AU2012203350A1 - Suspension culture of human embryonic stem cells - Google Patents

Abstract

This disclosure provides an improved system for culturing human embryonic stem cells. The cells are cultured in suspension so as to maximize the production capacity of the culture environment. The new culture system of this invention allows for bulk proliferation of hES cells in a more cost-effective manner, which facilitates commercial production of important products for use in human therapy.

Description

1 AUSTRALIA Patents Act 1990 GERON CORPORATION COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Suspension culture of human embryonic stem cells The following statement is a full description of this invention including the best method of performing it known to us:- SUSPENSION CULTURE OF HUMAN EMBRYONIC STEM CELLS OTHER PATENT APPLICATiONS 5 This is a divisional of AU2006262369, the entire contents of which were incorporated herein by reference. BACKGROUND 10 Regenerative medicine Is benefiting from recent advances relating to the Isolation, culture, and use of various types of progenitor cells. This disclosure provides further Improvements for the commercial development of human pluripotent stem cells and their derivatives. Embryonic stem cels have two very special properties: First, unlike other normal mammalian cell types, they can be propagated In culture almost Indefinitely, providing a virtually unlimited supply. Second, 15 they can be used to generate a variety of tissue types of Interest as a source of replacement cells and tissues for use In tissue therapy, or for use in the screening of pharmaceutical agents. Thomson at al. (U.S. Patent 5,843,780; Proo. NatI. Aced. Scl. USA 92:7844, 1995) were the first to successfully isolate and propagate pluripotent stem cells from primates. They subsequently derived human embryonic stem (hES) cell lines from human blastocysts (Science 282:114, 1998). Gearhart and coworkers 20 derived human embryonic germ (hEG) can lines from fetal gonadal issue (Shamblott et al., Proc. Nati. Aced. Sc. USA 95:13726, 1998; and U.S. Patent 5,090,622). Both hES and hEG cells have the long-sought characteristics of plurlpotent stem cells: they can be cultured extensively without differentiating, they have a normal karyotype, and they are capable of producing a number of important cell types. A significant challenge to the use of pluflpotent stem cells for therapy is that they are traditionally 25 cultured on a layer of feeder cells to prevent differentiation (U.S. 5,843,780;. U.S. 6,090,822). According to Thomson at al. (Science 282:114, 1998), hPS cells cultured without feeders soon die, or differentiate Into a heterogeneous population of committed cells. Leukemia Inhibitory factor (LIF) Inhibits differentiation of mouse ES cells, but it does not replace the role of feeder cells in preventing differentiation of human ES cells. U.S. Patent 6,800,480 (Geron Corp.) Is entitled Methods and materials for the growth of primate 30 derived primordial stem cells. International Patert Publication WO 01/51616 (Geron Corp.) Is entitled Techniques tbr gmwth and diffbrentaton of human pluripotent stem cells. An article by Xu at al. (Nature Biotechnology 19:971, 2001) Is entitled Feederfree growth of undlfferentleted human embryonic stem cells. An article by LebkowskI at al. (Cancer J. 7 Suppl. 2:583, 2001) Is entitled Human embryonic stem cells: culture, differentlation, and genetic modifloalon for regenerative medicine applications. International Patent 35 Publication WO 031020920 Is entitled Culture System for Rapid Expansion of Human Embryonic Stem Cells. An article by U at al. (Biotechnology and Bloengineering, Pubflshed Online: 21 Jun 2005) ta entitled 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. 1A The information provided in the section below further advances the science of hES cell culture that will facilitate growing and manipulating undifferentiated pluripotent stem cells, and help realize the full commercial potential of embryonic cell therapy. SUMMARY OF THE INVENTION 5 This disclosure provides an improved system for culturing and proliferating primate pluripotent stem (hES) cells. The suspension culture system of this invention enables the user to produce high-quality embryonic stem cells in a rapid and volume-efficient mode, for use in therapy and drug discovery. One aspect of this invention is a culture of human embryonic stem (hES) cells in suspension, 10 wherein the hES cells are substantially undifferentiated. The culture may contain one or more of the following: fibroblast growth factor at a high concentration, other medium additives such as TGF3, stem cell factor (SCF), or Flt3 ligand (FIt3L), one or more soluble or suspended extracellular matrix components such as laminin and/or fibronectin, or solid microparticles of various kinds, optionally porous in nature or coated with extracellular matrix. 15 Another aspect of the invention is a method for culturing hES cells, comprising: suspending the cells in a nutrient medium; maintaining the cells in suspension while culturing In a system such as described above; changing the medium periodically; optionally splitting the culture from time to time so as to reduce cell density; and finally harvesting cells from the culture. Another aspect of this invention is a system or kit for culturing hES cells in suspension, 20 comprising one or more of the components already referred to, or described below. These and other aspects of the invention will be apparent from the description that follows. DRAWINGS Figure 1 shows colonies of hES cells after six passages on a solid surface in unconditioned 25 medium supplemented with growth factors. (A) mEF conditioned ES medium (control) + bFGF (8 ng/mL); (B) X-VIVO' 10 + bFGF (40 ng/mL); (C) X-VIVO'M 10 + bFGF (40 ng/mL) + stem cell factor (SCF, Steel factor) (15 ng/mL); (D) X-VIVO"M 10 + bFGF (40 ng/mL) + Flt3 ligand (75 ng/mL); (E) QBSFM-60 + bFGF (40 ng/mL). All three base media (ES medium, X-VIVOTM 10, and QBSfTM-60) can be used to expand hES cells in feeder-free culture. In this illustration, the cells growing in combination shown in (C) expanded 8.2 30 fold per passage, whereas those in conditioned medium expanded 2.2-fold. Figure 2 shows the gene expression profile of hTERT and Oct3/4, measured by real time RT PCR, as described in Example 1. Figure 3 demonstrates that cells cultured in unconditioned medium retain their pluripotency. hES cells passaged 7 times in mEF conditioned medium, or unconditioned X-VIVOTM.10 medium containing 35 bFGF and SCF. The cells were then differentiated into embryoid bodies, plated, and analyzed by immunocytochemistry for phenotypic markers representing each of the three germ layers. The cells stain for a-fetoprotein (representing endoderm); muscle actin (representing mesoderm), and (p-tubulin IlIl (representing ectoderm). 2 Figure 4 shows the cell count of hES cells grown in suspension culture In spinner flasks (Example 3). After the culture became established, the cells continued to thrive at the same density (Upper Panel). When passaged back to standard surface culture they reverted to typical morphology of the undifferentiated phenotype: namely, distinct colonies of cells having the classic hES cell morphology (Lower Panel). 5 Figure 5 is taken from an experiment in which the cells grown in suspension (Figure 4) were differentiated Into embryold bodies, plated, and then analyzed by Immunocytochemistry for specific cell types (Top Row). Cells maintained throughout by standard surface culture are also shown (Bottom Row). The cells cultured In suspension maintained full plurlpotency, demonstrating the effectiveness of the suspension culture system In maintaining the Important properties of undifferentiated hES cells. 10 Figure 6 shows the cell count of another suspension culture In spinner flasks. Figure 7 shows cells from a different hES cell line maintained In suspension culture on a shaker device. After four weeks, the cells were plated back onto a solid surface, and showed classic undifferentiated hES cell morphology, as shown here. A culture was continued in this fashion for over three months, showing substantial proliferation of the cells in suspension. -5 DETAILED DESCRIPTION Previous technology for growing primate pluripotent stem (hES) cells has Involved culturing on a solid surface: either fibroblast feeder cells (U.S. Patent 6,200,806), or extracellular matrix (U.S. Patent 6,800,480). 20 The feeder-free technology can be optimized to allow for rapid expansion WO 03/020920, substantially reducing the cost of hES cell production for commercial purposes. The information disclosed below provides a new system that further advances the art of hES cell culture. Specifically, the production capacity of the culture is no longer constrained by the two dimensional size of the culture surface, and makes fuller use of the three-dimensions of the entire culture vessel. Growth 25 conditions have been identified that permit hES cells to be cultured In suspension for over three months (Example 4). hES cells cultured In suspension maintain phenotyplo 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 a three-dimensional space should make the bulking up of hES cells 30 an even more cost-effective process, and provides further opportunities to optimize the production capacity and growth rate of hES cell cultures. The use of suspension cultures also facilitates the adaptation of hES cell culture methods to a closed system, where cells and media are Introduced and recovered from the system In a sterile manner, but the system can otherwise be handled In a less scrupulous environment. Further advantages of the invention will be understood from the sections that follow. 35 Definitions Prototype "primate Pluripotent Stem cells" (pPS cells) are pluipotent cells derived from pre-embryonic, embryonic, or fetal tissue at any time after fertilization, and have the characteristic of being capable under the right conditions of producing progeny of several different cell types, pPS cells are capable of producing -3progeny that are derivatives of each of the three germ layers: endoderm, mesoderm, and ectoderm, according to a standard art-accepted test, such as the ability to form a teratoma in a suitable host, or the ability to differentiate into cells having markers for tissue types of all three germ layers In culture. Included In the definition of pPS cells are embryonic cells of various types, exemplified by hES cells, 5 defined below; embryonic stem cells from other primates, such as Rhesus or marmoset stem cells (Thomson et al., Proc. Nat. Acad. Sc. USA 92:7844, 1995; Developmental Biology 38:133, 1998); and human embryonic germ (hEG) cells (Shamblott et al., Proc. Nat]. Acad. Sc. USA 95:13726, 1998). Other types of pluripotent cells are also Included In the term. Any cells of primate origin that are capable of producing progeny that are derivatives of all three germinal layers are included, regardless of whether they were derived 10 from embryonic tissue, fetal tissue, or other sources. it Is beneficial to use pPS cells that are karyotypically normal and not derived from a malignant source. Prototype "human Embryonic Stem cells" (hES cells) are described by Thomson et al. (Science 282:1145, 1998; U.S. Patent 6,200,806). The scope of the term covers pluripotent stem cells that are derived from a human embryo at the blastocyst stage, or before substantial differentiation of the cells Into the three 15 germ layers. Those skilled In the art will appreciate that except where explicitly required otherwise, the term includes primary tissue and established lines that bear phenotypic characteristics of hES cells, and derivatives of such lines that still have the capacity of producing progeny of each of the three germ layers. hES cell cultures are described as "undifferentiated" when a substantial proportion of stem cells and their derivatives in the population display morphological characteristics of undifferentiated cells, clearly 20 distinguishing them from differentiated cells of embryo or adult origin. Undifferentiated hES cells are easily recognized by those skilled in the art, and typically appear in the two dimensions of a microscopic view with high nuclear/cytoplasmic ratios and prominent nucleoll. It Is understood that colonies of undifferentiated cells within the population will often be surrounded by neighboring cells that are differentiated, Nevertheless, the undifferentiated colonies persist when the population is cultured or passaged under appropriate conditions, 25 and Individual undifferentiated cells constitute a substantial proportion of the cell population. Cultures that are substantially undifferentiated contain at least 20% undifferentiated hES cells on an ongoing basis, and may contain at least 40%, 60%, or 80% in order of Increasing preference (in terms percentage of cells with the same genotype that are undifferentiated). Whenever a culture or cell population is referred to In this disclosure as proliferating "without 30 differentiation", what Is meant Is that after proliferation, the composition is substantially undifferentiated according to the preceding definition. Populations that proliferate through at least four passages (~20 doublings) without differentiation will contain 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 originating culture. 35 A "nutrient medium" is a medium for culturing cells containing nutrients that promote proliferation. The nutrient medium typically contains Isotonic saline, buffer, a protein source (in the form of one or more added proteins or amino acids), and potentially other exogenously added nutrients and growth factors. A "conditioned medium" is prepared by culturing a first population of cells in a medium, and then harvesting the medium. The conditioned medium (along with anything secreted into the medium by the cells) -4may then be used to support the growth of a second population of cells. Where a particular Ingredient or factor is described as having been added to the medium, what Is meant Is that the factor (or a cell or particle engineered to secrete the factor) has been mixed Into the medium by deliberate manipulation. A fresh medium' is a medium that has not been purposely conditioned by culturing with a different cell 5 type before being used with the cell type it Is ultimately designed to support. Otherwise, no limitations are Intended as to its manner of preparation, storage, or use. It Is added fresh (by exchange or Infusion) Into the ultimate culture, where It may be consumed or otherwise processed by the cell types that are present. General Technlaues 10 General methods In molecular genetics and genetic engineering are described in the current editions of Molecular Cloning: A Laboratory Manual, (Sambrook at al., Cold Spring Harbor); Gene Transfer Vectors for Mammalian Cells (Miller & Calos eds.); and Current Protocols In Molecular Biology (F.M. Ausubel at al. ads., Wiley & Sons). Cell biology, protein chemistry, and antibody techniques can be found In Current Protocols In Protein Science (J.E. Colligan et al. ads., Wiley & Sons); Current Protocols In Cell Biology (J.S. Bonlfacino at 15 al., Wiley & Sons) and Current Protocols In immunology (J.E. Coiligan et al. eds., Wiley & Sons.). Reagents, cloning vectors, and kits for genetic manipulation referred to in this disclosure are available from commercial vendors such as BloRad, Stratagene, InvItrogen, ClonTech, and Sigma-Aldrich Co. Cell culture methods are described generally In the current edition of Culture of Animal Cells: A Manual of Basic Technique (RI. Freshney ed., Wiley & Sons); General Techniques of Cell Culture (M.A. 20 Harrison & I.F. Rae, Cambridge Univ. Press), and EmbryonIc Stem Cells: Methods and Protocols (K. Turksen ed., Humana Press). Other references of Interest Include Culture Is Our Business (M. McLuhan, Ballantine Books, 1970); and Understanding Media (M. McLuhan, SIgnet, 1970). Tissue culture supplies and reagents are available from commercial vendors such as GibcoIBRL, Nalgene-Nunc Intemational, Sigma Chemical Co., and ICN Blomedicals. 25 Sources of stem cells Embryonic stem cells can be isolated from blastocysts of members of the primate species (U.S. Patent 5,843,780; Thomson at al., Proc. Neti. Aced. Sci. USA 92:7844, 1995). Human embryonic stem (hES) cells can be prepared from human blastocyst cells using primary mouse fibroblast feeder cells, according to the 30 techniques described by Thomson at al. (U.S. Patent 6,200,806; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998) and Reubinoff at al, Nature Blotech. 18:399,2000, hES cell lines can also be derived on human feeders (U.S. Patent 6,642,048), or in conditions entirely free of feeder cells (US 2002/0081724 Al). Equivalent cell types to hES cells Include their pluripotent derivatives, such as primitive ectoderm-like (EPL) cells, as outlined in WO 01/51610 (Bresagen). 35 The Illustrations provided in the Example section ensue from work done with hES cells. However, except where otherwise required, the invention can be practiced using other cells that meet the definition of primate plurlpotent stem cells. By no means does the practice of this invention require that a human blastocyst be disaggregated in order to produce the hES or embryonic stem cells for practice of this invention. hES cells can be obtained -5from established lines obtainable from public depositories (for example, the WICell Research Institute, Madison WI U.S.A., or the American Type Culture Collection, Manassas VA, U.S.A.). Human Embryonic Germ (hEG) cells can be prepared from primordial germ cells as described In Shamblott et al., Proc. Nati. Acad. Sci. U.S.A. 95:13726, 1998 and U.S. Patent 6,090,622. U.S. Patent Publication 200310113910 Al 5 reports plurlpotent stem cells derived without the use of embryos or fetal tissue. It may also be possible to reprogram other progenitor cells Into hES cells by using a factor that induces the plurfpotent phenotype (Chambers et al., Cell 113:643, 2003; Mitsui et al., Cell 113:631, 2003). Under appropriate conditions, any cell with appropriate proliferative and differentiation capacities can be used for the derivation of differentiated tissues for use according to this Invention. 10 Propagation of hES cells Initially, most scientists In the field preferred to culture hES cells a layer of feeder cells to prevent differentiation, as originally described by Thomson (U.S. 5,843,780; U.S. 6,090,622). Early on, scientists at Geron discovered culture systems In which the elements contributed by the 15 feeder cells to promote proliferation of the undifferentiated phenotype could be provided In another form. U.S. Patent 6,800,480, WO 01/51616 (Geron Corp.), and Xu et al. (Nature Biotechnology 19:971, 2001) describe a feeder-free culture environment that supports proliferation without dIfferentiatIon. One aspect of the feeder-free culture method Is to support the hES cells by culturing on an extracellular matrix. The matrix can be deposited by preculturing and lysing a matrix-forming cell line (U.S. 20 6,800,480), such as the STO mouse fibroblast line (ATCC AccessIon No. CRL-1503), or human placental fibroblasts. The matrix can also be coated directly Into the culture vessel with isolated matrix components. Matrlgel@ Is a soluble preparation from Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane. Other suitable extracellular matrix components may Include laminin, fibronectin, proteoglycan, vitronectin, entactin, heparan sulfate, and so on, alone or In various 25 combinations. The matrix components may be human, and/or produced by recombinant expression. Substrates that can be tested using the experimental procedures described herein include not only other extracellular matrix components, but also polyamines, hydrogels, and other commercially available coatings. Another aspect of the feeder-free culture system is the nutrient medium. The medium will generally contain the usual components to enhance cell survival, Including isotonic buffer (i.e., a buffer that Is Isotonic 30 when adjusted to working concentration), essential minerals, and either serum or a serum replacement of some kind. A direct way to introduce hES supportive factors Is to precondition the medium with primary mouse embryonic fibroblasts (mEF) which can be prepared as described in U.S. Patent 6,200,806 or WO 01/51616. Also suitable as feeder cells are telomerized cell lines, and human cell lines obtained from differentiating hES 35 cells (U.S. Patent 6,642,048) or other primitive cell types. hES cell medium can be conditioned by culturing the feeder cells (typically irradiated or otherwise Inactivated). Medium conditioned by culturing for 1-2 hours at 37 0 C contains a concentration of factors that support hES cell culture for about 1-2 days. However, the conditioning period can be adjusted upwards or downwards, determining empirically what constitutes an adequate period. -- 6-- As an alternative to conditioned medium, hES cells can be grown In fresh (non-conditioned) medium containing added factors that Invoke the appropriate signal transduction pathways In undifferentiated cells. A suitable base medium for use without conditioning can be identified empirically. The medium typically contains a neutral buffer (such as phosphate and/or high concentration bicarbonate) In isotonic solution; a 5 protein nutrient (e.g., serum such as FBS, serum replacement, albumin, or essential and non-essential amino acids such as glutamine). It also typically contains lipids (fatty acids, cholesterol, an HDL or LDL extract of serum) and other ingredients found In most stock media of this kind (such as insulin or transferrin, nucleosides or nucleotides, pyruvate, a sugar source such as glucose, selenium In any Ionized form or salt, a glucocorticoid such as hydrocortisone and/or a reducing agent such as S-mercaptoethanol). 10 Many suitable commercially available base media have been developed for culturing proliferative cell types like hematopoletic cells. Exemplary are X-VIVOM 10 expansion medium (Biowhittaker) and QBSFTO-60 (Quality Biological Inc.) (Example 1). See also WO 98/30079 (Life Technologies Inc.) and U.S. 5,405.772 (Amgen). The X-VIVO"' 10 formulation contains pharmaceutical grade human albumin, recombinant human insulin and pasteurized human transferrin. Exogenous growth factors, artificial 15 stimulators of cellular proliferation or undefined supplements are not Included In the X-VIVOm 10 medium. They are also devoid of any protein-kinase C stimulators. QBSF""-60 is a serum-free formulation that contains recombinant or pasteurized human proteins. Other potential alternatives are Ex-Cell VPROm medium made by JRH Blosciences, and HyQ CDM4"" made by Hyclone. The base medium is supplemented with additives that promote proliferation of the undifferentiated 20 phenotype while inhibiting differentiation. Fibroblast growth factor at high concentration is especially effective to promote hES cell proliferation without differentiation. Exemplary are basic FGF (FGF-2), and FGF-4, but. other members of the family can also be used. Equivalent forms are species homologs, artificial analogs, antibodies to the respective FGF receptor, and other receptor activating molecules. It has been determined from gene expression analysis that undifferentiated hES cells express receptors for acidic FGF (FGF-1). At a 25 high concentration, FGF alone is sufficient to promote growth of hES cells In an undifferentiated state (Examples I and 2). Concentrations of FGF effective for promoting undifferentiated hES cell growth on their own usually have a lower bound of about 20, 30, or 40 ng/mL, with a practical upper bound of about 200, 500, or 1000 ng/mL Concentrations of at least 60, 80, or 100 ngimL bFGF are both reliable and cost effective. Equivalent concentrations of other forms and analogs of FGF can be determined empirically by weaning 30 cultures from bFGF into the proposed substitute, and monitoring the culture for differentiation according to the marker system described below. Culturino hES Cells in Suspension it has now been discovered that hES cells can be grown in suspension culture, rather than letting the 35 cells grow on a solid substrate. hES cells expanded by another culture method (or obtained from a primary source) are inoculated into a vessel adapted to keep the cells in suspension. The vessel walls are typically Inert or resistant to adherence of undifferentiated hES cells. There is also a means for preventing the cells from settling out, such as a stirring mechanism like a magnetically or mechanically driven stir bar or paddle, a shaking -7mechanism (typically attached to the vessel by the outside), or an inverting mechanism (i.e., a device that rotates the vessel so as to change the direction of gravity upon the cells). Vessels suitable for suspension culture for process development include the usual range of commercially available spinner or shaker flasks. Fermentors suitable for commercial production are Ceillgen 5 Plus" (New Brunswick Scientific Co.) and the Stirred-Tank Reactorym (Applikon Inc.). These bloreactors can be continuously perfused with medium or used in a fed-batch mode, and come in various sizes. Nutrient medium that helps maintain the undifferentiated phenotype and supports growth is replaced as needed (for example, by letting the cells settle out, replacing the medium, and then resuspending the cells). Growth is monitored, and the culture is split when required to make room for further growth. After a 10 suitable culture period, the cells are harvested and used for their intended purpose. Media and other components designed for growing hES cells in the absence of feeders on a solid surface may also work in suspension cultures. Either conditioned or fresh media can be used (Example 4). However, the dynamics of suspension culture provide the user with a further opportunity to optimize the various components of the culture system. Without intending to be limited by theory, It is a hypothesis of this 15 invention that suspension culture will be enhanced if the hES cells are permitted to form small undifferentiated clusters (the three-dimensional equivalent of an undifferentiated colony on a solid surface), possibly surrounded by cells partly differentiated Into stromal cells - or If the hES cells are dispersed, but shielded from the dynamic fluid forces that otherwise might cause differentiation. Optimization of the suspension culture system can be accomplished by empirical testing. 20 Undifferentiated cells from a previous surface or suspension culture are passaged to the test condition, and cultured.for a week or more. The cells can be examined periodically for characteristics of hES cells, for example, using the marker system described in the next section, and illustrated in Example 1. The cells can also be passaged back to a well-established culture system, and evaluated for classic morphological features of undifferentiated cells (Example 3). If the hES cells are Intended ultimately for differentiation into a 25 particular tissue type, then the ultimate test may not be the marker profile of the undifferentiated culture, but the ability of the cells to differentiate as required. The plurpotency of hES suspension cultures can be confirmed by sampling the cells, and either producing teratomas In SCID mice, or by staining EB-derived cells for markers representing all three germ layers (Example 3). The user can thereby optimize the system to achieve a high growth rate while retaining the full pluripotency of the cells (or at least the ability of the cells to 30 differntiate into the Intended tissue of Interest). Aspects of the culture system that may benefit from further optimization Include the nutrient medium. Alternative base media and alternative FGF additives are listed in the previous section. It may also be advantageous to use one or more additional additives, such as the following: - stem cell factor (SCF, Steel factor), other ilgands or antibodies that dimerize c-kit, and other 35 activators of the same signal transduction pathway * ligands for other tyrosine kinase related receptors, such as the receptor for platelet-derived growth factor (PDGF), macrophage colony-stimulating factor, Flt-3 ligand and vascular endothelial growth factor (VEGF) 0 factors that elevate cyclic AMP levels, such as forskoiln -8- * factors that induce gpl 30, such as UF or Oncostatn-M: * hematopoletic growth factors, such as thrombopoletin (TPO) * transforming growth factors, such as TGF01 * other growth factors, such as epidermal growth factor (EGF) 5 * neurotrophlns, such as CNTF With a view to protecting the cells from adhering to each other, adhering to the vessel wall, or forming clusters that are too big, it may be beneficial to include an antI-clumping agent, such as those sold by Invitrogen (Cat # 0010057AE). While the cells have some capacity to form their own extracellular matrix to a limited extent, It may 10 also be beneficial to include one or more extracellular matrix components either dissolved or suspended In the medium. A suitable working range to keep laminin in suspension is about 10 to 33 pg/mL Other . candidate matrix components for suspension cultures Include some of those listed earlier, particularly fibronectin, proteoglycan, vltronectin, and artificial equivalents thereof. The extracellular matrix may assist the cells in forming small aggregates of an appropriate size. 15 Alternatively or in addition, the suspension culture may contain particulate carriers that create surfaces within the suspension, but still provide the benefits of culturing the cells in a three-dimensional space. The cells are cultured and passaged In the same way, except that the particles are retained in the vessel during medium exchange, and more particles are added when the cells are split. One type of microcarrier Is solid spherical or semi-spherical particles made from glass, plastic, dextran 20 having a positive charge to augment cell attachment (Cytodex), and so on. Another type is disk-shaped culture plastic, such as the Fibra-cel Disks T sold by New Brunswick Scientific Co, Inc. A gram of these disks provide a surface area of 1200 cm 2 . Solid carriers are optionally coated with an hES cell friendly extracellular matrix, such as laminin, so that the attached cells have the same microenvironment as cells plated onto a solid surface. 25 Another type of microcarrier is macroporous particles of various pore sizes that permit the cells to reside in the interior as well as the outside, to potentially enhance the protective effect In order to recover the hES cells with minimal disruption, it is beneficial to use particles made of a material such as agarose that can easily be dissolved or dispersed by gentle mechanical or enzymatic action, thereby releasing the cells for harvest or further culture. 30 Characteristics of undifferentiated hES cells Human ES cells cultured according to this invention have the characteristic morphological features of undifferentiated stem cells, In the two dimensions of a standard microscopic image, hES cells have high nuclear/cytoplasmic ratios In the plane of the image, prominent nucleoll, and compact colony formation with 35 poorly discernable cell junctions. Cell lines can be karyotyped using a standard G-banding technique (available at many clinical diagnostics labs that provides routine karyotyping services, such as the Cytogenetics Lab at Oakland CA) and compared to published human karyotypes. It Is desirable to obtain cells that have a "normal karyotype", which means that the cells are euploid, wherein all human chromosomes are present and are not noticeably altered. -9hES cells can be characterized by expressed cell markers detectable by antibody (flow cytometry or lmmunocytochemlstry) or by reverse transcriptase PCR. hES cells typically have antibody-detectable SSEA-4, Tra-1-60, and Tra-1-81, but flttle SSEA-1, and have alkaline phosphatase activity. Panels of suitable markers detectable at the mRNA level are listed in application US 2003/0224411 Al (Geron Corp.) 5 Exemplary are Cripto, gastrin-releasing peptide (GRP) receptor, podocalyxin-Ilke protein (PODXL), human telomerase reverse transcriptase (hTERT), and the POU transcription factor Oct 3/4. As already described, an important feature of propagated hES cells Is a potential to differentiate Into cells of all three germ layers: endoderm, mesoderm, and ectoderm. Pluripotency of hES cells can be confirmed by forming teratomas In SCID mice, and examining them for representative tissues of all three 10 germ layers. Alternatively, pluripotency can be determined by allowing hES cells to differentiate non specifically (for example, by forming embryold bodies), and then determining the cell types represented In the culture by Immunocytochemistry (Example 3). Potential of hES cells to differentiate into particular cell lines can be determined according to procedures described in the next section. 15 Uses of propagated hES cells This Invention provides a method by which large numbers of pluripotent cells can be produced on a commercial scale. The cells are useful for a number of research and commercial purposes In the undifferentiated form, or can be directed to differentiate into a particular cell type. Undifferentiated hES cells can be used to screen for factors (such as small molecule drugs, peptIdes, 20 polynucleotides, and the like) or condItions (such as culture conditions or manipulation) that affect the characteristics of hES cells in culture. hES cultures can also be used for the testing of pharmaceutical compounds in drug research. Assessment of the activity of candidate pharmaceutical compounds generally involves combining the differentiated cells of this Invention with the candidate compound, determining any resulting change, and then correlating the effect of the compound with the observed change. Cytotoxicity or 25 metabolic effects can be determined by cell viability, morphology, the expression or release of certain markers, receptors or enzymes, DNA synthesis or repair, and so on. hES cells cultured according to this invention can be used to make differentiated cells of various commercially and therapeutically important tissue types. 30 Liver Cells Hepatocytes can be differentiated from hES cells using an inhibitor of histone deacetylase, as described In U.S. Patent 6,458,689 and PCT publication WO 01/81549 (Geron Corporation). Undifferentiated hES calls are cultured in the presence of an inhibitor of histone deacetylase. Staged protocols for differentiating hES cells into hepatocytes are described. In US 2005/0037493 Al 35 (Geron Corp.). Cells are cultured with several combinations of differentiation and maturation agents in sequence, causing the hES cells to differentiate first Into early endoderm or hepatocyte precursors, and then to mature hepatocyte-Ilke cells. Briefly, differentiation into endoderm4lke cells can be initiated using either butyrate, DMSO or fetal bovine serum, optionally in combination with fibroblast growth factors. Differentiation can then continue using a commercially available hepatocyte culture medium, Including factors such as -10hepatocyte growth factor (HGF), epidermal growth factor (EGF), and/or bone morphogenic protein (e.g., BMP-2, 4, or 7) In various combinations. Final maturation may be enhanced by the presence of agents such as dexamethazone or Oncostatin M. Cells are obtained that express asialoglycoproteln, glycogen storage, cytochrome p450 enzyme expression: glucose-6-phosphatase activity, and morphological features of 5 hepatocytes. Nerve cells Neural cells can be generated from hES cells according to the method described in U.S. Patent 6,833,269; Carpenter et al., Exp Neurol. 2001;172(2):383-97; and WO 03/000868 (Geron Corporation). 10 Undifferentiated hES cells or embryold body cells are cultured in a medium containing one or more neurotrophins and one or more mitogens, generating a cell population in which at least -60% of the calls express A2B5, polyslalylated NCAM, or Nestin and which is capable of at least 20 doublings in culture. Exemplary mitogens are EGF, basic FGF, PDGF, and IGF-1. Exemplary neurotrophins are NT-3 and BDNF. The use of TGF-p Superfamily Antagonists, or a combination of cAMP and ascorbic acid, can be used to 15 increase the proportion of neuronal cells that are positive for tyrosine hydroxylase, a characteristic of dopaminergic neurons. The proliferating cells can then be caused to undergo terminal differentiation by culturing with neurotrophins in the absence of mitogen. Oligodendrocytes can be generated from hES cells by culturing them as cell aggregates, suspended in a medium containing a mitogen such as FGF, and oligodendrocyte differentiation factors such as 20 trilodothyronine, selenium, and retinoic acid. The cells are then plated onto a solid surface, the retinoic acid Is withdrawn, and the population is expanded. Terminal differentiation can be effected by plating on poly-L-lysine, and removing all growth factors. Populations can be obtained in which over 80% of the cells are positive for oligodendrocyte markers NG2 proteoglycan, A2B5, and PDGFRa, and negative for the neuronal marker NeuN. See PCT publication WO 04/007696 and Keirstead et al., J Neurosci. 25 2005;25(19):4694-705. Heart cells Cardiomyocytes or cardlomyocyte precursors can be generated from hES cells according to the method provided in WO 03/006950. The cells are cultured in suspension with fetal calf serum or serum 30 replacement, and optionally a cardlotrophic factor that affects DNA-methylatlon, such as 5-ezacytidine. Alternatively, cardlomyocyte clusters can be differentiated directly on a solid substrate using a combination of Activin A and Bone Morphogenic Protein 4: Spontaneously contracting cells can then be separated from other cells in the population, by density centrifugation. Further process steps can Include culturing the cells so as to form clusters known as cardiac bodies M, 35 removing single cells, and then dispersing and reforming the cardiac bodies' in successive iterations. Populations are obtained with a high proportion of cells staining positive for cTnl, cTnT, cardiac-specific myosin heavy chain (MHC), and the transcription factor Nkx2.5. See WO 03/006950, Xu at al., Ciro Res. 2002;91(6):501-8; and PCT/US2005/009081 (Geron Corporation). - 11 - Other cell types Islet cells can be differentiated from hES cells by initiating differentiation of hES cells by culturing in a medium containing a combination of several factors selected from Activin A, a histone deacetylase inhibitor (such as butyrate), a mitogen (such as bFGF); and a TGF-P Superfamily antagonist (such as noggin). The 5 cells can then be matured by culturing with nicolinamide, yielding a cell population In which at least 5% of the cells express Pdxl, Insulin, glucagon, somatostatn, and pancreatic polypeptide. See WO 031050249 (Geron Corp.). Hematopoletic cells can be made by coculturing hES cells with murine bone marrow cells or yolk sac endothelial cells was used to generate cells with hematopoletic markers (U.S. Patent 6,280,718). 10 Hematopoletic cells can also be made by culturing hES cells with hematogenic cytokines and a bone morphogenic protein, as described in US 2003/0153082 Al and WO 031050251 (Robarts Institute). Mesenchymal progenitors can be generated from hES cells according to the method described in WO 03/004605. The hES-derived mesenchymal cells can then be further differentiated Into osteoblest lineage cells in a medium containing an osteogenic factor, such as bone morphogenic protein (particularly 15 BMP-4), a ligand for a human TGF-3 receptor, or a lgand for a human vitamin D receptor (WO 03/004805; Sotle et al., Cloning Stem Cells 2003;5(2):149-55). Chondrocytes or their progenitors can be generated by culturing hES cells in microaggregates with effective combinations of differentiation factors listed in WO 03/050250 (Geron Corp.). Other differentiation methods known in the art or subsequently developed can be applied to hES cells 20 cultured according to this invention. hES derived cells can be used for drug screening, preparing pharmaceutical compositions, research, and many other worthwhile purposes. Commercial distribuion Components of the culture system of this invention may be offered for sale, sold, or otherwise 25 distributed from the place of manufacture for use by another entity for any purpose. Components may also be sold or distributed together in various useful combinations, such as two or more of the following: e media suitable for culturing hES cells in suspension factors * extracellular matrix components or thickeners present in or to be added to the medium * microcarriers present In or to be added to the medium 30 e vessels adapted for suspension culture * the hES cells themselves, either growing in the culture system, or stored In another form, but Intended for use In the culture system The products and product combinations are packaged In suitable containers, optionally in kit form, and 35 may be accompanied by written Information on the use of the materials according to this Invention - such as maintaining or expanding hES cells. The Information may be written in any language on any communication medium accessible and understandable by the Intended user. It may take the form of a label on the container or the kit, or a product Insert packaged with the container and distributed together. Equivalent forms are -12descriptions, instructions, or explanations written in hard copy or In electronic form available to the user or the Intended user as reference or resource material associated with the commercially distributed product. The examples that follow are Illustrations not meant to lmit the claimed invention 5 EXAMPLES Example 1: Growing pluripotent stem cells In rapid expansion medium A line of hES cells was obtained that had originally been grown on mouse embryonic fibroblast feeder 10 cells, and then expanded for 20 passages In a feeder-free environment comprising Matrigel@ extracellular matrix and conditioned medium, according to U.S. Patent 6,800,480; as elaborated In Xu et al., Stem Cells 2005;23(3):315-23. The hES cells were next weaned onto X-VIVO" 10 expansion medium from Blowhittaker; or QBSFT"-60 from Quality Biological Inc. For use In these experiments, the X-VIVOe 10 medium was 15 supplemented with the usual goodies: namely, 2 mM L-glutamlne, 1% non-essential amino acids (Gibco), 0.1 mM p-merceptoethanol, and 8 nglmL bFGF. The medium was further supplemented with 8 ng/mL or 40 ng/mL of bFGF (Gibco); 40 ng/mL of bFGF and 15 ng/mL of SCF (R & D System); or 40 ng/mL of bFGF and 75 ng/mL of Ft3 ligand (R & D System). QBSF m -60 medium was supplemented with 0.1 mM 1-mercaptoethanol, 1% non-essential amino acids (GIbco) and 40 nglmL of bFGF. hES cells cultured In mEF 20 conditioned medium were used as control In these experiments. The hES cells were first passaged onto MatrigeI coated plates using collagenase IV, and cultured for 2 days with conditioned medium. On day 2, the conditioned medium was replaced with 80% unconditioned ES medium plus 20% expansion medium. Cells were fed fresh daily and passaged weekly. The proportion of expansion medium was Increased by 20% approximately every 2 days until the cells were completely 25 weaned, and then grown until they had been passaged 6 more times. Figure 1 shows colonies of hES cell at the end of 6 passages (sufficient for full adaptation) in the following media: (A) mEF conditioned medium + bFGF (8ng/ml); (B) X-VIVOTm 10 + bFGF (40ng/mL): (C) X-VIVOm 10 + bFGF (40ng/mL) + stem cell factor (SCF, Steel factor) (15 ng/mL); (D) X-VIVO" h 10 + bFGF (40ng/mL) + FIt3 ligand (75 ng/mL); (E) QBSF

T

m-60 + bFGF (40ng/mL). 30 The following table shows the average total cell expansion per passage, for undifferentiated hES cells cultured for 4 passages in mEF conditioned medium, or for 7 passages In X-VIVOlm 10 or QBSFY&l-60. -13- Table 1: Growth Rates for ES Cell Cultures Medium Average Cell Expansion per Passage mEF conditioned medium 2.2 fold X-VIVO 10 + bFGF (40ng/mL) 6.0 fold X-VIVOm 10 + bFGF (40ng/mL) + SCF (15 ng/mL) 8.2 fold X-ViVOT 10 + bFGF (40ngmL) + F1t3 ligand (75 5.0 fold ng/mL) QBSF"-60 + bFGF (40ng/mL) 6.4 fold The average expansion of cells per passage In X-VIVOT 10 and QBSFm-60 was greater than the cells cultured In mEF conditioned medium culture. The cells In mEF conditioned medium were passaged on average every 7 days, while the cells In X-VIVOTm 10 and QBSFm-60 were passaged on average every 5 5 days. Thus, the rate of expansion In unconditioned X-VIVOT' 10 or QBSFM-60 was -3.2 to 5.2 times faster than In mEF conditioned ES medium. Figure 2 shows the gene expression profile of hTERT and Oct3/4. The RNA was Isolated from the cells using High Pure RNA Isolation Kit (Roche Diagnostics) and evaluated by Taqmanm assay (real time RT-PCR). The gene expression In each of the test condition is plotted relative to expression in the control 10 culture. Taking into consideration the instrument error and.assay variability, differences In expression between the test and control samples are only significant If greater than 2-fold. The analysis shows expression of hTERT and Oct-3/4 decreases somewhat upon adaptation to unconditioned X-VIVO"M 10 or QBSFm-60 medium (first four bars in each set), but returns to standard levels when the cells are passaged back Into mEF conditioned medium (last three bars in each set). 15 To confirm that cells cultured in unconditioned medium retain their pluripotency, embryold bodies were formed and analyzed by immunocytochemistry for phenotypic markers representing each of the three gemi layers. After passage 7 In expansion medium, the cells were dissociated into small clumps using 200 U/mL collagenase IV at 37'C for 10 min, placed In suspension culture In differentletion medium (DMEM + 10% FBS) for 4 days, then transferred onto poly-L-omlthine hydrobromide coated plates for a further 10 days. 20 They were fixed In 4% paraformaldehyde, permeabilized, and labeled by immunocytochemistry. Figure 3 shows the results. hES cells passaged 7 times In unconditioned X-VIVOm 10 medium stained for a-fetoproten (representing endoderm); muscle actin (representing mesoderm), and p-tubulln Ill (representing ectoderm). Thus, hES cells can be expanded in fresh (non-conditioned) media In a feeder-free environment at a 25 rapid rate suitable for commercial production - as much as 2- to 5-fold faster or more compared with growth in conditioned medium or on feeder cells. The cells retain the morphology of undifferentiated hES cells, and can be differentiated into derivative cells representing all three germ layers. -14- Example 2: Culture of hES cells in a defined system free of animal-based products hES cells cultured In MEF-CM on MatrIgel@ were passaged to a fresh (non-conditioned) serum free medium: X-VIVO" 10 supplemented with Glutamine, non-essential amino acids and p-mercaptoethanol, plus 80 ng/mL human basic FGF on Matrigel@, and then adapted to surfaces coated with human laminin. 5 Alternatively, cryopreserved cells were directly thawed into the same medium containing 80 ng/mL hbFGF. The cells were passaged every 5-8 days using Collagenase IV. Cultures grown under these conditions were similar or better then cultures on Matrigel@. (A) morphology for cells grown In mEF conditioned medium; (B) morphology fin defined medium on laminin; (C) Surface marker SSEA-4 expression in mEF-CM (H1p62) or defined medium (H1p34+28); (D) Expression of 10 surface marker Tra-1-60 in mEF-CM or defined medium. Culture performance in the defined medium on laminin was superior: very large ES cell colonies were observed, with colonies representing about 80% of the culture. Levels of marker expression were as follows: Table 2: Marker Expression in Defined Culture Conditions Passage no. Culture Surface marker Relative gene expression medium expression____________________ SSEA-4 Tra-1-80 hTERT Oct3/4 Cripto Experiment 1: HI p41 Conditioned 79% 93% 1.00 1.00 1.00 Hip3i+10 Defined 92% 87% 2.85*0.58' 0.74±0.04 1.82±0.62 Experiment 2: H1p44 Conditioned 81% 91% 1.00 1.00 1.00 Hip34+11 Defined 77% 84% 1.11 ± 0.38 0.5760.24 0.76k *0.39 Experiment 3: H1p47 Conditioned 78% 92% 1.00 1.00 1.00 H1p35+12 Defined 80% 86% 2.00±0.15 0.86 i0.20 3.12 ±0.91 * mean * SD for 3 RT-PCR detmnninations 15 Expression of other markers characteristic of undifferentiated hES cells was also comparable: Measured by real-time PCR, the levels of hTERT and Cripto were the same or greater In defined medium compared with mEF-CM, while the expression of Oct 3/4 was lower by about 28% (average of three experiments). TRAP analysis showed that the cells retained telomerese enzyme activity. Cells grown in completely defined culture system at p34+11 (75 days) were harvested and used to 20 make teratomas in SCID mice to evaluate pluripotency. The teratomas showed evidence for pigmented epithelium (endoderm); renal tissue (endoderm); mesenchymal tissue (mesoderm); and neural tubes (ectoderm). This confirms that the cells retained their pluripotency. -15- Examole 3: Culturina hES cells in suspension With a view to Increasing the yield of hES cells per culture volume, the cells were cultured In suspension, and then evaluated for morphology and their ability to form differentiated cells representative of 5 all three germ layers. H9 hES cells grown on Matrigel@ were harvested from 6-well plates and seeded into a spinner flask under the following condition e Vessel: 100 mL spinner flask 0 Inoculation (seeding) density: 3.6 x 105 cells/mL; 10 e Medium volume: 50 mL per spinner flask * Medium used: mEF conditioned medium containing bFGF (8 ng/mL) * Aglitation rate: 20 rpm (Belico carrier magnetic stirrer) . Atmosphere: 37"C C02 incubator 0 Medium exchange: Every' other day (exchanged by letting the cells settle down and 15 replacing the supernatant) H9 hES cells were maintained in the spinner flask under these conditions for 6 days. Figure 4 (upper panel) shows the results. Following an initial decline during which the culture was established, the cell number began to rise from day 2 through day 6. At this point, the cells were plated back into a 6-well plate coated with Matrigel@ to determine whether 20 they still had the phenotype of undifferentiated cells. The culture continued using mEF-CM medium containing bFGF (8 ng/mL). Figure 4 (lower panel) shows the results. After a single passage, the cells grew and exhibited morphology of undifferentiated cells. Pluripotency of the cells was evaluated by forming embryold bodies. The cells were harvested from 25 confluent culture using Collagenase IV, and transferred to a low attachment 6-well plate In DMEM + 20% FBS. EBs were formed and maintained for four days. The EBs were then replated onto polyornithine-coated chamber slides. After a further 11 days, the EB outgrowths were stained for a-fetoprotein (endoderm), muscle actin mesodermm) and p-tubulIn with neuron morphology (ectoderm). Figure 5 shows the results. The top row shows cells differentiated from the hES cells maintained In 30 suspension culture, and then plated back onto lamlnIn under standard conditions. The lower row shows cells differentiated from the same hES cell line that were maintained throughout as plated cells. As shown In these Images, hES cells maintained In suspension maintained their full capacity to differentiate into derivatives of all three-germ layers. --16- In another experiment, H9 hES cells were cultured under the following conditions: . Vessel: 100 mL spinner flask - Inoculation (seeding) density: 3.5 x 10" cells/mL . Medium volume: 50 mL per spinner flask 5 0 Medium used: mEF-CM+ bFGF (8 ng/mL) . Agitation rate: 20 rpm (as before) " Medium exchange: Once every three days Figure 6 shows the results. Once the culture was established, the cells were maintained happily for the full 12 day period. 10 Example 4: Long-term suspension culture This experiment was done with another hES cell line. The cells were cultured using a shaker flask Instead of a spinner flask under several different culture conditions, and the culture was extended for over two months. 15 e H1 hES cells were harvested from 6-well plates (growing on Matrlgel@ In mEF conditioned medium) and seeded Into shaker flasks under the following conditions: * Vessel: 100 mL Shaker Flask 0 Inoculation (seeding) density: 5.0 x 105 cells/mL * Medium volume: 15 mL per shaker flask 20 0 Agitation rate: 80 rpm (Labline rotator/shaker In a 37 0 C CO 2 incubator) a Medium exchange: Every other day Initially, once every 2-3 days later The media used and culture periods were as follows: A: mEF-CM + bFGF (8 ng/mL). Maintained for 98 days. B: mEF-CM + bFGF (8 ng/mL) + laminin (33pg/mL to begin with, -10 pg/mL thereafter for the 25 rest of the culture). Maintained for 49 days. C: X-VIVO 10 + FGF (40 ng/mL) + Ft-3 (75 ng/mL). Maintained for 11 days. D: X-VIVOm 10 +FGF (40 ng/mL) + Flt-3 (75 ng/mL) + laminin (33 pg/mL to begin with, -10 Pg/mL thereafter for the rest of the culture). Maintained for 11 days, Cell counts over the period of the culture are shown in the following Table. 30 -17- Table 3: Growth of hES Cells In Suspension Cultures Medium A Medium B Medium C Medium D (106 cells/mL) (10' cells/mL) (106 cells/mL) (10' camis/mL) 0 5.0 5.0 6.0 5.0 1 (not counted) 7.5 6.9 7.3 3 5.9 5.0 2.8 2.8 11 2.7 2.3 2.5 2.4 32 0.3 0.15 98 14 To determine whether the cells were maintaining the undifferentiated phenotype, cells cultured for 4 weeks in Medium A were plated beck onto Matrigell In mEF conditioned medium containing bFGF. 5 Figure 7 shows the results. After passaging, the cells derived from the suspension culture were shown to grow and exhibit characteristic undifferentiated hES cell colonies. These data Indicate that hES cells can be maintained in suspension culture for at least three months, potentially undergoing expansion by 3- to 40-fold after the culture becomes fully established. 10 Example 5: Suspension cultures using fresh medium The next experiment evaluates alternative additives for use with fresh (unconditioned) medium In suspension cultures. hES cells are harvested from a surface culture in fresh medium under standard conditions (substrate of human laminin from Sigma, coated onto 8-well plates at 2 pg/cm 2 ; X-VlVO 10T medium containing 80 15 ng/mL bFGF and 0.5 ng/mL TGF01). The harvested cells are passaged into suspension culture in 100 mL spinner flasks, using 50 mL per flask at an initial density of -5 x 106 cellsimL. The following medium alternatives are evaluated: 1) X-VIVOM10+ bFGF (80 ng/mL) 2) X-VIVOm10 + bFGF (50 ng/mL)+ TGF31 (0.5 ng/nL) 20 3) X-VlVOm10+ bFGF (40 ng/mL)4 +TGFS1 (0.5 ng/mL) 4) X-VVOM 10 + bFGF (80 ng/mL) + TGFD1 (0.5 ng/mL) + 10 pg/mL human laminin 5) X-VIVOM 10 + bFGF (80 ng/mL) + TGFp1 (0.5 ng/mL) + 50 pg/mL human serum albumin Each spinner flask is placed on a Belico carrier magnetic stirrer (Belico Biotechnology, Vineland NJ) In a 37*C CO 2 incubator, at an initial agitation rate to 20 rpm. The agitation rate is adjusted to keep the cells In 25 suspension and provide sufficient aeration, while minimizing shear forces. Medium Is changed every 2-3 days as before, monitoring cell count, and the flasks are split as needed. -18- At regular Intervals, cells are sampled from each flask and plated back onto a laminin coated surface to evaluate morphology, Cells returned to surface cultures and cells taken directly from the suspension cultures are tested for pluripotency by immunocytochemical staining of EB derived cells, as in Example 3 5 The compositions end procedures described above can be effectively modified without departing from the claimed invention and its equivalents. -19-

Claims (13)

1. A culture of human embryonic stem (hES) cells in suspension comprising FGF, wherein the hES cells are not grown on a solid substrate and wherein the hES cells are substantially undifferentiated. 5

2. The culture of claim 1, wherein the cells are cultured in a medium containing fibroblast growth factor at a concentration of at least about 40 ng/mL.

3. The culture of claim 1 or 2, wherein the cells are cultured in a medium containing one or more soluble 10 or suspended extracellular matrix components.

4. The culture of claim 3, wherein the extracellular matrix component(s) include human laminin and/or human fibronectin. 15

5. A method for culturing hES cells substantially undifferentiated, comprising: a) suspending the cells in a nutrient medium comprising FGF; b) maintaining the cells in suspension while culturing; c) changing the medium periodically; d) optionally splitting the culture from time to time so as to reduce cell density; and finally 20 e) harvesting cells from the culture.

6. The method of claim 5, wherein the cells are cultured in a medium containing fibroblast growth factor at a concentration of at least about 40 ng/mL. 25

7. The method of claim 5 or 6, wherein the cells are cultured in a medium containing one or more soluble or suspended extracellular matrix components.

8. The method of claim 7, wherein the extracellular matrix component(s) include human laminin and/or human fibronectin. 30

9. The method of any of claims 5 to 8, wherein the cells are cultured in suspension for at least two months.

10. The method of any of claims 5 to 9, wherein the cells undergo at least a 3-fold expansion while in 35 suspension culture. 20

11. The method of any of claims 5 to 10, further comprising plating the harvested cells back onto a solid surface and continuing to culture the cells so as to maintain a substantially undifferentiated cell population. 5

12. The method of any of claims 5 to 11, further comprising differentiating the harvested cells.

13. A kit when used for culturing hES cells undifferentiated in suspension according to claim 1, comprising a nutrient medium that contains at least 40 ng/mL fibroblast growth factor. 21