Novel method and culture medium for culturing pluripotent stem cells

Abstract

To provide a culture medium for a method for culturing pluripotent stem cells in suspension culture as single cells without aggregates, and also provide a method for expanding and maintaining undifferentiated pluripotent stem cells.SOLUTION: There is provided a culture medium comprising a basic fibroblast growth factor (bFGF) at concentration of at least 50 ng / ml and an IL6RIL6 chimera body. The concentration of the bFGF can be selected from a range of between 50 ng / ml to 150 ng / ml. The culture medium may include a serum replacement that is free of animal-derived contaminants. The IL6RIL6 chimera body may be used at concentration of 50 to 150 pg / ml.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention, in some embodiments thereof, relates to a method for culturing pluripotent stem cells in suspension culture as single cells free of aggregates, and isolated populations of pluripotent stem cells produced by said method, and more particularly, but not exclusively, to a novel culture medium capable of maintaining pluripotent stem cells in an undifferentiated state, and a method for culturing pluripotent stem cells in two-dimensional or three-dimensional culture systems while maintaining them in a proliferative, pluripotent, undifferentiated state. [Background technology]

[0002] The specific differentiation potential of human embryonic stem cells (hESCs) makes them the basis of one of the best models for studying early human development, lineage commitment, differentiation processes, and for use in industrial purposes and cell-based therapies.

[0003] Induced pluripotent (iPS) cells are somatic cells that can be reprogrammed into ESC-like cells with the ability to differentiate into representative tissues of the three embryonic germ layers both in vitro and in vivo. Mouse or human iPS cells were generated by overexpression of four transcription factors, c-Myc, Oct4, Klf4 and Sox2, in somatic cells. iPS cells have been shown to form the same colony morphology as ESCs and express several typical ESC markers, such as Myb, Kit, GDf3 and Zic3, but not minor markers, such as Dnmt3a, Dnmt3b, Utf1, Tcl1 and LIF receptor genes, thereby confirming that iPS cells are similar but not identical to ES cells [Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Meissner et al., 2007; Okita et al., 2007]. Yu Junying et al. (Science 318:1917-1920, 2007) found common gene expression patterns in fibroblast-derived iPS cells and hESCs.

[0004] Further studies have shown that iPS cells can be obtained by transforming somatic cells with Oct4, Sox2, Nanog and Lin28, while omitting the use of the oncogene C-Myc [Yu J. et al., 2007, Science, 318:1917-1920; Nakagawa et al., 2008]. Improvements in iPS cell derivation methods include the use of plasmids instead of viral vectors or derivation without genomic integration at all, which may simplify the future use of iPS cells in clinical applications [Yu J. et al., Science. 2009, 324:797-801].

[0005] Currently available iPS cells are derived from embryonic fibroblasts [Takahashi and Yamanaka, 2006; Meissner et al., 2007], fibroblasts generated from hESCs [Park et al., 2008], fetal fibroblasts [Yu et al., 2007; Park et al., 2008], foreskin fibroblasts [Yu et al., 2007; Park et al., 2008], adult skin and skin tissue [Hanna et al., 2007; Lowry et al., 2008], b-lymphocytes [Hanna et al., 2007], and adult liver and stomach cells [Aoi et al., 2008].

[0006] iPS cells, like hESCs, are traditionally cultured in two-dimensional culture with a feeder layer, which allows their continuous growth in an undifferentiated state. For example, iPS cells have been cultured on a feeder layer consisting of inactivated mouse embryonic fibroblasts (MEFs) or foreskin fibroblasts in the presence of a medium supplemented with fetal bovine serum (FBS) [Takahashi and Yamanaka, 2006; Meisnner et al., 2007]. Further improvements in the culture method include culturing iPS cells on MEF feeder layers in the presence of a more defined medium containing serum replacement and 10 ng / ml basic fibroblast growth factor (bFGF) (Park et al., 2008). However, for clinical applications (e.g. cell-based therapy) or industrial purposes, iPS cells need to be cultured under a defined xeno-free (e.g. animal-free) and scalable culture system with controlled processes.

[0007] PCT Publication WO 2007 / 026353 discloses a well-defined xeno-free medium containing a TGFβ isoform or a chimera formed between IL6 and a soluble IL6 receptor (hereinafter referred to as IL6RIL6) for the purpose of maintaining human embryonic stem cells in an undifferentiated state in a two-dimensional culture system.

[0008] US Patent Application Publication No. 2005 / 0233446 discloses a defined medium containing bFGF, insulin and ascorbic acid to maintain hESCs in an undifferentiated state when cultured on Matrigel™.

[0009] Ludwig TE et al., 2006 (Nature Biotechnology, 24:185-7) discloses TeSR1 defined medium for culturing hESCs on a matrix consisting of collagen IV, fibronectin, laminin and vitronectin.

[0010] US Patent Application Publication No. 2009 / 0029462 discloses methods for expanding pluripotent stem cells in suspension using microcarriers or cell encapsulation.

[0011] PCT Publication WO 2008 / 015682 discloses a method for expanding and maintaining human embryonic stem cells in suspension culture under culture conditions without substrate attachment.

[0012] US Patent Application Publication No. 2007 / 0155013 discloses a method for growing pluripotent stem cells in suspension using a carrier that adheres to the pluripotent stem cells.

[0013] U.S. Patent Application Publication No. 2008 / 0241919 (Parsons et al.) discloses a method for culturing pluripotent stem cells in suspension culture in a cell culture vessel containing an acellular matrix in a medium containing bFGF, insulin and ascorbic acid.

[0014] US Patent Application Publication No. 2008 / 0159994 (Mantalaris et al.) discloses a method for culturing pluripotent ES cells encapsulated inside alginate beads in three-dimensional culture in a medium containing serum replacement and bFGF.

[0015] US Patent Application Publication No. 2007 / 0264713 discloses a method for culturing undifferentiated stem cells in suspension on microcarriers in a vessel using conditioned medium.

[0016] PCT Publication WO 2006 / 040763 discloses isolated primate embryonic cells derived from expanded blastocysts (e.g., at least 9 days after fertilization) and methods of making and using the cells.

[0017] Additional background art includes U.S. Patent Application Publication No. 2009 / 0130759; Stankoff B. et al., J. Neuroscience 22:9221-9227, 2002; Ernst M. et al., Journal of Biological Chemistry, 271:30136-30143, 1996; Roeb E et al., Hepatology, 1993, 18:1437-42; U.S. Patent Application Publication No. 2004 / 0235160; Pera MF et al., 2000. Journal of Cell Science 113, 5-10. Human embryonic stem cells. Commentary. Summary of the Invention

[0018] In accordance with an aspect of some embodiments of the present invention, OCT4 + / TRA1-60 - / TRA1-81 - / SSEA1 + / SSEA4 - In one embodiment, an isolated population of human pluripotent stem cells is provided, the human pluripotent stem cells comprising at least 50% human pluripotent stem cells characterized by an expression signature of a gene encoding a pluripotent stem cell that is capable of differentiating into each of the germ layers of an embryo: endoderm, ectoderm and mesoderm.

[0019] According to an aspect of some embodiments of the present invention, there is provided a method of expanding and maintaining pluripotent stem cells (PSCs) in an undifferentiated state, comprising: (a) passaging the PSCs in suspension culture by mechanical dissociation of PSC clumps into single cells for at least two passages and up to ten passages, thereby obtaining a suspension culture of clump-free PSCs; and (b) passaging the suspension culture of clump-free PSCs without dissociation of the clumps, thereby expanding and maintaining the PSCs in the undifferentiated state.

[0020] According to some embodiments of the invention, the method further comprises culturing the PSCs under conditions that allow expansion of the pluripotent stem cells in the undifferentiated state.

[0021] According to an aspect of some embodiments of the present invention, there is provided a method of deriving an embryonic stem cell line, comprising: (a) obtaining embryonic stem cells (ESCs) from a pre-implantation stage blastocyst, a post-implantation stage blastocyst, and / or fetal reproductive tissue; and (b) passaging the ESCs in suspension culture by mechanical dissociation of ESC clumps into single cells for at least two passages and up to ten passages, thereby obtaining a clump-free suspension culture of ESCs; and (c) passaging the suspension culture of clump-free ESCs without dissociation of the clumps, thereby deriving the embryonic stem cell line.

[0022] According to some embodiments of the invention, the method further comprises culturing the ESCs under conditions that allow expansion of the undifferentiated embryonic single stem cells.

[0023] According to some embodiments of the invention, the passaging is performed under conditions that are free of enzymatic dissociation.

[0024] According to an aspect of some embodiments of the present invention there is provided a method of cloning pluripotent stem cells comprising culturing a single pluripotent stem cell obtained according to the methods of some embodiments of the present invention, or a single embryonic stem cell obtained according to the methods of some embodiments of the present invention, in suspension culture under conditions allowing expansion of said single pluripotent stem cell or expansion of said single embryonic stem cell, respectively, in an undifferentiated state, thereby expanding said single pluripotent stem cell or said single embryonic stem cell, respectively, into a clonal culture, thereby cloning said pluripotent stem cell.

[0025] According to some embodiments of the invention, the culturing is performed without dissociating the cell clumps.

[0026] According to an aspect of some embodiments of the present invention, there is provided a method of generating lineage specific cells from pluripotent stem cells, the method comprising: (a) culturing said pluripotent stem cells in suspension culture according to the method of some embodiments of the present invention, thereby obtaining clump-free expanded undifferentiated pluripotent stem cells; and (b) subjecting said clump-free expanded undifferentiated pluripotent stem cells to culture conditions suitable for differentiation and / or expansion of lineage specific cells, thereby generating said lineage specific cells from said pluripotent stem cells.

[0027] According to an aspect of some embodiments of the present invention, there is provided a method of producing embryoid bodies from pluripotent stem cells, the method comprising: (a) culturing said pluripotent stem cells in suspension culture according to the method of some embodiments of the present invention, thereby obtaining clump-free expanded undifferentiated pluripotent stem cells; and (b) subjecting said clump-free expanded undifferentiated pluripotent stem cells to culture conditions suitable for differentiating said pluripotent stem cells into embryoid bodies, thereby producing said embryoid bodies from said pluripotent stem cells.

[0028] According to an aspect of some embodiments of the present invention, there is provided a method of generating lineage-specific cells from pluripotent stem cells, the method comprising: (a) culturing said pluripotent stem cells in suspension culture according to the method of some embodiments of the present invention, thereby obtaining clump-free expanded undifferentiated pluripotent stem cells; (b) subjecting said clump-free expanded undifferentiated pluripotent stem cells to culture conditions suitable for differentiating said pluripotent stem cells into embryoid bodies; and (c) subjecting cells of the embryoid bodies to culture conditions suitable for differentiation and / or expansion of lineage-specific cells, thereby generating said lineage-specific cells from said pluripotent stem cells.

[0029] According to some embodiments of the invention, the clump-free suspension culture comprises single cells or small clusters, each cluster comprising up to about 200 pluripotent stem cells.

[0030] According to some embodiments of the invention, the culturing is performed under culture conditions that are free of substrate attachment.

[0031] According to some embodiments of the invention, the culture conditions do not include a Rho-associated kinase (ROCK) inhibitor.

[0032] According to some embodiments of the invention, the pluripotent stem cells are human pluripotent stem cells.

[0033] According to some embodiments of the invention, the human pluripotent stem cells are embryonic stem cells.

[0034] According to some embodiments of the invention, the human pluripotent stem cells are induced pluripotent stem cells.

[0035] According to an aspect of some embodiments of the present invention there is provided an isolated population of cell clump-free pluripotent stem cells obtainable according to the methods of some embodiments of the present invention and capable of differentiating into each of the germ layers of an embryo: endoderm, ectoderm and mesoderm.

[0036] According to an aspect of some embodiments of the present invention there is provided a method of producing mesenchymal stem cells in suspension culture comprising culturing pluripotent stem cells of some embodiments of the present invention in suspension culture under conditions suitable for differentiation of the pluripotent stem cells into mesenchymal stem cells, thereby producing said mesenchymal stem cells in said suspension culture.

[0037] According to an aspect of some embodiments of the present invention there is provided an isolated population of mesenchymal stem cells (MSCs) in suspension culture produced by the method of some embodiments of the present invention.

[0038] According to some embodiments of the invention, at least 40% of the cells are characterized by a CD73+ / CD31- / CD105+ expression signature.

[0039] According to some embodiments of the invention, the MSCs are capable of differentiation in suspension culture into a cell lineage selected from the group consisting of an adipogenic lineage, an osteoblastic lineage and a chondrogenic lineage.

[0040] According to an aspect of some embodiments of the present invention there is provided a method of producing neuronal progenitor cells in suspension culture comprising culturing pluripotent stem cells of some embodiments of the present invention in suspension culture under conditions suitable for differentiation of neuronal progenitor cells, thereby producing said neuronal progenitor cells in said suspension culture.

[0041] According to an aspect of some embodiments of the present invention there is provided an isolated population of neuronal progenitor cells in suspension culture produced by the method of some embodiments of the present invention.

[0042] According to an aspect of some embodiments of the present invention there is provided a method of producing endodermal cells in suspension culture comprising culturing pluripotent stem cells of some embodiments of the present invention in suspension culture under conditions suitable for differentiation of said pluripotent stem cells into endodermal cells, thereby producing said endodermal cells in said suspension culture.

[0043] According to an aspect of some embodiments of the present invention there is provided an isolated population of endoderm cells in suspension culture produced by the method of some embodiments of the present invention.

[0044] According to an aspect of some embodiments of the present invention there is provided a culture medium comprising interleukin 11 (IL11) and ciliary neurotrophic factor (CNTF).

[0045] According to an aspect of some embodiments of the present invention there is provided a culture medium comprising basic fibroblast growth factor (bFGF) at a concentration of at least 50 ng / ml and an IL6RIL6 chimera.

[0046] According to an aspect of some embodiments of the present invention there is provided a culture medium comprising an animal-derived contaminant-free serum replacement and an IL6RIL6 chimera.

[0047] According to an aspect of some embodiments of the present invention there is provided a cell culture comprising pluripotent stem cells and the culture medium of some embodiments of the present invention.

[0048] According to an aspect of some embodiments of the present invention there is provided a culture system comprising a matrix and a culture medium of some embodiments of the present invention.

[0049] According to an aspect of some embodiments of the present invention there is provided a cell culture comprising pluripotent stem cells and a serum-free culture medium, the culture medium comprising a soluble interleukin 6 receptor (sIL6R) and interleukin 6 (IL6), wherein a concentration of the sIL6R is at least 5 ng / ml and a concentration of the IL6 is at least 3 ng / ml.

[0050] According to an aspect of some embodiments of the invention there is provided a cell culture comprising pluripotent stem cells and a culture medium comprising interleukin 11 (IL11) and oncostatin.

[0051] According to an aspect of some embodiments of the present invention there is provided a method of expanding and maintaining pluripotent stem cells in an undifferentiated state, the method comprising culturing said pluripotent stem cells in a culture medium of some embodiments of the present invention, thereby expanding and maintaining said pluripotent stem cells in said undifferentiated state.

[0052] According to an aspect of some embodiments of the present invention there is provided a method of generating lineage specific cells from pluripotent stem cells comprising: (a) culturing said pluripotent stem cells according to the method of some embodiments of the present invention, thereby obtaining expanded undifferentiated stem cells; (b) subjecting said expanded undifferentiated stem cells to culture conditions suitable for differentiation and / or expansion of lineage specific cells, thereby generating said lineage specific cells from said pluripotent stem cells.

[0053] According to an aspect of some embodiments of the invention there is provided a cell culture comprising a population of pluripotent stem cells produced according to the method of some embodiments of the invention, said population comprising at least 1000 pluripotent stem cells per milliliter of medium.

[0054] According to an aspect of some embodiments of the invention there is provided a use of the cell culture of some embodiments of the invention for cell-based therapy.

[0055] According to an aspect of some embodiments of the present invention there is provided use of the cell culture of some embodiments of the present invention for pharmaceutical screening purposes.

[0056] According to an aspect of some embodiments of the invention there is provided use of the cell culture of some embodiments of the invention for the manufacture of a vaccine.

[0057] According to an aspect of some embodiments of the invention there is provided use of the cell culture of some embodiments of the invention for the production of a protein.

[0058] According to some embodiments of the invention, IL11 is used at a concentration of at least 0.1 ng / ml.

[0059] According to some embodiments of the invention, CNTF is used at a concentration of at least 0.1 ng / ml.

[0060] According to some embodiments of the invention, IL11 is used at a concentration of at least 1 ng / ml.

[0061] According to some embodiments of the invention, CNTF is used at a concentration of at least 1 ng / ml.

[0062] According to some embodiments of the present invention, the concentration of bFGF is selected from the range of between 50 ng / ml and 150 ng / ml.

[0063] According to some embodiments of the invention, the IL6RIL6 chimera is used at a concentration of at least 50 ng / ml.

[0064] According to some embodiments of the invention, the IL6RIL6 chimera is used at a concentration of at least 50 ng / ml.

[0065] According to some embodiments of the invention, the culture medium further comprises a serum replacement.

[0066] According to some embodiments of the invention, the serum replacement is used at a concentration of at least 10%.

[0067] According to some embodiments of the invention, the serum replacement is free of animal-derived contaminants.

[0068] According to some embodiments of the present invention, the IL6RIL6 chimera is used at a concentration of 50 ng / ml to 150 ng / ml.

[0069] According to some embodiments of the present invention, the IL6RIL6 chimera is used at a concentration of 50 pg / ml to 150 pg / ml.

[0070] According to some embodiments of the invention, the culture medium further comprises basic fibroblast growth factor (bFGF).

[0071] According to some embodiments of the invention, bFGF is used at a concentration of at least 4 ng / ml.

[0072] According to some embodiments of the invention, the culture medium further comprises ascorbic acid.

[0073] According to some embodiments of the invention, ascorbic acid is used at a concentration of 25 μg / ml to 100 μg / ml.

[0074] According to some embodiments of the invention, bFGF is used at a concentration of 100 ng / ml and IL6RIL6 is used at a concentration of 100 ng / ml.

[0075] According to some embodiments of the invention, bFGF is used at a concentration of 100 ng / ml and IL6RIL6 is used at a concentration of 100 pg / ml.

[0076] According to some embodiments of the invention, the culture medium further comprises TGFβ.

[0077] According to some embodiments of the invention, the TGFβ comprises TGFβ1.

[0078] According to some embodiments of the invention, the TGFβ comprises TGFβ3.

[0079] According to some embodiments of the invention, the culture medium is serum-free.

[0080] According to some embodiments of the invention, the culture medium is free of animal-derived contaminants.

[0081] According to some embodiments of the invention, expansion and maintenance of said pluripotent stem cells in an undifferentiated state is performed in suspension culture.

[0082] According to some embodiments of the invention, the culturing is carried out under conditions comprising static suspension culture.

[0083] According to some embodiments of the invention, the culturing is carried out under conditions comprising dynamic suspension culture.

[0084] According to some embodiments of the invention, the culturing is under conditions that allow the pluripotent stem cells to be expanded as single cells.

[0085] According to some embodiments of the invention, the culturing is performed under conditions that do not include enzymatic dissociation of cell clusters.

[0086] According to some embodiments of the invention, the expansion and maintenance of said pluripotent stem cells in an undifferentiated state is performed in a two-dimensional culture system.

[0087] According to some embodiments of the invention, the two-dimensional culture system comprises a matrix and the above-mentioned culture medium.

[0088] According to some embodiments of the invention, the pluripotent stem cells comprise embryonic stem cells.

[0089] According to some embodiments of the invention, the pluripotent stem cells comprise induced pluripotent stem (iPS) cells.

[0090] According to some embodiments of the invention, the embryonic stem cells are human embryonic stem cells.

[0091] According to some embodiments of the invention, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0092] According to some embodiments of the invention, the culture medium is capable of expanding the pluripotent stem cells in an undifferentiated state.

[0093] According to some embodiments of the invention, at least 85% of the pluripotent stem cells are in an undifferentiated state.

[0094] According to some embodiments of the invention, the culture conditions comprise a culture medium comprising interleukin 11 (IL11) and ciliary neurotrophic factor (CNTF).

[0095] According to some embodiments of the invention, the culture conditions comprise a culture medium comprising basic fibroblast growth factor (bFGF) at a concentration of at least 50 ng / ml and an IL6RIL6 chimera.

[0096] According to some embodiments of the invention, the culture conditions include a culture medium comprising an animal-derived contaminant-free serum replacement and an IL6RIL6 chimera.

[0097] According to some embodiments of the invention, the culture conditions include a serum-free culture medium containing soluble interleukin 6 receptor (sIL6R) and interleukin 6 (IL6), wherein the concentration of sIL6R is at least 5 ng / ml and the concentration of IL6 is at least 3 ng / ml.

[0098] According to some embodiments of the invention, the culture conditions comprise a culture medium comprising interleukin 11 (IL11) and oncostatin.

[0099] According to an aspect of some embodiments of the present invention there is provided a culture medium comprising serum and a serum replacement.

[0100] According to some embodiments of the invention, the serum replacement is used at a concentration of about 10%.

[0101] According to some embodiments of the invention, serum is used at a concentration of 10%.

[0102] According to some embodiments of the invention, the culture medium comprising serum and serum replacement does not comprise bFGF.

[0103] According to some embodiments of the invention, the culture medium comprising serum and serum replacement does not comprise IL6RIL6 chimera.

[0104] According to some embodiments of the invention, the culture medium comprising serum and serum replacement further comprises L-glutamine, β-mercaptoethanol and a non-essential amino acid stock solution.

[0105] According to some embodiments of the invention, the culture medium containing serum and serum replacement consists of 80% DMEM / F12, 10% Knockout Serum Replacement (SR), 10% FBS, 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock solution.

[0106] According to some embodiments of the present invention, the culture medium comprising serum and serum replacement is suitable for differentiation of pluripotent stem cells into mesenchymal stem cells in suspension.

[0107] According to some embodiments of the present invention, the conditions suitable for differentiation of pluripotent stem cells into mesenchymal stem cells comprise a culture medium comprising serum and serum replacements.

[0108] According to some embodiments of the invention, the method further comprises transporting the pluripotent stem cells of some embodiments of the invention as non-frozen, live cells.

[0109] According to some embodiments of the invention, the pluripotent stem cells remain viable, proliferative and in an undifferentiated state after the cells are transported as non-frozen, live cells.

[0110] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs.Methods and materials similar or equivalent to those described herein can be used in the practice or testing of this invention, but exemplary methods and / or materials are described below.In case of conflict, this patent specification, including definitions, will take precedence.In addition, materials, methods and examples are only illustrative and are not intended to be limiting.

[0111] Implementing the method and / or system of the present invention may involve performing or completing selected tasks manually, automatically, or a combination thereof. Furthermore, the actual device or apparatus of the device, method and / or system embodiments of the present invention may perform any number of selected steps using hardware, software, or firmware, or a combination thereof using an operating system.

[0112] For example, hardware for performing selected tasks according to embodiments of the present invention may be implemented as a chip or circuit. As software, selected tasks according to embodiments of the present invention may be implemented as software, such as a number of software instructions executed by a computer using a suitable operating system. In an exemplary embodiment of the present invention, one or more tasks according to exemplary embodiments of the methods and / or systems described herein are performed on a data processor, such as a computing platform that executes a number of instructions. Optionally, the data processor includes a volatile memory for storing instructions and / or data, and / or a non-volatile storage device (e.g., a magnetic hard disk and / or a removable recording medium) for storing instructions and / or data. Optionally, a network connection is also further provided. A display and / or a user input device (e.g., a keyboard or mouse) are also optionally further provided. [Brief description of the drawings]

[0113] Certain embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: Reference will now be made in particular detail to the drawings, stressing that the details shown are for the purpose of illustrating and discussing embodiments of the invention by way of example only, and in which the description given with reference to the drawings will make apparent to those skilled in the art how to practice the embodiments of the invention.

[0114] [Figure 1A-1C]1A-1C are photographs of I3 ESCs grown in suspension culture according to some embodiments of the present invention after live cell transport across the Atlantic (from Israel to Baltimore, USA) lasting 4 days. I3 cells were cultured in suspension for at least 20 passages before transport. FIG. 1A and FIG. 1B - Morphology of I3 3 days (FIG. 1A) and 1 day (FIG. 1B) after arrival and re-plating in suspension using CM100Fp culture medium. The cells demonstrate the typical sphere morphology composed of undifferentiated cells. FIG. 1C - Morphology of I3 3 days after arrival and re-plating with MEFs. The cells demonstrate the typical colony morphology of ESCs.

[0115] [Figures 2A-2D] Figures 2A-2D are fluorescent images of I3.2 hESC stained with antibodies against various markers of pluripotency (immunofluorescence staining). Cells cultured in the novel medium of some embodiments of the present invention (e.g., in this case CMTeSR2 medium) were examined for their pluripotency using the typical markers Oct4 (Figure 2A), SSEA4 (Figure 2B), Tra-160 (Figure 2C) and TRA-1-81 (Figure 2D). In this example, I3.2 at passage p19+83 (i.e., this I3.2 clonal cell line was derived from I3 cell line at passage 19, and the cells for analysis were at passage 83 after clonal isolation) were cultured in suspension for 5 passages in CMTeSR2 medium and then re-cultured on MEFs. Cells were found to be positive for all tested markers.

[0116] [Figure 3A] Figure 3A is a photograph of the I3.2 ESC line showing the morphology of cells cultured in suspension using the novel culture medium of some embodiments of the present invention. Figure 3A-p19+I3.2 at passage 87 (i.e., 87 passages after clonal isolation) was cultured in suspension using cmTeSR2 for 26 passages and then re-plated with MEFs. Typical ESC colony morphology is demonstrated. [Figure 3B]Figure 3B is a photograph of the I3.2 ESC line showing the morphology of cells cultured in suspension using the novel culture medium of some embodiments of the present invention. Figure 3B - J3 cells at passage 80 (p80) [a delayed (expanded) blastocyst cell line] were cultured in suspension for two passages using NCM100 medium, demonstrating the typical sphere morphology of undifferentiated cells. [Figure 3C] Figure 3C is a photograph of the I3.2 ESC line showing the morphology of cells cultured in suspension using the novel culture medium of some embodiments of the present invention. Figure 3C-H9.2 cells at p29+48 (i.e., H9 cell line at passage 29 was subjected to single cell cloning and the resulting clonal hESC line at passage 48 after isolation was used) were cultured in suspension for 5 passages using ILCNTF medium. The typical spherical morphology of undifferentiated cells is evident.

[0117] [Figure 4A] Figure 4A is a photograph of the H9 hESC line showing single cells in suspension culture. Figure 4A-H9 at p53 (passage 53) was cultured in suspension as single cells in static culture for 9 passages using CMrb100Fp medium. These results demonstrate that pluripotent stem cells cultured in suspension culture according to some embodiments of the present invention adopt a single cell growth pattern. [Figure 4B] Figure 4B is a photograph of human C2 iPS cell line showing single cells in suspension culture. Figure 4B - C2 iPS cells were cultured as single cells in spinner flasks (dynamic culture) using CM100Fp medium for 1.5 months. Cells were stained with trypan blue. Dead cells are stained blue. These results demonstrate that pluripotent stem cells cultured in suspension culture according to some embodiments of the present invention adopt a single cell growth pattern.

[0118] [Figure 5A-5C]Figures 5A-5C are micrographs showing pluripotent stem cells cultured in suspension under dynamic conditions using a Controlled Wave bioreactor (Biostat® Cultibag RM, Sartorius North America, Edgewood, New York, USA). Induced pluripotent stem cell line C2 was cultured as single cells (Figure 5A) or as sub-200 μm spheres (Figure 5B) in a wave-suppressed bioreactor for 5 days. Figure 5C - Cells grown in suspension as single cells were re-cultured on MEFs (Oct4 staining). The number of viable cells was increased by 64-fold while maintaining iPS cell characteristics such as Oct4 expression (Figure 5C).

[0119] [Figure 6A-6C] Figures 6A-6C are micrographs showing pluripotent stem cells after freeze / thaw and shearing of single cells cultured in suspension. C2 cell line (iPS derived from foreskin fibroblasts at passage 89 after derivation, the cells were cultured in suspension culture in the presence of cmrb100p culture medium for 48 passages) was frozen using the following freezing solutions: 90% serum replacement (SR) and 10% DMSO (Figure 6A); 20% SR, 20% fetal bovine serum (FBS) and 10% DMSO (Figure 6B); and serum-free freezing solution from Biological Industries (Beit HaEmek, Israel) (Figure 6C). After freezing in liquid nitrogen for 5 days, the cells were thawed and recultured in suspension culture. Cells are shown after freeze / thaw and reculture in suspension culture. Note that more than 70% of the cells survived this procedure and recovered directly into suspension culture.

[0120] [Figure 7A-7C]Figures 7A-7C are immunofluorescence staining images demonstrating directed differentiation of pluripotent stem cells into cells derived from the neural lineage. I6 cells cultured in suspension for more than 40 passages were induced to differentiate by addition of retinoic acid and stained for typical neural markers: nestin (Figure 7A), β-tubulin (Figure 7B) and polysialylated (PSA) neural cell adhesion molecule (NCAM) (Figure 7C). These specific markers are stained red and blue staining represents DAPI staining.

[0121] [Figure 8A-8B] Figures 8A-8B are FACS analyses demonstrating differentiation of pluripotent stem cells into the neural lineage. Figure 8A - FACS analysis using NCM FITC antibody showing 68% of cells positive for NCAM. Figure 8B - FACS analysis using NCAM IgG (isotype control).

[0122] [Figure 9A] 9A is a histogram showing the results of semi-quantitative RT-PCR analysis for neural-specific markers performed on cells cultured in suspension and induced into a neural lineage by retinoic acid, and on cells cultured in suspension as undifferentiated. [Figure 9B-9G] Figures 9B-9G are gel images showing the results of semi-quantified RT-PCR analysis for neural specific markers. RT-PCR analysis was performed on cells cultured in suspension and induced into the neural lineage by retinoic acid, and on cells cultured in suspension as undifferentiated. RT-PCR primers for OCT-4 gene (Figure 9B), PAX6 gene (Figure 9C), heavy chain neurofilament (HNF) gene (Figure 9D), nestin gene (Figure 9E) and LIM homeobox 2 (LHX2) gene (Figure 9F) and GAPDH gene (control gene, Figure 9G) are listed in Table 1 in the Examples section below. Results represent the average of three independent experiments. Lanes 1-3 are from three different biological repeats, and lane 4 is I6 undifferentiated cells cultured in suspension for 40 passages.

[0123] [Figure 10A-10B] 10A-10B are immunofluorescence images showing induction of pluripotent stem cells into cells of the endodermal lineage. Cells from the C2 cell line were induced to differentiate into the endodermal lineage. Ten days after induction of differentiation, cells were stained for PDX1 marker (a transcription factor associated with β-cells) (FIG. 10A, green) and also stained for DAPI (nuclear stain) (FIG. 10B, blue).

[0124] [Figure 11A-11B] Figures 11A-11B are two representative images showing the morphology of hESC colonies after replating on MEFs. CL1 (13E1) cells cultured in suspension as single cells for 17 passages were replated on MEFs and photographed using phase contrast. Note that when replated on feeder cells (MEFs), the cells form colonies characterized by the typical morphology of pluripotent cells with variable intercellular spacing, clear borders, and large nucleus-to-cytoplasm ratio.

[0125] [Figure 12A-12B] Figures 12A-12B are histograms showing FACS analysis of pluripotency markers. Human ESCs were grown on MEFs in two dimensions (2-D) and expression of TRA1-60, TRA1-81, SSEA1 and SSEA4 markers was assayed by FACS. H14 cells cultured in Figure 12A-2D and separated by TRA1-60 antibody (blue curve). Note that 74.9% of cells are TRA1-60 positive. H14 cells cultured in Figure 12B-2D and separated by TRA1-81 antibody (blue curve). Note that 71.2% of cells are TRA1-81 positive. The red curves in each of Figures 12A-B represent negative controls. [Fig. 12C-12D]Figures 12C-12D are histograms showing FACS analysis of pluripotency markers. Human ESCs were grown in suspension culture as cell clumps and the expression of TRA1-60, TRA1-81, SSEA1 and SSEA4 markers was assayed by FACS. Figure 12C - I3 cells cultured in suspension as cell clumps for more than 10 passages and separated by TRA1-60 antibody (blue curve). Note that 94.6% of the cells are TRA1-60 positive. Figure 12D - I3 cells cultured in suspension as cell clumps for more than 10 passages and separated by TRA1-81 antibody (blue curve). Note that 93% of the cells are TRA1-81 positive. The red curves in each of Figures 12C-D represent negative controls. [Fig. 12E-12F] Figures 12E-12F are histograms showing FACS analysis of pluripotency markers. Human ESCs were grown in suspension culture as single cells without cell clumps and the expression of TRA1-60, TRA1-81, SSEA1 and SSEA4 markers was assayed by FACS. Figure 12E - H14 cells cultured in suspension as single cells for more than 10 passages and separated by TRA1-60 antibody (blue curve). Note that only 0.65% of cells are TRA1-60 positive. Figure 12F - H14 cells cultured in suspension as single cells for more than 10 passages and separated by TRA1-81 antibody (blue curve). Note that only 0.7% of cells are TRA1-81 positive. The red curves in each of Figures 12E-F represent negative controls. [Fig. 12G-12H]Figures 12G-12H are histograms showing FACS analysis of pluripotency markers. Human ESCs were grown in suspension culture as cell clumps and the expression of TRA1-60, TRA1-81, SSEA1 and SSEA4 markers was assayed by FACS. Figure 12G - I3 cells cultured in suspension as cell clumps for more than 10 passages and separated by SSEA1 antibody (blue curve). Note that 11.1% of the cells are SSEA1 positive. Figure 12H - I3 cells cultured in suspension as cell clumps for more than 10 passages and separated by SSEA4 antibody (gray curve). Note that 98.4% of the cells are SSEA4 positive. The red curves in each of Figures 12G-H represent negative controls. [Fig. 12I-12J] Figures 12I-J are histograms showing FACS analysis of pluripotency markers. Human ESCs were grown in suspension culture as single cells without cell clumps and the expression of TRA1-60, TRA1-81, SSEA1 and SSEA4 markers was assayed by FACS. Figure 12I - H7 cells cultured in suspension as single cells for more than 10 passages and separated by SSEA1 antibody (blue curve). Note that 78.5% of the cells are SSEA1 positive. Figure 12J - H7 cells cultured in suspension as single cells for more than 10 passages and separated by SSEA4 antibody (blue curve). Note that only 5.43% of the cells are SSEA4 positive. The red curves in each of Figures 12I-J represent negative controls.

[0126] [Figure 13A]Figure 13A is a histogram showing RT-PCR analysis. The average fold change in gene expression by real-time PCR for H7 and CL1 pluripotent stem cells (three replicates from each) is shown. The average fold change was calculated relative to the expression level of the indicated genes in H7 and CL1 pluripotent stem cells when cultured on MEFs (shown as "1"). Figure 13A-shows results for Sox2, Rex1, Nanog and Oct4 pluripotency genes. Figure 13B-shows results for FBLN5, CTNNB1, PLXNA2, EGFR, ITGA7, IGTA6, ITGA2, CLDN18, CLDN6, CDH2, CDH1 and FN1 adhesion molecule genes. Blue bars = single cells cultured in suspension for more than 10 passages (SC); red bars = cell clumps cultured in suspension for more than 10 passages (Cl); green bars = pluripotent stem cells cultured on mouse embryonic fibroblasts (MEFs) in standard 2D culture. Note the slight decrease in Nanog expression in pluripotent single stem cells compared to pluripotent stem cells cultured on MEFs, whereas Oct4 expression is increased in the same cells cultured as single cells compared to cells cultured on MEFs or as cell clumps in suspension culture. [Figure 13B]Figure 13B is a histogram showing RT-PCR analysis. The average fold change in gene expression by real-time PCR for H7 and CL1 pluripotent stem cells (three replicates from each) is shown. The average fold change was calculated relative to the expression level of the indicated genes in H7 and CL1 pluripotent stem cells when cultured on MEFs (shown as "1"). Figure 13A-shows results for Sox2, Rex1, Nanog and Oct4 pluripotency genes. Figure 13B-shows results for FBLN5, CTNNB1, PLXNA2, EGFR, ITGA7, IGTA6, ITGA2, CLDN18, CLDN6, CDH2, CDH1 and FN1 adhesion molecule genes. Blue bars = single cells cultured in suspension for more than 10 passages (SC); red bars = cell clumps cultured in suspension for more than 10 passages (Cl); green bars = pluripotent stem cells cultured on mouse embryonic fibroblasts (MEFs) in standard 2D culture. Note the slight decrease in Nanog expression in pluripotent single stem cells compared to pluripotent stem cells cultured on MEFs, whereas Oct4 expression is increased in the same cells cultured as single cells compared to cells cultured on MEFs or as cell clumps in suspension culture.

[0127] [Figure 14] Figure 14 is an image showing the cloning efficiency of hESCs cultured in suspension as single cells. Single cell clones were formed by plating single cells of the H7 hESC lineage cultured in suspension as single cells without cell clumps. Each cell was plated individually in a well of a low-attachment 96-well plate and cultured in suspension. Note that the cloning efficiency of hESCs cultured in suspension as single cells is 95%.

[0128] [Figure 15]Figure 15 is an image showing the thawing efficiency of hESCs cultured in suspension as single cells. Human ESCs cultured as single cells in suspension were frozen using standard freezing solutions and then thawed in suspension. Cells recovered well, with at least 80% of cells remaining viable.

[0129] [Figure 16A-16B] Figures 16A-16B are images showing genetic manipulation of hESCs cultured in suspension as single cells. Human hESCs cultured in suspension as single cells were subjected to electroporation with a nucleic acid construct containing the GFP gene under the CMV promoter. Figure 16A - Phase contrast image of cells after genetic manipulation. Note that most of the cells (at least 90%) survived the electroporation procedure. Figure 16B - Fluorescence microscopy image of cells after genetic manipulation. Green signals correspond to cells expressing the recombinant construct (GFP under the transcriptional control of the CMV promoter).

[0130] [Figures 17A-17C] Figures 17A-17C are micrographs showing differentiation of human ESCs cultured in suspension as single cells into neural progenitors (NPs). Human ESCs cultured in suspension as single cells were induced to differentiate into a neuronal lineage. Figure 17A - astrocytes, GFAP (red); Figure 17B - oligodendrocytes, O4 (green); Figure 17C - neurons, β-tubulin (green) and nestin (red).

[0131] [Figures 18A-18C]Figures 18A-18C are histograms showing FACS analysis of MSCs isolated by differentiating hESCs grown in suspension culture as single cells. Figure 18A - MSCs derived from J3 hESC line grown in animal-derived free medium and separated by CD73 antibody (blue curve). Note that 82.5% are CD73 positive. Figure 18B - MSCs derived from J3 hESC line grown in animal-derived free medium and separated by CD31 antibody (blue curve). Note that only 4.83% are CD31 positive. Figure 18C - MSCs derived from J3 hESC line grown in serum-containing medium and separated by CD105 antibody (blue curve). Note that 99.3% are CD105 positive.

[0132] [Figures 19A-19D] Figures 19A-19D are images showing differentiation of hESCs cultured in suspension as single cells into MSCs. Single cells cultured in suspension as single cells can differentiate into potent MSCs both in suspension and in 2D. Figures 19A-19B - Phase contrast images of MSCs differentiated from human ESCs cultured in suspension as single cells. hESCs were re-plated in suspension culture and differentiated into MSCs with typical MSC morphology. Figure 19A - CL1 cells were differentiated in Fy enriched medium. Figure 19B - CL1 cells were differentiated in MeSusII medium. Figure 19C - Alizarin red staining of differentiated MSCs (formed by differentiation of hESCs grown in suspension as single cells) into bone lineage. Figure 19D - Oil red staining of differentiated MSCs (formed by differentiation of hESCs grown in suspension as single cells) into adipocytes.

[0133] [Figure 20A-20B]Figures 20A-20B are images showing the differentiation of hESCs cultured in suspension as single cells into endodermal germ layers. C2 cells were cultured as single cells in suspension for more than 10 passages. For endodermal differentiation, bFGF and IL6RIL6 chimeras were removed from the culture medium and Activin A was added at a concentration of 10 ng / ml for 48 hours in suspension culture. Ten days after Activin A exposure, cells were plated on Matrigel matrix or HFF matrix and stained for PDX1 expression using anti-PDX1 antibody (R&D Biosystems). Figure 20A - DAPI staining (nuclear staining) (blue); Figure 20B - PDX1 (red). Note that all cells stained with DAPI (nuclear staining) also stain for PDX1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0134] In some embodiments, the present invention relates to novel methods and culture media that can maintain pluripotent stem cells in an undifferentiated state, as well as novel pluripotent stem cells cultured in suspension culture as single stem cells free of cell aggregates, and more particularly, but not exclusively, to methods of culturing pluripotent stem cells in two-dimensional or three-dimensional culture systems while maintaining them in a proliferative, pluripotent, undifferentiated state.

[0135] Before describing at least one embodiment of the invention in detail, it should be understood that the invention is not necessarily limited in its application to the details set forth in the following description or illustrated by the examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it should be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

[0136] The inventors, after laborious experimentation, have discovered a defined medium that is serum-free and free of animal-derived contaminants and is capable of maintaining pluripotent stem cells, such as human iPS and ESC, in an undifferentiated state in the absence of feeder cell support while retaining their pluripotent ability to differentiate into all three embryonic germ layers.

[0137] Thus, as shown in the Examples section below, hESCs and iPS cells (e.g., derived from adult or foreskin fibroblasts) have been cultured in an undifferentiated state in the presence of serum-free defined culture media (e.g., yFIL25, CMrb100F, CMrb100Fp, ILCNTF) as well as in the presence of fully defined culture media (e.g., NCM100F, NCM100Fp, NCMrb100F, NCMrb100Fp, NILCNTF, CmHA13, CmHA13p) containing serum replacements free of animal-derived contaminants, on two-dimensional or three-dimensional culture systems (said media are suitable for clinical / therapeutic applications, since the human pluripotent stem cells cultured therein are completely devoid of animal-derived contaminants). Moreover, as shown in Example 4 of the Examples section below, pluripotent stem cells cultured in suspension can remain viable, proliferative and pluripotent as living cells during transport across countries. In culture, pluripotent stem cells exhibit undifferentiated morphology and molecular characteristics typical of iPS or hESC, such as normal karyotype, expression of markers of pluripotency (e.g., Oct4, SSEA4, TRA-1-81, TRA-1-60), and the ability to differentiate into all three embryonic germ layers both in vitro (by formation of embryoid bodies after at least 10 passages) and in vivo (by formation of teratomas after at least 20 passages). As shown in Figures 7-10 and described in Example 7 of the Examples section below, pluripotent stem cells have also been used to generate lineage-specific cells of neuronal, endodermal, and mesodermal cell lineages.

[0138] In addition, the inventors have discovered suitable culture conditions for maintaining undifferentiated pluripotent stem cells in suspension culture as single cells without cell aggregates, and have isolated a novel population of human pluripotent stem cells cultured in suspension culture as single cells.

[0139] Thus, as demonstrated in Example 3 of the Examples section below, the inventors cultured pluripotent stem cells (e.g., hESCs and human iPS cells) in suspension culture by mechanically passaging the cells (e.g., using a pipette) without using trypsin or ROCK inhibitor. After about 3-7 passages of mechanically dissociating the cell clumps into single cells, the pluripotent stem cells adopted a single-cell manner of expansion, which did not require any mechanical separation for culture passaging, thus enabling mass production of these cells. When the suspension cultures cultured as single cells were re-plated on MEFs, the cells formed colonies with the typical morphology of pluripotent stem cells (Figures 11A-11B). As further described in Example 8 of the Examples section below, human pluripotent stem cells cultured in suspension culture as single cells exhibit a more naive pattern of gene expression when compared to human ESCs cultured on MEFs or compared to hESCs cultured in suspension culture as cell clumps. Therefore, isolated populations of pluripotent stem cells cultured in suspension as single cells free of cell aggregates express SSEA4 - / TRA1-60 - / TRA1-81 - / SSEA1 + 12E, 12F, 12I and 12J; Table 3), which is typical of human ESCs cultured on MEFs or in suspension culture as cell aggregates. + / TRA1-60 + / TRA1-81 + / SSEA1 -The expression signature of hESCs differs from that of hESCs (Figures 12A, 12B, 12C, 12D, 12G, 12H; Table 3). In contrast, when cultured in suspension as single cells, pluripotent stem cells exhibit increased levels of OCT-4 (a marker of pluripotency) when compared to hESCs cultured on MEFs (2-D) or when compared to hESCs cultured in suspension as cell aggregates (Example 8, Figure 13A). In addition, pluripotent stem cells cultured in suspension as single cells were found to exhibit increased cloning efficiency (e.g., about 95% efficiency for hESCs) when compared to pluripotent stem cells cultured in 2-D (e.g., between 4% and 18%, depending on the use of ROCK inhibitors) (Example 9, Table 4), increased survival to freeze-thaw cycles (Example 9, Figure 15), and greater survival and higher efficiency of genetic manipulation (Example 9, Figures 16A-B). It has been shown that pluripotent stem cells cultured in suspension as single cells are capable of differentiation into all three germ layers of the embryo: ectoderm germ layer by forming neuronal progenitor cells expressing GFAP (glial fibrillary acidic protein) (a marker for astrocytes), O4 (a marker for oligodendrocytes), and β-tubulin and nestin (markers for neurons) (Example 10, Figures 17A-17C); mesoderm germ layer by forming mesenchymal stem cells with expression of CD73 and CD105 (Example 11, Figures 18A and 18C) and no expression of CD31 (Example 11, Figure 18B); endoderm germ layer by forming endoderm cells expressing PDX1 (Example 12, Figures 20A-20B). In addition, the present inventors have demonstrated for the first time the in vitro differentiation of pluripotent stem cells in suspension culture into mesenchymal stem cells (Example 11). These MSCs were capable of differentiating into the adipogenic (Example 11, FIG. 19D), osteogenic (Example 11, FIG. 19C) and chondrogenic (Example 11, data not shown) lineages.In summary, the novel pluripotent stem cells identified herein can be used as an unlimited source of pluripotent undifferentiated stem cells for a variety of cell-derived therapies, drug screening, vaccine production and / or protein production.

[0140] Thus, according to an aspect of some embodiments of the present invention, there is provided a method of expanding and maintaining pluripotent stem cells (PSCs) in an undifferentiated state, comprising: (a) passaging the PSCs in suspension culture by mechanical dissociation of PSC clumps into single cells for at least two passages and up to ten passages, thereby obtaining a suspension culture of clump-free PSCs; and (b) passaging the suspension culture of clump-free PSCs without dissociation of the clumps, thereby expanding and maintaining the PSCs in the undifferentiated state.

[0141] According to some embodiments of the invention, PSCs are passaged in suspension culture by mechanical dissociation of PSC aggregates into single cells for at least 2 and up to 9 passages; for at least 2 and up to 8 passages; for at least 2 and up to 7 passages; for at least 2 and up to 6 passages; for at least 2 and up to 5 passages; for at least 2 and up to 4 passages; for at least 3 and up to 9 passages; for at least 3 and up to 8 passages; for at least 3 and up to 7 passages; for at least 3 and up to 6 passages; for at least 3 and up to 5 passages.

[0142] According to some embodiments of the invention, the method further comprises culturing the PSCs under conditions that allow expansion of the pluripotent stem cells in the undifferentiated state.

[0143] As used herein, the phrase "pluripotent stem cells" refers to cells that have the ability to differentiate into all three embryonic germ layers (i.e., endoderm, ectoderm and mesoderm). The phrase "pluripotent stem cells" can refer to embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPS cells).

[0144] As used herein, the phrase "embryonic stem cells" refers to cells obtained from embryonic tissue formed after conception (e.g., blastocysts) (pre-implantation (i.e., pre-implantation blastocysts)), expanded blastocyst cells (EBCs) obtained from blastocysts at the late implantation / pre-gastrulation stage (see WO 2006 / 040763), and embryonic germ (EG) cells obtained from fetal reproductive tissue at any time during pregnancy, preferably prior to 10 weeks of gestation.

[0145] According to some embodiments of the invention, the pluripotent stem cells of the invention are embryonic stem cells, for example of human or primate (eg, monkey) origin.

[0146] The embryonic stem cells of the present invention can be obtained using well-known cell culture methods. For example, human embryonic stem cells can be isolated from human blastocysts. Human blastocysts are typically obtained from human in vivo preimplantation embryos or in vitro fertilized (IVF) embryos. Alternatively, single-cell human embryos can be expanded to the blastocyst stage. In the isolation of human ES cells, the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated by immunosurgery, in which the trophectoderm cells are lysed and removed from the intact ICM by gentle pipetting. The ICM is then plated in tissue culture flasks with an appropriate medium that allows its outgrowth. After 9-15 days, the outgrowth derived from the ICM is dissociated into clumps by either mechanical dissociation or enzymatic digestion, and the cells are then replated on fresh tissue culture medium. Colonies exhibiting undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and replated. The resulting ES cells are then split periodically every 4-7 days.For further details on the preparation of human ES cells, see Thomson et al., [U.S. Patent No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133, 1998; Proc. Natl. Acad. Sci. USA 92:7844, 1995]; Bongso et al., [Hum Reprod 4:706, 1989]; and Gardner et al., [Fertil. Steril. 69:84, 1998].

[0147] It will be appreciated that commercially available stem cells can be used in this aspect of the invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry (www: / / escr.nih.gov). Non-limiting examples of commercially available embryonic stem cell lines include BG01, BG02, BG03, BG04, CY12, CY30, CY92, CY10, TE03, TE04 and TE06.

[0148] Expanded blastocyst cells (EBCs) are available from blastocysts at the pregastrulation stage at least 9 days after fertilization. Prior to culturing the blastocysts, the zona pellucida is digested [e.g., with Tyrode's acid solution (Sigma Aldrich, St Louis, MO, USA)] to expose the inner cell mass. Blastocysts are then cultured in vitro as whole embryos using standard embryonic stem cell culture methods for at least 9 but not more than 14 days after fertilization (i.e., before the gastrulation event).

[0149] Embryonic germ (EG) cells are prepared from primordial germ cells obtained from fetuses at approximately 8-11 weeks of gestation (for human fetuses) using laboratory techniques known to those skilled in the art. The genital ridges are dissociated, cut into small pieces, and then separated into cells by mechanical dissociation. The EG cells are then grown in tissue culture flasks with appropriate medium. The cells are cultured in medium that is changed daily until a cell morphology consistent with EG cells is observed, typically for 7-30 days or 1-4 passages. For further details regarding the preparation of human EG cells, see Shamblott et al., [Proc. Natl. Acad. Sci. USA 95:13726, 1998] and U.S. Patent No. 6,090,622.

[0150] As used herein, the expression "induced pluripotent stem (iPS) cells" (or embryonic-like stem cells) refers to proliferative and pluripotent stem cells obtained by dedifferentiation of somatic cells (eg adult somatic cells).

[0151] According to some embodiments of the invention, iPS cells are characterized by a proliferative potential similar to that of ESCs, and therefore can be maintained and expanded in culture for an almost indefinite period of time.

[0152] IPS cells can be rendered pluripotent by genetic engineering to reprogram cells and obtain embryonic stem cell characteristics.For example, the iPS cells of the present invention can be generated from somatic cells by inducing the expression of Oct-4, Sox2, Kfl4 and c-Myc in somatic cells, essentially as described in Takahashi and Yamanaka, 2006; Takahashi et al., 2007; Meissner et al., 2007; and Okita K. et al., 2007, Nature 448:313-318).In addition, or alternatively, the iPS cells of the present invention can be generated from somatic cells by inducing the expression of Oct4, Sox2, Nanog and Lin28, essentially as described in Yu et al., 2007 and Nakagawa et al., 2008. It should be noted that genetic manipulation (reprogramming) of somatic cells can be performed using any known method, such as the use of plasmid or viral vectors, or by induction without any integration into the genome [Yu J. et al., Science. 2009, 324:797-801].

[0153] The iPS cells of the present invention can be obtained by inducing dedifferentiation of embryonic fibroblasts [Takahashi and Yamanaka, 2006; Meissner et al., 2007], fibroblasts formed from hESCs [Park et al., 2008], fetal fibroblasts [Yu et al., 2007; Park et al., 2008], foreskin fibroblasts [Yu et al., 2007; Park et al., 2008], adult skin and skin tissue [Hanna et al., 2007; Lowry et al., 2008], b-lymphocytes [Hanna et al., 2007], and adult liver and stomach cells [Aoi et al., 2008].

[0154] IPS cell lines are also available through cell banks such as the WiCell bank. Non-limiting examples of commercially available iPS cell lines include iPS foreskin clone 1 [WiCell catalog number: iPS(foreskin)-1-DL-1], iPSIMR90 clone 1 [WiCell catalog number: iPS(IMR90)-1-DL-1], and iPSIMR90 clone 4 [WiCell catalog number: iPS(IMR90)-4-DL-1].

[0155] According to some embodiments of the invention, the induced pluripotent stem cells are human induced pluripotent stem cells.

[0156] As used herein, the term "expand" refers to increasing the number of pluripotent stem cells (at least about 5%, 10%, 15%, 20%, 30%, 50%, 100%, 200%, 500%, 1000% or more) over the culture period. It will be understood that the number of pluripotent stem cells depends on the proliferation potential of pluripotent stem cells, since it can be obtained from only one pluripotent stem cell. The proliferation potential of pluripotent stem cells can be calculated by the doubling time of the cells (i.e., the time required for cells to undergo mitosis in culture) and the period that pluripotent stem cell culture can be maintained in an undifferentiated state (which is equal to the number of passages multiplied by the number of days between each passage).

[0157] According to some embodiments of the invention, the methods of some embodiments of the invention allow for the expansion of a single pluripotent stem cell (e.g., hESC or human iPS cell) at least 8-fold in 5 days (e.g., at least 16-fold in 5 days, e.g., at least 32-fold in 5 days, e.g., at least 64-fold in 5 days).

[0158] According to some embodiments of the invention, the method of some embodiments of the invention comprises culturing a single pluripotent stem cell (e.g., hESC or human iPS cell), or a small cluster of 2-100 cells, for at least 2 months in about one month. 8 To enlarge the image by a factor of 2 10 times, e.g., 2 14 times, e.g., 216 times, e.g., 2 18 times, e.g., 2 20 This allows for 2x the number of

[0159] As used herein, the term "aggregate" refers to a cluster of cells that adhere to one another in suspension.

[0160] According to some embodiments of the invention, the cell clumps remain intact when the medium of the suspension culture is changed (e.g., increased, decreased, or replaced) without any mechanical or enzymatic dissociation of the clumps.

[0161] According to some embodiments of the invention, each of the pluripotent stem cell aggregates comprises at least about 200 cells (e.g., about 200), e.g., at least about 500 cells (e.g., about 500), at least about 600 cells (e.g., about 600), at least about 700 cells (e.g., about 700), at least about 800 cells (e.g., about 800), at least about 900 cells (e.g., about 900), at least about 1000 cells (e.g., about 1000), at least about 1100 cells (e.g., about 1100), at least about 1200 cells (e.g., about 1200), at least about 1300 cells (e.g., about 1300), at least about 1400 cells (e.g., about 1400), at least about 1500 cells (e.g., about 1500), at least about 5×10 3 cells (e.g., about 5 × 10 3 pcs), at least about 1×10 4 cells (e.g., about 1 x 10 4 pcs), at least about 5×10 4 cells (e.g., about 5 × 10 4 pcs), at least about 1×10 5 cells (e.g., about 1 x 10 5 ) or more.

[0162] As used herein, the term "passaging" refers to splitting cells in a culture vessel into two or more culture vessels, which typically includes adding fresh medium. Passaging is typically performed when cells reach a certain density in culture.

[0163] According to some embodiments of the present invention, about 1×10 6 Cell cultures seeded at a concentration of cells / milliliter can be passaged in a static 3D culture system when the concentration of cells increases by approximately two or three-fold (e.g., approximately 2 × 10 6 cells / ml~3×10 6 cells / ml) and increased by up to about four-fold (e.g., about 4×10 6 The incubation is performed at a concentration of 100 μg / ml.

[0164] According to some embodiments of the present invention, about 1×10 6 Cell cultures seeded at a concentration of cells / milliliter can be passaged in a dynamic 3D culture system when the concentration of cells increases approximately 20- to 40-fold (e.g., approximately 20 × 10 6 cells / ml~40×10 6 cells / ml) and increased by up to about 50-fold (e.g., about 50×10 6 The incubation is performed at a concentration of 100 μg / ml.

[0165] According to some embodiments of the invention, passaging does not necessarily require dissociation of cell clumps in cell culture.

[0166] As used herein, the phrase "mechanical dissociation" refers to the separation of pluripotent stem cell aggregates into single cells by using physical forces rather than enzymatic activity.

[0167] As used herein, the phrase "single cell" refers to the state in which pluripotent stem cells do not form cell clusters in suspension culture, with each cluster containing more than about 200 pluripotent stem cells.

[0168] According to some embodiments of the invention, the pluripotent stem cells do not form cell clusters in suspension culture, each cluster comprising more than about 150, more than about 100, more than about 90, more than about 80, more than about 70, more than about 60, more than about 50, more than about 40, more than about 30, more than about 20, more than about 19, more than about 18, more than about 17, more than about 16, more than about 15, more than about 14, more than about 13, more than about 12, more than about 11, more than about 10, more than about 9, more than about 8, more than about 7, more than about 6, more than about 5, more than about 3, more than about 2, or more than about 1 pluripotent stem cells.

[0169] According to some embodiments of the invention, each of the plurality of pluripotent stem cells does not adhere to another pluripotent stem cell while in suspension culture.

[0170] For mechanical dissociation, pellets of pluripotent stem cells (which can be accomplished by centrifugation of the cells), or isolated pluripotent stem cell aggregates, can be dissociated by pipetting the cells up and down in a small volume of medium (e.g., 0.2 ml to 1 ml). For example, pipetting can be performed several times (e.g., between 3 and 20 times) using a 200 μl or 1000 μl pipette tip.

[0171] Additionally or alternatively, mechanical dissociation of large pluripotent stem cell aggregates can be performed using a device designed to break the aggregates into a predetermined size. Such a device can be obtained from CellArtis (Goteborg, Sweden). Additionally or alternatively, mechanical dissociation can be performed manually using a needle (e.g., a 27g needle (BD Microlance, Drogheda, Ireland)) while viewing the aggregates under an inverted microscope.

[0172] According to some embodiments of the invention, the passaging is performed under conditions that are free of enzymatic dissociation.

[0173] According to some embodiments of the invention, the culture in suspension is performed under conditions in the absence of enzymatic dissociation of cell clusters / aggregates.

[0174] According to some embodiments of the invention, the culture conditions do not include the use of an anti-apoptotic agent.

[0175] According to some embodiments of the invention, the culture conditions do not include the use of a Rho-associated kinase (ROCK) inhibitor.

[0176] According to some embodiments of the invention, the culture is in an undifferentiated pluripotent state for at least one passage, and for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 passages.

[0177] The inventors have discovered that when pluripotent stem cells in suspension culture are mechanically passaged without enzymatic dissociation of cell clusters for at least about 2 and up to about 10 passages, the pluripotent stem cells adopt a single-cell manner of cell growth (i.e., the pluripotent stem cells are expanded as single cells, not as cell clumps). Thus, as described in Example 3 of the Examples section below, cells cultured in suspension while passaged for the first 2-10 passages only by mechanical dissociation of cell clumps adopted a single-cell manner of expansion and grew without the need for further dissociation of cell clusters for at least about 15, 20 or 25 additional passages.

[0178] While the cells are cultured as single cells, they still have a cell concentration of approximately 1 × 10 6 Cells / milliliter (e.g., 5 x 10 per 5 ml Petri dish) 6 It should be noted that dilutions may be required when exceeding 100 μl of ...

[0179] As used herein, the phrase "suspension culture" refers to a culture in which pluripotent stem cells are suspended in the medium, rather than attached to a surface.

[0180] It should be noted that some protocols for culturing pluripotent stem cells (such as hESCs and iPS cells) involve microencapsulation of cells inside a semi-permeable hydrogel membrane that allows exchange of nutrients, gases and metabolites with the bulk medium surrounding the capsule (see, for details, e.g., U.S. Patent Application Publication No. 20090029462 (Beardsley et al.)).

[0181] According to some embodiments of the invention, pluripotent stem cells cultured in suspension culture are free of cell encapsulation.

[0182] According to some embodiments of the invention, the conditions for culturing pluripotent stem cells in suspension are no substrate attachment (e.g., no attachment to external substrates (e.g., components of the extracellular matrix, glass microcarriers or beads, etc.)).

[0183] According to some embodiments of the invention, the culture medium and / or conditions for culturing pluripotent stem cells in suspension do not include a protein carrier.

[0184] As used herein, the expression "protein carrier" refers to a protein that acts in the transport of proteins or nutrients (e.g., minerals such as zinc) to cells in culture. Such protein carriers can be, for example, albumin (e.g., bovine serum albumin), Albumax (lipid-enriched albumin) or plasmanate (isolated proteins of human plasma). Since these carriers are derived from either human or animal sources, their use in hESC of human iPS cell culture is limited by batch-specific variations and / or exposure to pathogens. Therefore, culture media that do not contain protein carriers (e.g., albumin) are very advantageous, since they allow truly defined media that can be produced from recombinant or synthetic materials.

[0185] Culturing in suspension culture according to the methods of some embodiments of the present invention is carried out by plating pluripotent stem cells in a culture vessel at a cell density that promotes cell survival and proliferation but limits differentiation. Typically, the cell density is about 1×10 3 cells / ml ~ approx. 2×10 6 A plating density (or seeding density) of between 1×10 cells / ml is used. When a bioreactor is used, the concentration of cells seeded in the bioreactor is about 1×10 4 cells / ml to about 10 6 It will be appreciated that although single cell suspensions of stem cells are typically seeded, small clusters (eg, 10-200 cells) may also be used.

[0186] To provide pluripotent stem cells with a sufficient and constant supply of nutrients and growth factors in suspension culture, the culture medium can be replaced daily or on a predetermined schedule, for example, every 2-3 days. For example, the culture medium can be replaced by subjecting the suspension culture of pluripotent stem cells to centrifugation at 80 g for about 3 minutes and resuspending the formed pluripotent stem cell pellet in fresh medium. Additionally or alternatively, a culture system can be used in which the culture medium is subjected to constant filtration or dialysis, resulting in a constant supply of nutrients or growth factors to the pluripotent stem cells.

[0187] The culture vessel used for culturing pluripotent stem cells in suspension according to the method of some embodiments of the present invention can be any tissue culture vessel (e.g., with a purity specification suitable for culturing pluripotent stem cells) with a surface designed such that the pluripotent stem cells cultured therein cannot attach or adhere to the inner surface (e.g., non-tissue culture treated cells to prevent attachment or adhesion to the surface). Preferably, to obtain a scalable culture, the culture according to some embodiments of the present invention is performed using a controlled culture system (preferably a computer-controlled culture system) in which various culture parameters (e.g., temperature, agitation, pH, and pO2, etc.) are automatically performed using suitable devices. Once the culture parameters are recorded, the system is set for automatic adjustment of the culture parameters required for the expansion of pluripotent stem cells.

[0188] According to some embodiments of the invention, the culturing is carried out under conditions that include static (i.e., non-dynamic) suspension culture.

[0189] For non-dynamic culture of pluripotent stem cells, pluripotent stem cells can be cultured in uncoated 58 mm Petri dishes (Greiner, Frickenhausen, Germany). For example, to start a suspension culture in a 58 mm Petri dish, pluripotent stem cells are cultured at 1 × 10 6 ~5 x 10 cells / dish 6 Cells are seeded at a cell density of 1000 cells / dish.

[0190] During non-dynamic suspension culture, pluripotent stem cells can be passaged every 5-7 days by dissociating the cell aggregates as described above and splitting the culture into additional culture vessels at a ratio of approximately 1:2 to 1:4.

[0191] According to some embodiments of the invention, the culturing is carried out under conditions that include dynamic suspension culture (eg, using a wave reactor or a stirred reactor).

[0192] For dynamic culture of pluripotent stem cells, the pluripotent stem cells can be cultured in spinner flasks [e.g., 200ml-1000ml spinner flasks, e.g., 250ml spinner flasks available from CellSpin (Integra Biosciences, Fernwald, Germany), 100ml spinner flasks available from Bellco (Vineland, NJ), or 125ml Erlenmeyer flasks (Corning Incorporated, Corning NY, USA)] that can be connected to a control device, thus resulting in a controlled culture system. The culture vessel (e.g., spinner flask, Erlenmeyer flask) is shaken continuously. According to some embodiments of the present invention, the culture vessel is shaken at 40-110 revolutions per minute (rpm) using a magnet plate and placed in an incubator. Additionally or alternatively, the culture vessel can be shaken using a shaker (S3.02.10L, ELMI ltd (Riga, Latvia)). According to some embodiments of the present invention, the culture medium is changed every 1-3 days, for example, every day. Other suitable controlled bioreactors that stir the medium by an impeller and can be used for dynamic culturing of pluripotent stem cells in the culture medium according to some embodiments of the present invention include Biostat® Aplus cell culture (Sartorius North America, Edgewood, New York, USA), Cell Optimizer controlled bioreactor with Cell Lift impeller (Infors HT, Rittergasse, Switzerland) (Wheaton Science Products, Millville, NJ, USA), Informs HT Multifors stirred reactor (Informs GA, CH-4103 Bottmingen, Switzerland).

[0193] Additionally or alternatively, dynamic culture of pluripotent stem cells can be achieved using controlled bioreactors where cell dynamics are achieved by wave-like motion, such as Biostat® Cultibag RM (Sartorius North America, Edgewood, New York, USA) (2 liter bags for 1 liter). Reactor parameters can include tilt speed: 10-16 revolutions per minute (rpm), angle: 7°, temperature: 37°C, pH: 7-7.4, O2 concentration: 50%. Another suitable bioreactor is the WavePod System 20 / 50 EH5 Wave Bioreactor (GE Healthcare, USA), which allows for a 70-fold expansion in 12 days while using the same parameters. A further suitable bioreactor is the 55 ml RWV / STLV bioreactor (Synthecon Incorporated, Houston, TX, USA), which allows for minimal shear forces within the reactor.

[0194] For example, to initiate suspension cultures under dynamic conditions, pluripotent stem cells were cultured at approximately 10 4 cells / ml~10 6 Cells are seeded at a density of 1000 cells / ml.

[0195] During dynamic suspension culture, pluripotent stem cells can be passaged every 5-7 days by dissociating the cell aggregates as described above. Because the bioreactor has a large capacity, the cell culture does not need to be further separated into additional culture vessels, and only the addition of medium and / or replacement of medium with fresh medium can be performed every 3-10 days.

[0196] The teachings of the present invention can be used to derive pluripotent stem cell lines.

[0197] The term "derive" as used herein refers to the production of an embryonic stem cell line or an induced pluripotent stem cell line from at least one embryonic stem cell or induced pluripotent cell.

[0198] As used herein, the phrase "embryonic stem cell line" refers to embryonic stem cells that are derived from a single embryonic stem cell or a population of embryonic stem cells of a single organism (e.g., a single human blastocyst) and are characterized by their ability to grow in culture while maintaining an undifferentiated state and pluripotent potential.

[0199] As used herein, the phrase "induced pluripotent stem cell line" refers to induced pluripotent stem cells that are derived from a single induced pluripotent stem cell or a population of pluripotent stem cells of a single organism and are characterized by their ability to grow in culture while maintaining their undifferentiated state and pluripotent potential.

[0200] According to an aspect of some embodiments of the present invention, there is provided a method of deriving an embryonic stem cell line, comprising: (a) obtaining embryonic stem cells (ESCs) from a pre-implantation stage blastocyst, a post-implantation stage blastocyst, and / or fetal reproductive tissue; and (b) passaging the ESCs in suspension culture by mechanical dissociation of ESC clumps into single cells for at least two passages and up to ten passages, thereby obtaining a clump-free suspension culture of ESCs; and (c) passaging the suspension culture of clump-free ESCs without clump dissociation, thereby deriving the embryonic stem cell line.

[0201] Embryonic stem cells can be obtained from pre-implantation stage blastocysts, post-implantation stage blastocysts and / or fetal reproductive tissues using methods known in the art and as described herein above.

[0202] According to an aspect of some embodiments of the invention, the method of deriving an embryonic stem cell line further comprises culturing the ESCs under conditions allowing expansion of the undifferentiated embryonic single stem cells.

[0203] According to an aspect of some embodiments of the present invention, there is provided a method of deriving an induced pluripotent stem cell (iPS cell) line, comprising inducing a somatic cell into a pluripotent stem cell, and expanding and maintaining said induced pluripotent stem cell in an undifferentiated state according to a method of some embodiments of the present invention (e.g., as described hereinabove and in the Examples section below), thereby deriving said induced pluripotent stem cell (iPS cell) line.

[0204] As noted above, and as described in Table 4 and Example 9 in the Examples section below, the cloning efficiency of pluripotent stem cells cultured in suspension as single cells is significantly higher than the cloning efficiency of the same cells when cultured in a two-dimensional culture system (e.g., when cultured on MEFs) without the use of anti-apoptotic agents (e.g., ROCK inhibitors, etc.).

[0205] According to an aspect of some embodiments of the present invention, a method for cloning pluripotent stem cells is provided, which comprises culturing a single pluripotent stem cell (i.e., one cell) obtained according to the method of some embodiments of the present invention, or a single embryonic stem cell (i.e., one cell) obtained according to the method of some embodiments of the present invention in suspension culture under conditions that allow the expansion of said single pluripotent stem cell in an undifferentiated state or the expansion of said single embryonic stem cell, thereby expanding said single pluripotent stem cell or said embryonic stem cell into a clonal culture, thereby cloning said pluripotent stem cell.

[0206] According to some embodiments of the invention, culturing of the single cell suspension cultures is performed without dissociating cell clumps.

[0207] As described in Example 9 of the Examples section below, under identical assay conditions, pluripotent stem cells cultured as single cells in suspension culture have greater tolerance to freeze-thaw cycles (e.g., approximately 80% survival) compared to the same cells cultured in 2-D (e.g., up to 50% survival when cultured on MEFs).

[0208] According to some embodiments of the invention, pluripotent stem cells can undergo at least 1 cycle, at least 2 cycles, at least 3 cycles, at least 4 cycles, at least 5 cycles, at least 6 cycles, at least 7 cycles, at least 8 cycles, at least 9 cycles, at least 10 cycles (e.g., up to 10 cycles) of freeze / thawing while retaining their pluripotent capacity and without interfering with the proliferative capacity of the cells in their undifferentiated state when the pluripotent stem cells are cultured as single cells in suspension culture.

[0209] As described in Example 8 of the Examples section below, pluripotent stem cells cultured according to the methods of some embodiments of the present invention as single cells in suspension culture exhibit a unique expression pattern that is slightly different from that of hESCs but similar to that of mouse ESCs (TRA1-60 - / TRA1-81 - / SSEA1 + / SSEA4 - ; see Pera MF et al., 2000. Journal of Cell Science, 113, 5-10 (human embryonic stem cells, review). Thus, as shown in Table 3 and Figure 13A, pluripotent stem cells cultured in suspension as single cells (without cell clumps) express OCT4 (a marker of pluripotency) when compared to levels of OCT4 RNA in pluripotent stem cells cultured on MEFs or (e.g., approximately 200-1 x 10 per clump). 5 The OCT4 RNA levels are expressed at significantly greater levels (e.g., approximately 8-fold greater RNA levels) when compared to levels of OCT4 RNA in pluripotent stem cells cultured in suspension culture as cell aggregates (with aggregates having more than 10 cells).

[0210] Cells cultured according to the methods of some embodiments of the present invention can be further isolated.

[0211] Thus, according to an aspect of some embodiments of the present invention there is provided an isolated population of pluripotent stem cells produced according to the methods of some embodiments of the present invention and capable of differentiating into each of the germ layers of an embryo: endoderm, ectoderm and mesoderm.

[0212] As shown in Figures 12A-12J, and as described in Example 8 of the Examples section below, pluripotent stem cells cultured in suspension as single cells do not express TRA1-60, TRA1-81 or SSEA-4, but do express SSEA1.

[0213] Thus, in accordance with an aspect of some embodiments of the present invention, OCT4 + / TRA1-60 - / TRA1-81 - / SSEA1 + / SSEA4 -At least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70% (e.g., 70%), at least about 75% (e.g., 75%), at least about 80% (e.g., 80%), at least about 81% (e.g., 81%), at least about 82% (e.g., 82%), at least about 83% (e.g., 83%), at least about 84% (e.g., 84%), at least about 85% (e.g., 85%), at least about 86% (e.g., 86%), at least about 87% (e.g., 87%), at least about 88% (e.g., , 88%), at least about 89% (e.g., 89%), at least about 90% (e.g., 90%), at least about 91% (e.g., 91%), at least about 92% (e.g., 92%), at least about 93% (e.g., 93%), at least about 94% (e.g., 94%), at least about 95% (e.g., 95%), at least about 96% (e.g., 96%), at least about 97% (e.g., 97%), at least about 98% (e.g., 98%), at least about 99% (e.g., 99%), e.g., 100%, wherein the human pluripotent stem cells are capable of differentiating into each of the germ layers of an embryo, including endoderm, ectoderm and mesoderm.

[0214] According to some embodiments of the invention, the isolated cell population comprises cells that express Rex1, Sox2, EGFR, TGA7, TGA6, ITGA2, CTNNB1, CDH1 at levels comparable (within the same order of magnitude) to hESCs cultured on MEFs, and cells that express significantly greater levels of FBLN5 and PLXNA2 when compared to hESCs cultured on MEFs under the same assay conditions.

[0215] As described in Examples 1 and 2 of the Examples section below, the inventors have discovered novel culture media that can be used to maintain and expand pluripotent stem cells in a proliferative, undifferentiated state.

[0216] According to an aspect of some embodiments of the present invention there is provided a defined culture medium suitable for maintaining and expanding pluripotent stem cells in a proliferative, pluripotent and undifferentiated state in a two-dimensional or three-dimensional culture system in the absence of a feeder cell support.

[0217] As used herein, the phrase "culture medium" refers to a liquid substance used to support the growth of pluripotent stem cells and maintain them in an undifferentiated state. The culture medium used by the present invention according to some embodiments can be a water-based medium, which contains a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins, e.g., cytokines, growth factors and hormones, all of which are necessary for cell growth and have the ability to maintain pluripotent stem cells in an undifferentiated state. For example, the culture medium according to aspects of some embodiments of the present invention may be a synthetic tissue culture medium, such as Ko-DMEM (a product of Gibco-Invitrogen corporation, Grand Island, NY, USA), DMEM / F12 (Biological Industries, Beit Haemek, Israel), Mab ADCB medium (HyClone, Utah, USA), Nutrisem™ (Biological Industries, Beit HaEmek, Israel; also known as Stemedia™ NutriStem™ XF / FF Culture Medium, STEMGENT, USA), TeSR™ (StemCell Technologies) and TeSR2™ (StemCell Technologies), supplemented with necessary additives as further described below.

[0218] According to some embodiments of the invention, the culture medium comprises DMEM / F12 at a concentration in the range of 80-90%, for example about 85%.

[0219] According to some embodiments of the invention, the culture medium is serum-free.

[0220] As used herein, the phrase "serum-free" refers to the absence of human or animal serum.

[0221] It should be noted that the function of serum in culture protocols is to provide the cultured cells with an environment similar to that which exists in vivo (i.e., inside the organism from which the cells are derived, e.g., the blastocyst of an embryo). However, the use of serum from either animal sources (e.g., bovine serum) or human sources (human serum) is limited by significant differences in serum components between donor individuals (from whom the serum is obtained) and the risk of having xenogeneic contaminants (if animal serum is used).

[0222] According to some embodiments of the present invention, the serum-free culture medium does not contain serum or any part thereof.

[0223] According to some embodiments of the invention, the serum-free culture medium of the invention does not contain serum albumin (e.g., albumin purified from human serum or animal serum).

[0224] According to some embodiments of the invention, the culture medium comprises a serum replacement.

[0225] As used herein, the phrase "serum replacement" refers to defined preparations that replace the function of serum by providing pluripotent stem cells with components required for proliferation and survival.

[0226] A variety of serum replacement formulations are known in the art and are commercially available.

[0227] For example, GIBCO™ Knockout™ Serum Replacement (Gibco-Invitrogen Corporation, Grand Island, NY, USA, Catalog No. 10828028) is a defined serum-free formulation optimized to grow and maintain undifferentiated ES cells in culture. It should be noted that the GIBCO™ Knockout™ Serum Replacement formulation contains Albumax (a lipid-rich bovine serum albumin) derived from an animal source (International Patent Publication No. WO 98 / 30679 to Price PJ et al.). However, a recent publication by Crook et al., 2007 (Crook JM et al., 2007, Cell Stem Cell, 1:490-494) describes six clinical grade hESC lines that were generated using FDA-approved clinical grade foreskin fibroblasts in Knockout™ Serum Replacement (Invitrogen Corporation, USA, Catalogue No. 04-0095) produced under cGMP.

[0228] Another commercially available serum replacement is B27 supplement without vitamin A, available from Gibco-Invitrogen Corporation, Grand Island, NY USA, catalog number 12587-010. B27 supplement is a serum-free formulation that contains d-biotin, fatty acid free fraction V bovine serum albumin (BSA), catalase, L-carnitine HCl, corticosterone, ethanolamine HC1, D-galactose (Anhyd.), glutathione (reduced), recombinant human insulin, linoleic acid, linolenic acid, progesterone, putrescine-2-HCl, sodium selenite, superoxide dismutase enzyme, T-3 / albumin complex, DL-α-tocopherol, and DL-α-tocopherol acetate. However, the use of B27 supplement is limited because it contains albumin from animal sources.

[0229] According to some embodiments of the invention, the serum replacement is free (completely free) of animal-derived contaminants, which may be pathogens capable of infecting human cells, or cellular or acellular components (fluids) of the animal.

[0230] It should be noted that when serum replacements that are free of animal-derived contaminants are used to culture human cells, such serum replacements are designated as being "xeno-free."

[0231] The term "xeno" is a prefix based on the Greek word "xenos," or stranger. As used herein, the phrase "xeno-free" indicates free of any components / contaminants derived from a xenos (i.e., non-identical, foreign) species.

[0232] For example, a xeno-free serum replacement for use with human cells (i.e., a serum replacement free of animal-derived contaminants) may include a combination of insulin, transferrin, and selenium. Additionally or alternatively, the xeno-free serum replacement may include human or recombinantly produced albumin, transferrin, and insulin.

[0233] Non-limiting examples of commercially available xeno-free serum replacement compositions include ITS (insulin, transferrin and selenium) premix available from Invitrogen corporation (ITS, Invitrogen, Catalog No. 51500-056); Serum replacement 3 (Sigma, Catalog No. S2640), which contains human serum albumin, human transferrin and human recombinant insulin, and does not contain growth factors, steroid hormones, glucocorticoids, cell adhesion factors, detectable Ig and mitogens; KnockOut™ SR XenoFree (Catalog Nos. A10992-01, A10992-02, Part Nos. 12618-012 or 12618-013, Invitrogen GIBCO), which contains only human-derived or human recombinant proteins.

[0234] According to some embodiments of the present invention, ITS (Invitrogen corporation) or SR3 (Sigma) xeno-free serum replacement formulations are diluted in a 1:100 ratio to reach a working concentration of ×1.

[0235] According to some embodiments of the present invention, the concentration of the serum replacement (e.g., KnockOut™ SR XenoFree (Invitrogen)) in the culture medium is in the range of about 1% [volume / volume (v / v)] to about 50% (v / v), such as in the range of 5% (v / v) to about 40% (v / v), such as in the range of about 5% (v / v) to about 30% (v / v), such as in the range of about 10% (v / v) to about 30% (v / v), such as in the range of about 10% (v / v) to about 25% (v / v), such as in the range of 10% (v / v) to about 20% (v / v), such as about 10% (v / v), such as about 15% (v / v), such as about 20% (v / v), such as about 30% (v / v).

[0236] According to some embodiments of the invention, the culture medium is capable of maintaining pluripotent stem cells in a proliferative, pluripotent, undifferentiated state for at least 5 passages, for at least 10 passages, for at least 15 passages, for at least 20 passages, for at least 25 passages, for at least 30 passages, for at least 35 passages, for at least 40 passages, for at least 45 passages, for at least 50 passages (e.g., for at least 25, 50, 75, 100 or 250 days in culture).

[0237] According to some embodiments of the invention, the culture medium is capable of expanding pluripotent stem cells in an undifferentiated state.

[0238] For example, as described in Example 1 of the Examples section below, hESCs or human iPS cells could be maintained in an undifferentiated state when cultured in suspension for at least 20 passages in a two-dimensional culture system or at least 50 passages in a three-dimensional culture system. Assuming that each passage occurs every 5-7 days (e.g., 144 hours) and that the observed doubling time is approximately 25-36 hours, a single hESC or human iPS cell cultured under these conditions could be maintained in an undifferentiated state for at least 20 passages in a two-dimensional culture system or at least 50 passages in a three-dimensional culture system. 4 ~2 5 It should be noted that when cultured in a controlled bioreactor, the expansion capacity of pluripotent stem cells increases approximately 64-fold within 5 days. Thus, within one month (i.e., 720 hours) of culture, a single pluripotent stem cell could be expanded to 2 20 (1×10 6 ) hESCs or human iPS cells.

[0239] The inventors have discovered that a combination of growth factors, namely interleukin 11 (IL11) and ciliary neurotrophic factor (CNTF), or interleukin 11 (IL11) and oncostatin, can be used to support the growth and expansion of pluripotent stem cells in a proliferative undifferentiated pluripotent state.

[0240] According to an aspect of some embodiments of the present invention there is provided a culture medium comprising interleukin-11 (IL11) and ciliary neurotrophic factor (CNTF), or a culture medium comprising interleukin-11 and oncostatin.

[0241] As used herein, the term "interleukin-11" refers to a protein member of the gp130 family of cytokines (also known as AGIF and IL-11). Interleukin-11 (e.g., human IL-11 polypeptide, GenBank Accession No. NP_000632.1 (SEQ ID NO: 32); human IL-11 polynucleotide, GenBank Accession No. NM_000641.2 (SEQ ID NO: 33)) can be obtained from a variety of commercial sources, such as from R&D Systems, or PeproTech.

[0242] As used herein, the term "ciliary neurotrophic factor" (also known as HCNTF; CNTF) refers to a polypeptide hormone whose action is limited to the nervous system, where it promotes the synthesis of neurotransmitters and the growth of neurites in certain neuronal populations. This protein is a potent survival factor for neurons and oligodendrocytes, and may be involved in reducing tissue destruction during inflammatory attacks. CNTF [e.g., human CNTF polypeptide, GenBank Accession No. NP_000605.1 (SEQ ID NO: 34); human CNTF polynucleotide, GenBank Accession No. NM_000614 (SEQ ID NO: 35)] can be obtained from various commercial sources, such as R&D Systems, or PeproTech.

[0243] As used herein, the term "oncostatin" (also known as OSM oncostatin M (OSM)) refers to a polypeptide member of the cytokine family that includes leukemia inhibitory factor, granulocyte colony stimulating factor, and interleukin 6. Oncostatin (e.g., human oncostatin polypeptide, GenBank Accession No. NP_065391.1 (SEQ ID NO: 36) or P13725 (SEQ ID NO: 37); human polynucleotide, GenBank Accession No. NM_020530.3 (SEQ ID NO: 38)) can be obtained from a variety of commercial sources, for example, from R&D Systems (e.g., R&D Systems catalog number 295-OM-010).

[0244] According to some embodiments of the invention, the culture medium does not contain a glycogen synthase kinase 3 (GSK3) inhibitor.

[0245] Non-limiting examples of GSK3 inhibitors include inhibitors of GSK-alpha or GSK-beta, such as CHIR 98014, CHIR 99021, AR-AO144-18, SB216763 and SB415286. Examples of GSK3 inhibitors are described in Bennett C et al., J. Biological Chemistry, Vol. 277, No. 34 (August 23, 2002), pp. 30998-31004, and Ring DB et al., Diabetes, Vol. 52 (March 2003), pp. 588-595 (each of which is fully incorporated herein by reference).

[0246] According to some embodiments of the invention, IL11 is used at a concentration of at least about 0.1 ng / ml and up to about 10 ng / ml, such as at a concentration of at least about 0.2 ng / ml, such as at a concentration of at least about 0.3 ng / ml, for example at a concentration of at least about 0.4 ng / ml, for example at a concentration of at least about 0.5 ng / ml, at a concentration of at least about 0.6 ng / ml, for example at a concentration of at least about 0.7 ng / ml, such as at a concentration of at least about 0.8 ng / ml, for example at a concentration of at least about 0.9 ng / ml, for example at a concentration of at least about 1 ng / ml, for example at a concentration of about 1 ng / ml.

[0247] According to some embodiments of the present invention, IL11 is used at a concentration of between about 0.5 ng / ml and about 5 ng / ml.

[0248] According to some embodiments of the invention, CNTF is used at a concentration of at least about 0.1 ng / ml and up to about 10 ng / ml, such as at a concentration of at least about 0.2 ng / ml, such as at a concentration of at least about 0.3 ng / ml, such as at a concentration of at least about 0.4 ng / ml, such as at a concentration of at least about 0.5 ng / ml, such as at a concentration of at least about 0.6 ng / ml, such as at a concentration of at least about 0.7 ng / ml, such as at a concentration of at least about 0.8 ng / ml, such as at a concentration of at least about 0.9 ng / ml, such as at a concentration of at least about 1 ng / ml, such as at a concentration of about 1 ng / ml.

[0249] According to some embodiments of the present invention, CNTF is used at a concentration of between about 0.5 ng / ml and about 5 ng / ml.

[0250] According to some embodiments of the invention, oncostatin is used at a concentration of at least about 0.1 ng / ml and up to about 10 ng / ml, such as at a concentration of at least about 0.2 ng / ml, such as at a concentration of at least about 0.3 ng / ml, such as at a concentration of at least about 0.4 ng / ml, such as at a concentration of at least about 0.5 ng / ml, such as at a concentration of at least about 0.6 ng / ml, such as at a concentration of at least about 0.7 ng / ml, such as at a concentration of at least about 0.8 ng / ml, such as at a concentration of at least about 0.9 ng / ml, such as at a concentration of at least about 1 ng / ml, such as at a concentration of about 1 ng / ml.

[0251] According to some embodiments of the present invention, oncostatin is used at a concentration of between about 0.5 ng / ml and about 5 ng / ml.

[0252] According to some embodiments of the present invention, the medium comprising IL11 and CNTF, or the medium comprising IL11 and oncostatin, further comprises a serum replacement (e.g., a serum replacement free of animal-derived contaminants) at a concentration of between about 10% and about 20%, for example, at a concentration of about 15%.

[0253] According to some embodiments of the invention, the culture medium comprising IL11 and CNTF, or the culture medium comprising IL11 and oncostatin, further comprises basic fibroblast growth factor (bFGF).

[0254] Basic fibroblast growth factor (bFGF, also known as FGF2 or FGF-β) is a member of the fibroblast growth factor family. bFGF [(e.g., human bFGF polypeptide GenBank Accession No. NP_001997.5 (SEQ ID NO: 39); human bFGF polynucleotide GenBank Accession No. NM_002006.4 (SEQ ID NO: 40) may be obtained from a variety of commercial sources, such as Cell Sciences® (Canton, MA, USA) (e.g., catalog numbers CRF001A and CRF001B), Invitrogen Corporation products (Grand Island NY, USA) (e.g., catalog numbers: PHG0261, PHG0263, PHG0266, and PHG0264), ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel) (e.g., catalog number: CYT-218), and Sigma (St Louis, MO, USA) (e.g., catalog number: F0291).

[0255] The concentration of bFGF in a culture medium containing IL11 and CNTF, or in a culture medium containing IL11 and oncostatin, can be at least about 4 ng / ml and up to 100 ng / ml, for example, at least about 5 ng / ml, for example, at least about 6 ng / ml, for example, at least about 7 ng / ml, for example, at least about 8 ng / ml, for example, at least about 9 ng / ml, for example, at least about 10 ng / ml.

[0256] Non-limiting examples of culture media containing IL11 and CNTF include ILCNTF medium, NILCNTF medium, described in the Examples section below, which have been shown to be capable of supporting the growth of hESCs and iPS cells in a proliferative, pluripotent, undifferentiated state for at least 12 passages in two-dimensional culture systems and at least 10 passages in suspension culture.

[0257] The present inventors have discovered that IL6RIL6 chimeras can be used in culture media that is completely free of animal-derived contaminants to support the growth of human pluripotent stem cells in an undifferentiated state.

[0258] Thus, according to an aspect of some embodiments of the present invention there is provided a culture medium comprising an animal-derived contaminant-free serum replacement and an IL6RIL6 chimera.

[0259] As used herein, the phrase "IL6RIL6 chimera" refers to a chimeric polypeptide comprising a soluble portion of the interleukin-6 receptor [IL-6-R, e.g., human IL-6-R as represented by GenBank Accession No. AAH89410 (SEQ ID NO: 41); e.g., a portion of the soluble IL6 receptor as represented by amino acids 112 to 355 of GenBank Accession No. AAH89410 (SEQ ID NO: 42)] and interleukin-6 (IL6; e.g., human IL-6 as represented by GenBank Accession No. CAG29292 (SEQ ID NO: 43)] or a biologically active fraction thereof (e.g., a receptor binding domain).

[0260] When constructing an IL6RIL6 chimera, it should be noted that the two functional parts (i.e., IL6 and its receptor) can be directly fused to each other (e.g., linked or translationally fused, i.e., encoded by only one open reading frame), or can be conjugated (linked or translationally fused) via a suitable linker (e.g., a polypeptide linker). According to some embodiments of the present invention, the IL6RIL6 chimera polypeptide exhibits an amount and pattern of glycosylation similar to that of naturally occurring IL6 and IL6 receptor. For example, a suitable IL6RIL6 chimera is as shown in SEQ ID NO: 19 and FIG. 11 of International Publication WO99 / 02552 (Revel M. et al.), which is incorporated herein by reference in its entirety.

[0261] It should be noted that given that the serum replacement is completely free of animal-derived contaminants, further culture medium components can also be selected to be free of animal-derived contaminants (e.g., can be synthetic, recombinant, or purified from human origin), so that the entire culture medium is free of animal-derived contaminants and can be used as a xeno-free medium for culturing human pluripotent stem cells, suitable for clinical / therapeutic purposes.

[0262] The inventors have discovered that IL6RIL6 chimeras can be used at either high concentrations (i.e., between 50 ng / ml and 150 ng / ml) or low concentrations (i.e., between 50 pg / ml and 150 pg / ml) while still maintaining that the medium can support the growth of pluripotent stem cells in an undifferentiated state.

[0263] According to some embodiments of the present invention, the concentration of the IL6RIL6 chimera is at least about 50 ng / ml and up to about 350 ng / ml, for example, between about 50 ng / ml and 200 ng / ml, for example, in the range of about 55 ng / ml to about 195 ng / ml, for example, in the range of about 60 ng / ml to about 190 ng / ml, for example, in the range of about 65 ng / ml to about 185 ng / ml, for example, in the range of about 70 ng / ml to about 180 ng / ml, for example, in the range of about 75 ng / ml to about 175 ng / ml, for example, in the range of about 80 ng / ml to about 170 ng / ml. for example, about 85 ng / ml to about 165 ng / ml, for example, about 90 ng / ml to about 150 ng / ml, for example, about 90 ng / ml to about 140 ng / ml, for example, about 90 ng / ml to about 130 ng / ml, for example, about 90 ng / ml to about 120 ng / ml, for example, about 90 ng / ml to about 110 ng / ml, for example, about 95 ng / ml to about 105 ng / ml, for example, about 98 ng / ml to about 102 ng / ml, for example, about 100 ng / ml of IL6RIL6 chimera.

[0264] Non-limiting examples of animal-derived contaminant-free culture media containing between about 50 ng / ml and 200 ng / ml of IL6RIL6 chimera include cmTeSR2, NCMrb100F, NCM100F, cmV5b and cmHA13.

[0265] According to some embodiments of the present invention, the concentration of the IL6RIL6 chimera is at least about 50 pg / ml and at most about 150 pg / ml, for example, between about 50 pg / ml and 200 pg / ml, for example, in the range of about 55 pg / ml to about 195 pg / ml, for example, in the range of about 60 pg / ml to about 190 pg / ml, for example, in the range of about 65 pg / ml to about 185 pg / ml, for example, in the range of about 70 pg / ml to about 180 pg / ml, for example, in the range of about 75 pg / ml to about 175 pg / ml, for example, in the range of about 80 pg / ml to about 170 pg / ml. for example, about 85 pg / ml to about 165 pg / ml, for example, about 90 pg / ml to about 150 pg / ml, for example, about 90 pg / ml to about 140 pg / ml, for example, about 90 pg / ml to about 130 pg / ml, for example, about 90 pg / ml to about 120 pg / ml, for example, about 90 pg / ml to about 110 pg / ml, for example, about 95 pg / ml to about 105 pg / ml, for example, about 98 pg / ml to about 102 pg / ml, for example, about 100 pg / ml of an IL6RIL6 chimera.

[0266] Non-limiting examples of xeno-free culture media containing between about 50 pg / ml and 200 pg / ml of IL6RIL6 chimeras include cmTeSR2p, NCMrb100Fp, NCM100Fp, cmV5bp and cmHA13p.

[0267] For example, IL6RIL6 chimeras can be added to TeSR™2 Animal Protein-Free Medium (StemCell Technologies, Catalog #05860 / 05880) culture medium. TeSR™2 medium is a defined, animal protein-free, serum-free complete formulation containing recombinant human basic fibroblast growth factor (rhbFGF) and recombinant human transforming growth factor β (rhTGFβ).

[0268] According to some embodiments of the present invention, the animal-derived contaminant-free culture medium comprising the IL6RIL6 chimera further comprises bFGF.

[0269] bFGF can be used in either low concentrations (eg, between about 4 ng / ml and 20 ng / ml) or high concentrations (eg, between 50 ng / ml and 150 ng / ml).

[0270] According to some embodiments of the present invention, the culture medium containing the serum replacement free of animal-derived contaminants and the IL6RIL6 chimera further contains bFGF at a concentration of at least about 4ng / ml, for example at a concentration of at least about 5ng / ml, for example at a concentration of at least about 6ng / ml, for example at a concentration of at least about 7ng / ml, for example at a concentration of at least about 8ng / ml, for example at a concentration of at least about 9ng / ml, for example at a concentration of at least about 10ng / ml, for example at a concentration of at least about 15ng / ml, for example at a concentration of at least about 20ng / ml. Non-limiting examples of such culture medium include cmV5b, NCM100Fp, NCM100F and cmV5bp.

[0271] According to some embodiments of the present invention, the culture medium containing a serum substitute free of animal-derived contaminants and an IL6RIL6 chimera further contains bFGF at a concentration of at least about 50 ng / ml to about 1 μg, for example, at a concentration of about 60 ng / ml to about 1 μg / ml, for example, at a concentration of about 70 ng / ml to about 500 ng / ml, for example, at a concentration of about 80 ng / ml to about 500 ng / ml, for example, at a concentration of about 90 ng / ml to about 250 ng / ml, for example, at a concentration of about 50 ng / ml to about 200 ng / ml, for example. For example, at a concentration of about 50ng / ml to about 150ng / ml, for example, at a concentration of about 50ng / ml, for example, at a concentration of about 60ng / ml, for example, at a concentration of about 70ng / ml, for example, at a concentration of about 80ng / ml, for example, at a concentration of about 90ng / ml, for example, at a concentration of about 100ng / ml, for example, at a concentration of about 110ng / ml, for example, at a concentration of about 120ng / ml, for example, at a concentration of about 130ng / ml, for example, at a concentration of about 140ng / ml, for example, at a concentration of about 150ng / ml. Non-limiting examples of such culture media include NCMrb100F, NCMrb100Fp, cmTeSR2, and cmTeSR2p.

[0272] According to some embodiments of the present invention, the animal-derived contaminant-free culture medium comprising the IL6RIL6 chimera further comprises ascorbic acid.

[0273] Ascorbic acid (also known as vitamin C) is a sugar acid (C6H8O6; molecular weight, 176.12 grams / mole) with antioxidant properties. The ascorbic acid used by the culture medium of some embodiments of the present invention can be natural ascorbic acid, synthetic ascorbic acid, ascorbate salts (e.g., sodium ascorbate, calcium ascorbate, potassium ascorbate), ester forms of ascorbic acid (e.g., ascorbyl palmitate, ascorbyl stearate), functional derivatives thereof (molecules derived from ascorbic acid that exhibit the same activity / function when used in the culture medium of the present invention), or analogs thereof (e.g., functional equivalents of ascorbic acid that exhibit similar activity to that observed for ascorbic acid when used in the culture medium of the present invention). Non-limiting examples of ascorbic acid formulations that can be used in the culture medium of some embodiments of the present invention include L-ascorbic acid and ascorbic acid 3-phosphate.

[0274] Ascorbic acid can be obtained from a variety of manufacturers, for example, from Sigma (St. Louis, MO, USA) (e.g., catalog numbers: A2218, A5960, A7506, A0278, A4403, A4544, A2174, A2343, 95209, 33034, 05878, 95210, 95212, 47863, 01-6730, 01-6739, 255564, A92902, W210901).

[0275] According to some embodiments of the present invention, the concentration of ascorbic acid in the animal-derived contaminant-free culture medium containing the IL6RIL6 chimera is between about 25 μg / ml and 200 μg / ml, for example, between 25 μg / ml and 150 μg / ml, for example, between 30 μg / ml and 150 μg / ml, for example, between about 40 μg / ml and 120 μg / ml, for example, between about 40 μg / ml and 100 μg / ml, for example, between about 40 μg / ml and 80 μg / ml, for example, between about 40 μg / ml and 60 μg / ml, for example, about 50 μg / ml. Non-limiting examples of such culture media include cmHA13p medium and cmHA13 medium described in the Examples section below.

[0276] According to some embodiments of the present invention, the animal-derived contaminant-free culture medium comprising the IL6RIL6 chimera further comprises an isoform of transforming growth factor beta (TGFβ).

[0277] As used herein, the phrase "transforming growth factor beta (TGFβ)" refers to any of the isoforms of transforming growth factor beta (β) that function through the same receptor signaling system in controlling growth, differentiation, and other functions in many cell types. TGFβ acts in inducing transformation and also acts as a negative autocrine growth factor.

[0278] According to some embodiments of the invention, the term TGFβ refers to TGFβ1 [human TGFβ1 mRNA sequence: GenBank Accession No. NM_000660.4 (SEQ ID NO: 44), polypeptide sequence: GenBank Accession No. NP_000651.3 (SEQ ID NO: 45)], TGFβ2 [human TGFβ2 mRNA sequence: GenBank Accession No. NM_001135599.1 isoform 1 (SEQ ID NO: 46) or GenBank Accession No. NM_003238.2 isoform 2 (SEQ ID NO: 47), polypeptide sequence: GenBank Accession No. NP_001129071.1 isoform 2 (SEQ ID NO: 48) or GenBank Accession No. NP_003229.1 isoform 2 (SEQ ID NO: 49)], or TGFβ3 [human TGFβ3 mRNA sequence: GenBank Accession No. NM_003239.2 (SEQ ID NO: 50), polypeptide sequence: GenBank Accession No. NP_003230.1 (SEQ ID NO: 51)]. TGFβ isoforms can be obtained from a variety of commercial sources, such as R&D Systems (Minneapolis, MN, USA) and Sigma (St Louis, MO, USA).

[0279] According to some embodiments of the invention, the TGFβ contained in the culture medium is TGFβ1.

[0280] According to some embodiments of the present invention, the concentration of TGFβ1 in the culture medium is in the range of about 0.05 ng / ml to about 1 μg / ml, for example, 0.1 ng / ml to about 1 μg / ml, for example, about 0.5 ng / ml to about 100 ng / ml.

[0281] According to some embodiments of the invention, the concentration of TGFβ1 in the culture medium is at least about 0.5 ng / ml, such as at least about 0.6 ng / ml, such as at least about 0.8 ng / ml, for example, at least about 0.9 ng / ml, such as at least about 1 ng / ml, for example, at least about 1.2 ng / ml, such as at least about 1.4 ng / ml, for example, at least about 1.6 ng / ml, such as at least about 1.8 ng / ml, for example, about 2 ng / ml.

[0282] Non-limiting examples of animal-derived contaminant-free culture media containing IL6RIL6 chimera, bFGF and TGFβ1 are cmV5b, cmV5bp, cmTeSR2 and cmTeSR2p described in the Examples section below.

[0283] According to some embodiments of the invention, the TGFβ contained in the culture medium is TGFβ1.

[0284] According to some embodiments of the present invention, the concentration of TGFβ3 in the culture medium ranges from about 0.05 ng / ml to about 1 μg / ml, for example, from 0.1 ng / ml to about 1 μg / ml, for example, from about 0.5 ng / ml to about 100 ng / ml.

[0285] According to some embodiments of the invention, the concentration of TGFβ3 in the culture medium is at least about 0.5 ng / ml, such as at least about 0.6 ng / ml, such as at least about 0.8 ng / ml, for example, at least about 0.9 ng / ml, such as at least about 1 ng / ml, for example, at least about 1.2 ng / ml, such as at least about 1.4 ng / ml, for example, at least about 1.6 ng / ml, such as at least about 1.8 ng / ml, for example, about 2 ng / ml.

[0286] According to an aspect of some embodiments of the present invention, a culture medium is provided that includes bFGF at a concentration of at least about 50ng / ml (e.g., between 50ng / ml and 200ng / ml) and IL6RIL6 chimera at either a high concentration (e.g., between 50ng / ml and 200ng / ml) or a low concentration (e.g., between 50pg / ml and 200pg / ml). Non-limiting examples of such culture media include CMrb100F, CMrb100Fp, NCMrb100F and NCMrb100Fp culture media that have been shown to be capable of maintaining hESC and iPS cells in a proliferative, pluripotent, undifferentiated state for at least five passages in a two-dimensional culture system and at least 15 passages in a three-dimensional culture system.

[0287] The inventors have discovered that culture media containing high concentrations of soluble interleukin 6 receptor (sIL6R) and interleukin 6 (IL6) can be used to support the growth of pluripotent stem cells in a proliferative undifferentiated pluripotent state.

[0288] Thus, according to an aspect of some embodiments of the present invention, there is provided a culture medium comprising sIL6R and IL6, wherein the concentration of sIL6R is at least about 5 ng / ml and the concentration of IL6 is at least about 3 ng / ml.

[0289] According to some embodiments of the invention, the concentration of sIL6R is at least about 5 ng / ml, e.g., at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, e.g., in the range of 10 ng / ml to 50 ng / ml, e.g., between 20 ng / ml to 40 ng / ml, e.g., about 25 ng / ml.

[0290] According to some embodiments of the invention, the concentration of IL6 is at least about 3 ng / ml, e.g., at least about 4 ng / ml, at least about 5 ng / ml, at least about 6 ng / ml, at least about 7 ng / ml, at least about 8 ng / ml, at least about 9 ng / ml, at least about 10 ng / ml, at least about 15 ng / ml, at least about 20 ng / ml, at least about 25 ng / ml, e.g., in the range of 10 ng / ml to 50 ng / ml, e.g., between 20 ng / ml to 40 ng / ml, e.g., about 25 ng / ml.

[0291] According to some embodiments of the present invention, the medium comprising sIL6R and IL6 further comprises bFGF at a concentration of at least about 4 ng / ml and at a concentration of up to 100 ng / ml, such as at a concentration of at least about 5 ng / ml, such as at a concentration of at least about 6 ng / ml, such as at a concentration of at least about 7 ng / ml, such as at a concentration of at least about 8 ng / ml, such as at a concentration of at least about 9 ng / ml, such as at least about 10 ng / ml.

[0292] According to some embodiments of the present invention, the medium containing sIL6R and IL6 further contains a serum replacement at a concentration between 10% and 30%, for example, at a concentration of about 15%. It should be noted that the concentration of the serum replacement may vary depending on the type of serum replacement used.

[0293] Non-limiting examples of culture media containing sIL6R and IL6 include yFIL25 medium, described in the Examples section below.

[0294] According to some embodiments of the invention, the culture medium further comprises insulin, which can be obtained from Invitrogen (Carlsbad, CA, USA), Sigma (St Louis, MO, USA).

[0295] The concentration of insulin in the culture medium can be between 0.0001 grams / liter and 1 gram / liter (e.g., between about 0.001 μg / μl and about 0.1 μg / μl, e.g., between about 0.005 μg / μl and about 0.05 μg / μl, e.g., about 0.01 μg / μl).

[0296] According to some embodiments of the invention, the culture medium further comprises albumin. Albumin can be obtained from Sigma (St Louis, MO, USA).

[0297] The concentration of albumin in the culture medium can be between about 0.1% and about 5%.

[0298] According to some embodiments of the invention, the culture medium further comprises transferrin, which can be obtained from Invitrogen (Carlsbad, CA, USA), Sigma (St Louis, MO, USA).

[0299] According to some embodiments of the invention, the culture medium further comprises a lipid mixture.

[0300] As used herein, the phrase "lipid mixture" refers to a defined (e.g., chemically defined) lipid composition required to culture pluripotent stem cells. It should be noted that lipid mixtures are typically added to culture media that do not contain serum or serum replacement, thereby replacing the lipids that are typically added to serum or serum replacement formulations.

[0301] A non-limiting example of a commercially available lipid mixture that can be used in the culture medium of some embodiments of the present invention includes Chemically Defined Lipid Concentrate available from Invitrogen (catalog number 11905-031).

[0302] According to some embodiments of the invention, the concentration of the lipid mixture in the culture medium is from about 0.5% [volume / volume (v / v)] to about 3% v / v, such as from about 0.5% v / v to about 2% v / v, such as from about 0.5% v / v to about 1% v / v, such as about 1% v / v.

[0303] According to some embodiments of the invention, the culture medium further comprises sodium bicarbonate, which can be obtained from Biological Industries (Beit HaEmek, Israel).

[0304] According to some embodiments of the present invention, the concentration of sodium bicarbonate in the culture medium is about 5% to about 10%, such as about 6% to about 9%, such as about 7% to about 8%, for example, about 7.5%.

[0305] According to some embodiments of the present invention, the culture medium further comprises L-glutamine. The concentration of L-glutamine in the culture medium can be from about 0.5 millimolar (mM) to about 10 mM, such as about 1 mM to 5 mM, e.g., 2 mM.

[0306] According to some embodiments of the present invention, the culture medium further comprises a non-essential amino acid. The non-essential amino acid can be obtained from various sources, such as Invitrogen Corporation products (Grand Island, NY, USA) as a 10 mM stock solution. The concentration of the non-essential amino acid in the culture medium can be about 0.1% to 10%, such as about 0.2% to 5%, such as 0.5% to 2%, such as about 1%.

[0307] According to some embodiments of the invention, the culture medium further comprises a reducing agent, such as beta-mercaptoethanol (β-mercaptoethanol), in a concentration range of between about 0.01 mM and 1 mM, for example, 0.1 mM.

[0308] As mentioned, any of the proteinaceous factors (e.g., interleukin 11, CNTF, oncostatin, bFGF, IL6RIL6 chimera, TGFβ1, TGFβ3, insulin, albumin, transferrin) used in the culture medium of the present invention can be recombinantly expressed or biochemically synthesized. In addition, naturally occurring proteinaceous factors (e.g., bFGF and TGFβ, etc.) can be purified from biological samples (e.g., human serum, cell culture) using methods widely known in the art. It should be noted that in order to prepare a culture medium free of animal-derived contaminants, proteinaceous factors are preferably purified from human sources or recombinantly expressed.

[0309] Biochemical synthesis of the proteinaceous factors of the present invention (e.g., IL6RIL6 chimeras) can be carried out using standard solid-phase techniques, including exclusive solid-phase synthesis, partial solid-phase synthesis, fragment condensation and classical solution synthesis.

[0310] Recombinant expression of the proteinaceous factors of the invention may be accomplished by the use of recombinant methods such as those described by Bitter et al. (1987) Methods in Enzymol. 153:516-544; Studier et al. (1990) Methods in Enzymol. 185:60-89; Brisson et al. (1984) Nature 310:511-514; Takamatsu et al. (1987) EMBO J. 6:307-311; Coruzzi et al. (1984) EMBO J. 3:1671-1680; Brogli et al. (1984) Science 224:838-843; Gurley et al. (1986) Mol. Cell. Biol. 6:559-565 and Weissbach and Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463. In particular, IL6RIL6 chimeras may be produced as described in PCT Publication WO 99 / 02552 (Revel M. et al. and Chebath J. et al., 1997), which is incorporated herein by reference in its entirety.

[0311] Thus, according to an aspect of some embodiments of the present invention, there is provided a cell culture comprising pluripotent stem cells of some embodiments of the present invention (e.g., hESCs or iPSCs cultured in suspension as single cells without cell clumps; hESCs or iPSCs cultured in suspension as cell clumps; pluripotent stem cells cultured in a two-dimensional culture system, etc.) and a culture medium of some embodiments of the present invention.

[0312] According to some embodiments of the invention, the cell culture is feeder cell-free (does not contain a feeder cell support).

[0313] The expression "feeder cell support" as used herein refers to the ability of feeder cells (e.g., fibroblasts) to maintain pluripotent stem cells in a proliferative and undifferentiated state when the pluripotent stem cells are co-cultured on the feeder cells or when the pluripotent stem cells are cultured on a matrix (e.g., extracellular matrix, synthetic matrix) in the presence of conditioned medium produced by the feeder cells. The support of the feeder cells depends on the structure of the feeder cells while in culture (e.g., a three-dimensional matrix formed by culturing the feeder cells in tissue culture plates), the function of the feeder cells (e.g., secretion of growth factors, nutrients and hormones by the feeder cells, the proliferation rate of the feeder cells, the expansion ability of the feeder cells before senescence), and / or the attachment of the pluripotent stem cells to the feeder cell layer.

[0314] As used herein, the phrase "absence of a feeder cell support" refers to a culture medium and / or cell culture that does not contain feeder cells, and / or the conditioned medium produced thereby.

[0315] According to some embodiments of the invention, the pluripotent stem cells comprised in the cell culture of some embodiments of the invention exhibit a stable karyotype (chromosomal stability) during the culture period, such as over at least 2 passages, such as at least 4 passages, such as at least 8 passages, such as at least 15 passages, such as at least 20 passages, such as at least 25 passages, such as at least 30 passages, such as at least 35 passages, such as at least 40 passages, such as at least 45 passages, such as at least 50 passages.

[0316] According to some embodiments of the invention, the cell cultures of the invention exhibit a doubling time of at least 20 hours, e.g., a doubling time of between 20 and 40 hours (e.g., about 36 hours), thereby representing non-tumorigenic, genetically stable pluripotent stem cells (e.g., hESCs and iPS cells).

[0317] According to some embodiments of the invention the cell cultures of the invention are characterised by at least 40%, at least 50%, at least 60%, such as at least 70%, such as at least 80%, such as at least 85%, such as at least 90%, such as at least 95% undifferentiated pluripotent stem cells.

[0318] The cell culture of some embodiments of the present invention comprises at least 1000 pluripotent undifferentiated stem cells per milliliter (ml) of culture medium. For some applications, for example, for single cell cloning of pluripotent stem cells, the concentration of cells can be about 1 per 100 μl to 200 μl of medium, and it should be noted that each cell is plated in a separate dish, preferably in an uncoated dish (e.g., a non-culture-treated dish) to prevent adhesion of the cells to the dish.

[0319] The differentiated or undifferentiated state of pluripotent stem cells cultured in 2-D or in suspension as cell aggregates can be determined using known methods (e.g., as described in Thompson et al. (1998)). For example, the differentiated state can be determined using various techniques, including, for example, morphological evaluation (e.g., as shown in Figures 1A-1C and 3A-3C), and / or detecting the expression pattern of markers typical of an undifferentiated state using immunological techniques (e.g., flow cytometry for membrane-bound markers, immunohistochemistry or immunofluorescence for extracellular and intracellular markers, and enzyme immunoassays for secreted molecular markers, etc.). For example, immunofluorescence used on hESCs or human iPS cells cultured in culture medium according to some embodiments of the present invention revealed the expression of Oct4, stage-specific embryonic antigen (SSEA) 4, tumor rejection antigen (TRA)-1-60, and TRA-1-81 (e.g., Figures 2A-2D). In addition, transcript levels of specific undifferentiated (e.g., Oct4, Nanog, Sox2, Rex1, Cx43, FGF4) or differentiated (e.g., albumin, glucagon, α-cardiac actin, β-globulin, Flk1, AC133 and neurofilament) markers can be detected using RNA-based techniques, such as RT-PCR analysis and / or cDNA microarray analysis.

[0320] Determination of ES cell differentiation can also be performed by measuring alkaline phosphatase activity. Undifferentiated human ES cells have alkaline phosphatase activity that can be detected by fixing the cells with 4% paraformaldehyde and developing with the Vector Red substrate kit according to the manufacturer's instructions (Vector Laboratories, Burlingame, California, USA).

[0321] According to some embodiments of the present invention, the cell culture comprises pluripotent stem cells and a xeno-free medium, and thus the medium does not contain any contaminants from a species different from the species of the pluripotent stem cells.For example, when the cell culture comprises human pluripotent stem cells, the medium does not contain animal-derived contaminants.Similarly, when the cell culture comprises primate pluripotent stem cells (e.g., monkeys), the culture medium does not contain contaminants from other animals or humans.

[0322] According to an aspect of some embodiments of the present invention there is provided a method of expanding and maintaining pluripotent stem cells in a pluripotent undifferentiated state.

[0323] According to some embodiments of the present invention, the method of expanding and maintaining pluripotent stem cells in an undifferentiated state is carried out by culturing the pluripotent stem cells in any of the novel culture media of the present invention (as described herein).

[0324] According to some embodiments of the invention, expanding and maintaining pluripotent stem cells in an undifferentiated state is performed in suspension culture.

[0325] According to some embodiments of the invention, culturing the pluripotent stem cells in suspension culture is performed in a serum-free and feeder cell-free culture medium.

[0326] Since large clusters of pluripotent stem cells can give rise to cell differentiation, various measures are taken to avoid large pluripotent stem cell aggregates. According to some embodiments of the present invention, the formed pluripotent stem cell aggregates are dissociated every 5-7 days, and single cells, or small aggregates of cells, are either separated (i.e., passaged) into additional culture vessels or remain in the same culture vessel but with additional culture medium.

[0327] According to some embodiments of the invention, the culturing is performed under conditions that allow the pluripotent stem cells to be expanded as single cells.

[0328] As described hereinabove, passaging of pluripotent stem cells can be carried out using mechanical dissociation of cell clumps.

[0329] Additionally and / or alternatively, pluripotent stem cells can be passaged in suspension culture using enzymatic digestion, with or without subsequent mechanical dissociation.

[0330] Enzymatic digestion of the pluripotent stem cell aggregates can be performed by subjecting the aggregates to an enzyme (e.g., type IV collagenase (Worthington biochemical corporation, Lakewood, NJ, USA) and / or dispase (Invitrogen Corporation products, Grand Island, NY, USA)). The incubation time with the enzyme depends on the size of the cell aggregates present in the suspension culture. Typically, when the pluripotent stem cell aggregates are dissociated every 5-7 days during suspension culture, a 20-60 minute incubation with 1.5 mg / ml type IV collagenase results in small cell aggregates that can be further cultured in an undifferentiated state. Alternatively, the pluripotent stem cell aggregates can be subjected to about 25 minutes of incubation with 1.5 mg / ml type IV collagenase, followed by a 5 minute incubation with 1 mg / ml dispase. It should be noted that passaging human ESCs with trypsin can lead to chromosomal instability and abnormalities (see, e.g., Mitalipova MM. et al., Nature Biotechnology, 23:19-20, 2005, and Cowan CA. et al., N. Eng. J. of Med., 350:1353-1356, 2004). According to some embodiments of the present invention, passaging hESCs or iPS cells with trypsin should be avoided.

[0331] According to some embodiments of the invention, after enzymatic or mechanical dissociation of large cell clumps, the dissociated pluripotent stem cell clumps are further broken down into smaller clumps using a 200 μl Gilson pipette tip (e.g., by pipetting the cells up and down).

[0332] According to some embodiments of the invention, the method of expanding and maintaining pluripotent stem cells in an undifferentiated state is carried out in a two-dimensional culture system.

[0333] The two-dimensional culture system can include a matrix or feeder cell layer.

[0334] For example, culture on a two-dimensional culture system can be performed by plating pluripotent stem cells onto a matrix or feeder cell layer at a cell density that promotes cell survival and proliferation but limits differentiation, typically around 15,000 cells / cm. 2 ~about 3,000,000 cells / cm 2 Plating densities between 0.1 and 0.5 are used.

[0335] Single cell suspensions of pluripotent stem cells are typically seeded, although it will be appreciated that small clumps can also be used. For this reason, the enzymatic digestion (e.g. with type IV collagenase) used to disrupt clumps (see "General Materials and Experimental Methods" in the Examples section below) is terminated before the stem cells are completely disaggregated, and the cells are triturated with a pipette to the extent that clumps (i.e., 10-200 cells) are formed. However, measures are taken to avoid large clumps that may induce cell differentiation.

[0336] According to some embodiments of the invention, a culture system comprises a matrix and a culture medium of some embodiments of the invention.

[0337] The term "matrix" as used herein refers to any material to which pluripotent stem cells can attach and thus replace the cell attachment function of feeder cells. Such matrices typically contain extracellular components to which pluripotent stem cells can attach and thus provide a suitable culture substrate.

[0338] According to some embodiments of the invention, the matrix comprises an extracellular matrix.

[0339] The extracellular matrix may consist of components derived from basement membranes or extracellular matrix components that form part of adhesion molecule receptor-ligand coupling. MATRIGEL® (Becton Dickinson, USA) is an example of a commercially available matrix suitable for use in the present invention. MATRIGEL® is a soluble preparation derived from Engelbreth-Holm-Swarm tumor cells that gels at room temperature and forms a reconstituted basement membrane, while MATRIGEL® is also available as a growth factor reduced preparation. Other extracellular matrix components and component mixtures suitable for use in the present invention include foreskin matrix, laminin matrix, fibronectin matrix, proteoglycan matrix, entactin matrix, heparan sulfate matrix, collagen matrix, and the like, either alone or in various combinations thereof.

[0340] According to some embodiments of the invention, the matrix is ​​free of animal-derived contaminants (is a xeno-free matrix for culturing human pluripotent stem cells).

[0341] If completely animal-free culture conditions are desired, the matrix is ​​preferably derived from human sources or synthesized using recombinant technology as described above. Such matrices include, for example, human-derived fibronectin, recombinant fibronectin, human-derived laminin, foreskin fibroblast matrix or synthetic fibronectin matrix. Human-derived fibronectin can be derived from plasma fibronectin or cellular fibronectin, both of which are available from Sigma (St. Louis, MO, USA). Human-derived laminin and foreskin fibroblast matrix are available from Sigma (St. Louis, MO, USA). Synthetic fibronectin matrix is ​​available from Sigma (St. Louis, MO, USA).

[0342] If a feeder cell layer is desired, the human pluripotent stem cells can be cultured on a human foreskin fibroblast feeder cell layer.

[0343] The present inventors have discovered that pluripotent stem cells can be shipped as live, non-frozen cells and still remain viable, undifferentiated and pluripotent.

[0344] According to some embodiments of the invention, the cells remain viable, undifferentiated and pluripotent after transport (air or sea) lasting at least 4 days.

[0345] The inventors have discovered that the novel culture media of the present invention can be used to derive new pluripotent stem cell lines.

[0346] According to some embodiments of the invention, the pluripotent stem cell line is an embryonic stem cell line, and a method of deriving the embryonic stem cell line comprises: (a) obtaining embryonic stem cells from a pre-implantation stage blastocyst, a post-implantation stage blastocyst and / or fetal reproductive tissue; and (b) culturing the embryonic stem cells in a culture medium of some embodiments of the invention, thereby deriving the embryonic stem cell line.

[0347] According to some embodiments of the invention, the pluripotent stem cell line is an induced pluripotent stem cell (iPS cell) line, and a method of deriving an iPS cell line comprises: (a) inducing a somatic cell into a pluripotent stem cell; and (b) culturing the pluripotent stem cell in a culture medium of some embodiments of the invention, thereby deriving an induced pluripotent stem cell line.

[0348] Once obtained, the ESCs or iPS cells are further cultured in any of the culture media described hereinabove that allow for the expansion of pluripotent stem cells in an undifferentiated state, essentially as described hereinabove.

[0349] It will be appreciated that established pluripotent stem cell lines (e.g., embryonic stem cell lines or induced pluripotent stem cell lines) can be subjected to freeze / thaw cycles without interfering with the proliferation capacity of the cells in an undifferentiated state while retaining their pluripotent capacity. For example, as shown in Figures 6A-6C and described in Example 6 of the Examples section below, when serum replacement (10%-95%) and dimethyl sulfoxide (DMSO; 5%-10%) were used, hESCs or human iPS cells were successfully frozen and thawed, with over 70% of the cells surviving and directly recovering in suspension culture.

[0350] It should be noted that any of the novel culture media described hereinabove can be used to culture, maintain and expand pluripotent undifferentiated stem cells in suspension culture as single cells without cell aggregates.

[0351] According to some embodiments of the present invention, culture conditions for expanding and maintaining undifferentiated pluripotent stem cells in suspension culture as single cells without cell clumps comprise a culture medium comprising interleukin 11 (IL11) and ciliary neurotrophic factor (CNTF).

[0352] According to some embodiments of the present invention, culture conditions for expanding and maintaining undifferentiated pluripotent stem cells in suspension culture as single cells without cell clumps comprise a culture medium comprising basic fibroblast growth factor (bFGF) at a concentration of at least 50 ng / ml and an IL6RIL6 chimera.

[0353] According to some embodiments of the present invention, culture conditions for expanding and maintaining undifferentiated pluripotent stem cells in suspension culture as single cells without cell clumps comprise a culture medium comprising an animal-derived contaminant-free serum replacement and an IL6RIL6 chimera.

[0354] According to some embodiments of the present invention, culture conditions for expanding and maintaining undifferentiated pluripotent stem cells in suspension culture as single cells without cell clumps comprise a serum-free culture medium containing soluble interleukin 6 receptor (sIL6R) and interleukin 6 (IL6), wherein the concentration of sIL6R is at least 5 ng / ml and the concentration of IL6 is at least 3 ng / ml.

[0355] According to some embodiments of the present invention, culture conditions for expanding and maintaining undifferentiated pluripotent stem cells in suspension culture as single cells without cell clumps comprise a culture medium comprising interleukin 11 (IL11) and oncostatin.

[0356] Below is a non-limiting description of methods for producing differentiated cell lineages from pluripotent stem cells according to some embodiments of the present invention.

[0357] As described in Example 2 of the Examples section below, hESCs and human iPS cells expanded and maintained in any of the culture media described hereinabove are pluripotent (i.e., capable of differentiating into all cell types of the three germ layers of the embryo (ectoderm, endoderm and mesoderm)) as evidenced in vitro (by the formation of EBs) and in vivo (by the formation of teratomas) after extended culture periods (e.g., for a period of at least 10 or 30 passages) in two-dimensional culture systems (e.g., feeder-free matrix) or three-dimensional culture systems (e.g., static or dynamic suspension cultures).

[0358] Thus, hESCs or human iPS cells cultured according to the teachings of the present invention can be used as a source for generating differentiated lineage-specific cells. Such cells can be obtained directly from pluripotent stem cells by subjecting ESCs to various differentiation signals (e.g., cytokines, hormones, growth factors) or indirectly by the formation of embryoid bodies and subsequent differentiation of cells from EBs into lineage-specific cells.

[0359] Thus, according to an aspect of some embodiments of the present invention, there is provided a method of generating embryoid bodies from pluripotent stem cells, the method being carried out by the steps of (a) culturing pluripotent stem cells of some embodiments of the present invention according to the methods of some embodiments of the present invention, thereby obtaining expanded undifferentiated pluripotent stem cells, and (b) subjecting the expanded undifferentiated pluripotent stem cells to culture conditions suitable for differentiation of the stem cells into embryoid bodies, thereby generating embryoid bodies from the pluripotent stem cells.

[0360] The expression "embryoid body" as used herein refers to a morphological structure consisting of a population of ESCs, expanded blastocyst cells (EBCs), embryonic germ cells (EGCs) and / or induced pluripotent stem cells undergoing differentiation. The formation of EBs is initiated following removal of differentiation blocking factors from pluripotent stem cell cultures. In the first step of EB formation, pluripotent stem cells proliferate into small cell clusters and then proceed to differentiate. In the first phase of differentiation, after 1-4 days in culture for either human ESCs or human iPS cells, an endodermal cell layer forms on the outer layer of the cluster, resulting in "simple EBs". In the second phase, after 3-20 days following differentiation, "complex EBs" are formed. Complex EBs are characterized by extensive differentiation of ectodermal and mesodermal cells and derivative tissues.

[0361] Thus, the method according to some embodiments of the present invention comprises culturing the pluripotent stem cells of some embodiments of the present invention in any of the above culture media to obtain expanded undifferentiated pluripotent stem cells (e.g., in suspension culture as cell clumps or single cells without cell clumps, or in a two-dimensional culture system), and subsequently placing the expanded undifferentiated pluripotent stem cells (e.g., ESCs or iPS cells) under culture conditions suitable for differentiation of the pluripotent stem cells into embryoid bodies. Such differentiation-promoting culture conditions are substantially free of differentiation inhibitors, such as TGFβ1, TGFβ3, ascorbic acid, IL-11, CNTF, oncostatin, bFGF and / or IL6RIL6 chimeras, that are used when the pluripotent stem cells are to be propagated in an undifferentiated state.

[0362] In the formation of EBs, pluripotent stem cells (ESCs or iPS cells) are removed from their feeder-free culture system or suspension culture and transferred to suspension culture in the presence of culture medium containing serum or serum replacement and not containing differentiation inhibitors. For example, a culture medium suitable for the formation of EBs may include a basic medium (e.g., Ko-DMEM or DMEM / F12) supplemented with 20% FBSd (HyClone (Utah, USA)), 1 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% non-essential amino acid stock.

[0363] Monitoring the formation of EBs is within the capabilities of one of ordinary skill in the art and can be performed by morphological evaluation (e.g., histological staining) and by determining the expression of differentiation-specific markers (e.g., using immunological techniques or RNA-based analyses (e.g., RT-PCR, cDNA microarrays)).

[0364] It will be appreciated that to obtain lineage-specific cells from the EBs, the cells of the EBs can be further subjected to culture conditions suitable for lineage-specific cells.

[0365] According to some embodiments of the invention for generating lineage-specific cells from pluripotent stem cells, the method further comprises the step of (c) subjecting cells of the embryoid body to culture conditions suitable for differentiation and / or expansion of the lineage-specific cells, whereby the lineage-specific cells are generated from the embryonic stem cells.

[0366] As used herein, the phrase "culture conditions suitable for differentiation and / or expansion of lineage-specific cells" refers to a combination of a culture system, e.g., a feeder-free matrix or suspension culture, and a culture medium suitable for differentiation and / or expansion of specific cell lineages derived from cells of the EBs. Further non-limiting examples of such culture conditions are described below.

[0367] According to some embodiments of the invention, the method of this aspect of the invention further comprises the step of isolating the lineage specific cells after step (b).

[0368] As used herein, the phrase "isolate lineage-specific cells" refers to the enrichment of a mixed population of cells in culture with cells that exhibit at least one characteristic primarily associated with a specific lineage phenotype. It will be understood that all cell lineages are derived from three embryonic germ layers. Thus, for example, hepatocytes and pancreatic cells are derived from endoderm, bone, cartilage, elastic fiber connective tissue, muscle cells, cardiomyocytes, bone marrow cells, vascular cells (i.e., endothelial and smooth muscle cells), and hematopoietic cells are differentiated from embryonic mesoderm, and neural, retinal and epidermal cells are derived from embryonic ectoderm.

[0369] According to some preferred embodiments of the invention, isolation of lineage-specific cells is performed by sorting cells of the EBs with a fluorescence activated cell sorter (FACS).

[0370] Methods for isolating differentiated cells from EBs by FACS analysis are known in the art. According to one method, EBs are separated using a solution of trypsin and EDTA (0.025% and 0.01%, respectively), washed with 5% fetal bovine serum (FBS) in phosphate-buffered saline (PBS), and incubated on ice for 30 minutes with fluorescently labeled antibodies specific to cell surface antigens characteristic of specific cell lineages. For example, endothelial cells are isolated by adhering to an antibody specific to platelet endothelial cell adhesion molecule-1 (PECAM1), such as the fluorescently labeled PECAM1 antibody (30884X) available from PharMingen (PharMingen, Becton Dickinson Bio Sciences (San Jose, CA, USA)), as described in Levenberg S. et al. ("Endothelial cells derived from human embryonic stem cells." Proc.Natl.Acad.Sci.USA. 2002, 99:4391-4396). Hematopoietic cells are isolated using fluorescently labeled antibodies such as CD34-FITC, CD45-PE, CD31-PE, CD38-PE, CD90-FITC, CD117-PE, CD15-FTTC, Class I-FITC (all of which are available in IgG1 from PharMingen), CD133 / 1-PE(IgG1) (available from Miltenyi Biotec, Auburn, CA), and glycophorin A-PE(IgG1) (available from Immunotech, Miami, FL). Live cells (i.e., without fixation) are analyzed on a FACScan (Becton Dickinson Bio Sciences) using propidium iodide, and dead cells are removed using either PC-LYSIS or CELLQUEST software.It will be appreciated that the isolated cells can be further enriched using magnetically labeled secondary antibodies and magnetic separation columns (MACS, Miltenyi) as described by Kaufman DS et al. ("Hematopoietic colony-forming cells derived from human embryonic stem cells." Proc. Natl. Acad. Sci. USA. 2001, 98:10716-10721).

[0371] According to some embodiments of the invention, isolation of lineage-specific cells is performed by mechanical dissociation of cells, tissues and / or tissue-like structures contained within EBs.

[0372] For example, beating cardiomyocytes can be isolated from EBs as disclosed in US Patent Application Publication No. 2003 / 0022367 (issued to Xu et al.). Four-day-old EBs of the present invention are transferred to gelatin-coated plates or chamber slides and allowed to attach and differentiate. Spontaneously contracting cells observed on day 8 of differentiation are mechanically dissociated and collected in 15 mL tubes containing low calcium media or PBS. Cells are dissociated using collagenase activity-dependent digestion with collagenase B for 60-120 min at 37°C. Dissociated cells are then resuspended in differentiation KB medium (85 mM KCl, 30 mM K2HPO4, 5 mM MgSO4, 1 mM EGTA, 5 mM creatine, 20 mM glucose, 2 mM Na2ATP, 5 mM pyruvate, and 20 mM taurine, buffered to pH 7.2; Maltsev et al., Circ. Res. 75:233, 1994) and incubated for 15-30 minutes at 37°C. After dissociation, cells are seeded into chamber slides and cultured in differentiation medium to generate single, beating cardiomyocytes.

[0373] According to some embodiments of the invention, isolation of lineage-specific cells is performed by subjecting EBs to a differentiation factor, thereby inducing differentiation of the EBs into lineage-specific differentiated cells.

[0374] Below is a non-limiting description of procedures and approaches for inducing differentiation of EBs into lineage-specific cells.

[0375] To differentiate EBs into neural precursors in some embodiments of the present invention, 4-day-old EBs are cultured for 5-12 days in tissue culture dishes containing DMEM / F-12 medium (ITSFn medium, Okabe S. et al., 1996, Mech. Dev. 59:89-102) with 5 mg / ml insulin, 50 mg / ml transferrin, 30 nM selenium chloride, and 5 mg / ml fibronectin. Furthermore, the resulting neural precursors can be transplanted and neural cells generated in vivo (Bruestle O. et al., 1997, "In vitro-generated neural precursors participate in mammalian brain development." Proc. Natl. Acad. Sci. USA. 94:14809-14814). It will be appreciated that neural precursors are trypsinized and triturated into a single cell suspension in the presence of 0.1% DNase prior to their transplantation.

[0376] EBs of some embodiments of the invention may differentiate into oligodendrocytes and myelinate cells by culturing the cells in modified SATO medium, i.e., DMEM with bovine serum albumin (BSA), pyruvate, progesterone, putrescine, thyroxine, triiodothyronine, insulin, transferrin, sodium selenite, amino acids, neurotrophin 3, ciliary neurotrophic factor, and Hepes (Bottenstein JE and Sato GH, 1979, Proc. Natl. Acad. Sci. USA 76, 514-517; Raff MC, Miller RH, and Noble M., 1983, Nature 10, 111-115). 303:390-396]. Briefly, EBs are dissociated using 0.25% trypsin / EDTA (5 min at 37°C) and triturated into a single cell suspension. The suspended cells are plated in flasks with SATO medium supplemented with 5% horse serum and 5% fetal calf serum (FCS). After 4 days in culture, the flasks are gently shaken to suspend the less adherent cells (mainly oligodendrocytes) while the astrocytes remain attached to the flask and produce more conditioned medium. Primary oligodendrocytes are transferred to new flasks with SATO medium for another 2 days. After a total of 6 days in culture, oligospheres are either partially dissociated and resuspended in SATO medium for cell transplantation or completely dissociated and plated in the oligosphere conditioned medium resulting from the preceding shaking step [Liu S. et al. (2000) "Embryonic stem cells differentiate into oligodendrocytes and myelinate in culture and after spinal cord transplantation.'' Proc. Natl. Acad. Sci. USA. 97:6126-6131].

[0377] For mast cell differentiation, 2-week-old EBs of some embodiments of the present invention are transferred to tissue culture dishes containing DMEM medium supplemented with 10% FCS, 2 mM L-glutamine, 100 units / ml penicillin, 100 mg / ml streptomycin, 20% (v / v) WEHI-3 cell conditioned medium and 50 ng / ml recombinant rat stem cell factor (rrSCF, Tsai M. et al., 2000, "In vivo immunological function of mast cells derived from embryonic stem cells: An approach for the rapid analysis of even embryonic lethal mutations in adult mice in vivo." Proc Natl Acad Sci USA. 97:9186-9190). Cultures are expanded weekly by transferring cells to new flasks and replacing half of the medium.

[0378] To generate hemato-lymphoid cells from EBs of some embodiments of the invention, 2-3 day old EBs are transferred to gas permeable culture dishes in the presence of 7.5% CO2 and 5% O2 using an incubator with adjustable oxygen content. After 15 days of differentiation, cells are harvested and dissociated by gentle digestion with collagenase (0.1 units / mg) and dispase (0.8 units / mg) (both available from F. Hoffman-La Roche Ltd, Basel, Switzerland). CD45 positive cells are isolated using anti-CD45 monoclonal antibody (mAb) M1 / 9.3.4.HL.2 conjugated to goat anti-rat immunoglobulin and paramagnetic microbeads (Miltenyi) as described in Potocnik AJ et al. (Immunology Hemato-lymphoid in vivo reconstruction potential of subpopulations derived from in vitro differentiated embryonic stem cell. Proc. Natl. Acad. Sci. USA. 1997, 94:10295-10300). Isolated CD45 positive cells can be further enriched using a single pass over a MACS column (Miltenyi).

[0379] It will be appreciated that culture conditions suitable for differentiation and expansion of isolated lineage-specific cells include a variety of tissue culture media, growth factors, antibiotics, amino acids, etc., and it is within the ability of one of skill in the art to determine which conditions should be applied to expand and differentiate a particular cell type and / or cell lineage.

[0380] As mentioned above, lineage-specific cells can be obtained by directly inducing expanded undifferentiated pluripotent stem cells, such as ESCs or iPS cells, into culture conditions suitable for differentiation of specific cell lineages.

[0381] For example, as described in Examples 10, 11, and 12 of the Examples section below, pluripotent stem cells expanded and maintained in suspension culture as single cells free of cell clumps are pluripotent as evidenced in vitro by the direct differentiation of pluripotent stem cells to neuronal progenitors of the ectodermal cell lineage (Figures 17A-C, Example 10), to mesenchymal stem cells of the mesodermal lineage (Figures 18A-C, Example 11), and to PDX1-expressing cells of the endodermal cell lineage (Figures 20A-B, Example 12).

[0382] According to an aspect of some embodiments of the present invention, there is provided a method of generating lineage-specific cells from pluripotent stem cells, the method being carried out by the steps of (a) culturing pluripotent stem cells according to the methods of some embodiments of the present invention, thereby obtaining expanded undifferentiated stem cells, and (b) subjecting the expanded undifferentiated stem cells to culture conditions suitable for differentiation and / or expansion of lineage-specific cells, whereby the lineage-specific cells are generated from the pluripotent stem cells.

[0383] Below are non-limiting examples of culture conditions suitable for differentiation and / or expansion of lineage-specific cells from pluripotent stem cells (eg, ESCs and iPS cells).

[0384] Mesenchymal stromal cells that are CD73 positive and SSEA-4 negative can be generated from hESCs by mechanically expanding the fraction of fibroblast-like differentiated cells formed in cultures of hESCs essentially as described in Trivedi P and Hematti P. Exp Hematol. 2008, 36(3):350-9. Briefly, to induce differentiation of hESCs, the interval between medium changes is extended to 3-5 days and the cells at the periphery of the ESC colonies become spindle-shaped fibroblast-like cells. After 9-10 days under these conditions, when about 40-50% of the cells in culture acquire a fibroblast-like appearance, the undifferentiated part of the ESC colony is physically removed and the remaining differentiated cells are passaged under the same conditions into new culture plates.

[0385] To induce differentiation of hESCs into dopaminergic (DA) neurons, cells may be co-cultured with mouse stromal cell lines PA6 or MS5, or cultured with a combination of stromal cell-derived factor 1 (SDF-1 / CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2) and ephrinBl (EFNB1), essentially as described in Vazin T. et al., PLoS One. 2009 Aug. 12;4(8):e6606; and Elkabetz Y. et al., Genes Dev. 2008 Jan. 15;22:152-165.

[0386] To generate midbrain dopamine (mesDA) neurons, hESCs can be engineered to express the transcription factor Lmx1a (e.g., using a lentiviral vector harboring the PGK promoter and Lmx1a) essentially as described in Friling S. et al., Proc Natl Acad Sci USA. 2009, 106:7613-7618.

[0387] To generate lung epithelium (type II alveolar epithelium) from hESCs, ESCs may be cultured in the presence of commercially available cell culture medium (Small Airway Growth Medium; Cambrex, College Park, MD) as described in Rippon HJ et al., Proc Am Thorac Soc. 2008;5:717-722, or alternatively, in the presence of conditioned medium obtained from a lung cell line (e.g., the A549 human lung adenocarcinoma cell line).

[0388] To induce differentiation of hESCs or human iPS cells into neural cells, pluripotent stem cells can be cultured in the presence of serum replacement medium supplemented with a TGF-b inhibitor (SB431542, Tocris; e.g., 10 nM) and Noggin (R&D; e.g., 500 ng / ml) essentially as described in Chambers SM et al., Nat Biotechnol. 2009, 27:275-280 for about 5 days, after which the cells are cultured with increasing amounts (e.g., 25%, 50%, 75%, changed every 2 days) of N2 medium (Li XJ et al., Nat Biotechnol. 2005, 23:215-21) in the presence of 500 ng / mL Noggin.

[0389] To induce differentiation of hESCs or human iPS cells into neural progenitors, cells were cultured in suspension, differentiation inhibitors were then removed from the culture medium, and 5 × 10 -5 M retinoic acid is added for 21 days. Cells are then transferred to fibronectin-coated plates and cultured for an additional 5 days before cells are harvested for analysis. q-PCR and immunostaining confirm the presence of neuronal progenitor cells (see Example 7 in the Examples section below).

[0390] To induce differentiation of hESC or human iPS cells into endoderm cells (including insulin-producing cells), differentiation inhibitors are removed from the culture medium of pluripotent stem cells, and cells are exposed to 10ng / ml activin for 48 hours in medium containing cAMP-increasing agents (e.g., forskolin, 8-bromo cAMP, GABA, IBMX and DBC, etc.). After 10 days, cells are analyzed for endodermal markers. q-PCR for Sox17 reveals a significant increase in Sox17 expression in treated cells compared to untreated controls (see Example 7 in the Examples section below).

[0391] To induce differentiation of hESCs or human iPS cells into mesenchymal stem cells (MSCs), the pluripotent stem cells are transferred to serum-containing medium for 14 days and then plated on either gelatin or Matrigel. After 7-14 days, the cells are allowed to differentiate into MSCs, which can be frozen or passaged using trypsin.

[0392] In addition to lineage-specific primary cultures, the EBs of the invention can be used to generate lineage-specific cell lines that have the potential for indefinite expansion in culture.

[0393] The cell lines of the present invention can be generated by immortalizing EB-derived cells by methods known in the art, including, for example, expression of the telomerase gene in the cells (Wei W. et al., 2003, Mol Cell Biol. 23:2859-2870) or co-culturing the cells with NIH3T3 hph-HOXll retrovirus producer cells (Hawley RG et al., 1994, Oncogene 9:1-12).

[0394] As described in Example 11 of the Examples section below, pluripotent stem cells cultured in suspension as single cells without cell clumps can be further differentiated into cells of the mesodermal lineage in suspension culture (3-D) or in two-dimensional culture systems.

[0395] As described in Example 11 of the Examples section below, the present inventors have discovered a novel method for differentiating pluripotent stem cells into mesenchymal stem cells in suspension.

[0396] According to an aspect of some embodiments of the present invention, there is provided a method of producing mesenchymal stem cells in suspension culture, the method comprising culturing pluripotent stem cells of some embodiments of the present invention (e.g., PSCs cultured in suspension as single cells without cell clumps, or PSCs cultured in suspension as cell clumps) in suspension culture under conditions suitable for differentiation of the pluripotent stem cells into mesenchymal stem cells, thereby producing mesenchymal stem cells in suspension culture.

[0397] Any known culture medium suitable for differentiating pluripotent stem cells into MSCs can be used.

[0398] The present inventors have found that the following culture media are suitable for differentiation of pluripotent stem cells into mesenchymal stem cells: (1) Fy-enriched medium; consisting of 80% DMEM / F12 (Biological Industries, Beit Haemek, Israel) containing 10% knockout serum replacement, 10% fetal bovine serum (FBS; HyClone or Biological Industries), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, and 1% non-essential amino acid stock solution (all obtained from Invitrogen Corporation products (Grand Island, NY, USA) unless otherwise indicated); (2) MeSus I medium; consisting of 80% DMEM (Biological Industries, Beit Haemek, Israel), containing 20% ​​FBS (HyClone or Biological Industries), 2 mM L-glutamine (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated); (3) MeSus II medium; consisting of 80% αMEM (Biological Industries, Beit Haemek, Israel), containing 20% ​​FBS (HyClone or Biological Industries), 2 mM L-glutamine (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated); (4) MeSus III medium; consisting of DMEM / F12 (Biological Industries, Beit Haemek, Israel), 1% ITS (Invitrogen), and 2 mM L-glutamine (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated).

[0399] The inventors have found that the culture conditions must include gradually transferring the pluripotent stem cells from suspension culture in undifferentiated medium to suspension culture in MSC differentiation medium. The following is a non-limiting method for transferring the pluripotent stem cells to differentiation medium: I. (i) 25% differentiation medium / 75% pCM100F for one passage; (ii) 50% differentiation medium / 50% pCM100F for one passage; (iii) 75% differentiation medium / 25% pCM100F for one passage; (iv) 100% differentiation medium. II. (i) 50% differentiation medium / 50% pCM100F for one passage; (ii) 75% differentiation medium / 25% pCM100F for one passage; (iii) 100% differentiation medium. III. (i) 50% differentiation medium / 50% pCM100F for one passage; (ii) 100% differentiation medium.

[0400] According to an aspect of some embodiments of the present invention there is provided an isolated population of mesenchymal stem cells (MSCs) in suspension culture produced by the method of some embodiments of the present invention.

[0401] According to some embodiments of the invention, at least about 30% (e.g., 30%), at least about 35% (e.g., 35%), at least about 40% (e.g., 40%), at least about 45% (e.g., 45%), at least about 50% (e.g., 50%), at least about 55% (e.g., 55%), at least about 60% (e.g., 60%), at least about 65% (e.g., 65%), at least about 70% (e.g., 70%), at least about 75% (e.g., 75%), at least about 80% (e.g., 80%), at least about 81% (e.g., 81%), at least about 82% (e.g., 82%), at least about 83% (e.g., 83%), at least about 84% (e.g., 84%), at least about 85% (e.g., 85%), at least about 86% (e.g., 86%), at least about 87% (e.g., 87%), at least about 88% (e.g., 88%), at least about 89% (e.g., 89%), at least about 90% (e.g., 90%), at least about 91% (e.g., 91%), at least about 92% (e.g., 92%), at least about 93% (e.g., 93%), at least about 94% (e.g., 94%), at least about 95% (e.g., 95%), at least about 96% (e.g., 96%), at least about 97% (e.g., 97%) at least about 85% (e.g., 85%), at least about 86% (e.g., 86%), at least about 87% (e.g., 87%), at least about 88% (e.g., 88%), at least about 89% (e.g., 89%), at least about 90% (e.g., 90%), at least about 91% (e.g., 91%), at least about 92% (e.g., 92%), at least about 93% (e.g., 93%), at least about 94% (e.g., 94%), at least about 95% (e.g., 95%), at least about 96% (e.g., 96%), at least about 97% (e.g., 97%), at least about 98% (e.g., 98%), at least about 99% (e.g., 99%), e.g., 100% are characterized by a CD73+ / CD31- / CD105+ expression signature.

[0402] According to some embodiments of the invention, the MSCs are capable of differentiation in suspension culture into a cell lineage selected from the group consisting of an adipogenic lineage, an osteoblastic lineage and a chondrogenic lineage.

[0403] As described in Example 10 of the Examples section below, pluripotent stem cells cultured in suspension as single cells without cell clumps can be further differentiated into cells of the ectodermal lineage in suspension culture (3-D) or in two-dimensional culture systems.

[0404] According to an aspect of some embodiments of the present invention, there is provided a method of producing neuronal progenitor cells in suspension culture, the method comprising culturing pluripotent stem cells of some embodiments of the present invention (e.g., pluripotent stem cells cultured in suspension culture as single cells without cell clumps) in suspension culture under conditions suitable for differentiation into neuronal progenitor cells, thereby producing neuronal progenitor cells in suspension culture.

[0405] Any known culture medium suitable for differentiating pluripotent stem cells into neuronal progenitor cells can be used. Non-limiting examples include retinoic acid (100 mM NaCl), acetylcholine (10 mM NaCl), or acetylcholine (10 mM MgCl), essentially as described in "General Materials and Experimental Methods." -3 M) or medium containing noggin (10 ngr / ml).

[0406] According to an aspect of some embodiments of the present invention there is provided an isolated population of neuronal progenitor cells in suspension culture produced by the method of some embodiments of the present invention.

[0407] As described in Example 12 of the Examples section below, pluripotent stem cells cultured in suspension as single cells without cell clumps can be further differentiated into cells of the endodermal lineage in suspension culture (3-D) or in two-dimensional culture systems.

[0408] According to an aspect of some embodiments of the present invention, there is provided a method of producing endodermal cells in suspension culture, the method comprising culturing pluripotent stem cells of some embodiments of the present invention (e.g., pluripotent stem cells cultured in suspension culture as single cells without cell clumps) in suspension culture under conditions suitable for differentiation of the pluripotent stem cells into endodermal cells, thereby producing endodermal cells in suspension culture.

[0409] Any known culture medium suitable for differentiating pluripotent stem cells into endoderm cells can be used. Non-limiting examples include medium containing activin A (e.g., at a concentration of 10 ng / ml) for 24 to 48 hours, essentially as described in "General Materials and Experimental Methods."

[0410] According to an aspect of some embodiments of the present invention there is provided an isolated population of endoderm cells in suspension culture produced by the method of some embodiments of the present invention.

[0411] It will be appreciated that since the lineage-specific cells or cell lines obtained according to the teachings of the present invention develop by a differentiation process similar to that occurring naturally in the human embryo, they may further be used for human cell-derived therapies and tissue regeneration.

[0412] Thus, the present invention contemplates the use of expanded and / or differentiated lineage-specific cells or cell lines of some embodiments of the present invention to treat disorders requiring cell replacement therapy (cell-derived therapy).

[0413] For example, oligodendrocyte precursors can be used to treat myelin disorders (Treatment of Myelin Disease: Strategies and Advances in Animal Models, Molecular Medicine Today, 1997, pp. 554-561), chondrocytes or mesenchymal cells can be used in the treatment of bone and cartilage defects (U.S. Pat. No. 4,642,120), and cells of epithelial lineage can be used in wound or burn skin regeneration (U.S. Pat. No. 5,716,411).

[0414] For certain disorders, such as genetic disorders in which a particular gene product is missing (e.g., lack of the CFTR gene product in cystic fibrosis patients (Davies JC, 2002, New Therapies for Cystic Fibrosis Lung Disease, JR Soc. Med., 95 Suppl., 41:58-67)), the ESC- or iPS-derived cells are preferably engineered to overexpress the mutant gene prior to their administration to the individual. For other disorders, it will be appreciated that the ESC- or iPS-derived cells must be engineered to exclude certain genes.

[0415] Gene overexpression or elimination can be achieved using knock-in and / or knock-out constructs [see, e.g., Fukushige, S. and Ikeda, JE: Capture of mammalian promoters by Cre-lox site-specific recombination, DNA Res, 3 (1996), 73-50; Bedell, MA, Jerkins, NA and Copeland, NG: Mouse models of human disease Part I: techniques and resources for genetic analysis in mice, Genes and Development, 11 (1997), 1-11; Bermingham, JJ, Scherer, SS, O'Connell, S., Arroyo, E., Kalla, KA, Powell, FL and Rosenfeld, MG: Tst-1 / Oct-6 / SCIP regulates unique steps in peripheral myelination and is required for normal respiration, Genes Dev, 10 (1996), 1751-62].

[0416] The lineage-specific cells of some embodiments of the present invention can be utilized to produce large amounts of proteins (such as hormones, cytokines, growth factors, and drugs) (mass production). For example, to produce a protein, the cells must be induced to overexpress the protein, for example, by transfection, and after expansion, the protein can be isolated from the culture medium.

[0417] The lineage-specific cells of some embodiments of the present invention can be used to prepare a cDNA library. mRNA is prepared from the lineage-specific cells by standard techniques and further reverse transcribed to form cDNA. The cDNA preparation can be subjected to subtraction with nucleotides from embryonic fibroblasts and other cells of undesired specificity to generate a subtracted cDNA library by techniques known in the art.

[0418] The lineage-specific cells of some embodiments of the invention can be used to screen for factors (e.g., small molecule drugs, peptides and polynucleotides, etc.) or conditions (e.g., culture conditions or manipulations, etc.) that affect the differentiation of lineage precursors into terminally differentiated cells (e.g., drug screening). For example, growth influencers, toxins or potential differentiation factors can be examined by their addition to the culture medium.

[0419] The lineage-specific cells of some embodiments of the present invention can be used to prepare vaccines. For example, pluripotent stem cells or cells differentiated from pluripotent stem cells can be inoculated with viral particles and cultured in a suitable medium until cell lysis occurs and the newly produced viral particles are released into the medium. These cells can be used for the production of attenuated viruses belonging to the Poxviridae family: specifically, canarypox virus, fowlpox virus and vaccinia virus (e.g., natural or recombinant vaccinia virus [e.g., modified vaccinia virus Ankara (e.g., MVA available under ATCC number VR-1508) or other orthopoxviruses]. For further description, see US Patent Publication No. 20040058441, which is incorporated herein by reference in its entirety.

[0420] The cell cultures of some embodiments of the invention, or the lineage-specific cells produced therefrom, can be genetically engineered using either infection or transfection with a polynucleotide of interest. The polynucleotide can be included in a nucleic acid construct under the control of a promoter.

[0421] Methods for introducing polynucleotides into cells are described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1989, 1992), Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Maryland (1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, MI (1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor, MI (1995), and Vectors, A Survey of Molecular Cloning Vectors and Their Uses, Butterworths, Boston, MA (1988)], and Gilboa et al. [Biotechniques, 4(6):504-512 (1986)], and such methods include, for example, stable or transient transfection, lipofection, electroporation and infection with recombinant viral vectors (e.g., using retroviruses, adenoviruses (e.g., the adenovirus-derived vector Ad-TK, Sandmair et al., 2000, Hum Gene Ther. 11:2197-2205), chimeric adenovirus / retrovirus vectors combining retroviral and adenoviral components (Pan et al., Cancer Letters 184:179-188, 2002)). In addition, see U.S. Pat. No. 4,866,042 for vectors involving the central nervous system. Also see US Pat. Nos. 5,464,764 and 5,487,992 for positive / negative selection methods for inducing homologous recombination.

[0422] In some embodiments, the numbers set forth therein are prefaced with about.

[0423] The term "ng" refers to nanograms. The term "pg" refers to picograms. The term "ml" refers to milliliters. The term "mM" refers to millimole. The term "μM" refers to micromole.

[0424] As used herein, the term "about" refers to ±10%.

[0425] The terms "comprises, comprising, includes, including," "having," and their conjugations mean "including but not limited to."

[0426] The term "consisting of" means "including and limited to."

[0427] The phrase "consisting essentially of" means that the composition, method, or structure may include additional ingredients, steps, and / or moieties only if the additional ingredients, steps, and / or moieties do not materially alter the basic and novel characteristics of the claimed composition, method, or structure.

[0428] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" can include a plurality of compounds, including mixtures thereof.

[0429] Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Thus, the description of a range should be considered to have all possible subranges specifically disclosed, as well as individual numerical values ​​contained within that range. For example, the description of a range such as 1-6 should be considered to have specifically disclosed subranges (e.g., 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc.), as well as individual numerical values ​​contained within that range (e.g., 1, 2, 3, 4, 5, and 6). This applies regardless of the breadth of the range.

[0430] Whenever a range of numerical values ​​is given herein, it is meant to include any recited numbers (fractional or integer) that are included in the given range. The phrases "ranging between" a first given number and a second given number, and "ranging from" a first given number to a second given number, are used interchangeably and are meant to include the first given number, the second given number, and all fractional and integer numbers therebetween.

[0431] The term "method" as used herein refers to manner, means, techniques, and procedures for accomplishing a given task, including, but not limited to, such manner, means, techniques, and procedures that are known to a practitioner in the art of chemistry, pharmacology, biology, biochemistry, and medicine or that are readily developed by a practitioner in the art of chemistry, pharmacology, biology, biochemistry, and medicine from known manner, means, techniques, and procedures.

[0432] As used herein, the term "treating" includes canceling, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating the clinical or cosmetic symptoms of a condition, or substantially preventing the appearance of clinical or cosmetic symptoms of a condition.

[0433] It will be appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as otherwise suitable in other described embodiments of the invention. Certain features described in the context of various embodiments should not be construed as essential features of those embodiments, unless the embodiment is inoperable without those elements.

[0434] Each of the various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below finds experimental support in the following examples. EXAMPLES

[0435] Reference is now made to the following examples, which together with the above descriptions illustrate the invention in a non-limiting fashion.

[0436] The nomenclature used herein and the laboratory procedures utilized in the present invention broadly include molecular biochemistry, microbiology and recombinant DNA techniques, which are explained thoroughly in the literature. See, for example, the following references: Molecular Cloning: A laboratory Manual, Sambrook et al., (1989); Current Protocols in Molecular Biology, volumes I-III, edited by Ausubel, RM (1994); Ausubel et al., Current Protocols in Molecular Biology, John Wiley and Sons, Baltimore, MD, USA (1989); Perbal, A Practical Guide to Molecular Cloning, John Wiley & Sons, New York, USA (1988); Watson et al., Recombinant DNA, Scientific American Books, New York, USA; Birren et al., eds., Genome Analysis: A Laboratory Manual Series, volumes 1-4, Cold Spring Harbor Laboratory Press, New York, USA (1998); the methods described in U.S. Patent Nos. 4,666,828, 4,683,202, 4,801,531, 5,192,659 and 5,272,057; Cell "Biology: A Laboratory Handbook", vols. I-III, edited by Cellis, JE (1994); "Current Protocols in Immunology", vols. I-III, edited by Coligan, JE (1994); "Basic and Clinical Immunology", 8th ed., Stites et al., Appleton & Lange, Norwalk, CT, USA (1994); "Selected Methods in Cellular Immunology", edited by Mishell and Shiigi, WH Freeman and Co., New York, USA (1980); available immunoassay methods are described extensively in the patent and scientific literature, e.g., U.S. Patent Nos. 3,791,932, 3,839,153, 3,850,752, 3,850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984,533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521; "Oligonucleotide Synthesis," edited by Gait, MJ (1984); "Nucleic Acid Hybridization," Hames, B.D. and Higgins (1985); "Transcription and Translation" Hames, BD and Higgins SJ eds. (1984); "Animal Cell Culture" Freshney, RI ed. (1986); "Immobilized Cells and Enzymes" IRL Press (1986); "A Practical Guide to Molecular Cloning" Perbal, B. (1984) and "Methods in Enzymology" vols. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, Calif., USA (1990); Marshak et al., "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this specification. The methods described therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained in those documents is incorporated herein by reference.

[0437] General Materials and Experimental Methods Induced pluripotent stem (iPS) cell lines - iPS cell lines J1.2-3 (derived from foreskin fibroblasts; Park et al., Nature, 451:P141-147, 2008), C2 and C3 (derived from foreskin fibroblasts; Germanguz et al., JCMM, 2009), iF4 (derived from adult skin fibroblasts) [Park et al., 2008; Germanguz et al., 2009], KTN7 and KTN3 (derived from keratinocytes; Novac-Petraro Cellular Reprogramming, in press), and KTR13 and KTR13.4 (derived from keratinocytes; Novac-Petraro Cellular Reprogramming, in press) were cultured with inactivated MEFs as previously described [Park et al., 2008].

[0438] Human embryonic stem cell (hESC) lines - human ESC lines H9.2, I3, I3.2 and I6.2 (described in Amit et al., J. Anatomy (2002)) and human ESC lines H14, H7, H9 (Winsconsin cell line) were cultured as previously described [Amit et al., 2000].

[0439] Human expanded blastocyst cell (hEBC) lines - Human expanded blastocyst cell lines J3 and J6 (described in International Publication WO 2006 / 040763) were cultured as described in Amit et al., Dev Biol (2000).

[0440] Culture Media - The following culture media combinations were examined for their ability to support the growth of iPS, hESC and hEBC lines in adherent (2D) or suspension cultures (three dimensional, 3D):

[0441] yF10 - a basal culture medium consisting of 85% DMEM / F12 (Biological Industries, Beit Haemek, Israel) containing 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock solution and 10 ng / ml basic fibroblast growth factor (bFGF) (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated). This basal culture medium was used as a control and for the routine culture of iPS cells and hESCs using inactivated MEFs or foreskin fibroblasts as feeder layers in 2D cultures.

[0442] yFIL25 - basal medium (yF10) supplemented with 25ng / ml interleukin 6 (IL6) and IL6 soluble receptor (R&D Biosystems, Minneapolis, MN, USA). It should be mentioned that any gp130 agonist, e.g., oncostatin, IL11, etc., can be used instead of IL6.

[0443] Instead of NCM100F-Knockout serum replacement, the serum replacement was an animal-derived-free serum replacement (Invitrogen corporation, Knockout SR (xeno-free), catalog number 12618) in the base medium (yF10). In addition, NCM100F medium contained 100ng / ml IL6RIL6 [IL6-IL6 receptor chimera (SEQ ID NO: 19; described in Chebath J. et al. (1997) and International Publication WO99 / 02552 (Revel M. et al.)). 85Kda IL6RIL6 was produced and purified (Serono International SA, Geneva, Switzerland) and was donated by the Merck-Serono group (Nes-Ziona (Israel) and Geneva (Switzerland)).

[0444] Instead of NCM100Fp-Knockout serum replacement, the serum replacement was an animal-derived free serum replacement (Invitrogen corporation, Knockout SR (xeno-free), catalog number 12618) in the base medium (yF10). In addition, NCM100Fp medium contained 100 pg / ml IL6RIL6.

[0445] ILCNTF-basal medium (yF10) supplemented with 1 ng / ml interleukin 11 (IL11; R&D Biosystems, catalog no. 18-IL) and ciliary neurotrophic factor (CNTF; R&D Biosystems, catalog no. 257-NT).

[0446] Instead of NILCNTF-Knockout serum replacement, the serum replacement was an animal-derived free serum replacement (Invitrogen corporation, Knockout SR (xeno-free), catalog number 12618), and the basal medium (yF10) was supplemented with 1 ng / ml IL11 and CNTF (R&D Biosystems).

[0447] 10 ng / ml bFGF (Invitrogen corporation), 100 ng / ml IL6IL6 receptor chimera in cmV5b-Nutristem medium (Biological Industries).

[0448] 10 ng / ml bFGF (Invitrogen corporation), 100 pg / ml IL6IL6 receptor chimera in cmV5bp-Nutristem (Biological Industries).

[0449] IL6IL6 receptor chimera at 100 ng / ml in cmTeSR-mTeSR medium (StemCell Technologies).

[0450] IL6IL6 receptor chimera at 100 pg / ml in cmTeSRp-mTeSR (StemCell Technologies).

[0451] cmTeSR2-100 ng / ml IL6IL6 receptor chimera in TeSR2 (StemCell Technologies).

[0452] cmTeSR2p IL6IL6 receptor chimera at 100 pg / ml in TeSR2 (StemCell Technologies).

[0453] cmHA13 85% DMEM / F12 (Biological Industries, Beit Haemek, Israel) containing 1% SR3 serum replacement (Sigma), 2 mM L-glutamine, ascorbic acid (50 μg / ml), 1% lipid mixture and 10 ng / ml bFGF, and IL6IL6 receptor chimera at 100 ng / ml. 85 Kda IL6RIL6 was produced and purified as described and was a gift from the Merck-Serono group. (All are obtained from Invitrogen Corporation products (Grand Island, NY, USA) unless otherwise indicated.)

[0454] cmHA13p 85% DMEM / F12 (Biological Industries, Beit Haemek, Israel) containing 1% SR3 serum replacement (Sigma), 2 mM L-glutamine, ascorbic acid (50 μg / ml), 1% lipid mix and 10 ng / ml bFGF, and IL6IL6 receptor chimera at 100 pg / ml. 85 Kda IL6RIL6 was produced and purified as described and was a gift from the Merck-Serono group. (All are obtained from Invitrogen Corporation products (Grand Island, NY, USA) unless otherwise indicated.)

[0455] CMrb100F - basal medium (yF10) containing: bFGF concentration increased to 100ng / ml; 100ng / ml IL6RIL6 (IL6-IL6 receptor chimera; described in Chebath J. et al. (1997) and International Publication WO99 / 02552 (Revel M. et al.)). 85Kda IL6RIL6 was produced and purified (Serono International SA, Geneva, Switzerland) and was a gift from the Merck-Serono group (Nes-Ziona, Israel and Geneva, Switzerland).

[0456] CMrb100Fp-basal medium (yF10) containing: bFGF concentration increased to 100ng / ml; 100pg / ml IL6RIL6 (IL6-IL6 receptor chimera; described in Chebath J. et al. (1997) and International Publication WO99 / 02552 (Revel M. et al.)). 85Kda IL6RIL6 was produced and purified (Serono International SA, Geneva, Switzerland) and was a gift from the Merck-Serono group (Nes-Ziona, Israel and Geneva, Switzerland).

[0457] Instead of NCMrb100F-knockout serum replacement, serum replacement was animal-origin-free serum replacement (Invitrogen corporation, Knockout SR (xeno-free), Cat. No. 12618) and bFGF concentration was increased to 100ng / ml in basal medium (yF10). In addition, 100ng / ml IL6RIL6 (IL6-IL6 receptor chimera; described in Chebath J. et al. (1997) and International Publication WO99 / 02552 (Revel M. et al.)). 85Kda IL6RIL6 was produced and purified (Serono International SA, Geneva, Switzerland) and was donated by the Merck-Serono group (Nes-Ziona (Israel) and Geneva (Switzerland)).

[0458] Instead of NCMrb100Fp-knockout serum replacement, serum replacement was animal-origin-free serum replacement (Invitrogen corporation, Knockout SR (xeno-free), catalog number 12618) and the basic medium (yF10) was increased to 100ng / ml bFGF concentration. In addition, NCMrb100Fp medium contained 100pg / ml IL6RIL6 (IL6-IL6 receptor chimera; described in Chebath J. et al. (1997) and International Publication WO99 / 02552 (Revel M. et al.)). 85Kda IL6RIL6 was produced and purified (Serono International SA, Geneva, Switzerland) and was donated by the Merck-Serono group (Nes-Ziona (Israel) and Geneva (Switzerland)).

[0459] Culture in 2D Culture Systems - For feeder layer-free culture systems, the extracellular matrix Matrigel (BD Biosciences) or human fibronectin (Millipore, Billerica, MA) was used.

[0460] Initiation of suspension cultures - To initiate suspension cultures, iPS or ES cells were removed from their culture dishes using 1.5 mg / ml type IV collagenase (Worthington biochemical corporation, Lakewood, NJ, USA) or using a scraper, further broken into small clumps using a 200 μl-1000 μl Gilson pipette tip, and then diluted to 1 × 10 6 ~5 x 10 cells / dish 6Cells were cultured in suspension in 58 mm Petri dishes (Greiner, Frickenhausen, Germany) at a cell density of 1000 cells / dish. Petri dishes were placed in an incubator at 37°C with 5% CO2. Medium in suspension cultures was changed daily and cells were passaged every 5-7 days either by manual disruption of clumps using a 27 g needle (only in the 1st to 3rd passages) or by gentle pipetting using a 200 μl to 1000 μl Gilson pipette tip. Alternatively, cells were passaged using trypsin / EDTA (0.25%, Biological Industries, Beit Haemek, Israel) in combination with a 1 hour treatment with 10 M ROCK inhibitor (EMD Biosciences, Inc., La Jolla, CA, USA) prior to incubation with trypsin.

[0461] Culture in spinner flasks - Cell clumps cultured in Petri dishes for at least one passage were transferred to 250 ml spinner flasks in test medium and placed in an incubator with constant shaking at 40-110 revolutions per minute (rpm) using a magnetic plate. The medium was changed every 1-3 days. Every 5-7 days, the clumps were split in a ratio of 1:2-1:4.

[0462] Cultivation in controlled bioreactors - Cells were cultivated in Biostat® Cultibag RM (Sartorius North America, Edgewood, New York, USA) controlled bioreactors (2 liter bags for 1 liter). Reactor parameters included: tilt speed: 16 revolutions per minute (rpm), angle: 7°, temperature: 37°C, pH: 7-7.4, O2 concentration: 50%.

[0463] Immunohistochemistry - For immunofluorescence staining, undifferentiated hESCs grown in suspension or recultured on MEFs were fixed with 4% paraformaldehyde and exposed to primary antibodies overnight at 4°C. Cys3-conjugated antibodies (Chemicon International, Temecula, CA, USA) were used as secondary antibodies (dilution 1:200). Primary antibodies (dilution of 1:50) included SSEA1, SSEA3 and SSEA4 (Hybridoma Bank, Iowa, USA), TRA1-60 and TRA1-81 (Chemicon International, Temecula, CA, USA), Oct4 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), oligodendrocyte marker (O4; obtained from R&D Biosystems), glial fibrillary acidic protein (GFAP; obtained from Millipore, Billerica, MA, USA), β-tubulin (Covance, Princeton, NJ, USA), nestin (Chemicon Intnl, Inc., Temecula, CA, USA), PDX1 (primary antibody was goat anti-human PDX1; two secondary antibodies were used: rabbit anti-goat IgG conjugated to FLUOR (green), or donkey anti-goat NL557 (red), all obtained from R&D Biosystems).

[0464] Flow cytometry analysis - Spheres of hPSCs cultured in suspension were dissociated into single cells using trypLE (Invitrogen Corporation products, Grand Island, NY, USA). Single cells were pipetted up and down using a 200 μl pipette tip. Cells were stained with anti-h / mSSEA4 Ab, anti-h / mSSEA1 Ab, h / mTRA1-160 Ab, h / mTRA1-81 Ab conjugated to phycoerythrin, and phycoerythrin-conjugated rat IgG2B was used as an isotype control (all antibodies were purchased from R&D systems, Minneapolis, MN, USA, unless otherwise stated). Stained cells were then analyzed by a FACS calibur flow cytometer (Becton Dickinson, San Jose, CA, USA) using CellQuest software according to the manufacturer's instructions. Anti-CD73 (BD ​​Pharmingen), CD146 (BD Pharmingen), CD105 (BioScience), CD44 (BioScience), CD45 and CD31 (BD Pharmingen) antibodies.

[0465] Karyotyping - Karyotyping (G-banding) was performed on at least 10 cells from each sample (two samples per study) as previously described [Amit et al., 2003]. Karyotypes were analyzed and reported according to the “International System for Human Cytogenetic Nomenclature” (ISCN).

[0466] Embryoid body (EB) formation - For EB formation, hESCs and iPS cells were passaged as described and transferred to 58 mm Petri dishes (Greiner, Frickenhausen, Germany). EBs were grown in medium consisting of 80% DMEM / F12 (Biological Industries, Beit Haemek, Israel) supplemented with 10% fetal bovine serum (FBS) (HyClone, Utah, USA), 10% serum replacement (SR), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol and 1% non-essential amino acid stock solution (Invitrogen Corporation, Grand Island, NY, USA). 10-14 day old EBs were collected for RNA isolation and histological examination. For histological analysis, EBs were fixed in 10% neutral buffered formalin, dehydrated in graded alcohols (70%-100%) and embedded in paraffin. Sections of 1 μm to 5 μm were deparaffinized and stained with hematoxylin / eosin (H&E).

[0467] Reverse transcription polymerase chain reaction (RT-PCR) - Total RNA was isolated from hESCs and iPSCs grown in suspension (three-dimensional, 3D) or two-dimensional (2D) in the tested media for at least five passages, and from 10- to 21-day-old EBs (formed from cells grown in suspension or cultured in 2D) using Tri-Reagent (Sigma, St. Louis, MO, USA) according to the manufacturer's instructions. Complementary DNA (cDNA) was synthesized from 1 μg of total RNA using MMLV reverse transcriptase RNase H minus (Promega, Madison, WI, USA). The PCR reaction included the following steps: denaturation at 94°C for 5 minutes, followed by repeated cycles of the following steps (cycle numbers are shown in Table 1 below): denaturation at 94°C for 30 seconds, annealing at a specific annealing temperature (as shown in Table 1 below) and in the presence of a specific MgCl2 concentration (as shown in Table 1 below) for 30 seconds, and extension at 72°C for 30 seconds. The PCR primers and reaction conditions used are listed in Table 1. The PCR products were size fractionated using 2% agarose gel electrophoresis. DNA markers were used to confirm the sizes of the resulting fragments. TIFF0007663644000001.tif222135TIFF0007663644000002.tif196133TIFF0007663644000003.tif96134

[0468] Real-time RT-PCR - RNA was extracted from cells grown in 2-D, suspension culture as cell clumps, or suspension culture as single cells using TriReagent (Talron). RNA was then subjected to real-time RT-PCR using RT mix (Applied Biosystems) and the primers provided in Table 2 herein below (Applied Biosystems) according to the manufacturer's instructions. TIFF0007663644000004.tif154132

[0469] Teratoma formation - Cells from four to six 58 mm dishes (three to six wells in a six-well plate) or from 20 ml of suspension culture were pooled and injected into the hind leg muscle of 4-week-old male severe combined immunodeficiency (SCID) beige mice. Ten weeks after injection, the resulting teratomas were collected and prepared for histological analysis using the same method described for EBs.

[0470] Testing cloning efficiency of pluripotent stem cells (PSCs) - PSCs were cultured in 2-D, in suspension as cell aggregates, or in suspension as single cells and tested for their cloning potential as described below, with six replicates of 96 cells for each treatment group.

[0471] Cloning from 2D culture with MEFs - H7 cells were trypsinized with 0.05% trypsin / 0.53 mM EDTA (Invitrogen) to single cells. Each individual cell was plated in a separate well in a 96-well plate (Nunc) covered with mitotically inactivated MEFs. 96 cells in each biological replicate were cloned with the addition of 10 μM / ml Rock inhibitor. The number of colonies that arose 10 days after plating was calculated. Three colonies were picked by passaging using 1 mg / ml collagenase type IV and 10 mg / ml dispase (both from GibcoBRL). Cloned cultures were grown in pCM100F medium [containing 85% DMEM / F12 (Biological Industries, Beit Haemek, Israel), 15% knockout serum replacement (SR), 2 mM L-glutamine, 0.1 mM b-mercaptoethanol, 1% non-essential amino acid stock solution, and 4 ng / ml bFGF (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated), supplemented with 100 pg / ml IL6RIL6 chimera] and routinely passaged every 5-7 days with 1 mg / ml collagenase type IV. After expansion in culture, the three resulting clones were examined for ESC characteristics.

[0472] Cloning from 3D cultures as single cells - H7 cells cultured as single cells in suspension were used. Each individual cell was plated in a separate low-attachment well in a 96-well plate (Nunc) or on a plate covered with MEFs. 96 cells in each biological replicate were cloned with or without the addition of 10 μM / ml of Rock inhibitor as described in Table 4 in Example 9 of the Examples section below. Ten days after plating, the number of resulting colonies was calculated. Three colonies were picked by 200 μL tip. Cloned cultures were grown in pCM100F medium containing serum replacement, IL6IL6 receptor chimera and 4 ng / ml bFGF and routinely passaged every 5-7 days by pipette. After expansion in culture, the three resulting clones were examined for ESC characteristics.

[0473] Freeze-thaw efficiency - Cells were frozen using one of the following freezing solutions: 1. Serum and animal freezing solutions (Biological Industries). 2. DMEM supplemented with 10% DMSO and 20% FBS. 3. DMEM supplemented with 10% DMSO and 30% SR.

[0474] After 1-7 days in a -80°C freezer (using a freezer box), the vials were transferred to liquid nitrogen. Cells were thawed and viability was checked by trypan blue staining. Three separate experiments were performed.

[0475] Genetic manipulation - Cells were transfected using the following vector: CMV promoter-GFP (based on the N1 plasmid). The following method was used: 1. Electroporation using a BTX ECM2001 electroporator with the following parameters: 40 μgr DNA, 10 7 cells, 3mSc~6mSc, 220V. 2. Transfection with Fugene6 (Roche) or Lipofectamine (Invitrogen) reagents according to the manufacturer's instructions (10 6 40μgr DNA per cell).

[0476] Neuronal Differentiation - To induce neuronal differentiation, single cells cultured in suspension were transferred to medium without bFGF and IL6RIL6 chimeras. -3 Either 10ngr / ml or noggin (10ngr / ml) was added for 3-7 days. Three weeks after induction of differentiation, cells were plated for staining with fibronectin. Cells were stained for O4 (oligodendrocyte marker), GFAP (glial fibrillary acidic protein), nestin, and β-tubulin.

[0477] Media used for differentiation of suspension MSCs into adipogenic, osteogenic and chondrogenic lineages: Adipogenic medium - 10% FBS, 1 mM L-glutamine, 0.5 mM IBMX, 10 μg / ml insulin, 10 -6 DMEM F12 supplemented with 0.1 mM dexamethasone and 0.1 mM indomethacin.

[0478] Osteogenic medium - GMEM BHK-21 supplemented with 10% FBS, 1% sodium pyruvate, 1% non-essential amino acids, 50 μg / ml L-ascorbic acid, 0.1 mM β-mercaptoethanol, 10 mM β-glycerol phosphate and 0.1 μM dexamethasone.

[0479] Chondrogenic medium-10 -7 DMEM supplemented with 1 mM dexamethasone, 1% ITS, 50 μg / ml L-ascorbic acid, 1 mM sodium pyruvate, 4 mM L-proline, and 10 ng / ml TGFβ3.

[0480] Differentiation protocol of suspension MSCs into adipogenic, osteogenic and chondrogenic lineages: Differentiation procedure for adipogenic differentiation and Oil Red O staining - MSCs were cultured at 20,000 cells / cm in 6-well plates. 2 and grown in adipogenic medium for 4 weeks with medium changes twice a week.

[0481] Adipogenic differentiation was assessed by observing the intracellular accumulation of lipid-rich vacuoles after Oil Red O staining.

[0482] Oil Red O staining - Cells were rinsed once with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min, rinsed again, and stained with Oil Red O solution for 10 min at room temperature. The staining solution was removed, and cells were washed five times with water.

[0483] Differentiation procedure for osteogenic differentiation and Alizarin Red staining - MSCs were cultured at 2000 cells / cm in 6-well plates. 2 ~3000 cells / cm 2 Cells were seeded at a density of 100x100cm and grown in osteogenic medium for 4 weeks with medium changes twice weekly. Cell cultures were assayed for mineral content by Alizarin Red staining.

[0484] Alizarin red staining - Cells were rinsed once with PBS, fixed with 4% paraformaldehyde (PFA) for 20 min, rinsed again, and stained with 2% Alizarin red solution for 15 min at room temperature. The staining solution was removed, and cells were washed several times with water.

[0485] Differentiation procedure for chondrogenic differentiation, hematoxylin-eosin staining and Alcian blue staining - For chondrogenic differentiation, 2 x 10 5 The MSCs were centrifuged in a 15 ml polypropylene Falcon tube at 300 g for 5 min to form a cell pellet. The cells were grown in chondrogenic medium for 9 weeks with medium changes twice a week without disturbing the cell mass. Cell sections were prepared after the cell pellets were fixed with 4% PFA and embedded in low melting point agarose (1.5%).

[0486] Hematoxylin and eosin (H&E) and Alcian blue staining - these were performed in the pathology laboratory of the Rambam Medical Center.

[0487] Differentiation protocol for MSCs in suspension - Using the same adipogenic, osteogenic and chondrogenic media (described hereinabove), MSCs were differentiated in suspension without seeding them in a 2-D culture system.

[0488] Example 1 Suspension culture of pluripotent stem cells in novel culture media of some embodiments of the present invention Cultivation of pluripotent cells in suspension has significant advantages over conventional culture, especially when large amounts of cells are to be obtained for cell and tissue transplantation. A number of growth factors and cytokines have been used to initiate suspension cultures from pluripotent cells grown with MEFs or under feeder-layer-free conditions [Amit et al., 2004]. Pluripotent cells from different sources have been used: iPS cells from neonates (foreskin fibroblasts), iPS cells from adults (fibroblasts), and hESCs.

[0489] Experimental Results Suspension culture - After 24 hours in suspension culture in the presence of the following culture media: yFIL25, NCM100F, NCM100Fp, ILCNTF, NILCNTF, cmV5b, cmV5bp, cmTeSR, cmTeSRp, cmTeSR2, cmTeSR2p, cmHA13, CMrb100F, CMrb100Fp, NCMrb100F or NCMrb100Fp, pluripotent cells gave rise to spherical aggregates or disc-like structures that revealed a homogenous population of small cells with large nuclei on histological examination. The spheroids grew and allowed the expansion of suspension cultures by mechanically dividing every 5-7 days while maintaining their morphology. All of these types of media were found to be favorable for culturing ESCs and iPS cells in suspension as single cells or as small aggregates of less than 100 cells.

[0490] Alternatively, by using trypsin-EDTA and ROCK inhibitor treatment, the suspended cells could be dissociated into single cells and still formed spheroids of the same morphology and characteristics, thus allowing efficient cell expansion. Cells subjected to suspension culture with the tested culture media showed similar behavior and spheroid morphology and histology. When transferred back to 2D culture with MEF or fibronectin after at least five passages in suspension, all of the spherical aggregates attached to the MEF or fibronectin matrix, respectively, and after 24-48 hours manifested typical pluripotent cell colony morphology, showing a large nucleus-to-cytoplasm ratio with prominent presence of one to three nucleoli and typical intercellular spacing.

[0491] Maintenance of undifferentiated stem cell phenotype - Several surface markers typical of undifferentiated primate ESCs and iPS cells were examined using immunofluorescence staining essentially as described in Thomson et al. (1998), Bhattacharya et al. (2004), Kristensen et al. (2005), each of which is incorporated herein by reference in its entirety. Human pluripotent cells cultured in suspension in test medium for at least five passages were found to remain strongly positive for SSEA4, TRA-1-60 and TRA-1-81, and Oct4. As in other primate ESCs [Thomson et al., 1995 and 1996] and similar to cells cultured with MEFs, staining for SSEA3 was weak and staining for SSEA1 was negative. Staining for stem cell markers remained high when cells cultured in suspension were returned to 2D culture on MEF feeder cell layers. RT-PCR analysis showed that pluripotent cells cultured in suspension for at least five passages expressed genetic markers of pluripotency, including Oct4, Nanog, Sox2, Rex1, and FGF4, similar to cells cultured with MEFs [King et al., 2006]. No significant differences in gene expression were detected between cells cultured in suspension or for cells recultured with MEFs after continuous culture in suspension.

[0492] Karyotype maintenance - Karyotype analysis by Giemsa banding was performed on cells after at least seven passages in suspension. Cells were found to exhibit a normal 46,XY or 46,XX karyotype. Thus, the karyotype of the suspension cell cultures remained stable.

[0493] Pluripotency-After prolonged expansion in suspension culture with the test medium, the pluripotent cells retained their pluripotent differentiation potential. The developmental potential of the cells was first examined in vitro by the formation of EBs. When pluripotent cells cultured in suspension for more than five passages were transferred to serum-containing medium without added growth factors, the formation of cystic EBs was observed after 7-10 days (similar to cells cultured with MEFs, where hollowed-out EBs appeared after 10 days in culture [Itskovitz et al., 2000]), and cystic EBs were observed after 14-20 days. Within these EBs were cell types representing the three germ layers of the embryo, typical for pluripotent cell differentiation.

[0494] The pluripotency of suspension pluripotent cells was further demonstrated in vivo by the formation of teratomas. Cells cultured in suspension for approximately 10 passages were injected into SCID-beige mice, and tumors formed after 10 weeks. Within these teratomas, tissues representing the three germ layers were observed.

[0495] Shaking suspension culture - Pluripotent cells were cultured in suspension in spinner flasks for at least one month using the tested medium. Examination after one month showed that morphologically, the spherical aggregates formed by the cells remained similar to those observed for cells cultured statically using Petri dishes. When recultured on MEFs, the cells in the aggregates reattached and again formed typical colonies of pluripotent cells. The karyotype of cells cultured for one month in spinner flasks was found to be normal.

[0496] Example 2 Two-dimensional culture of pluripotent stem cells in novel culture media according to some embodiments of the present invention Cultivation of pluripotent cells in 2D culture using serum-free and xeno-free support layer-free system - Several possible medium combinations were tested for their ability to support feeder layer-free or animal-origin-free (xeno-free) culture of pluripotent cells (e.g., using foreskin fibroblasts as feeders). All tested media (i.e., yFIL25, NCM100F, NCM100Fp, ILCNTF, NILCNTF, cmV5b, cmV5bp, cmTeSR, cmTeSRp, cmTeSR2, cmTeSR2p, cmHA13, CMrb100F, CMrb100Fp, NCMrb100F or NCMrb100Fp) were found to be suitable for supporting the culture of undifferentiated pluripotent cells. The pluripotent cells were continuously cultured for at least five passages while maintaining their stemness characteristics including undifferentiated proliferation, karyotype stability and pluripotency. No morphological differences could be observed between colonies grown in the culture systems tested and those grown on MEFs with basal medium, and correspondingly, morphological features remained unchanged at the single-cell level, with cells being small and round, exhibiting a large nuclear-to-cytoplasmic ratio with prominent presence of one to three nucleoli and typical intercellular spacing. Similar to cells grown on MEFs, cells were routinely passaged every 5-7 days at the same ratio of 1 / 2 or 1 / 3, which showed similar population doubling times. Cells reached approximately 1 million cells / 10 cm. 2 were passaged at the same seeding efficiency of 100%, which had the same viability of over 90%.

[0497] Pluripotent stem cells cultured in the 2-D culture system in the presence of the novel culture medium of some embodiments of the present invention maintain the expression pattern of undifferentiated cells - several surface markers typical of undifferentiated primate ESCs and iPS cells were examined using immunofluorescence staining essentially as described in Thomson et al. (1995, 1996, 1998) (each of which is incorporated herein by reference in its entirety). Cells cultured in the test medium for at least seven passages (e.g., 10, 15 passages) were found to be strongly positive for the surface markers SSEA4, TRA-1-60, TRA-1-81 and Oct4. As in the case of other primate ESCs, staining for SSEA3 was weak and negative for SSEA1.

[0498] Pluripotent stem cells cultured in the 2-D culture system in the presence of the novel culture medium of some embodiments of the present invention are capable of in vitro and in vivo differentiation into cell lineages derived from the three germ layers of the embryo - the developmental capacity of the cells after prolonged culture in the tested conditions was examined in vitro by the formation of embryoid bodies (EBs). Pluripotent cells cultured in the tested conditions formed EBs similar to those generated by ESCs grown on MEFs. Within these EBs, the stem cells differentiated into cell types representative of the three germ layers of the embryo (data not shown).

[0499] In addition, these pluripotent stem cells were shown to be capable of differentiation in vivo: cells cultured under the conditions tested thus formed teratomas containing cell types representative of the three germ layers of the embryo (i.e., ectoderm, endoderm and mesoderm) after their injection into SCID beige mice (data not shown).

[0500] Example 3 Culturing pluripotent stem cells as single cells in suspension without enzymatic passaging Experimental Results Culture in single cell suspension culture - Pluripotent cells were cultured as single cells in suspension in spinner flasks or petri dishes using all of the media tested (yFIL25, NCM100F, NCM100Fp, ILCNTF, NILCNTF, cmV5b, cmV5bp, cmTeSR, cmTeSRp, cmTeSR2, cmTeSR2p, cmHA13, CMrb100F, CMrb100Fp, NCMrb100F or NCMrb100Fp) for at least one month. Testing after one month showed that the cells exhibited characteristics of pluripotent cells including stable karyotype, expression of specific markers and differentiation potential. Cells were passaged without the use of ROCK inhibitors and without the use of trypsin and were mechanically separated using a pipette. This is the first time that it has been shown that human ESCs or iPS can be cultured in suspension as single cells without the need for enzymatic passaging. This is because the cells are in a single cell culture format. This system can be used for industrial processes without passaging.

[0501] Human ESCs cultured in suspension as single cells can be re-plated in 2D culture systems, which demonstrate typical hESC morphology - CL1(13E1) cultured for 17 passages in suspension as single cells were re-plated with inactivated MEFs. Colony morphology is not evident during the first passage. After several weeks, cells formed colonies with intercellular spacing, clear borders, and large nucleus-to-cytoplasm ratios of pluripotent cell morphology (Figure 11A-B).

[0502] Example 4 Human ESCs and iPS cells can be transported in suspension culture Transport of live cells - Cells cultured in suspension as cell aggregates using the described method tolerate transport at room temperature or at 0°C-15°C. Using 50 ml tubes containing 20 ml-40 ml of culture medium (aerated or non-aerated), 2-10 million cells per tube can be transported. At least 50% of the cells survived and continued to grow while maintaining all pluripotent characteristics. Other tube sizes may be used. The medium may be supplemented with antioxidants and RoCK agents or other anti-apoptotic agents.

[0503] Example 5 Expansion of pluripotent stem cells under dynamic culture conditions in the presence of novel culture media of some embodiments of the present invention The inventors have investigated whether the novel culture media of some embodiments of the present invention can support the growth and expansion of pluripotent stem cells (such as, for example, iPSCs and ESCs) under dynamic culture conditions when the pluripotent stem cells are cultured as single cells (without cell clumps) or in suspension with cell clumps.

[0504] Experimental Method Cell line and seeding concentration: The C2 IPS cell line was used at passage 77, with 37 of those passages being in suspension prior to seeding into dynamic culture conditions. IPSCs were cultured at 3.7 × 10 4 The cells were inoculated at a concentration of 100 cells / ml.

[0505] Culture medium and conditions: The following culture medium was used for dynamic suspension culture: CM100Fp. Cells were cultured in spinner flasks or controlled bioreactors for 5 consecutive days. When cultured in bioreactors, the medium was not changed during the culture process. When cultured in spinner flasks, the medium was changed daily.

[0506] Culture conditions for dynamic growth in suspension: Cultivation in a controlled bioreactor - Cells were cultivated in a controlled bioreactor Biostat® Cultibag RM (Sartorius North America, Edgewood, New York, USA) (2 liter bags for 1 liter). Reactor parameters included: tilt speed: 16 revolutions per minute (rpm), angle: 7°, temperature: 37°C, pH: 7-7.4, O2 concentration: 50%.

[0507] Culture in spinner flasks - Cell clumps cultured in Petri dishes for at least one passage were transferred to 250 ml spinner flasks in test medium and placed in an incubator with constant shaking at 40-110 revolutions per minute (rpm) using a magnetic plate. The medium was changed every 1-3 days. Every 5-7 days, the clumps were split in a ratio of 1:2 to 1:4.

[0508] Experimental Results Expansion of pluripotent stem cells in suspension culture using culture media according to some embodiments of the present invention - Pluripotent stem cells subjected to dynamic culture conditions expanded in cell number by about 26-fold within 11 days of culture in spinner flasks when grown in suspension culture with cell clumps, and expanded in cell number by about 50-fold within 11 days of culture in spinner flasks when grown in suspension culture as single cells without cell clumps. In addition, pluripotent stem cells expanded in cell number by about 64-fold within 5 days of culture in a controlled bioreactor when grown as single cells (Figures 5A-C, data not shown). These results demonstrate that the novel culture media of some embodiments of the present invention can support the expansion of pluripotent cells when they are cultured in suspension under dynamic culture conditions.

[0509] Example 6 Pluripotent stem cells cultured in suspension recover well from freeze / thaw cycles To examine whether pluripotent stem cells cultured in suspension in the presence of the novel culture medium of some embodiments of the present invention can recover from a refreeze / thaw cycle, the cells were frozen in liquid nitrogen by using the following freezing solution: (1)10%DMSO(Sigma), 10%FBS(HyClone)10%SR(Invitrogen corporation), 70%DMEM (2)5%DMSO, 10%FBS, 10%SR, 75%DMEM (3) 10% DMSO, 90% SR (4) 5% DMSO, 95% SR (5) Commercially available serum-free freezing solution (Biological Industries, Beit HaEmek, Israel)

[0510] The frozen cells were initially frozen in a -80°C freezer, and after 12 hours to 3 days, they were transferred to a liquid nitrogen tank for storage.

[0511] Experimental Results Pluripotent stem cells were subjected to freezing conditions using the freezing solutions described above, then thawed and recultured in suspension. Figures 6A-6C demonstrate C2 cells that were thawed using three different freezing solutions and then cultured in suspension in cmrb100p medium for 48 passages.

[0512] Example 7 Generation of lineage-specific cells from pluripotent stem cells Neuronal differentiation - Cells from the four tested cell lines (I3, I4, I6 and H9.2) were cultured in suspension with cell clumps for at least 25 passages. Factors were then removed from the culture medium and 5 × 10 -5M retinoic acid was added for 21 days. Afterwards, cells were transferred to fibronectin-coated plates and cultured for an additional 5 days before cells were collected for analysis. Quantitative RT-PCR, immunostaining (immunofluorescence and FACS) were performed. The results show the expression of neuronal lineage genes (e.g., PAX6, HNF, nestin, β-tubulin and PSA-NCAM, etc.) (Figure 7A-C, Figure 8A-B, Figure 9A-G).

[0513] Differentiation into endoderm cells - Suspension-cultured cells with cell clumps obtained from C2 cell line (iPS cell line derived from foreskin fibroblasts) were cultured in suspension for at least 10 passages. Factors were then removed from the culture medium and cells were exposed to 10 ng / ml activin in medium containing cAMP-increasing agents (e.g., forskolin, 8-bromo cAMP, GABA, IBMX and DBC, etc.) for 48 hours. After 10 days, cells were analyzed for markers of endoderm. Quantitative RT-PCR for Sox17 reveals a significant increase in Sox17 expression in treated cells compared to untreated controls (data not shown). As shown in Figure 10A-B, differentiated cells express PDX1 (transcription factor), indicating differentiation into endoderm lineages (mainly β-cells).

[0514] Differentiation into mesenchymal stem cells (MSCs) - Cells cultured in suspension in clumps were transferred to serum-containing medium for 14 days and then plated on either gelatin or Matrigel. After 7-14 days, the resulting MSCs were either frozen or passaged using trypsin.

[0515] Example 8 Characterization of expression patterns of human pluripotent embryonic stem cells cultured in suspension as single cells Study design to characterize novel hESCs cultured in suspension as single cells Three groups of cultured pluripotent stem cells (PSCs) were examined: 1. hESCs cultured with MEFs in standard conditions in two dimensions (2D). 2. hESCs grown as clumps (spheroids, >200 cells) in suspension culture (3D). 3. hESCs cultured as single cells (SCs, less than 50 cells, most of them as single cells) in suspension (3D).

[0516] Cells were examined for expression of pluripotency markers using flow cytometry after at least 15 passages in the above conditions.

[0517] Experimental Results Human ESCs cultured in suspension as single cells display unique expression patterns similar to those of "naive" mouse ESCs - As shown in Figures 12A-J, FACS analysis of pluripotent stem cells cultured in suspension as single cells reveals altered expression patterns when compared to hESCs cultured in 2D or in suspension as cell aggregates. For example, the majority of hESCs cultured in 2D or in suspension as cell aggregates express pluripotency markers TRA1-60 (Figure 12A,C), TRA1-81 (Figure 12B,D) and SSEA4 (Figure 12H), whereas the majority of pluripotent hESCs cultured in suspension as single cells do not express the TRA1-60 (Figure 12E), TRA1-81 (Figure 12F) and SSEA4 (Figure 12J) markers. In contrast, only 11% of hESCs cultured in 2D or in suspension culture as cell aggregates express SSEA1 (Figure 12G), while the majority of hESCs cultured in suspension culture as single cells express SSEA1 (Figure 12I). Thus, hESCs cultured in suspension as single cells show an altered expression pattern when compared to hESCs cultured in 2D or in suspension culture as cell aggregates. Such an expression pattern resembles that of more "naive" mouse ESC cells (which do not express TRA1-60, TRA1-81 and SSEA4, but do express SSEA1).

[0518] Table 3 herein below summarizes the results of the FACS analysis. TIFF0007663644000005.tif49133

[0519] Cells cultured in suspension as single cells show increased levels of OCT-4 - Real-time RT-PCR analysis was performed on hESCs cultured in 2-D, suspension culture as cell aggregates, or suspension culture as single cells using the primers listed in Table 2 in "General Materials and Experimental Methods" hereinabove. As shown in Figure 13A, the expression level of Nanog is slightly reduced in single cell suspension cultures when compared to hESCs grown in 2-D. On the other hand, the expression of OCT4 was found to be increased by about 8-fold in hESCs cultured in suspension as SCs when compared to hESCs cultured in 2D.

[0520] Example 9 Characterization of the cloning efficiency of human pluripotent embryonic stem cells cultured in suspension as single cells Experimental Results Human ESCs cultured in suspension as single cells or hESCs cultured in 2-D were examined for their cloning efficiency. Cells cultured in 2-D were trypsinized and plated as single cells in wells of MEF-covered 96-well plates (as described in "General Materials and Experimental Methods" hereinabove), and cells grown as single cells in suspension were plated individually in wells of low-attachment 96-well plates (as described in "General Materials and Experimental Methods" hereinabove).

[0521] Human ESCs cultured in suspension as single cells exhibit significantly greater cloning efficiency when compared to hESCs cultured in 2-D - As shown in Table 4, significantly greater cloning efficiency was observed for hESCs cultured in suspension as single cells (95.63%) compared to hESCs cultured in 2-D (4.33%). In addition, the addition of ROCK inhibitor increased the cloning efficiency of hESCs cultured in 2-D, while the cloning efficiency of hESCs cultured in suspension as single cells was not increased in the presence of ROCK inhibitor. TIFF0007663644000006.tif73132

[0522] Human ESCs cultured in suspension as single cells exhibit greater survival to freeze-thaw cycles when compared to hESCs cultured in 2-D - To investigate whether pluripotent stem cells can survive freeze-thaw cycles, hESCs (cultured in suspension as single cells) were subjected to freezing cycles using one of the following freezing solutions: I. Serum and animal freezing solutions (Biological Industries). II. DMEM supplemented with 10% DMSO and 20% FBS. III. DMEM supplemented with 10% DMSO and 30% SR (serum replacement).

[0523] After freezing at -80°C for approximately 1-7 days, the vials were transferred to liquid nitrogen. The cells were thawed and viability was examined by trypan blue staining. The survival of hESCs to freeze-thaw cycles was approximately 80% for hESCs cultured in suspension as single cells. This is significantly greater than the survival of hESCs cultured in 2-D to freeze-thaw cycles under identical assay conditions (up to 50%, data not shown). Figure 15 shows a representative image of human ESCs cultured as single cells in suspension culture after freeze-thaw cycles.

[0524] Human ESCs cultured in suspension as single cells show greater survival and efficiency of genetic manipulation when compared to hESCs cultured in 2-D - Cells were transfected with a CMV promoter-GFP nucleic acid construct (based on N1 plasmid) as described in "General Materials and Experimental Methods". After genetic manipulation, cell survival was assessed using phase contrast microscopy. As shown in Figure 16A, more than 90% of the suspended single cells survived the procedure. In contrast, no significant differences were observed (10% GFP, GFP, or GFP-GFP) from 2-D cells cultured with MEFs. 7 Only a maximum of 17 cells (out of 100 cells) were recovered (data not shown). Moreover, none of the hESCs cultured in 2-D were green (data not shown), whereas a minority of the hESCs cultured in 3-D as single cells were green, i.e., expressed the transgene CMV-GFP construct (Figure 16B).

[0525] Example 10 Human pluripotent embryonic stem cells cultured in suspension as single cells are capable of differentiation into neural lineages Experimental Results Human ESCs cultured in suspension as single cells are capable of differentiation into neuronal lineages - To induce neural differentiation, hESCs cultured in suspension as single cells were incubated with retinoic acid (10 -3 Differentiation was performed on 100 cells / cm2 of cultured cells transferred to neuronal differentiation medium (which does not contain bFGF and IL6RIL6 chimeras) containing either 100 mM (1M) or noggin (10 ng / ml). 2The cells were induced either in 2-D by plating on human plasma fibronectin (HPF) coated plates (at a concentration of 50 μgr per HFP) or in suspension culture. Three weeks after differentiation induction, the cells were plated for staining for fibronectin, O4, GFAP, nestin and β-tubulin. As shown in Figure 17A-C, the cells differentiated into neuronal progenitor cells that stained positive for GFAP (glial fibrillary acidic protein) (a marker for astrocytes), O4 (a marker for oligodendrocytes), and β-tubulin and nestin (markers for neurons). These results conclusively show that hESCs cultured in suspension as single cells can differentiate into the germ layer of the embryo, the ectoderm.

[0526] Example 11 A novel method for differentiation of mesenchymal stem cells in suspension The present inventors have developed a novel method for differentiating pluripotent stem cells into mesenchymal stem cells in suspension, as described below.

[0527] To induce differentiation into MSCs, single cells cultured in suspension were gradually transferred into one of the following media: (1) Fy-enriched; consists of 80% DMEM / F12 (Biological Industries, Beit Haemek, Israel) containing 10% knockout serum replacement (SR), 10% FBS (HyClone or Biological Industries), 2 mM L-glutamine, 0.1 mM β-mercaptoethanol, 1% non-essential amino acid stock solution (all obtained from Invitrogen Corporation products (Grand Island, NY, USA) unless otherwise indicated). (2) MeSus I: consists of 80% DMEM (Biological Industries, Beit Haemek, Israel), containing 20% ​​FBS (HyClone or Biological Industries), 2 mM L-glutamine (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated). (3) MeSus II: consists of 80% αMEM (Biological Industries, Beit Haemek, Israel), containing 20% ​​FBS (HyClone or Biological Industries), 2 mM L-glutamine (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated). (4) MeSus III: consists of DMEM / F12 (Biological Industries, Beit Haemek, Israel), 1% ITS (Invitrogen), 2 mM L-glutamine (all obtained from Invitrogen Corporation products, Grand Island, NY, USA, unless otherwise indicated).

[0528] Human ESCs grown in suspension as single cells were gradually transferred into MSC differentiation medium using one of the following methods: I. (i) 25% differentiation medium / 75% pCM100F for one passage; (ii) 50% differentiation medium / 50% pCM100F for one passage; (iii) 75% differentiation medium / 25% pCM100F for one passage; (iv) 100% differentiation medium. II. (i) 50% differentiation medium / 50% pCM100F for one passage; (ii) 75% differentiation medium / 25% pCM100F for one passage; (iii) 100% differentiation medium. III. (i) 50% differentiation medium / 50% pCM100F for one passage; (ii) 100% differentiation medium.

[0529] All of the above media and transfer methods resulted in efficient differentiation into MSCs.

[0530] The cells were then cultured in suspension (Petri dishes, spinner flasks and / or bioreactors) and passaged every 5-10 days by pipetting. After the cells were cultured in differentiation medium for at least one passage, the MSCs were characterized. Figures 19A-B show images of MSCs differentiated from PSCs cultured in suspension as single cells for at least 10 passages. When the cells were replated on gelatin, they demonstrate typical MSC morphology. Figure 19A shows CL1 cells differentiated in Fy-enriched medium, and Figure 19B shows CL1 cells differentiated in MeSusII medium.

[0531] To enrich the MSC population, magnet-activated cell sorting (MACS) was used with anti-CD73 antibody (Milteniy) according to the manufacturer's instructions. CD73-MACS resulted in enrichment of MSCs from approximately 40% CD73-positive cells to more than 80% CD73-positive cells.

[0532] MSCs, when generated by differentiation of hESCs cultured in suspension as single cells, show the typical MSC expression pattern - as shown in Figures 18A-C, FACS analysis shows that 82.5% of MSCs are CD73 positive and only 4.83% are CD31 positive when cells are grown in animal-derived free medium. In addition, 99.3% are CD105 positive when MSCs are grown in serum-containing medium.

[0533] Differentiation of Suspension MSCs to Adipogenic Lineage - MSCs in suspension were subjected to the differentiation protocol to adipogenic lineage either in 2-D culture system or in suspension culture as described in "General Materials and Experimental Methods" hereinabove. Briefly, MSCs were cultured at 20000 cells / cm in 6-well plates. 2 or in suspension culture at a density of 1 x 10 6 cells / ml~5×106 MSCs were seeded at a concentration of 10 ...

[0534] Differentiation of Suspension MSCs to Osteogenic Lineage - MSCs in suspension were subjected to the differentiation protocol to osteogenic lineage either in 2-D culture system or in suspension culture as described in "General Materials and Experimental Methods" hereinabove. Briefly, MSCs were cultured at 2000 cells / cm in 6-well plates. 2 ~3000 cells / cm 2 or in suspension culture at a density of 1 x 10 6 cells / ml~5×10 6 MSCs were seeded at a concentration of 10 ...

[0535] Differentiation of Suspension MSCs to the Chondrogenic Lineage - MSCs in suspension were subjected to a differentiation protocol to the chondrogenic lineage either in a 2-D culture system or in suspension culture as described in "General Materials and Experimental Methods" hereinabove. Briefly, 2×10 5 The MSCs were centrifuged in 15 ml polypropylene Falcon tubes at 300 g for 5 min to form a cell pellet. The cells were grown in chondrogenic medium as pellets in the tubes for 9 weeks with medium changes twice a week without disturbing the cell mass. Cell sections were made after the cell pellets were fixed with 4% PFA and embedded in low melting point agarose (1.5%). The cells were stained with Alcian Blue, which stains the matrix of chondrocytes of the chondrogenic lineage (data not shown). This demonstrates that MSCs can differentiate into the chondrogenic lineage.

[0536] Example 12 Human pluripotent embryonic stem cells cultured in suspension as single cells are capable of differentiation along endodermal lineages Experimental Results C2 cells were cultured in suspension in pCM100F culture medium as single cells for more than 10 passages. For endoderm differentiation, bFGF and IL6RIL6 chimera were removed from the culture medium, and Activin A at a concentration of 10 ng / ml was added in suspension culture for 48 hours. Ten days after exposure to Activin A, cells were plated on Matrigel™ or HFF (human foreskin fibroblast) matrix, or cultured in a 3D culture system (in suspension), and stained for PDX1. When the expression level of SOX17 gene was examined by real-time PCR, an increase could be observed during the differentiation period from day 2 to day 10 after exposure to Activin A (data not shown). Figure 20A-B shows the expression of PDX1 in cells, which demonstrates differentiation into endoderm cells.

[0537] While the present invention has been described in terms of specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, the present invention is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0538] All publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication, patent, and patent application was specifically and individually indicated to be incorporated by reference. Furthermore, citation or identification in this application should not be construed as an admission that any material is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

[0539] TIFF0007663644000007.tif193135TIFF0007663644000008.tif204130

Claims

1. A culture medium comprising basic fibroblast growth factor (bFGF) at a concentration of at least 50 ng / ml and an IL6RIL6 chimera, said culture medium being capable of expanding and maintaining human induced pluripotent stem cells characterized by an expression signature of OCT4 + / TRA1-60 - / TRA1-81 - / SSEA1 + / SSEA4 - as single cells in suspension culture in a pluripotent, proliferative state for at least five passages.

2. The culture medium according to claim 1, wherein the concentration of the bFGF is selected from the range of 50 ng / ml to 150 ng / ml.

3. 3. The culture medium of claim 1, further comprising a serum replacement.

4. 4. The culture medium of claim 3, wherein the serum replacement is free of animal-derived contaminants.

5. The culture medium according to any one of claims 1 to 4, wherein the IL6RIL6 chimera is used at a concentration of 50 ng / ml to 150 ng / ml.

6. The culture medium according to any one of claims 1 to 4, wherein the IL6RIL6 chimera is used at a concentration of 50 pg / ml to 150 pg / ml.

7. The culture medium according to any one of claims 1 to 5, wherein the bFGF is used at a concentration of 100 ng / ml, and the IL6RIL6 is used at a concentration of 100 ng / ml.

8. The culture medium according to any one of claims 1 to 4 and 6, wherein the bFGF is used at a concentration of 100 ng / ml and the IL6RIL6 is used at a concentration of 100 pg / ml.

9. A cell culture comprising pluripotent stem cells and the culture medium according to any one of claims 1 to 8.

10. A culture system comprising a matrix and a culture medium according to any one of claims 1 to 8.

11. 9. A method for expanding and maintaining pluripotent stem cells in an undifferentiated state, comprising culturing the pluripotent stem cells in a culture medium according to any one of claims 1 to 8, thereby expanding and maintaining the pluripotent stem cells in the undifferentiated state.

12. The method of claim 11, wherein the expansion and maintenance of the pluripotent stem cells in the undifferentiated state is performed in suspension culture.

13. The method of claim 11 or 12, wherein the culturing is carried out under conditions that allow the pluripotent stem cells to be expanded as single cells.

14. 1. A method for generating lineage-specific cells from pluripotent stem cells, comprising: (a) culturing the pluripotent stem cells according to the method of any one of claims 11 to 13, thereby obtaining expanded undifferentiated stem cells; (b) subjecting the expanded undifferentiated stem cells to culture conditions suitable for differentiation and / or expansion of lineage-specific cells, thereby generating said lineage-specific cells from the pluripotent stem cells; The method includes:

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