METHODS FOR LARGE SCALE GENERATION OF STEM CELLS

FIELD AND BACKGROUND OF THE INVENTION

The invention, in some embodiments thereof, relates to methods for large scale generation of stem cells.

A considerable amount of interest has been generated in the fields of regenerative medicine and gene therapy by recent work relating to the isolation and propagation of stem cells. The ability of stem cells to be propagated indefinitely in culture combined with their ability to generate a variety of tissue types makes the therapeutic potential from these cells almost limitless.

Pluripotent stem cells are ideal candidates for the treatment of a variety of human diseases, including diabetes, myocardial infarction, ischemic stroke, various autoimmune diseases, etc. Exemplary stem cells, including but not limited to, ES or ESCs (embryonic stem cells), human embryonic stem cells (hESC) derived from either surplus pre-implantation genetic diagnosis (PGD) in vitro fertilization (ivf) embryos or surplus ivf embryos, human induced pluripotent stem cells (hiPSCs), human pluripotent stem cells (hPSC; hESCs and hiPSC are jointly called hPSC) neural stem cells, ADSCs (adipocyte stem cells), non-human primate ES cells, etc., are useful for downstream therapeutic applications such as cell therapy. Effective cell therapy could potentially require vast amounts of these cells. As cell therapies progress through clinical development, scale and cost of manufacturing become looming issues. Most clinical trials have been done on a small scale, where the required cell number was achieved through traditional cell culture techniques where cells grow adherently in 2D on treated tissue culture surfaces. However, these standard 2D culture methods are too labor intensive and inefficient to meet the potential large scale needs of several cell therapy applications. Looking ahead toward scale-up and commercialization, it is likely that approximately 10 ⁹ -10 ¹² or more cells, derived from a single source, which may be required for effective therapeutic batch use, especially during treatments requiring multiple doses. This amount of stem cells is greater than what can be delivered using current manufacturing protocols. In addition, for human cell therapy, stem cell culture systems need to completely exclude animal serum during culture for a regulatory compliant end-product. Therefore, despite the existence of systems for growing stem cells in culture, there remain many difficulties in obtaining large scale systems for stem cells growth which maintain their sternness, and are effective for therapeutic use.

The large scale differentiation of stem cells into other useful cell types is also of major importance. For example, pancreatic islet cells and insulin producing cells are required to conduct clinical trials for diabetes, drug discovery and also to develop potential future cell therapies. Since human pluripotent stem cells (hPSC) are pluripotent and can differentiate to all germ layers, hPSC can provide a source of pancreatic islet cells and other cell types for these uses. So far, few hPSC derived pancreatic islet cell differentiation protocols have been described by the scientific community, but the scalability of the proposed bioprocesses is not clear. The invention seeks to solve these and other problems in the art.

SUMMARY OF THE INVENTION

According to one aspect of the present invention, there is provided a method of expanding pluripotent stem cells to high density in suspension, the method comprising:

(a) cultivation of the pluripotent stem cells in feeder-free conditions;

(b) splitting said pluripotent stem cells into single cells;

(c) attaching said pluripotent stem cells to microcarriers pre-coated with extracellular matrix (ECM) attachment substrate;

(d) culturing said pluripotent stem cells on the microcarriers pre-coated with ECM attachment substrate in a container, while the container is kept substantially still, followed by constant agitation; and

(f) harvesting said pluripotent stem cells from the microcarriers and reattaching said pluripotent stem cells to a new volume of microcarriers pre- coated with ECM attachment substrate while the container is kept substantially still, followed by constant agitation.

According to some embodiments of the invention, the pluripotent cells are embryonic stem cells (ESCs) or induced pluripotent stem cell (iPSCs).

According to some embodiments of the invention, the feeder-free conditions utilize an ECM substrate selected from the group consisting of Matrigel, CELLstart, laminin, gelatin, vitronectin and fibronectin. According to some embodiments of the invention, the ECM attachment substrate is selected from the group consisting of Matrigel, CELLstart, laminin, gelatin, vitronectin and fibronectin.

According to some embodiments of the invention, the feeder-free conditions utilize a serum free media (SFM) selected from the group consisting of STEMPRO ^® hESC SFM, E8, E6, AF NutriStem ^® hESC XF and KO-SR XF.

According to some embodiments of the invention, the ECM attachment substrate is feeder cell-free.

According to some embodiments of the invention, the container is a bioreactor or an Erlenmeyer.

According to some embodiments of the invention, the agitation is performed at a speed of between 20 and 200 rpm.

According to some embodiments of the invention, the culturing continues for at least four days.

According to some embodiments of the invention, the expanding continues for at least three passages.

According to some embodiments of the invention, the SFM is added every one to two days.

According to some embodiments of the invention, the pluripotent stem cells are attached to the microcarriers in a medium containing a Rock inhibitor.

According to some embodiments of the invention, the pluripotent stem cells are attached to the new volume of microcarriers in a medium containing a Rock inhibitor.

According to some embodiments of the invention, the pluripotent stem cells are dissociated into single cells by a reagent selected from the group consisting of EDTA, EGTA, BAPTA, TrypLE, Accutase and Versene.

According to some embodiments of the invention, the pluripotent stem cells are removed from the microcarriers by a reagent selected from the group consisting of EDTA, EGTA, BAPTA, TrypLE, Accutase and Versene.

According to some embodiments of the invention, the pluripotent stem cells are removed from the new volume of microcarriers by a reagent selected from the group consisting of EDTA, EGTA, BAPTA, TrypLE, Accutase and Versene. According to some embodiments of the invention, the microcarriers consist of a positively charge cross-linked dextran matrix (Cytodex 1).

According to some embodiments of the invention, the microcarriers consist of a collagen covalent bound to a cross-linked dextran matrix (Cytodex 3).

According to some embodiments of the invention, the container is kept substantially still for two to three days.

According to some embodiments of the invention, the method further comprising the step of inducing differentiation of the stem cells obtained after step (f), wherein the method comprises placing the microcarrier-stem cell complexes under conditions which induce the differentiation of the stem cells.

According to some embodiments of the invention, after step (f) the method comprises the step of separating stem cells from the microcarriers and culturing the separated stem cells in non-microcarrier culture under conditions which induce differentiation of the stem cells.

According to some embodiments of the invention, the method comprises inducing islet cell differentiation.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings and images. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-C illustrate Matrigel coating layer on the microcarriers 1A: Positive to Anti Laminin antibody, IB: Control-using only the secondary antibody, 1C: Control- microcarriers without Matrigel coating layer.

FIG. 2 illustrates the growth of aggregates of cells on Cytodex 3 or Cytodex 1 microcarriers at the end of seeding stage (day 2) and at the end of the passage during static phase. FIG. 3 illustrates cell concentration, cell pluripotency and cell expansion factor of KS-4 hESC cell line on Cytodex 1 or Cytodex 3 microcarriers seeded with single cells or cell clumps. The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIG. 4 illustrates thawing of KS-4 frozen cells on Matrigel coated Cytodex 3 or Cytodex 1 microcarriers. Morphology of the cell/microcarrier aggregates thawed on Matrigel coated plate during four cultivation days with STEMPRO hESC SFM (Life Technologies Corporation, USA).

FIG. 5 illustrates comparison of different types of growth media and splitting procedures: StemPro versus NutriStem and CoUagenase versus TrypLE. Morphology of the cell/microcarrier aggregates; growth, pluripotency and specific Glucose consumption rates curves during two passages with different growth media and different splitting methodologies. The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIGs. 6A-B illustrate comparison of different splitting methodologies: Dispase versus Mechanical splitting. The morphology of the cell/microcarrier aggregates are shown during 2-3 passages. 6A- the cells were grown in an Erlenmeyer; 6B- the cells were grown in a bioreactor.

FIGs. 7A-C illustrate splitting procedures of Matrigel coated Cytodex 3 microcarrier/cells aggregates using different EDTA concentrations. The morphology of the cell/microcarrier aggregates before (7 A) and after (7B) EDTA treatment is presented. 7C- Curves of cell concentration, splitting efficiency, viability, glucose consumption and pluripotency during three passages. The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIG. 8 illustrates the morphology of the cell/microcarrier aggregates; growth, attachment efficiency and pluripotency curves of cell cultures seeded from single cells (using EDTA splitting) versus cell clumps (using Dispase splitting) during two passages.

FIG. 9 illustrates growth, expansion factor, attachment efficiency and pluripotency curves of KS-4 cells grown on Matrigel coated Cytodex 3 microcarriers, comparing daily fed batches versus 80% repeated batches every two days (control) during two passages. The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIGs. lOA-C illustrate morphology of the cell/microcarrier aggregates (1 OA- IB); growth, expansion factor and pluripotency curves (IOC) during two passages in two different experiments and two different working cell banks of KS-4 on Cytodex 1 microcarrier seeded with 10% human serum and daily fed. The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIGs. 11A-B illustrate morphology of KS-2 and KS-4 cells grown on matrigel coated microcarriers (11 A) and their growth, pluripotency and metabolism curves (11B) during three passages on two microcarrier types (Cytodex 1/ Cytodex 3). The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIG. 12 illustrates morphology of the cell/microcarrier aggregates as well as growth, pluripotency and metabolism curves during two passages using two agitate cultivation systems (Bioreactor versus Erlenmeyer). The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIG. 13 is a process flow chart, which illustrates the scale up process in Erlenmeyer and Bioreactor. The actual accumulative cell number and expansion factor were measured during four to five passages, in two separate experiments using KS-4 cells. The results are shown as mean values (±SD) of samples obtained from two different experiments performed in duplicates.

FIG. 14 is a scheme describing the differentiation protocol of hESCs towards the pancreatic phenotype through step wise, well defined stages of pancreatic development.

FIG. 15 illustrates the expression of insulin and glucagon in the differentiated hESC cells as measured by quantitative Real time PCR (qRT-PCR). qRT-PCR was conducted for insulin and glucagon gene expression analyzed on three time points; Days 10, 15 and 23. The results are relative to the expression on day 10 (RQ=1). Results were normalized to the expression of the house keeping gene HPRT.

FIGs. 16A-B illustrate insulin expression and C-peptide content in differentiated hESC grown on microcariers. Figure 16A demonstrates immunofluorescence staining for insulin expression (Guinea pig DAKO, 1 : 100). DAPI: Nuclear staining.

Figure 16B demonstrates C-peptide content in hESC-derived insulin producing cells. C-peptide content was measured by ELISA (Mercodia) in two time points; Days 24 and 32. The results were normalized to total protein content.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a method for large scale generation of stem cells. The present invention is directed to compositions and methods for cultivating stem cells in large-scale culture.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Thus, according to one aspect of the present invention there is provided a method of expanding pluripotent stem cells to high density in suspension, the method comprising:

(a) cultivation of the pluripotent stem cells in feeder-free conditions;

(b) splitting said pluripotent stem cells into single cells;

(c) attaching said pluripotent stem cells to microcarriers pre-coated with ECM attachment substrate;

(d) culturing said pluripotent stem cells on the microcarriers pre-coated with ECM attachment substrate in a container, while the container is kept substantially still, followed by constant agitation; and

(f) harvesting said pluripotent stem cells from the microcarriers and reattaching said pluripotent stem cells to a new volume of microcarriers pre-coated with ECM attachment substrate while the container is kept substantially still, followed by constant agitation.

For example, human embryonic stem cells can be isolated from human blastocysts or delayed blastocyst stage (as described in WO2006/040763). Human blastocysts are typically obtained from human in-vivo preimplantation embryos or from in-vitro fertilized (IVF) embryos. Alternatively, a single cell human embryo can be expanded to the blastocyst stage. For the isolation of human ES cells the zona pellucida is removed from the blastocyst and the inner cell mass (ICM) is isolated from the intact ICM by gentle pipetting. The ICM is then plated in a tissue culture plate containing the appropriate medium which enables its outgrowth. Following 9 to 15 days, the ICM derived outgrowth is dissociated into clumps either by a mechanical dissociation or by an enzymatic degradation and the cells are then re -plated on a fresh tissue culture vessel. Colonies demonstrating undifferentiated morphology are individually selected by micropipette, mechanically dissociated into clumps, and re- plated. Resulting ES cells are then routinely split every 3-7 days. For further details on methods of preparation human ES cells see Thomson et al., [U.S. Pat. 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]; Gardner et al, [Fertil. Steril. 69: 84, 1998].

It will be appreciated that commercially available stem cells can also be used with this aspect of the invention. Human ES cells can be purchased from the NIH human embryonic stem cells registry (<http://escr.nih.gov>). Non-limiting examples of commercially available embryonic stem cell lines are BG01, BG02, BG03, SA01, TE03 (13), TE04, TE06 (16), HES-1, HES-2, HES-3, UCOl, UC06, WA01, WA07 and WA09.

Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells, which can be prepared by introducing certain particular nuclear reprogramming substances to somatic cells in the form of DNA or protein, and have properties almost equivalent to those of ES cells, such as pluripotency and growth capacity by self-renewal (K. Takahashi and S. Yamanaka (2006) Cell, 126: 663-676; K. Takahashi et al. (2007) Cell, 131 : 861-872; J. Yu et al. (2007) Science, 318: 1917- 1920; M. Nakagawa et al. (2008) Nat. BiotechnoL, 26: 101-106; WO 2007/069666). The nuclear reprogramming substances may be genes specifically expressed in ES cells, or genes or gene products thereof that play important roles for maintaining the undifferentiated state of ES cells. The nuclear reprogramming substances are not restricted, and examples thereof include Oct3/4, Klf4, Klfl, Klf2, Klf5, Sox2, Soxl, Sox3, Soxl5, Soxl7, Soxl8, c-Myc, L-Myc, N-Myc, TERT, SV40 Large T antigen, HPV16 E6, HPV16 E7, Bmil, Lin28, Lin28b, Nanog, Salll, Sall4, Glisl, Esrrb, Esrrg, Nr5a2 and Tbx3. These reprogramming substances may be used in combination when iPS cells are to be established. For example, the combination may contain at 2 0 least one, two or three of the above reprogramming substances, and the combination preferably contains four of the above reprogramming substances. The information on the nucleotide sequences of mouse and human cDNAs of the above-described respective nuclear reprogramming substances, and the amino acid sequences of the proteins encoded by the cDNAs can be obtained by referring to the NCBI (National Center for Biotechnology Information) accession numbers described in WO 2007/069666. Further, the information on the mouse and human cDNA sequences and amino acid sequences of L-Myc, Lin28, Lin28b, Esrrb and Esrrg can be obtained by referring to the NCBI accession numbers described below. Those skilled in the art can prepare desired nuclear reprogramming substances by a conventional method based on the information on the cDNA sequences or amino acid sequences.

These nuclear reprogramming substances may be introduced into somatic cells in the form of protein by a method such as lipofection, binding to a cell membrane- permeable peptide, or microinjection, or in the form of DNA by a method such as use of a vector including a virus, plasmid and artificial chromosome; lipofection; use of liposomes; or microinjection. Examples of the virus vector include retrovirus vectors, lentivirus vectors (these are described in Cell, 126, pp. 663-676, 2006; Cell, 131, pp. 861-872, 2007; and Science, 318, pp. 1917-1920, 2007), adenovirus vectors (Science, 322, 945-949, 2008), adeno-associated virus vectors and Sendai virus vectors (Proc Jpn Acad Ser B Phys Biol Sci. 85, 348-62, 2009). Examples of the artificial chromosome vector include human artificial chromosomes (HACs), yeast artificial chromosomes (YACs) and bacterial artificial chromosomes (BACs, PACs) (WO 2010/038904). Examples of the plasmid which may be used include plasmids for mammalian cells (Science, 322:949-953, 2008). The vectors may contain a regulatory sequence(s) such as a promoter, enhancer, ribosome binding sequence, terminator and/or polyadenylation site. Examples of the promoter to be used include the EF1. alpha, promoter, CAG promoter, SR.alpha. promoter, SV40 promoter, LTR promoter, CMV (cytomegalovirus) promoter, RSV (Rous sarcoma virus) promoter, MoMuLV (Moloney murine leukemia virus) LTR and HSV-TK (herpes simplex virus thymidine kinase) promoter. Among these, the EF1. alpha, promoter, CAG promoter, MoMuLV LTR, CMV promoter, SR. alpha, promoter and the like are preferred. The vectors may further contain, as required, a sequence of a selection marker such as a drug resistance gene (e.g., kanamycin-resistant gene, ampicillin-resistant gene or puromycin-resistant gene), thymidine kinase gene or diphtheria toxin gene; a gene sequence of a reporter such as the green-fluorescent protein (GFP), .beta.- glucuronidase (GUS) or FLAG; or the like. Further, in order to remove, after introduction of the above vector into somatic cells, the genes encoding the nuclear reprogramming substances, or both the promoters and the genes encoding the reprogramming substances linked thereto, the vector may have loxP sequences in the upstream and the downstream of these sequences. In another preferred mode, a method may be employed wherein, after incorporation of the transgene(s) into a chromosome(s) using a transposon, transposase is allowed to act on the cells using a plasmid vector or an adenovirus vector, thereby completely removing the transgene(s) from the chromosome(s). Preferred examples of the transposon include piggyBac, which is a transposon derived from a lepidopteran insect (Kaji, K. et al., Nature, 458: 771-775 (2009); Woltjen et al, Nature, 458: 766-770 (2009); WO 2010/012077). Further, the vector may contain the origin of lymphotrophic herpes virus, BK virus or Bovine papillomavirus and sequences involved in their replication, such that the vector can replicate without incorporation into the chromosome and exist episomally. Examples of such a vector include vectors containing EBNA-1 and oriP sequences and vectors containing Large T and SV40ori sequences (WO 2009/115295; WO 2009/157201; WO 2009/149233). Further, in order to introduce plural nuclear reprogramming substances at the same time, an expression vector which allows polycistronic expression may be used. In order to allow polycistronic expression, the sequences encoding the genes may be linked to each other via IRES or the foot-and- mouth disease virus (FMDV) 2A coding region (Science, 322:949-953, 2008; WO 2009/092042 2009/152529).

Regardless of their origin, stem cells used in accordance with the present invention are at least 50 % purified, 75 % purified or at least 90 % purified. When human embryonic stem cell lines are used, the human ES cell colonies are separated from their feeder layer (x-ray irradiated fibroblast-like cells) such as by mechanical and/or enzymatic means to provide substantially pure stem cell populations. The cells used for the assay of the present invention may be freshly generated or frozen cells. Typically, if the cells are frozen they are thawed in medium comprising growth factors (e.g. bFGF and EGF).

An "adherent culture" refers to a culture in which cells in contact with a suitable growth medium are present, and can be viable or proliferate while adhered to a substrate. A "non-adherent culture" refers to a culture in which cells are typically in suspension with a suitable growth medium, and can be viable or proliferate while not being adhered to a substrate.

Microcarrier culture involves growing adherent cells on the surface of small, micron range diameter particles which are usually suspended in culture medium by gentle stirring. Microcarrier suspension culture systems are readily scalable and make it possible to achieve yields of several million cells per milliliter. Microcarrier culture has made it more economically feasible to use adherent cells for production of vaccines, protein production by animal cells and some other biotechnical products. Cells can be grown on microcarriers in a variety of formats such as suspended in spinner flasks and STR bioreactors, packed in column beds, in a fluidized bed bioreactor or even on microcarriers in micro titer plate wells.

Commercial microcarriers are often produced using cross linked polymers of dextran, cellulose, polyethylene or other polymers. Some such products feature polymeric coatings on glass or other net negative charged surfaces. Most cells exhibit significant net negative charge due to abundant surface carboxylic acid groups. This makes it easier for them to attach and grow at net positive charged surfaces. In many cases growth surfaces are modified with positively charged entities to promote carrier surface adherence of cells. Examples include commercially available Cytodex 1 microcarriers, prepared from cross linked dextran particles coated with diethylaminoethyl (DEAE) groups, as well as DEAE modified cotton (Cytopore 1) bead carriers from GE Healthcare Biosciences AB. In addition to non-specific (e.g. electrostatic) interactions, affinity or other interactions may hold cells to surfaces modified with appropriate affinity substances. Cytodex 3 microcarriers are prepared from cross linked dextran beads coated with a collagen protein layer designed to mimic the protein coated surfaces which cells bind to in the body (Microculture Cell Carrier Principles and Methods, GE). Containers suitable for the method of the present invention include Erlenmeyers, bioreactors, spinners, and other containers suitable for cultivating cells. The term "bioreactor" refers to a container suitable for the cultivation of eukaryotic cells, preferably animal cells, and more preferably for the cultivation of mammalian cells. The term "controlled bioreactor" refers to bioreactor systems, wherein at least the oxygen partial pressure and the pH in the culture medium can be measured and regulated using known devices. Suitable devices for measuring the oxygen partial pressure and pH are, for instance, oxygen and pH electrodes. The oxygen partial pressure and pH can be regulated via the amount and the composition of the selected gas mixture (e.g., air or a mixture of air and/or oxygen and/or nitrogen and/or carbon dioxide). Suitable devices for measuring and regulating the oxygen partial pressure are described by Bailey, J E. (Bailey, J E., Biochemical Engineering Fundamentals, second edition, McGraw-Hill, Inc. ISBN 0-07-003212-2 Higher Education, (1986)) or Jackson A T. Jackson A T., Verfahrenstechnik in der Biotechnologie, Springer, ISBN 3540561900 (1993)). A typical cultivation volume of a controlled bioreactor is between 500 ml and 30,000 L. Other suitable containers include spinners. Spinners are regulated or unregulated bioreactors, which can be agitated using various agitator mechanisms, such as glass ball agitators, impeller agitators, and other suitable agitators. The cultivation volume of a spinner is typically between 20 ml and 500 ml.

"Large scale", as used herein with regard to cell cultivation and expansion, refers to the cultivation of SC under conditions which permit at least the doubling of cells after four weeks. The term may be used to denote cultures of both undifferentiated pluripotent stem cells and cultures of differentiated cells derived from stem cells (either by directed differentiation or by spontaneous differentiation).

"Long term" as used herein with regard to cell cultivation and expansion, refers to the cultivation of SC for at least three weeks, preferably, for at least ten weeks and more preferably, for at least 20 weeks.

As used herein, the term "single cell suspension" or equivalents thereof refers to a hES cell single cell suspension or a hES -derived single cell suspension by any mechanical or chemical means. Several methods exist for dissociating cell clusters to form single cell suspensions from primary tissues, attached cells in culture, and aggregates, e.g., physical forces (mechanical dissociation such as cell scraper, trituration through a narrow bore pipette, fine needle aspiration, vortex disaggregation and forced filtration through a fine nylon or stainless steel mesh), enzymes (enzymatic dissociation such as trypsin, collagenase, Accutase and the like), EDTA, Versene or a combination of both. Further, methods and culture media conditions capable of supporting single-cell dissociation of hES cells is useful for expansion, cell sorting, and defined seeding for multi-well plate assays and enable automatization of culture procedures and clonal expansion. Thus, one embodiment of the invention provides methods for generating a stable single-cell dissociation hES cell or hES-derived cell culture system capable of supporting long-term maintenance and efficient expansion of undifferentiated, pluripotent hES cell or differentiated hES cells.

As used herein, the term "survival" is meant any process by which a cell avoids death. The term survival, as used herein, also refers to the prevention of cell loss as evidenced by necrosis, apoptosis, or the prevention of other mechanisms of cell loss. Increasing survival as used herein indicates a decrease in the rate of cell death by at least 10%, 25%, 50%>, 75%, 100%, or more relative to an untreated control. The rate of survival may be measured by counting cells capable of being stained with a dye specific for dead cells (e.g., propidium iodide) in culture.

As used herein, the term "to a greater extent" is meant between 2 to 100 fold. As used herein, the term "flow cytometry" refers to an assay in which the proportion of a material (e.g. hES comprising a particular maker) in a sample is determined by labeling the material (e.g., by binding a labeled antibody to the material), causing a fluid stream containing the material to pass through a beam of light, separating the light emitted from the sample into constituent wave lengths by a series of filters and mirrors, and detecting the light.

A multitude of flow cytometers are commercially available including for e.g. Becton Dickinson FACScan and FACScalibur (BD Biosciences, Mountain View, CA). Antibodies that may be used for FACS analysis are taught in Schlossman S, Boumell L, et al, [Leucocyte Typing V. New York: Oxford University Press; 1995] and are widely commercially available.

Examples of dispersing agents include, but are not limited to EDTA, dispase, collagenase, accutase, TrypLE and trypsin. Alternatively, or additionally trituration may also be performed to increase the dispersal of the cells.

As used herein, the term "differentiate" refers to the production of a cell type that is more differentiated than the cell type from which it is derived. The term therefore encompasses cell types that are partially and terminally differentiated. Differentiated cells derived from hES cells are generally referred to as hES -derived cells or hES-derived cell aggregate cultures, or hES-derived single cell suspensions, or hES-derived cell adherent cultures and the like.

As used herein, the term "differentiation" refers to a change that occurs in cells to cause those cells to assume certain specialized functions and to lose the ability to change into certain other specialized functional units. Cells capable of differentiation may be any of totipotent, pluripotent or multipotent cells. Differentiation may be partial or complete with respect to mature adult cells.

As used herein, the term "pluripotent cell" refers to a cell derived from an embryo produced by activation of a cell containing DNA of all female or male origin that can be maintained in vitro for prolonged, theoretically indefinite period of time in an undifferentiated state that can give rise to different differentiated tissue types, i.e., ectoderm, mesoderm, and endoderm. The pluripotent state of the cells may be maintained by culturing inner cell mass or cells derived from the inner cell mass of an embryo produced by androgenetic or gynogenetic methods under appropriate conditions, for example, by culturing on a fibroblast feeder layer or another feeder layer. The pluripotent state of such cultured cells can be confirmed by various methods, e.g., (i) confirming the expression of markers characteristic of pluripotent cells; (ii) production of chimeric animals that contain cells that express the genotype of the pluripotent cells; (iii) injection of cells into animals, e.g., SCID mice, with the production of different differentiated cell types in vivo; and (iv) observation of the differentiation of the cells (e.g., when cultured in the absence of feeder layer) into embryoid bodies and other differentiated cell types in vitro.

The pluripotent state of the cells produced by the present invention can be confirmed by various methods. For example, the cells can be tested for the presence or absence of characteristic ES cell markers. In the case of human ES cells, examples of such markers are identified surface markers, and include TRA-1-60, SSEA-4, SSEA-3, and TRA-1-81 and are known in the art.

As used herein, the phrase "endoderm cells" refers to a population of cells wherein at least 50 % thereof, more preferably at least 70 % thereof express at least one of the two markers Sox 17 or FoxA2. According to a preferred embodiment, less than 20 % of the cells, more preferably less than 10 % of the cells express markers for pluripotency, e.g. Oct4. Methods of determining expression levels of Soxl7, FoxA2 or Oct4 are known in the art and include for example RT-PCR, Immunohistochemistry and the like.

Methods of generating endoderm cells from pluripotent stem cells are known in the art and include for example use of Nodal (NM_018055; NP_060525.3) and small molecules (see for example Borowiak et al Cell Stem Cell, Volume 4, Issue 4, 348-358, 3 April 2009). Alternatively, endoderm cells may be generated via embryoid bodies. Specifically, hES cells may be cultured in suspension without FGF to generate embryoid bodies. The endodermal cells may be selected out of the EBs, see for example (Segev, Fischman, Ziskind et al, Stem cells, 2004;22(3):265-74.

According to one embodiment the differentiation into endodermal cells is carried out in the presence of activin A.

Exemplary concentration ranges of activin A include 1-500 ng/ml, more preferably 1-250 ng/ml, more preferably 50-200 ng/ml, such as for example 100 ng/ml.

According to a particular embodiment of this aspect of the present invention, the pluripotent stem cells are differentiated into endodermal cells by initial culture (e.g. for about 2 days) in a medium comprising activin A and a Wnt-3 ligand and subsequent culture (e.g. 1 day) in a medium comprising activin A, but devoid of Wnt- 3.

Typically, in the first culture medium there may be a lower concentration of serum, relative to the second culture medium. Increasing the serum concentration in the second culture medium increases the survival of the cells, or, alternatively, may enhance the proliferation of the cells. The serum concentration of the first medium may be in the range of about 0 % to about 10 %. Alternatively, the serum concentration of the first medium may be in the range of about 0 % to about 2 %. Alternatively, the serum concentration of the first medium may be in the range of about 0 % to about 1 %. Alternatively, the serum concentration of the first medium may be about 0.5 %.

According to a particular embodiment, both the first culture medium and the second culture medium are devoid of serum. Typically, in place of serum a replacement is added. Such replacements may be provided at various concentrations, such as a concentration of at least 0.1 %, e.g., a concentration of at least 0.2 %, at least 1 %, at least 1.5 % or at least 2 %. Serum replacements are widely available - for example from Invitrogen (knock-out serum replacement™ and Sigma-Aldrich). An additional agent that may be used to replace serum is albumin, for example human recombinant albumin.

As used herein, the phrase "pancreatic progenitor cells" refers to a population of cells which are not fully differentiated into pancreatic cells, yet are committed to differentiating towards at least one type of pancreatic cell - e.g. beta cells that produce insulin; alpha cells that produce glucagon; delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide.

Typically, pancreatic progenitor cells express some of the phenotypic markers that are characteristic of pancreatic lineages (e.g. GLUT2, PDX-1 Ηηθβ, PCl/3, Beta2, Nkx2.2 and PC2). Typically, they do not produce progeny of other embryonic germ layers when cultured by themselves in vitro, unless dedifferentiated or reprogrammed. It will be appreciated that it is not implied that each of the cells within the population have the capacity of forming more than one type of progeny, although individual cells that are multipotent pancreatic progenitor cells may be present.

As used herein, the phrase "islet cells" refers to a cell that synthesizes at least one of the following islet-specific polypeptide hormones - insulin, glucagon, somatostatin and pancreatic polypeptide. Thus, the islet cells generated according to the methods of the present invention may be construed as beta cells that produce insulin; 2) alpha cells that produce glucagon; 3) delta cells (or D cells) that produce somatostatin; and/or F cells that produce pancreatic polypeptide.

Typically the islet cells of this aspect of the present invention store the hormones in secretary vesicles in the form of secretory granules.

As mentioned herein above, the present inventors have shown that using the methods of the present invention populations of islet cells may be generated, the relative amounts of each cell type reflecting those in naturally occurring islets (i.e. two thirds insulin producing cells and one third glucagon producing cells).

As used herein "diabetes" refers to a disease resulting either from an absolute deficiency of insulin (type 1 diabetes) due to a defect in the biosynthesis or production of insulin, or a relative deficiency of insulin in the presence of insulin resistance (type

2 diabetes), i.e., impaired insulin action, in an organism. The diabetic patient thus has absolute or relative insulin deficiency, and displays, among other symptoms and signs, elevated blood glucose concentration, presence of glucose in the urine and excessive discharge of urine.

As used herein "extracellular matrix (ECM) substrates" refer to a surface beneath cells which supports growth. For example, such ECM substrates include, but are not limited to, Matrigel, laminin, gelatin, vitronectin and fibronectin substrates. In a related aspect, such substrates may comprise collagen IV, entactin, heparin sulfate proteoglycan, to include various growth factors (e.g., bFGF, epidermal growth factor, insulin-like growth factor- 1, platelet derived growth factor, nerve growth factor, and TGF-.beta.-l).

As used herein, the term "substantially" refers to a great extent or degree, e.g. "substantially similar" in context is used to describe one method which is to a great extent or degree similar to or different than another method. However, as used herein, the term "substantially free", e.g., "substantially free" or "substantially free from contaminants," or "substantially free of serum" or "substantially free of insulin or insulin like growth factor" or equivalents thereof, means that the solution, media, supplement, excipient and the like, is at least 98%, or at least 98.5%, or at least 99%, or at least 99.5%, or at least 100% free of serum, contaminants or equivalent thereof. In one embodiment, there is provided a defined culture media with no serum, or 100% serum-free, or substantially free of serum. Conversely, as used herein, the term "substantially similar" or equivalents thereof means that the composition, process, method, solution, media, supplement, excipient and the like is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar to that previously described in the specification herein, or in a previously described process or method incorporated herein in its entirety.

In certain embodiments of the present invention, the term "enriched" refers to a cell culture that contains more than approximately 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the desired cell lineage.

As used herein, the term "express" refers to the transcription of a polynucleotide or translation of a polypeptide in a cell, such that levels of the molecule are measurably higher in a cell that expresses the molecule than they are in a cell that does not express the molecule. Methods to measure the expression of a molecule are well known to those of ordinary skill in the art, and include without limitation, Northern blotting, RT-PCR, in situ hybridization, Western blotting, and immunostaining.

As used herein when referring to a cell, cell line, cell culture or population of cells, the term "isolated" refers to being substantially separated from the natural source of the cells such that the cell, cell line, cell culture, or population of cells are capable of being cultured in vitro. In addition, the term "isolating" is used to refer to the physical selection of one or more cells out of a group of two or more cells, wherein the cells are selected based on cell morphology and/or the expression of various markers.

Accordingly, the cell culture environments and methods of the present invention comprise plating the cells in an adherent culture. As used herein, the terms "plated" and "plating" refer to any process that allows a cell to be grown in adherent culture. As used herein, the term "adherent culture" refers to a cell culture system whereby cells are cultured on a solid surface, which may in turn be coated with an insoluble substrate that may in turn be coated with another surface coat of a substrate, such as those listed below, or any other chemical or biological material that allows the cells to proliferate or be stabilized in culture. The cells may or may not tightly adhere to the solid surface or to the substrate. The substrate for the adherent culture may comprise any one or combination of polyornithine, laminin, poly-lysine, purified collagen, gelatin, fibronectin, tenascin, vitronectin, entactin, heparin sulfate proteoglycans, poly glycolytic acid (PGA), poly lactic acid (PLA), hyaluronan hydrogel and poly lactic-glycolic acid (PLGA). Furthermore, the substrate for the adherent culture may comprise the matrix laid down by a feeder layer, or laid down by the pluripotent human cell or cell culture. As used herein, the term "extracellular matrix" encompasses solid substrates such as but not limited to those described above, as well as the matrix laid down by a feeder cell layer or by the pluripotent human cell or cell culture. In one embodiment, the cells are plated on matrigel-coated plates or microcarriers. In another embodiment, the cells are plated on vitronectin-coated plates. In another embodiment, serum can be placed in the medium for up to 24 hours to allow cells to plate to the plastic. If using serum to promote the attachment of the cells, the media is then removed and the compositions, which are essentially serum-free, are added to the plated cells.

The compositions and methods of the present invention contemplate that the differentiable cells are cultured in conditions that are essentially free of a feeder cell or feeder layer. As used herein, a "feeder cell" is a cell that grows in vitro, that is co- cultured with a target cell and stabilizes the target cell in its current state of differentiation. As used herein, a "feeder cell layer" can be used interchangeably with the term "feeder cell." As used herein, the term "essentially free of a feeder cell" refers to tissue culture conditions that do not contain feeder cells, or that contain a de minimus number of feeder cells. By "de minimus", it is meant that number of feeder cells that are carried over to the instant culture conditions from previous culture conditions where the differentiable cells may have been cultured on feeder cells. In one embodiment of the above method, conditioned medium is obtained from a feeder cell that stabilizes the target cell in its current state of differentiation. In another embodiment, the defined medium is a non-conditioned medium, which is a medium that is not obtained from a feeder cell.

As used herein, the term "stabilize," when used in reference to the differentiation state of a cell or culture of cells, indicates that the cells will continue to proliferate over multiple passages in culture, and preferably indefinitely in culture, where most, if not all, of the cells in the culture are of the same differentiation state. In addition, when the stabilized cells divide, the division typically yield cells of the same cell type or yield cells of the same differentiation state. A stabilized cell or cell population in general, does not further differentiate or de-differentiate if the cell culture conditions are not altered, and the cells continue to be passaged and are not overgrown. In one embodiment, the cell that is stabilized is capable of proliferation in the stable state indefinitely, or for at least more than 2 passages. In a more specific embodiment, the cells are stable for more than 3 passages, 4 passages, 5 passages, 6 passages, 7 passages, 8 passages, 9 passages, more than 10 passages, more than 15 passages, more than 20 passages, more than 25 passages, or more than 30 passages. In one embodiment, the cell is stable for greater than approximately 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, or 11 months of continuous passaging. In another embodiment, the cell is stable for greater than approximately 1 year of continuous passaging. In one embodiment, stem cells are maintained in culture in a pluripotent state by routine passage in the defined medium until it is desired that they be differentiated. As used herein, the term "proliferate" refers to an increase in the number cells in a cell culture. In general, the cell medium compositions of the present invention are refreshed at least once every day, but the medium can be refreshed more often or less often, depending of the specific needs and circumstances of the suspension culture. In vitro, cells are usually grown in culture media in a batch mode and exposed to various media conditions. As described herein, the cells exist in a dish-culture as either adherent cultures or as microcarrier/cell aggregates in suspension, and maintained in contact with a surrounding culture medium. In general, the culture medium may be refreshed about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours, or any fraction thereof. In additional examples, the medium may be refreshed less often such as, but not limited to, every 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9 or every 2 or more days, or any time frame in between.

In general, the cells that are cultured in suspension in the medium compositions of the present invention are "split" or "passaged" every week or so, but the cells can be split more often or less often, depending on the specific needs and circumstances of the suspension culture. For example, the cells may be split every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more days, or any time frame in between. As used herein, the term "split" or "passaged" in the context of cell culture is used as it is in the art. Namely, cell culture splitting, or passaging, is the collection of cells from a previous culture and subsequent transfer the collected (harvested) cells into a new cell culture vessel. In general, passaging cells allows the cells to continue to grow in a healthy cell culture environment. One of skill in the art will be familiar with the process and methods of cell culture passaging, which may, but not necessarily, involve the use of enzymatic or non-enzymatic methods that may be used to disaggregate cells that have clumped together during their growth expansion.

It is contemplated that the cells can be passaged using enzymatic, non- enzymatic, or manual dissociation methods prior to and/or after contact with the defined medium of the invention. Non-limiting examples of enzymatic dissociation methods include the use of proteases such as trypsin, TrypLE, collagenase, dispase, and accutase. When enzymatic passaging methods are used, the resultant culture can comprise a mixture of singlets, doublets, triplets, and clumps of cells that vary in size depending on the enzymatic method used. A non-limiting example of a non-enzymatic dissociation method is a cell dispersal buffer or EDTA. Manual passaging techniques have been well described in the art, such as in Schulz et al., 2004 Stem Cells, 22(7): 1218-38. The choice of passaging method may be influenced by the choice of extracellular matrix, if one is present, and is easily determined by one of ordinary skill in the art.

The disaggregation solution used in the methods of the present invention can be any disaggregation solution capable of breaking apart or disaggregating the cells into single cells, without causing extensive toxicity to the cells. Examples of disaggregation solutions include, but are not limited to, accutase, 0.25% Trypsin/EDTA, TrypLE, 0.05% EDTA or VERSENE™ (EDTA) and trypsin. The methods of the present invention need not result in every cell of the confluent layer or suspension being disaggregated into single cells, provided that at least a few single cells are disaggregated and capable of being re-cultured.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

As used herein the term "about" refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

Throughout this application, various embodiments of this 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. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is 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 sub-combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al, "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8 ^th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. 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" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (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 document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

MATERIALS AND METHODS Culture of Human ES cell lines (hESCs)

Human ES cell lines (KS-4 & KS-2 cell lines, passages 20-42) and IPS cells were cultured on a feeder layer of inactivated (γ-irradiated),, human foreskin fibroblast (HFF). Cells were maintained in growth medium consisting of 84% KnockOut® DMEM, 14% (v/v) KnockOut® SR, 1 mM Glutamax, 0.1 mM -β- mercaptoethanol, 1% nonessential amino acids (all from Gibco, Life Technologies, USA), and 8 ng/ml bFGF (R&D Systems, USA); the medium was replaced every other day. Under these conditions the cells were kept undifferentiated. The cells were harvested every 4-7 days in a ratio of 1 : 2-4 by a treatment with lmg/ml CoUagenase type IV (Gibco, Life Technologies, USA) followed by mechanical dissociation to achieve small cell clumps, and reseeded on freshly prepared HFF feeders.

Culture of hESCs at Feeder Free Conditions

Undifferentiated hESCs and IPS grown to confluency were adapted to grow at feeder free conditions. The cells were removed from their feeder layer by a treatment with CoUagenase type IV (O.lml/cm ² ) and mechanically dissociated into small clumps. Cells were re-seeded as colonies in a concentration of -50 colonies /cm ² onto plates coated with matrix (BD Matrigel™ (BD Biosciences, USA), Vitronectin (Peprotech) or Cellstart (Life technologies, USA) and cultured with serum free medium (STEMPRO ^® hESC SFM (Life Technologies Corporation, USA) with 0.1 mM -β-mercaptoethanol and 8 ng/ml bFGF, AF NutriStem® hESC XF (Biological Industries, Israel), E8 (Life technologies, USA) or KO-SR XF (Life technologies, US A)). The medium was replaced every other day. The cells were harvested every 4-6 days in a ratios of 1 to 2-4 by a treatment with Dispase (Gibco, Life Technologies, USA) or 0.05% EDTA (Biological Industries, Israel) or TrypLE Select (Gibco Life Technologies, USA), followed by mechanical dissociation to achieve small cell clumps, and reseeded on freshly prepared plates coated with matrix (up to 15 passages). Under these conditions cells were kept undifferentiated.

Cell Culture on Microcarrier

Adapted undifferentiated hESCs and IPS grown to confluency were used to grow the cells on microcarriers at feeder free conditions. The cells fed with fresh medium including Rock inhibitor (Y-27632, Sigma) 4-6 hr prior the split. The cells were removed from their feeder free matrix by treatment with 0.05% EDTA or TrypLE Select followed by mechanical dissociation to achieve single cells. Cells were re-seeded on Cytodex 1 and Cytodex 3 microcarriers coated with feeder free matrix, at cell concentration of 0.08±0.04X10 ⁶ viable cell/cm ² , and cultured with the relevant serum free medium. Rock inhibitor (10μΜ) was added at seeding. The medium was added every day at ~10%> of the working volume, up to total volume of ~30%> of the working volume. At day 2-3 full amount of bFGF and β-mercaptoethanol was added. The cells were harvested every 4-7 days with 0.01-0.05% EDTA or TrypLE Select followed by mechanical dissociation to achieve small cell/microcarrier aggregates and reseeded on freshly prepared feeder free microcarriers (up to 10 Passages). Under these conditions cells were kept undifferentiated.

Dynamic Cell Expansion on Microcarrier

The dynamic process was confirmed in three cultivation systems - non adherent 6-well plates, Erlenmeyers (125ml, 250ml and 500ml, Corning Incorporated, Corning NY, USA) and Bioreactor (500ml, CellSpin of Integra Biosciences, Fernwald, Germany). The shaking and agitation values were adjusted with the cultivation system and the working volume to the range of 65-95RPM. The expansion process included static seeding period of two days followed by 2-5 days of growth period.

Cryopreservation and thawing hESC/microcarriers aggregates

For freezing: the hESC/microcarriers aggregates (KS#4, P35PFF4E8) were collected, washed, and re-suspended in fresh StemPro medium. The hESC/microcarriers were treated similar to cryopreserved hESC colonies, with the exception that the cells were not removed from the microcarriers prior to freezing. hESC/microcarriers were suspended in DMSO (10%), KO-SR (30%), Rock inhibitor (ΙΟμΜ) and 60% HESM at 3X10 ⁶ cells/mL (0.11 and 0.27X10 ⁶ cells/cm ² , Cytodex 1 or Cytodex 3, respectively). The cells were divided into cryovials, frozen inside the Mr. Frosty Freezing container in a freezer (-80°C), and transferred into liquid nitrogen within 24 hrs.

For thawing: the cryovials were quickly thawed in a 37°C water bath. Fresh

StemPro medium and Rock inhibitor were added into the vials to dilute the cryoprotectants. The cells were then pelleted and washed in the culture medium. Finally, the cells were re-suspended in StemPro and Rock inhibitor and plated onto a Matrigel coated culture plates, without detached from the microcarriers prior to re- plating.

Microcarrier coating with BD Matrigel™

To prepare Matrigel-coated microcarriers, the microcarriers were prepared and sterilized, following the manufacturer's instructions. Briefly, the dry Cytodex 1 or Cytodex 3 microcarriers (GE healthcare, USA) were swollen in PBS for at least three hours at room temperature. The supernatant was decanted and the microcarriers were washed twice for a few minutes in fresh PBS, and sterilized by autoclaving. At this stage the sterile microcarriers may be stored at 4°C for about two months (based on the manufacturer's recommendation). Prior to use, the sterilized microcarriers were allowed to settle and the PBS was decanted. For coating, 1050μ1 cold BD Matrigel™ was dissolved in cold DMEM and added to 70-210 mg (dry weight) cold, sterile microcarriers. DMEM was added to bring the final volume to 70 ml. Then the mixture was transferred into the 125ml erlenmeyer and shacked continuously overnight at 70 RPM. The Matrigel coated microcarriers may be stored at 4°C up to seven days. The Matrigel coated microcarriers were washed with DMEM/F12 before seeding of hESCs or IPS.

Cell Concentration, Viability, Morphology, Attachment efficiency and Splitting efficiency

Homogenously suspended cell/microcarrier aggregates were harvested in ~lml growth medium (at-least two separate samples for each cultivation vessel). The samples were analyzed for total cell concentration with NucleoCounter ^® NC-100 (ChemoMetec, Denmark) while attached to the microcarriers. For viability measurement the medium was removed, cell/microcarrier aggregates were washed with PBS and incubated for five minutes with two ml TrypLE Select (Gibco, Life technologies, USA) at 37°C and re-suspended to achieve a single cell suspension. Cells' viability was determined by NucleoCounter ^® NC-100. For attachment efficiency, utilized medium from day two was collected and the cell concentration (of cells which were not attached to the microcarrier) was determined by NucleoCounter ^® NC-100 and calculated based on the viable cell concentration at seeding. For splitting efficiency, total cell number after the splitting process (before re-seeding) was analyzed with NucleoCounter ^® NC-100 and calculated based on the total cell number before splitting. All samples were prepared in duplicates and the results were represented as mean values (±SD). Morphology of the cell/microcarrier aggregates were analyzed using the OLYMPUS CKX41 inverted microscope.

Medium Sampling and Analysis

For medium sampling, the cultivation systems were removed from the shaker to allow the cell/microcarrier aggregates to settle. Samples from the utilized medium were immediately analyzed for pH. Determination of glucose and lactic acid concentrations was performed by using RQfiexlO analyzer (MERCK, Germany). Glucose consumption rates (GCR) and lactate formation rates (LFR) were calculated.

Live/Dead Fluorescent Immuno-staining

For fluorescent immunostaining of live/dead cells attached to the microcarriers, staining was performed according to manufacturer's protocol using LIVE/DEAD ^® Viability/Cyto toxicity Kit *for mammalian cells* (Life technologies, USA) and analyzed using the OLYMPUS 1X70 fluorescent microscope.

FACS Analysis

Single cell suspension was obtained from cell/microcarrier aggregates by dissociation with TrypLE Select for five minutes at 37°C. Cells were re-suspended in FACS buffer - PBS with 0.5% BSA (Sigma). FACS analysis was performed using PE-conjugated anti-human TRA-1-60, FITC-conjugated anti-human SSEA1, PerCP- Cy5/5- conjugated anti-human EpCAM, APC- conjugated anti-human SSEA4, PE- Cy7- conjugated anti-human CXCR4 (BioLegend, USA). Karyotype Analysis

Cell division was blocked in mitotic metaphase using colcemid-spindle formation inhibitor (Biological Industries, Israel). Nuclear membranes were disrupted by treatment with hypotonic solution 0.28 grams Potassium chloride (Sigma) in 50 ml KPW. The rest of the Karyotype analysis was performed in Rabin Medical Center, Israel.

For the chromosome visualization, G-band standard staining with Giemsa (Merck, Darmstadt, Germany) was used. The karyotypes were analyzed and reported according to the International System for Human Cytogenetic Nomenclature.

Pancreatic Endocrine Differentiation

hESCs grown on cytodex microcarriers as described in example 1 1 , were washed once with Day 1 differentiation media without differentiation factors. Following gravity sedimentation (1-2 minutes) the supernatant was discarded and the cells were dissolved in Day 1 differentiation media including Activin A (100 ng/ml) and Wnt3a (25 ng/ml) (R&D), in a concentration of lxl 0 ⁶ cells/ml. The cells were then transferred to low adherent 6 well plates (Corning) in static conditions (2xl0 ⁶ cells/well). Media change was done according to Table 1 by tilting the plate, sedimentation of cells and replacing about 90% of the media.

Table 1

Day 1 Differentiation Media: DMEM/F12, 2mg/ml HSA, 100 ng/ml, 2 mM Glutamine, lx NEAA, 1 mM Pyruvate, 0.1 mM BMEtOH, 2% KOSR, lxPS+ 2% Human Serum, NEAA. 100 ng/ml Activn A and 25 n /ml Wnt3a.

No c ange

Day 6 Differentiation Media DMEM/F12, 50 ng/ml FGF10, 10 uM FRSK, 1% B27, 2mg/ml HSA, 2 mM Glutamine, lxPS.

No chan e

Measurement of human insulin and C-peptide

The content of human insulin C-peptide was measured with an ELISA kit (Mercodia,Upsalah, Sweden, ultrasensitive c-peptide kit (minimal detection 5pM) or Mercodia c-peptide ELISA (minimal detection 90pM). Samples in M-PER were diluted 3 fold, or more in the kit buffer, and 20 μΐ of the 1/3 dilution were assayed. Results were expressed per mg total protein (as measured by Bradford assay) and per million cells (counted in a NucleoCounter). Other cell pellets were suspended in 0.7 ml buffer RLT (RNAeasy, Qiagen) per million cells, for RNA extraction. Human insulin mRNA was quantitated by RT-qPCR (Taq-Man, Applied Biosystems, Step One) using the Hypoxanthine-guanine phosophoribosyl transferase (HPRT) gene as reference.

EXAMPLE 1

Microcarriers coating procedure

Propagation of hESCs in feeder free conditions was conducted on 2D feeder free plates coated with 1 ml of Matrigel diluted (1 :70) per well (9.6cm ² ). The coating procedure was developed using Matrigel/DMEM solution at dilution ratio of 1 :70. Cytodex 3 (2.7cm ² /mg) or Cytodex 1 (4.4cm ² /mg) microcarriers were coated at a concentration of 1 mg/ml, at a volume of 70 ml in Erlenmeyer (125 ml). The dynamic conditions were 70 RPM, at 37°C overnight. The coated microcarriers were stored at 4°C for up to one week. The high dilution ratio and the slow shaking rate led to a very thin and homogenous Matrigel coating layer as shown in Figure 1. EXAMPLE 2

Comparison of different types of microcarrier in a static cultivation system

Static ultra-low attachment 6-well plates were inoculated with 0.5xl0 ⁶ cells/4mg microcarriers (Cytodex 1 or Cytodex 3). 0.03-0.05xl0 ⁶ cells/cm ² were seeded into each well. As control, the cells were seeded at the same concentrations into 2D- 6-well plate covered with Matrigel. As shown in Figure 2 large aggregates were established due to static conditions. Most of the cells are a live (>65%).

EXAMPLE 3

Comparison of different seeding procedures in a static cultivation system

Single cells were harvested from colonies grown at least three passages on Matrigel/Stempro 2D feeder free growth system, using TrypLE in the presence of ROCK inhibitor, while small clumps were harvested using TrypLE (dilutedl :5) in the presence of ROCK inhibitor. Figure 3 shows that there is no advantage of seeding cell clumps as compared to single cells. In contrary, the diluted TrypLE was not able to dissociate efficiently the cell colonies to cell clumps. In the Cytodex 3 and Cytodex 1 growth systems, which were seeded with single cells, there were no distinct differences in terms of cell expansion and pluripotency. Nevertheless, the 2D system, seeded with single cells which had the same growth area of -lOcm ² as the Cytodex 3 system, was inferior in terms of cell expansion, although the pluripotency remained similar to the pluripotency of cells grown on the microcarriers systems.

EXAMPLE 4

Freezing and thawing of the Microcarriers/cell aggregates

Cells frozen on Matrigel coated Cytodex 1 or Cytodex 3 microcarriers were efficiently thawed on 2D Matrigel coated plates with over 60% thawing yield (Figure 4). Expansion factors of passages PI & P2 were 3.3 & 6.7, respectably. Pluripotency remained high (Tra-1-60 over 85% and EpCAM over 90%) on both cytodex types and rose to 98%o and 99%, respectively, following two passages. After two passages the cells grown on one well were collected for RNA extraction.

EXAMPLE 5

Comparison between different medium types and different splitting procedures in a dynamic cultivation system

Cells were harvested using TrypLE in the presence of ROCK inhibitor for receiving single cells, and using collagenase for receiving small clumps from colonies grown on 2D feeder free (Matrigel) plates. Erlenmeyers (125 ml) were inoculated with 0.02xl0 ⁶ cell/cm ² seeded on Matrigel coated Cytodex 3 (3 mg/ml) and the dynamic growth phase was started following two days of the static seeding stage. Figure 5 presents the morphology of the cell/microcarrier aggregates at day six. The cells were grown in two different media (StemPro® hESC SFM versus NutriStem® hESC XF) and two different splitting methodologies (single cells versus cell clumps) were used. The curves of cell growth, Glucose consumption rate and pluripotency during two passages on the same two media and using the same splitting methodologies are also presented. The single cell seeding gave rise to smaller aggregates. The smaller aggregates presented higher cell concentration, higher expansion, lower doubling time (data not show), higher specific Glucose consumption rate and higher pluripotency. Although the Nutristem medium presented better expansion ratio and growth rate (up to 20 folds and td = 35hr) the pluripotency was not preserved and the cultivation system collapsed due to lack of cells attachment to the microcarriers at P2.

In order to avoid karyotype abnormality risks which may occur when the

TrypLE splitting method was used, several other splitting methodologies were tested including mechanical breaking of the microcarrier/cell aggregates and dispase splitting that gave rise to cell clamps instead of single cells. As shown in Figures 6A-

B, while Dispase enabled the splitting and growing of the cells on fresh microcarriers, even though with very low efficiency, the mechanical breaking method was not efficient and did not break the aggregates to small and non-limited oxygen penetration size.

Anther splitting methodology which was tested and finally elected was the use of EDTA in different conditions and concentrations in order to obtain single cells or very small aggregates. Several EDTA concentrations as well as several splitting procedures were tested. As shown in Figure 7, using very mild concentrations of 0.01- 0.05% EDTA for 3-4 minutes and medium neutralization was found to be ideal to re- growing of the cells on new microcarriers. KS-4 were seeded on Matrigel coated Cytodex 3 and cultivated during three passages. The splitting efficiency was high when using all EDTA concentrations and reached up to 80%. As shown in figures 7A-

C, the cells remained on the beads but the aggregates were broken and were used as a small nucleus to the next passage. This procedure led to less dense aggregates at the end of the passage. The cells from the 2D plates (splitted with 0.05% EDTA) were propagated for ten passages in two different experiments and were sent to the karyotype test. Normal karyotypes were found in both experiments.

EXAMPLE 6

Testing the influence of the splitting and growing methodologies used during 2D cultivation on 3D dynamic cell propagation

The influence of the splitting and growing methodologies used during the 2D cultivation on the 3D dynamic cell propagation was tested. Two different splitting procedures were tested on 2D plates:

A) using EDTA (0.01-0.05%) splitting procedure to obtain single cells, and B) using Dispase splitting procedure to obtain small clamps. The seeding on 3D- microcarriers in both experiments was with single cells established by using EDTA (0.01%-0.05%) in mild conditions. As shown in Figure 8, the use of the EDTA splitting procedure during 2D cultivation led to smaller aggregates. Nevertheless, higher cell concentration, expansion factor and pluripotency were established from the 2D cell colonies grown following the Dispase splitting procedure.

EXAMPLE 7

Comparison between two feeding methodologies -repeated batch versus daily fed batches

Daily fed batches were tested in comparison with repeated batches (removing most of the conditioned medium every 2-3 days). Figure 9 showed a clear advantage to the daily fed batches, probably due to the lack of some ingredients after 24 hrs. in the repeated batches, which may be the cause of the linear growth curve during passage 1 instead of a logarithmic growth curve.

EXAMPLE 8

Attachment and growth on non-coated Cytodex 1 microcarriers using a medium supplemented with ten percent human serum

In order to avoid Matrigel and collagen coating, the cells were attached to non- coated Cytodex 1 microcarriers in the presence StemPro medium supplemented with ten percent human serum at seeding. Three different experiments with two working cell banks of KS-4 were tested on non-coated Cytodex 1 microcarriers. As shown in Figures lOA-C, this procedure led to high differentiation and low expansion of the cells.

EXAMPLE 9

Comparison between different microcarrier types in a dynamic cultivation system

Cell growth on Matrigel coated Cytodex 1 or Cytodex 3 microcarriers was tested using two different cell lines (KS-2 and KS-4). The optimal conditions used were as described below: the cell seeding concentration was 0.08±0.04X10 ⁶ viable cells/cm ² , StemPro medium was used, the microcarrier concentration was 1-20 mg/ml, the seeding duration was two days at static conditions followed by constant agitation (65-95 RPM), a working volume of 10-30%, daily fed-batches was used, the passage duration was 4-6 days and EDTA (0.01-0.05%) was used for splitting. Figure 11A presents the morphology of the cell/microcarrier aggregates and Figure 11B demonstrates the growth and pluripotency curves during three passages using the two microcarrier types. It was found that the cell/microcarrier aggregate size was depended on two parameters: the propagation duration and the splitting efficiency. When the splitting efficiency was lower, the new seeded aggregates were larger. When the passage duration was longer, the cell/microcarrier aggregates were larger and denser, and it was harder to break them prior to the next passage. The cell concentration was 0.8xl0 ⁶ cell/cm ² following eight days in culture, and 0.4-0.6 cell/cm ² following five days of culture (at seeding concentration of 0.04X10 ⁶ viable cells/cm ² ₎ . The cells displayed high growth rate (doubling time of -40 hours) and high expansion factor of -10 with potential of -15. The pluripotency remained over 80% during the four passages. The metabolism conditions were kept in the following levels: Glucose level over 1.0 gr/L in a passage of five days; lactate level lower than 12mM and pH over 6.5, in order to avoid oxygen limitation in the cell/microcarrier aggregates and high acidification of the medium.

EXAMPLE 10

Comparison of the expansion rate of KS-4 hESCs grown on pre-coated microcarriers in Bioreactor versus Erlenmeyer

In order to test the agitation technology of a stirred tank bioreactor, the cells were seeded into 500 ml glass ball impeller CELLSPIN (Integra). As a control the cells were seeded into 125 ml Erlenmeyer. Mechanical splitting with high RPM was used. All the other growth parameters remained as described in Example 9.

Figure 12 presents the morphology of the cell/microcarrier aggregates and the growth; metabolism and pluripotency at day 7, during two passages, in the two cultivation systems. Although the bioreactor presented larger aggregates, the expansion factor at PI was higher and the doubling time was lower (data not shown), due to higher surface area/volume ratio and a better medium replacement methodology, as was detected by the higher levels of glucose and lower levels of lactate in the spent media. Nevertheless, the decrease in the pluripotency was dramatic at both systems and can be referred to the non-efficient mechanical splitting rather than the cultivation systems.

EXAMPLE 11

Scale up of KS-4 on Matrigel coated Cytodex 3 microcarriers

Flow chart of the scale up process started from one ampule on 2D 6-well plate following by seeding on Matrigel coated microcarriers in Erlenmeyers, as well as accumulative cell number and expansion factor during 4 to 5 passages in two separate experiments with KS-4 cells is presented in Figure 13. Since the figure demonstrated the accumulated amount of cells in the Erlenmeyer alone, seeded with 3xl0 ⁶ viable cells/Erlenmeyer, the actual cell expansion, including cell expansion at the 2D stage during 1-3 passages from thawing (~0.5xl0 ⁶ /cm ² on 200cm ² = ~10xl0 ⁶ cells), can reach to 3-4 times higher.

The scale up process flow chart may comprise the following steps:

1. Adaptation and cultivation of pluripotent hESCs in feeder- free growth conditions: Matrigel coated 2D vessels; Stempro media including 8ng bFGF; passaged every 2-8 days using Dispase; high pluripotency (over 85% EpCAM and Tra-1-60)

2. Splitting the cells into single hESCs using EDTA (0.01-0.05%).

3. Seeding the cells (0.08±0.04X10 ⁶ viablecell/cm ² ) on Matrigel coated Cytodex 1 or Cytodex 3 microcarriers, in a medium containing Rock inhibitor.

4. Growing the cells in a dynamic system (Erlenmeyer, bioreactor or nonadherent 6-well plate) for two days of static seeding phase, followed by 2- 5 days of dynamic growth phase; daily fed batch of 10-15% media; 65- 95RPM; 10-50% working volume; pH over 6.5; glucose over lgr/1, lactate under 12mM; high pluripotency (over 65% EpCAM and Tra-1-60).

5. Splitting the cells from 3D microcarriers using EDTA (0.01-0.05%) into small cell/microcarrier aggregates.

6. Re-seeding the cells on fresh Matrigel coated Cytodex 1 or Cytodex 3 microcarriers added to the cell/microcarrier aggregates in a medium containing Rock inhibitor.

7. Re-growing the cells in a dynamic system (Erlenmeyer, bioreactor or nonadherent 6-well plate) under the same conditions, up to seven passages.

EXAMPLE 12

Pancreatic differentiation of KS-4 and KS-2 on Matrigel coated Cytodex 1&3 microcarriers

The pancreatic differentiation potential of hESCs grown on microcarriers was analyzed by applying a step wise differentiation protocol developed to manipulate differentiation of hESCs towards mature pancreatic endocrine phenotype. In this protocol cells go through several well characterized developmental stages, yielding mature endocrine hormone producing cells, similar to human islets. A flow chart of the differentiation protocol is described in figure 14. The differentiated islet like cells exhibit mature β-cell characteristics manifested by the expression of insulin mRNA (Figure 15) and the ability to produce and store insulin/C-peptide (Figures 16 A and B). In addition to insulin mRNA, Glucagon mRNA is also detected, implying that the pancreatic endocrine differentiation protocol leads also to the generation of glucagon producing a-cells.

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

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference 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.