the rest of the culture). The maintenance period is 49 days.

C：X-VIVO^TM10+ FGF (40ng/mL) + Flt-3(75 ng/mL). The maintenance period is 11 days.

D：X-VIVO^TM10+ FGF (40ng/mL) + Flt-3(75ng/mL) + laminin (33 μ g/mL initially, followed by 10 μ g/mL for the remainder of the culture). The maintenance period is 11 days.

The number of cells during the culture is shown in the following table.

To determine whether cells maintained an undifferentiated phenotype, cells cultured for 4 weeks with Medium A were inoculated backmEF conditioned medium containing bFGF.

Fig. 7 shows the results. After passaging, cells derived from suspension cultures showed growth and were characterized as colonies of undifferentiated hES cells.

These data indicate that hES cells can be cultured in suspension for at least 3 months, possibly expanding 3-40 fold after the culture is fully established.

Example 5: suspension culture with fresh Medium

The next experiment evaluated another additive for suspension culture in fresh (unconditioned) medium. In standard conditions (substrate of human laminin from Sigma, at 2. mu.g/cm)²Coated onto 6-well plate, X-VIVO 10 containing 80ng/mL bFGF and 0.5ng/mL TGF β 1^TMMedia) was harvested from surface cultures in fresh media. The harvested cells were passaged into suspension cultures in 100mL spinner flasks using 50mL of culture per flask at an initial density of-5X 10⁵Individual cells/mL. The following media substitutes were evaluated:

1)X-VIVO^TM10+bFGF(80ng/mL)

2)X-VIVO^TM10+bFGF(80ng/mL)+TGFβ1(0.5ng/mL)

3)X-VIVO^TM10+bFGF(40ng/mL)+TGFβ1(0.5ng/mL)

4)X-VIVO^TM10+ bFGF (80ng/mL) + TGF β 1(0.5ng/mL) + 10. mu.g/mL human laminin

5)X-VIVO^TM10+ bFGF (80ng/mL) + TGF β 1(0.5ng/mL) + 50. mu.g/mL human serum albumin

Placing each rotary culture flask at 37 deg.C CO₂On a Bell carrier magnetic stirrer (Bellco Biotechnology, Vineland, N.J.) in an incubator, the initial stirring rate was 20 rpm.The agitation rate was adjusted to keep the cells in suspension and to provide sufficient air while minimizing shear forces. The medium was changed every 2-3 days (as before), the cell number was monitored, and flask culture was performed as needed.

Cell samples were taken from each flask at regular intervals and inoculated back onto the laminin-coated surface to assess morphology. Pluripotency of cells reverted to surface culture and cells removed directly from suspension cultures were monitored by immunocytochemical staining of EB-derived cells, as described in example 3.

The above compositions and methods may be usefully modified without departing from the scope of the present invention and its equivalents.

Claims (7)

1. A suspension culture of human embryonic stem (hES) cells, wherein said hES cells are substantially undifferentiated, said hES cells being from an established hES cell line.

2. The culture of claim 1, wherein said cells are cultured in a medium comprising basic fibroblast growth factor at a concentration of at least about 20 ng/mL.

3. The culture of claim 2, wherein the medium further comprises transforming growth factor beta (TGF β), Stem Cell Factor (SCF), or Flt3 ligand (Flt 3L).

4. The culture of any preceding claim, wherein the cells are cultured in a medium comprising one or more soluble or suspendable extracellular matrix components.

5. The culture of claim 4, wherein the extracellular matrix component comprises human laminin and/or human fibronectin.

6. The culture of any preceding claim, wherein the cells are cultured in a suspension comprising solid microparticles.

7. The culture of claim 7, wherein the microparticles are coated with one or more soluble or suspended extracellular matrix components.