Differentiation of human embryonic stem cells

[0001] The present invention provides methods for promoting the differentiation of pluripotent stem cells into insulin producing cells. In particular, the present invention provides a method for producing a cell population, wherein more than 85% of the cells in the population express markers characteristic of a fully developed endoderm line.

BACKGROUND [0002] Advances in cell replacement therapy for type I diabetes and the lack of transplantable Langehnas islets have focused interest in developing sources of insulin-producing cells or β-cells suitable for implantation. One approach is to generate functional β cells from pluripotent stem cells, such as, for example, embryonic stem cells.

[0003] In the embryonic development of vertebrates, a pluripotent cell gives rise to a group of cells containing three germ layers (ectoderm, mesoderm and endoderm) in a process known as gastrulation. Tissues such as, for example, the thyroid gland, thymus, pancreas, intestine and liver develop from the endoderm through the intermediate stage. The intermediate stage in this process is the creation of a fully developed endoderm. Cells of fully developed endoderms express a number of markers such as HNF3 beta, GATA4, MIXL1, CXCR4 and SOX17.

[0004] Pancreatic formation arises from the differentiation of a fully developed endoderm to a pancreatic endoderm. Pancreatic endoderm cells express the pancreatic duodenal homeobox gene, PDX1. In the absence of PDX1, the pancreas does not develop beyond the formation of abdominal and dorsal buds. Thus, PDX1 expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among others, cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.

[0005] Cells bearing the characteristics of islet cells are allegedly derived from mouse embryonic cells. For example, Lumelsky et al. (Science 292: 1389, 2001) describes the differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49: 157, 2000) describes that insulin secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin-induced diabetic mice.

[0006] In one example, Hori et al. (PNAS 99: 16105, 2002) discloses that treatment of murine embryonic stem cells with phosphoinositide 3-kinase inhibitors (LY294002) produces cells that resemble β cells.

[0007] In another example, Blyszczuk et al. (PNAS 100: 998, 2003) describes the generation of insulin producing cells from mouse embryonic stem cells constitutively expressing Pax4.

[0008] Micallef et al. Describes that retinoic acid regulates the orientation of embryonic stem cells to form PDX1 positive pancreatic endoderm. Retinoic acid is most effective in inducing Pdx1 expression when added to culture on day 4 of embryonic stem cell differentiation during the period corresponding to the end of gastrulation in the embryo (Diabetes 54: 301, 2005).

[0009] Miyazaki et al. Describes mouse embryonic stem cell lines overexpressing Pdx1. Their results show that the exogenous expression of Pdx1 clearly enhances the expression of the insulin, somatostatin, glucokinase, neurogenin3, p48, Pax6 and Hnf6 genes in the differentiated cells obtained (Diabetes 53: 1030, 2004).

[0010] Skoudy et al. describes that activin A (a member of the TGF-β superfamily) stimulates the expression of pancreatic exocrine genes (p48 and amylase) and endocrine genes (Pdx1, insulin and glucagon) in mouse embryonic stem cells. The maximum effect was observed with 1 nM activin A. They also observed that insulin and Pdx1 mRNA expression levels were not affected by retionic acid; however, treatment of 3 nM FGF7 results in increased levels of Pdx1 transcript (Biochem. J. 379: 749,2004). [0011] Shiraki et al. studied the effects of growth factors that specifically enhance the differentiation of embryonic stem cells into PDX1 positive cells. They observed that TGF-e2 repeatedly gives a higher proportion of PDX1 positive cells (Genes Cells. 2005 Jun; 10 (6): 503-16.).

[0012] Gordon et al. Demonstrated that induction of endoderm cells [brachy positive / HNF3 beta positive] from mouse embryonic stem cells in the absence of serum and in the presence of activin together with the Wnt signaling inhibitor (US 2006 / 0003446A1) .

[0013] Gordon et al. (PNAS, volume 103, page 16806, 2006) states "Wnt and TGF-beta / nodal / activin signal transduction was simultaneously required to generate the anterior primary streak."

[0014] However, the mouse embryonic stem cell development model may not exactly mimic the development program in higher mammals such as, for example, humans.

[0015] Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282: 114, 1998). Simultaneously, Gearhart et al. Derived human embryonic cell lines (hEG) from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). Unlike mouse embryonic stem cells, which can be protected against differentiation simply by culturing with leukemia inhibitory factor (LIF), human embryonic stem cells must be kept under very special conditions (US Patent No. 6,200,806; WO 99/20741; WO 01/51616 ).

[0016] D'Amour et al. describes the production of enriched cultures of fully developed endoderm derived from human embryonic stem cells in the presence of high levels of activin and low serum (Nature Biotechnology 2005). Transplantation of these cells under the renal capsule of mice results in differentiation into more mature cells with the characteristics of some endodermal organs. Fully developed endoderm cells derived from human embryonic stem cells can further differentiate into PDX1 positive cells after the addition of FGF-10 (US 2005 / 0266554A1).

[0017] D'Amour et al. (Nature Biotechnology - 24, 1392 - 1401 (2006)) state: "We have developed a differentiation process that converts human germ cells (hES) to endocrine cells capable of synthesizing pancreatic hormones insulin, glucoagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics pancreatic organogenesis in vivo by directing cells through stages resembling fully developed endoderm, intestinal endoderm, pancreatic endoderm and endocrine precursor on the path to cells that express endocrine hormones. "

[0018] In another example, Fisk et al. describes a system for producing islet cells from human germ cells (US2006 / 0040387A1). In this case, the differentiation pathway is divided into three stages. Human embryonic stem cells were first differentiated into endoderm using a combination of sodium butyrate and activin A. Cells were then cultured with TGF-β antagonists such as Noggin in combination with EGF or betacellulin to generate PDX1 positive cells. Final differentiation was induced by nicotinamide.

[0019] WO 2007/143193 describes a method for generating a fully developed endoderm and pancreatic endoderm cells using defined media in the absence of feeder cells.

[0020] WO 2009/154606 describes methods for generating endoderm line type cells derived from human pluripotent cells, such as human embryonic stem cells, by various means, including GDF8, GDF11 and GSK3beta inhibitors.

[0021] There remains a significant need to develop in vitro methods for generating a functional insulin-expressing cell that closely resembles a β cell. The present invention takes an alternative approach to improve the efficiency of differentiating human embryonic stem cells towards insulin-expressing cells by generating a cell population in which more than 85% of the cells in the population express markers characteristic of a fully developed endoderm line.

SUMMARY [0022] The invention provides a method of generating a cell population expressing markers characteristic of a fully developed endoderm line, comprising the steps of:

and. cultivating a population of human pluripotent stem cells, b. differentiation of the population of human pluripotent stem cells to the population of cells expressing markers characteristic of a fully developed endoderm line in serum-free medium supplemented with BSA and a factor selected from the group consisting of insulin and IGF-1, the medium further supplemented with GDF-8 and 14prop- 2-en-1-yl-3,5,7,14,17,23,27-heptaazatetracyclo [19.3.1.1 ~ 2.6 ~ .1 ~ 8.12 ~] heptacose-1 (25), 2 (27 ), 3,5,8 (26), 9,11,21,23-nonaen-16-one.

[0023] In one embodiment, the present disclosure provides a population of cells, wherein more than 85% of the cells in the population express markers characteristic of a fully developed endoderm line.

[0024] In one embodiment, pluripotent stem cell populations are differentiated into a population of cells expressing markers characteristic of a fully developed endoderm line by culturing pluripotent stem cells in BSA supplemented medium and an agent selected from the group consisting of insulin and IGF1. In one embodiment of the disclosure, the differentiation of a pluripotent stem cell population towards a population of cells expressing markers characteristic of a fully developed endoderm line is achieved by treatment of pluripotent stem cells with activin A and a Wnt ligand.

[0025] In one embodiment, differentiation of the population of pluripotent stem cells towards the population of cells expressing markers characteristic of a fully developed endoderm line is achieved by treatment of pluripotent stem cells GDF-8 and at least one other factor was selected from the group consisting of: aniline pyridine triazine, cyclic aniline-pyridine triazine, N- {[1- (phenylmethyl) azepan-4-yl] methyl} -2-pyridin-3-ylacetamide, 4 - {[4- (4 - {[2- (pyridin-2-ylamino) ethyl] amino} -1,3,5triazyn-2-yl) pyridin-2-yl] oksylobutan-1-ol, 3 - ({3- [4 - ({2- [methyl (pyridin-2-yl) amino] ethyl} amino) -1,3,5-triazin-2-yl] pyridin-2-yl} amino) propan-1 ol, N ~ 4 ~ - [2- (3-fluorophenyl) ethyl] -N ~ 2 ~ - [3- (4-methylpiperazin-1-yl) -propyl] -pyrido [2,3-d] pyrimidine-2,4diaminy. 1-methyl-N - [(4-pyridin-3-yl-2 - {[3- (trifluoromethyl) phenyl] amino} -1,3-thiazol-5-yl) methyl] piperidine-4-carboxamide, 1,1-dimethylethyl {2- [4 - ({5- [3- (3-hydroxypropyl) phenyl] -4H-1,2,4-triazol-3-yl {amino) phenyl] ethyl} carbamate, {[3 - ({5- [5- (3-hydroxypropyl) -2- (methyloxy) phenyl] -1,3-oxazol-2-yl {amino) phenyl] methyl {1,1-dimethylethyl carbamate, 1 - ({5- [6 - ({4 - [(4-methylpiperazin-1-yl) sulfonyl] phenyl} amino) pyrazin-2-yl] thiophen-2-yl {methyl) piperidin-4-ol, 1- ({4- [6 - ({4 - [(4-methylpiperazin-15-yl) sulfonyl] phenyl {amino) pyrazin-2-yl] thiophen-2-yl {methyl) piperidine-4-carboxamide and 2 { [4- (1-methylethyl) phenyl] amino {N- (2-thiophen-2-yl-ethyl) -7,8-dihydropyrido [4,3-d] pirymidyno6 (5H) -carboxamide.

BRIEF DESCRIPTION OF THE DRAWINGS [0026]

Figure 1 shows real-time PCR analysis of gene expression indicated in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 1.

Figure 2 shows FACS analysis of proteins indicated in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 1.

Figure 3 shows real-time PCR analysis of gene expression indicated in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 2.

Figure 4 shows SOX17 expression by immunofluorescence in cells of human H1 embryonic stem cell lines differentiated according to the methods disclosed in Example 2.

Figure 5 shows FACS analysis of proteins indicated in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 2.

Figure 6 shows real-time PCR analysis of gene expression indicated in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 3.

Figure 7 shows SOX17 expression by immunofluorescence in cells of human H1 embryonic stem cell lines differentiated according to the methods disclosed in Example 3.

Figure 8 shows SOX17 expression by immunofluorescence in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 3.

Figure 9 shows real-time PCR analysis of gene expression indicated in human H1 embryonic stem cell line cells differentiated according to the methods disclosed in Example 5.

DETAILED DESCRIPTION [0027] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections, which describe or illustrate certain features, embodiments or uses of the present invention.

Definitions [0028] Stem cells are undifferentiated cells defined by their ability at the level of a single cell to both self-renew and to differentiate to produce daughter cells, including self-renewing progenitors, non-renewing progenitors and ultimately differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of different cellular origin from different germ layers (endoderms, mesoderms and ectoderms), as well as to the formation of tissues with many germ layers after transplantation, and to contribute essentially to the majority, if not all, tissue after injection into blastocysts.

[0029] Stem cells are classified according to their development potential as: (1) totipotent, which means capable of giving rise to all embryonic and non-embryonic cell types; (2) pluripotent, which means capable of giving rise to all types of germ cells; (3) multipotential, which means capable of giving rise to a subset of cell lines, but all within a particular tissue, an organ or physiological system (for example, hematopoietic stem cells (HSCs) can produce offspring, which includes HSC (self-renewal), oligopotential progenitors limited to blood cells and all cell types and elements (e.g., shallow), which are normal blood components); (4) oligopotential, which means capable of giving rise to a more limited subset of cell lines than multipotent stem cells; and (5) unipotential, which means capable of giving rise to a single cell line (e.g., spermatogenic stem cells).

[0030] Differentiation is the process by which non-specialized ("non-targeted") or less specialized cells acquire the characteristics of specialized cells, such as, for example, a nerve cell or a muscle cell. A differentiated or induced differentiation cell is the one that has taken a more specialized ("targeted") position within the cell line. The term "targeted", when used for the differentiation process, refers to a cell that follows the differentiation pathway to the point where, under normal circumstances, it continues to differentiate into a specific cell type or subset of the cell type, and cannot normally differentiate into a different cell type or return to a less diverse cell type. Differentiation refers to the process by which cells return to a less specialized (or targeted) position in a cell line. As used herein, the cell line defines the heredity of the cell, i.e., which cells are derived and which cells can give rise to. The cell line places the cell in the pattern of inheritance of development and differentiation. The line-specific marker refers to a feature specifically related to the cell phenotype of the line of interest and can be used to assess the differentiation of an undirected cell to a line of interest.

[0031] "Cells expressing markers characteristic of a fully developed endoderm line" or "stage 1 cells" or "stage 1" as used herein refer to cells expressing at least one of the following markers: SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, mix-like homeobox protein, FGF4 CD48, eomezodermine (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99 or OTX2. Cells expressing markers characteristic of a fully developed endoderm line include primitive band precursor cells, mesoderm cells and fully developed endoderm cells.

[0032] "Cells expressing markers characteristic of the pancreatic endoderm line" as used herein refer to cells expressing at least one of the following markers: PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6, HNF4 alpha, SOX9, HB9 or PROX1. Cells expressing markers characteristic of pancreatic endoderm linna include pancreatic endoderm cells, primary intestinal cells, and posterior frontal intestinal cells.

[0033] A "fully developed endoderm", as used herein, refers to cells that carry the characteristics of cells formed from epiblast during gastrulation, and which form the gastrointestinal tract and its derivatives. Cells of fully developed endoderm express the following markers: HNF3 beta, GATA4, SOX17, Cerberus, OTX2, goosecoid, CKit, CD99 and MIXL1.

[0034] "Markers" as used herein are nucleic acid or polypeptide molecules that are expressed differently in a cell of interest. In this context, differential expression means increased levels for positive markers and reduced levels for negative markers. The detectable level of the marker nucleic acid or polypeptide is sufficiently higher or lower in the cells of interest compared to other cells, such that the cells of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.

[0035] "Pancreatic endocrine cell" or "pancreatic hormone expressing cell" or "cells expressing markers characteristic of the pancreatic endocrine line" as used herein refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin and pancreatic polypeptide.

Isolation, expansion and culture of pluripotent stem cells Characterization of pluripotent stem cells [0036] Pluripotent stem cells may express one or more of stage-specific embryonic antigens (SSEA) 3 and 4 and detectable markers using antibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science 282: 1145, 1998). In vitro differentiation of pluripotent stem cells results in loss of SSEA-4, Tra1-60 and Tra1-81 expression (if present) and increased SSEA-1 expression. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity that can be detected by fixing cells with 4% paraformaldehyde and then developing with Vector Red as a substrate, as described by the manufacturer (Vector Laboratories, Burlingame Calif.). Undifferentiated pluripotent stem cells also typically express Oct-4 and TERT as detected by RT-PCR.

[0037] Another desirable phenotype of propagated pluripotent stem cells is the potential for cell differentiation of all three germ layers: endoderm, mesoderm and ectoderm. The pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into mice with severe combined immunodeficiency (SCID), fixing the formed teratats using 4% paraformaldehyde, and then histologically examining them for evidence of cell types from the three germ layers. Alternatively, pluripotency can be determined by forming embryoid bodies and assessing embryoid bodies for the presence of markers associated with the three germ layers.

[0038] Propagated pluripotent stem cell lines can be karyotyped using standard G-band technique and compared with published karyotypes of the respective primate species. It is desirable to obtain cells that have a "normal karyotype", which means that the cells are euploid, with all human chromosomes present and not noticeably changed.

Sources of pluripotent stem cells [0039] Examples of pluripotent stem cells are established human embryonic stem cell lines or human embryonic generative cell lines, such as, for example, H1, H7 and H9 human embryonic stem cell lines (WiCell). The use of the compositions of this disclosure is also contemplated during the initial establishment or stabilization of such cells, in which case the source cells would be primary pluripotent cells taken directly from the source tissues. Cells harvested from pluripotent stem cell populations already cultured in the absence of feeder cells are also suitable. Mutant human embryonic stem cell lines are also suitable, such as, for example, the BG01v reference cell line (BresaGen, Athens, GA).

Culture of Pluripotent Stem Cells [0040] In one embodiment, pluripotent stem cells are cultured on a layer of nutrient cells that support pluripotent stem cells in a variety of ways. Alternatively, pluripotent stem cells are grown in a culture system that is essentially free of feeder cells, but nevertheless promotes the proliferation of pluripotent stem cells without undergoing substantial differentiation. Growth of pluripotent stem cells in a nutrient-free culture without differentiation is promoted by using a conditioned medium by prior culturing with another type of cell. Alternatively, the growth of pluripotent stem cells in a nutrient-free culture without differentiation is promoted by the use of a chemically defined medium.

[0041] In one embodiment, pluripotent stem cells can be cultured on a layer of mouse embryonic fibroblast nutrient cells according to the methods disclosed in Reubinoff et al. (Nature Biotechnology 18: 399-404 (2000)). Alternatively, pluripotent stem cells can be cultured on a layer of mouse embryonic fibroblast nutrient cells according to the methods disclosed in Thompson et al. (Science 6 November 1998: volume 282. No. 5391 1145 - 1147). Alternatively, pluripotent stem cells can be cultured on any of the layers of feeder cells disclosed in Richards et al. (Stem Cells 21: 546-556, 2003).

[0042] In one embodiment, pluripotent stem cells can be cultured on a layer of human feeder cells according to the methods disclosed in Wang et al. (Stem Cells 23: 1221-1227, 2005). In an alternative embodiment, pluripotent stem cells can be cultured on a layer of human feeder cells disclosed in Stojkovic et al. (Stem Cells 2005 23: 306-314, 2005). Alternatively, pluripotent stem cells can be cultured on a layer of human feeder cells disclosed in Miyamoto et al. (Stem Cells 22: 433-440, 2004). Alternatively, pluripotent stem cells can be cultured on a layer of human feeder cells disclosed in Amit et al. (Biol. Reprod 68: 2150-2156, 2003). Alternatively, pluripotent stem cells can be cultured on a layer of human feeder cells disclosed in Inzunza et al. (Stem Cells 23: 544-549, 2005).

[0043] In one embodiment, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in US20020072117. Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in US6642048. Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in WO2005014799. Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in Xu et al. (Stem Cells 22: 972-980, 2004). Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in US20070010011. Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in US20050233446. Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in US6800480. Alternatively, pluripotent stem cells can be cultured in culture media derived according to the methods disclosed in WO2005065354.

[0044] In one embodiment, pluripotent stem cells can be cultured according to the methods disclosed in Cheon et al. (BioReprod DOI: 10.1095 / biolreprod. 105.046870, 19 October 2005). Alternatively, pluripotent stem cells can be cultured according to the methods disclosed in Levenstein et al. (Stem Cells 24 :. 568-574, 2006). Alternatively, pluripotent stem cells can be cultured according to the methods disclosed in US20050148070. Alternatively, pluripotent stem cells can be cultured according to the methods disclosed in US20050244962. Alternatively, pluripotent stem cells can be cultured according to the methods disclosed in WO2005086845. [0045] Pluripotent stem cells can be seeded in suitable culture medium. In one embodiment, a suitable culture medium is an extracellular matrix component, such as, for example, those originating from the basement membrane, or which may be part of the receptor-ligand adhesion receptor coupling. In one embodiment, a suitable culture medium is MATRIGEL® (Becton Dickenson). MATRIGEL® is a soluble preparation of Engelbreth-Holm-Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.

[0046] Other components of the extracellular matrix and mixtures of components are suitable as an alternative. Depending on the proliferating cell type, they may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate and the like, alone or in various combinations.

[0047] Pluripotent stem cells are seeded in a suitable distribution and in the presence of a medium that promotes cell survival, propagation and maintenance of desired characteristics. All these features benefit from paying particular attention to the spreading pattern and can be easily determined by a person skilled in the art.

[0048] Suitable culture media can be made from the following ingredients, such as, e.g, Dulbecco's modified Eagle's medium (DMEM), Gibco # 11965092; Knockout Eagle's Modified Dulbecco's Medium (KO DMEM), Gibco # 10829-018; F12 Hama / 50% DMEM basal medium; 200 mM L-Glutamine, Gibco # 15039027; endogenous amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256029.

Formation of cells expressing markers characteristic of a fully developed endoderm line from pluripotent stem cells [0049] The present invention provides methods for creating a population of cells expressing markers characteristic of a fully developed endoderm line from a pluripotent stem cell population. In one embodiment, the present invention provides methods for further differentiating cells expressing markers characteristic of a fully developed endoderm line to cells expressing markers of the pancreatic endocrine line. In one embodiment, this is achieved using a staged differentiation protocol, wherein pluripotent stem cell populations are first differentiated into a population of cells expressing markers characteristic of a fully developed endoderm line. Then, the population of cells expressing markers characteristic of a fully developed endoderm line is then differentiated to the population of cells expressing markers characteristic of the pancreatic endoderm line. Then, cell populations expressing markers characteristic of the pancreatic endoderm line are then differentiated to a population of cells expressing markers characteristic of the pancreatic endocrine line.

[0050] The present disclosure provides a population of cells, where more than 85% of the cells in the population express markers characteristic of a fully developed endoderm line. The cell population can be further treated to form a population of cells expressing markers characteristic of the endoderm pancreatic line. The population of cells expressing markers characteristic of the pancreatic endoderm line can then be treated to form a population of cells expressing markers characteristic of the pancreatic endocrine line.

[0051] The efficiency of differentiation can be determined by exposing the treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the desired cell type.

[0052] Methods for assessing the expression of protein markers and nucleic acids in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blot, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et al., Eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of the specimen material, Western blot and for markers that are available in intact cells, cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).

[0053] Features of pluripotent stem cells are well known to those skilled in the art and additional features of pluripotent stem cells are still identified. Markers of pluripotent stem cells include, for example, the expression of one or more of the following: ABCG2, cripto, FOXD3, connexin43, connexin45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, Tra 1- 60, Tra 1-81. [0054] After treating pluripotent stem cells with the methods of the present invention, differentiated cells can be purified by exposing the treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker, such as CXCR4, expressed by cells expressing markers characteristic of fully developed endoderm line.

[0055] Pluripotent stem cells cited for reference are the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line H1 (NIH code: WA01), the human embryonic stem cell line H7 (NIH code: WA07) and the line human embryonic stem cells SA002 (Cellartis, Sweden). Also suitable for use in the present invention are cells that express at least one of the following markers characteristic of pluripotent cells: ABCG2, cripto, CD9, FOXD3, connexin 43, connexin 45, OCT4, SOX2, NANOG, hTERT, UTF 1, ZFP42, SSEA-3, SSEA-4, Tra 1-60 and Tra 1-81.

[0056] Markers characteristic of a fully developed endoderm line were selected from the group consisting of SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, homeobox-like protein, FGF4 CD48, eomezodermine (EOMES), DKK4, FGF17 , GATA6, CXCR4, C-Kit, CD99 and OTX2. A cell suitable for use in the present invention is that expressing at least one of the markers characteristic of a fully developed endoderm line. In one aspect of the present invention, the cell expressing markers characteristic of a fully developed endoderm line is a primitive precursor streak cell. In an alternative aspect, the cell expressing markers characteristic of a fully developed endoderm line is a mesoderm cell. In an alternative aspect, the cell expressing markers characteristic of a fully developed endoderm line is a cell of fully developed endoderm.

[0057] Markers characteristic of the pancreatic endoderm line were selected from the group consisting of PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6, HNF4 alpha, SOX9, HB9 and PROX1. A cell suitable for use in the present invention is that expressing at least one of the markers characteristic of the pancreatic endoderm line. In one aspect of the present invention, the cell expressing markers characteristic of the pancreatic endoderm line is a pancreatic endoderm cell. [0058] Markers characteristic for the pancreatic endocrine line are selected from the group consisting of NGN3, NEUROD, ISL1, PDX1, NKX6.1, PAX4, NGN3 and PTF1 alpha. In one embodiment, the pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin and pancreatic polypeptide. A cell suitable for use in the present invention is that expressing at least one of the markers characteristic of the pancreatic endocrine line. In one aspect of the present invention, the cell expressing markers characteristic of the pancreatic endocrine line is a pancreatic endocrine cell. The endocrine pancreatic cell may be a hormone expressing pancreatic cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell.

[0059] In one aspect of the present invention, the pancreatic endocrine cell is a cell that expresses markers characteristic of a β cell line. A cell expressing markers characteristic of the β cell line expresses PDX1 and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISL1, HNF3 beta, MAFA, PAX4 and PAX6. In one aspect of the present invention, the cell expressing markers characteristic of the β cell line is a β cell.

Formation of cells expressing markers characteristic of a fully developed endoderm line from pluripotent stem cells [0060] In one aspect of the present invention, pluripotent stem cell populations can be differentiated into a population of cells expressing markers characteristic of a fully developed endoderm line by culturing pluripotent stem cells in serum-free medium supplemented with BSA and a factor selected from the group consisting of insulin and IGF-1. In one embodiment of the disclosure, the differentiation of a pluripotent stem cell population towards a population of cells expressing markers characteristic of a fully developed endoderm line is achieved by treatment of pluripotent stem cells with activin A and a Wnt ligand.

[0061] In an alternative embodiment, differentiation of the population of pluripotent stem cells towards the population of cells expressing markers characteristic of a fully developed endoderm line is achieved by treatment of pluripotent stem cells GDF-8 and at least one other factor was selected from the group consisting of: aniline-pyridine triazine, cyclic aniline-pyridine triazine, N- {[1- (phenylmethyl) azepan-4-yl] methyl} -2-pyridin-3-ylacetamide, 4 - {[4- (4 - {[2- (pyridin-2-ylamino) ethyl] amino} -1,3,5-triazin-2-yl) pyridin-2-yl] oxy} butan-1-ol 3 - ({3- [4 - ({2- [methyl (pyridin-2-yl) amino] ethyl} amino) -1,3,5-triazin-2-yl] pyridin-2-yl} amino) propan- 1olu, N ~ 4 ~ - [2- (3-fluorophenyl) ethyl] -N ~ 2 ~ - [3- (4-methylpiperazin-1-yl) propyl] pyrido [2,3-d] pyrimidine-2,4-diamine, 1-methyl-N - [(4-pyridin-3-yl-2 - {[3- (trifluoromethyl) phenyl] amino} 1,3-thiazol-5-yl) methyl] piperidine-4-carboxamide, 1,1-dimethylethyl {2- [4 - ({5- [3- (3-hydroxypropyl) phenyl] 4H-1,2,4-triazol-3-yl {amino) phenyl] ethyl} carbamate, {[3 - ({5- [5- (3-hydroxypropyl) -2- (methyloxy) phenyl] -1,3-oxazol-2-yl {amino) phenyl] methyl {1,1-dimethylethyl carbamate, 1 - ({5- [6 - ({4 - [(4-methylpiperazin-1-yl) sulfonyl] phenyl} amino) pyrazin-2-yl] thiophen-2-yl {methyl) piperidin-4-ol, 1 - ({4- [6 - ({4 - [(4-methylpiperazin-1-yl) sulfonyl] phenyl {amino) pyrazin-2-yl] thiophen-2-yl {methyl) piperidine-4-carboxamide and 2 {[ 4- (1-methylethyl) phenyl] amino {N- (2-thiophen-2-yl-ethyl) -7,8-dihydropyrido [4,3-d] pirymidyno6 (5H) -carboxamide. Examples of agents suitable for use can be found in US Patent Application Serial No. 12 / 494.789. In one embodiment, at least one factor is 14-prop-2-en-1-yl-3,5,7,14,17,23,27 heptaazatetracyclo [19.3.1.1 ~ 2.6 ~ .1 ~ 8.12 ~] heptakoza-1 (25), 2 (27), 3,5,8 (26), 9,11,21,23nonaen-16-one.

[0062] The pluripotent stem cell population can be cultured in serum free medium supplemented with BSA and an agent selected from the group consisting of insulin and IGF1 for about one day to about seven days. Alternatively, the pluripotent stem cell population can be cultured in serum free medium supplemented with BSA and an agent selected from the group consisting of insulin and IGF-1 for about one day to about six days. Alternatively, the pluripotent stem cell population can be cultured in serum free medium supplemented with BSA and an agent selected from the group consisting of insulin and IGF-1 for about one day to about five days. Alternatively, the pluripotent stem cell population can be cultured in serum free medium supplemented with BSA and an agent selected from the group consisting of insulin and IGF-1 for about one day to about four days. Alternatively, the pluripotent stem cell population can be cultured in serum-free medium supplemented with BSA and a factor selected from the group consisting of insulin and IGF-1 for about four days.

[0063] In one embodiment, GDF-8 is used at a concentration of from about 5 ng / ml to about 500 ng / ml. In an alternative embodiment, GDF-8 is used at a concentration of from about 5 ng / ml to about 50 ng / ml. In an alternative embodiment, GDF-8 is used at a concentration of about 5 ng / ml to about 25 ng / ml. In an alternative embodiment, GDF-8 is used at a concentration of about 25 ng / ml.

[0064] Activin A can be used at a concentration of from about 1 pg / ml to about 100 μg / ml. In an alternative embodiment, the concentration may be from about 1 pg / ml to about 1 μg / ml. In another alternative embodiment, the concentration may be from about Lg / ml to about 100ng / ml. In another alternative embodiment, the concentration may be from about 50ng / ml to about 100ng / ml. In another alternative embodiment, the concentration may be 100ng / ml.

[0065] The Wnt ligand may be selected from the group consisting of Wnt-1, Wnt-3a, Wnt-5a and Wnt-7a. In one embodiment, the Wnt ligand is Wnt-1. In an alternative embodiment, the Wnt ligand is Wnt-3a.

[0066] The Wnt ligand may be used at a concentration of from about 1 ng / ml to about 1000 ng / ml. In an alternative embodiment, the Wnt ligand may be used at a concentration of from about 10 ng / ml to about 100 ng / ml. In one embodiment, the Wnt ligand concentration is about 20 ng / ml.

[0067] In one embodiment, insulin is used at a concentration of about 1 ng / ml to about 100 ng / ml.

[0068] In one embodiment, IGF-1 is used at a concentration of from about 1 ng / ml to about 200 ng / ml.

Formation of Cells Expressing Markers Specific to the Pancreatic Endoderm Line [0069] In one embodiment, the populations of cells expressing markers characteristic of the fully developed endoderm line formed by the methods of the present invention are further differentiated to the cell population expressing markers characteristic of the pancreatic endoderm line by any method in the art. .

[0070] For example, cell populations expressing markers characteristic of the fully developed endoderm line obtained according to the methods of the present invention can be further differentiated into a population of cells expressing markers characteristic of the pancreatic endoderm line by treating a population of cells expressing markers characteristic of the fully developed endoderm line according to the methods disclosed in D'Amour et al. Nature Biotechnology 24, 1392 - 1401 (2006).

[0071] For example, cell populations expressing markers characteristic of a fully developed endoderm line obtained according to the methods of the present invention can be further differentiated to a population of cells expressing markers characteristic of the pancreatic endoderm line by treating a population of cells expressing markers characteristic of a fully developed endoderm line according to of the methods disclosed in US Patent Application Serial No. 11 / 736,908.

Formation of cells expressing markers characteristic of the pancreatic endocrine line [0072] In one embodiment, the populations of cells expressing markers characteristic of the pancreatic endoderm line are further differentiated to the population of cells expressing markers characteristic of the pancreatic endocrine line by any method in the art.

[0073] For example, populations of cells expressing markers characteristic of the pancreatic endoderm line can be further differentiated into a population of cells expressing markers characteristic of the pancreatic endocrine line by treating a population of cells expressing markers characteristic of the pancreatic endoderm line according to the methods disclosed in D 'Amour et al., Nature Biotechnology, 2006.

[0074] For example, populations of cells expressing markers characteristic of the pancreatic endoderm line can be further differentiated to a population of cells expressing markers characteristic of the pancreatic endocrine line by treating a population of cells expressing markers characteristic of the pancreatic endoderm line according to the methods disclosed in D 'Amour et al., Nature Biotechnology, 2006.

[0075] For example, populations of cells expressing markers characteristic of the pancreatic endoderm line can be further differentiated to a population of cells expressing markers characteristic of the pancreatic endocrine line by treating a population of cells expressing markers characteristic of the pancreatic endoderm line according to the methods disclosed in US Patent Application Serial No. 11 / 736.908. [0076] For example, populations of cells expressing markers characteristic of the pancreatic endoderm line can be further differentiated to a population of cells expressing markers characteristic of the pancreatic endocrine line by treating a population of cells expressing markers characteristic of the pancreatic endoderm line according to the methods disclosed in US Patent Application Serial No. 11 / 779.311. [0077] For example, populations of cells expressing markers characteristic of the pancreatic endoderm line can be further differentiated to a population of cells expressing markers characteristic of the pancreatic endocrine line by treating a population of cells expressing markers characteristic of the pancreatic endoderm line according to the methods disclosed in US Patent Application Serial No. 60 / 953.178. [0078] For example, populations of cells expressing markers characteristic of the pancreatic endoderm line can be further differentiated to a population of cells expressing markers characteristic of the pancreatic endocrine line by treating a population of cells expressing markers characteristic of the pancreatic endoderm line according to the methods disclosed in US Patent Application Serial No. 60 / 990.529. [0079] The present invention is further illustrated, but not in a limiting manner, by the following examples.

EXAMPLES

Reference example 1

The role of insulin in the differentiation of human pluripotent stem cells into cells expressing markers characteristic of a fully developed endoderm line: Cluster spacing [0080] Previous studies have shown that high FBS concentration is detrimental to the formation of fully developed endoderm (DE) from embryonic stem cells. See, for example, D'Amour et al., Nature Biotechnology, 2005, where the induction of fully developed endoderms from human embryonic stem cells was significantly increased when FBS concentration was reduced from 10% FBS to 0.5-2% FBS. Similar observations have been reported with the addition of 25 ng / ml IGF or 200 ng / ml insulin to 2% FBS to ES cells grown in MEF-CM (media conditioned with mouse embryonic fibroblasts) reduced SOX17 expression by approximately 70% after treatment with activin A. See McLean et al., Stem Cells 25: 2938, 2007.

[0081] The inhibitory effect observed was probably due to the presence of insulin or IGF in FBS, triggering the phosphatidylinositol 3-kinase pathway. See McLean et al., Stem Cells 25: 29-38, 2007. Blockade of the PI-3 kinase signal transduction pathway increased the percentage of Soxl7 positive cells in human ES cells grown in MEF-CM (media conditioned with mouse embryonic fibroblasts). See McLean et al., Stem Cells 25: 2938, 2007.

[0082] These data suggest that the addition of such a small amount as 25 ng / ml IGF or 200 ng / ml insulin to the medium containing activin A and low FBS (0.5-2% FBS) was expected to block formation completely an educated endoderm. Typical IGF and insulin concentrations in FBS are approximately 70 ng / ml, respectively (J. Clin. Invest. 76: 4, 1985) and approximately 60 ng / ml (In Vitro Cell Dev Biol. 32: 8-12, 1996). This means approximately 1.4 ng / ml IGF and approximately 1.2 ng / ml insulin in 2% FBS.

[0083] Cells of human H1 embryonic stem cell lines (p40-p52) were grown in MATRIGEL® coated dishes (1:30 dilution) (BD Biosciences; Cat. No. 356231) in MEF-CM (mouse embryonic fibroblast conditioned media) supplemented with 16 ng / ml FGF2 (catalog number 100-18B, PeproTech, NJ) and differentiated into cells expressing markers characteristic of a fully developed endoderm line as follows:

a. RPMI medium supplemented with 2% fatty acid-free BSA (catalog number 68700, Proliant, IA) and 100 ng / ml activin A (R&D Systems, MN) plus 20 ng / ml WNT-3a (catalog number 1324-WN-002 , R&D Systems, MN) for one day, then

b. RPMI medium supplemented with 2% BSA and 100 ng / ml activin A for an additional three days.

[0084] In some cultures, the cells were treated with the following dilution ITS-X (catalog number 51500-056, Invitrogen, CA): 0, 1:10⁶, 1: 5 X 10⁵, 1:10⁵, 1:10⁴. ITS-X is a supplemented replacement serum composed of 1 mg / ml insulin, 0.55 mg / ml transferrin, 0.00067 mg / ml sodium selenite and 0.2 mg / ml ethanolamine. The dilution range of ITS-X corresponds to 0, 1 ng / ml, 2 ng / ml, 10 ng / ml and 100 ng / ml insulin. As a control, 0.2% FBS (catalog number SH30070.03, Hyclone, UT) was used for day 1 differentiation, 0.5% FBS on day 2 and 2% FBS was used on days 3-4. FBS-treated cultures were not supplemented with ITS-X. [0085] On day 4, samples were taken for FACS analysis and gene expression using real-time PCR. Surprisingly, as shown in Figure 1, the addition of 1-100 ng / ml insulin to the medium used for cell differentiation did not significantly affect the expression of markers associated with fully developed endoderm (FOXA2, SOX17 and CXCR4), mesenchyme-related markers (T, known also as Brach) or extra-embryonic markers (SOX7, AFP). In addition, cultures treated with 2% BSA supplemented medium showed significantly higher expression of markers associated with fully developed endoderm than cultures treated with 0.5-2% FBS supplemented medium.

[0086] These observations are further supported by the expression of CXCR4 and CD9, as determined by FACS, for various treatment groups. See figure 2. The CXCR4 cell surface receptor has previously been shown to be a marker for a fully developed endoderm. CD9 is a marker for undifferentiated ES cells. Consequently, an increase in CXCR4 expression and a decrease in CD9 expression in the cell population indicates the formation of a fully developed endoderm. As summarized in Table 1, no significant change was observed in the expression of CXCR4 or CD9 in cells treated with BS supplemented medium A at any insulin concentration tested. These data suggest that insulin has no inhibitory effect at the concentrations tested in the culture medium used in these studies. Table I.

<td>Treatment</td><td>% CXCR4 + CD9-</td><td>% CXCR4-CD9 +</td><td>% CXCR4-CD9-</td>

<td>FBS</td><td>56</td><td>27</td><td>9</td>

<td>BSA</td><td>69</td><td>13</td><td>13</td>

<td>BSA + 1 ng / ml insulin</td><td>70</td><td>13</td><td>10</td>

<td>BSA + 5 ng / ml insulin</td><td>67</td><td>15</td><td>12</td>

<td>BSA + 10 ng / ml insulin</td><td>69</td><td>13</td><td>13</td>

<td>BSA + 100 ng / ml insulin</td><td>73</td><td>12</td><td>9</td>

Reference example 2

The role of insulin in the differentiation of human pluripotent stem cells into cells expressing markers characteristic of a fully developed endoderm line: Plating of single cells [0087] Cells of human embryonic stem cell lines H1 (p40-p52) were plated as single cells at a density of 100,000 cells / cm² on MATRIGEL® coated dishes (1:30 dilution) (BD Biosciences; cat.no. 356231) in MEF-CM (media conditioned with mouse embryonic fibroblasts) supplemented with 16 ng / ml FGF2 (catalog numbers 100-18B, PeproTech, NJ) and 10 μΜ Y27632 (Rock inhibitor, catalog number Y0503, Sigma, MO). 72 h after sowing, the cultures were differentiated into a fully developed endoderm (DE) as follows:

a. MCDB-131 medium (catalog number 10372-019, Invitrogen, CA) supplemented with 2%

BSA without fatty acids (catalog number 68700, Proliant, IA), 0.0025 g / ml sodium bicarbonate (catalog number S3187, Sigma, MO), 1X GlutaMax ™ (catalog number 35050-079, Invitrogen, Ca) and 100 ng / ml activin A (R&amp;D Systems, MN) plus 20 ng / ml WNT-3a (catalog number 1324-WN-002, R&amp;D Systems, MN) for one day, then

b. MCDB-131 medium supplemented with 2% BSA, sodium bicarbonate, Glutamax and 100 ng / ml activin A for an additional three days.

[0088] In some cultures, the cells were treated with the following concentrations of insulin (catalog number I9278, Sigma, MO): 0, 1, 10, 100, 1000 or 10,000 ng / ml. On day 4, samples were taken for FACS analysis and gene expression using real-time PCR.

[0089] The addition of 1-100 ng / ml insulin to the medium used for cell differentiation did not significantly affect the expression of markers associated with fully developed endoderm (FOXA2, SOX17, CER1 and CXCR4). Similarly, expression of embryonic markers (NANOG) or non-embryonic markers (SOX7, AFP) was not affected. See figure 3. However, the addition of 1-10 μg / ml insulin did not increase NANOG expression. This data is further supported by immunofluorescence (IF) staining for the fully developed SOX17 endoderm marker (catalog number AF1924, R &amp; D systems, MN) (Figure 4).

[0090] Figure 5 shows the expression profile of CXCR4 and CD9 of different treatments, as measured by FACS analysis. As summarized in Table II, only above physiological insulin concentrations (1-10 μg / ml) there was a decrease in the percentage of CXCR4 + CD9- cells and an increase in the expression fraction of CXCR4-CD9 +. These data suggest that under the conditions in this study, only above physiological insulin concentrations inhibit the formation of a fully developed endoderm.

Table II

<td>Treatment</td><td>% CXCR4 + CD9-</td><td>% CXCR4-CD9 +</td><td>% CXCR4-CD9-</td>

<td>BSA</td><td>96</td><td>1.3</td><td>0.9</td>

<td>BSA + 1 ng / ml insulin</td><td>96</td><td>1.5</td><td>0.7</td>

<td>BSA + 10 ng / ml insulin</td><td>95</td><td>1.4</td><td>0.7</td>

<td>BSA + 100 ng / ml insulin</td><td>90</td><td>4.1</td><td>2</td>

<td>BSA + 1 μg / ml insulin</td><td>90</td><td>3.6</td><td>2.2</td>

<td>BSA + 10 μg / ml insulin</td><td>84</td><td>6.6</td><td>4.7</td>

Reference example 3

The role of IGF in differentiating human pluripotent stem cells to cells expressing markers characteristic of a fully developed endoderm line: Plating of single cells [0091] Cells of human embryonic stem cell lines H1 (p40-p52) were plated as single cells at a density of 100,000 cells / cm² on coated dishes

MATRIGEL® (1:30 dilution) (BD Biosciences; Cat. No. 356231) in MEF-CM (mouse embryonic fibroblast conditioned media) supplemented with 16 ng / ml FGF2 (catalog numbers 100-18B, PeproTech, NJ) and 10 μΜ Y27632 ( Rock inhibitor, catalog number Y0503, Sigma, MO). 72 h after sowing, the cultures were differentiated into a fully developed endoderm (DE) as follows:

and. MCDB-131 medium (catalog number 10372-019, Invitrogen, CA) supplemented with 2% BSA without fatty acids (catalog number 68700, Proliant, IA) 0.0025 g / ml sodium bicarbonate (catalog number S3187, Sigma, MO) 1X GlutaMax ™ (catalog number 35050-079, Invitrogen, Ca) and 100 ng / ml GDF8 (catalog number 120-00, PeproTech, NJ) plus 2.5 μΜ of GSK3B 14-prop-2-en-1-yl-3,5,7,14,17,23,27heptaazatetracyclo [19.3.1.1 ~ 2.6 ~ .1 ~ 8.12 ~ ] heptacose1 (25), 2 (27), 3.5.8 (26), 9,11,21,23-nonaen-16-one for one day, next

b. MCDB-131 medium supplemented with 2% BSA, sodium bicarbonate, Glutamax and 100 ng / ml

GDF-8 for an additional three days.

[0092] In some cultures, the cells were treated with the following IGF concentrations (catalog number AF100, PeproTech, NJ): 0, 1, 10, 50 or 200 ng / ml. As a control, instead of BSA, 0.2% FBS (catalog number SH30070.03, Hyclone, UT) was used for day 1 differentiation and 2% FBS was used on days 2-4. Some of the FBS-treated cultures were also treated with different concentrations of IGF.

[0093] On day 4, samples were taken for FACS analysis and gene expression using real-time PCR.

[0094] Surprisingly, the addition of 1-200 ng / ml IGF to BSA-treated cultures did not significantly affect the expression of markers associated with fully developed endoderm (FOXA2,

SOX17, CER1 and CXCR4), compared to untreated IGF control samples (Figure 6). Similar results were observed with extra-embryonic markers (SOX7, AFP). The addition of 50-200 ng / ml IGF did not increase the expression of the NANOG embryo marker.

[0095] Cultures treated with FBS supplemented medium were much more sensitive to the IGF inhibitory effect. In these cultures, SOX17, HNF3B and CXCR4 expression decreased with increasing IGF concentration. These observations are further supported by immunofluorescence (IF) staining for the DE SOX17 marker (catalog number AF1924, R&amp;D Systems, MN) (Figures 8-9).

[0096] As summarized in Table III, only above physiological IGF concentrations (50-200 ng / ml) in BSA-treated cultures there was a decrease in CXCR4 + CD9- cell expression and an increase in CXCR4-CD9 + expression fraction. However, with increasing doses of IGF, FBS-treated cultures show a more significant decrease in CXCr4 + CD9- expression when compared to BSA-treated cultures. Together, the above examples show that the absence of FBS, physiological concentration of IGF or insulin do not have an inhibitory effect on the induction of DE markers.

Table III:

<td>Treatment</td><td>% CXCR4 + CD9-</td><td>% CXCR4-CD9 +</td><td>% CXCR4-CD9-</td>

<td>BSA</td><td>92</td><td>0.9</td><td>2.6</td>

<td>FBS</td><td>93</td><td>2.7</td><td>5.9</td>

<td>BSA + 1 ng / ml IGF</td><td>92</td><td>0.8</td><td>3</td>

<td>FBS + 1 ng / ml IGF</td><td>89</td><td>3</td><td>3.6</td>

<td>BSA + 10 ng / ml IGF</td><td>90</td><td>1.3</td><td>5.3</td>

<td>FBS + 10ng / ml IGF</td><td>87</td><td>6.4</td><td>3.3</td>

<td>Treatment</td><td>% CXCR4 + CD9-</td><td>% CXCR4-CD9 +</td><td>% CXCR4-CD9-</td>

<td>BSA + 50 ng / ml IGF</td><td>87</td><td>6</td><td>3.5</td>

<td>FBS + 50 ng / ml IGF</td><td>78</td><td>6.8</td><td>13.2</td>

<td>BSA + 200 ng / ml IGF</td><td>79</td><td>10</td><td>8</td>

<td>FBS + 200 ng / ml IGF</td><td>70</td><td>13.4</td><td>12.9</td>

Example 4

IGF concentrations in various FBS batches [0097] The IGF-1 kit was purchased from Diagnostic Systems Laboratories (DSL) (cat. DSL-10-2800) 5 and used for detection. Twenty microliters (20 μΙ) of serum (two replicates) were pretreated and then 20 μΙ of the diluted sample was used for the test. For medium samples, 20 μΙ samples were directly used for the test. The test was carried out according to the instructions provided in the kit. This kit can detect both human and cattle

IGF-1 because the 2 monoclonal antibodies used in the kit are against 10 homologous peptide sequences.

[0098] Test samples: The following test samples were used:

<td>4A / 5A:</td><td>Hyclone neonatal calf serum; batch AKM12868</td>

<td>4B / 5B:</td><td>NIH-FBS (from parts after -20 C)</td>

<td>4C / 5C:</td><td>Hyclone FBS, batch: ATK33398</td>

<td>4D / 5D:</td><td>Hyclone FBS, batch: AUK 54924</td>

<td>4E / 5E:</td><td>Human serum, batch: A70184, from Valley Biomedical Inc.</td>

<td>4F / 5F:</td><td>Knockout serum; Invitrogen, batch: 557914</td>

<td>4F / 5F:</td><td>F12 DMEM, Invitrogen, batch: 692281</td>

<td>4H / 5H:</td><td>MEF conditioned media, batch: 011410 (day 5)</td>

[0099] Control samples: The following control samples were used:

Background: IGF-1 "0" (negative control, from kit); sample F12 mentioned above also serves as a negative control.

positive controls (127 ng / ml and 241 ng / ml; from kit) [0100] Results: The test sensitivity for the serum was greater than 10 ng / ml and for the medium was greater than 0.1 ng / ml.

<td>Known positive (ng / ml)</td><td>Test concentration (ng / ml)</td><td>SE</td>

<td>127 ng / ml</td><td>126.8</td><td>8.2</td>

<td colspan="2">Known positive (ng / ml)</td><td colspan="2">Test concentration (ng / ml)</td><td>SE</td>

<td colspan="2">241 ng / ml</td><td colspan="2">233.8</td><td>4.2</td>

<td>Two reps</td><td colspan="2">samples</td><td>IGF-1 (ng / ml)</td><td>SE</td>

<td>4A / 5A</td><td colspan="2">Neonatal calf serum HyClone; batch AKM12868</td><td>29.78</td><td>0.41</td>

<td>4B / 5B</td><td colspan="2">NIH-FBS (from parts after -20 C)</td><td>49.16</td><td>2.68</td>

<td>4C / 5C</td><td colspan="2">Hyclone FBS, batch: ATK33398</td><td>80.29</td><td>5.39</td>

<td>4D / 5D</td><td colspan="2">Hyclone FBS, batch: AUK 54924</td><td>76.45</td><td>1.99</td>

<td>4E / 5E</td><td colspan="2">Human serum, batch: A70184, from Valley Biomedical Inc.</td><td>55.65</td><td>2.28</td>

<td>4F / 5F</td><td colspan="2">Knockout serum; Invitrogen, batch: 557914</td><td>12.07</td><td>0.15</td>

<td>4G / 5G</td><td colspan="2">F12 DMEM, Invitrogen, batch: 692281</td><td>Well*</td><td>Well</td>

<td>4H / 5H</td><td colspan="2">MEF conditioned medium, batch: 011410 (day 5)</td><td>1.33 **</td><td>0.07</td>

Reference example 5

The role of insulin / IGF and FBS in differentiating human pluripotent stem cells to cells expressing markers characteristic of a fully developed endoderm line: Plating of single cells [0101] Cells of human embryonic stem cell lines H1 (p40-p52) were seeded as single cells at a density of 100,000 cells / cm² on MATRIGEL® coated dishes (1:30 dilution) (BD Biosciences; cat.no. 356231) in MEF-CM (media conditioned with mouse embryonic fibroblasts) supplemented with 16 ng / ml FGF2 (catalog numbers 100-18B, PeproTech, NJ) and 10 μΜ Y27632 (Rock inhibitor, catalog number Y0503, Sigma, MO). 72 h after sowing, the cultures were differentiated into a fully developed endoderm (DE) as follows:

a. MCDB-131 medium (catalog number 10372-019, Invitrogen, CA) supplemented with 0.2% FBS (catalog number SH30070.03, Hyclone, UT), 0.0025 g / ml sodium bicarbonate (catalog number S3187, Sigma, MO), 1X GlutaMax ™ (catalog number 35050-079, Invitrogen,

Ca) and 100 ng / ml GDF8 (catalog number 120-00, PeproTech, NJ) plus 2.5 μΜ of the GSK3B 14-prop-2-en-1-yl-3,5,7,14,17,23 inhibitor, 27heptaazatetracyklo [19.3.1.1 ~ 2,6 ~ .1 ~ 8,12 ~] heptakoza1 (25), 2 (27), 3,5,8 (26), 9,11,21,23-nonaen-16-one for one day, then

b. MCDB-131 medium supplemented with FBS, sodium bicarbonate, Glutamax and 100 ng / ml GDF8 for an additional three days.

[0102] 0.5% FBS was used on day 2 and 2% FBS was used on days 3-4. In addition to regular FBS, heat treated FBS (catalog number F4135, Sigma, MO) and FBS treated with charcoal stripped (catalog number F6765, Sigma, MO) were also tested. Some of the FBS-treated cultures were also treated with different concentrations of IGF (10-100 ng / ml) or insulin (10100 ng / ml).

[0103] On day 4, samples were taken for real-time FACS and PCR analysis. In contrast to BSA-treated cultures (see previous Examples) in the presence of FBS, the addition of insulin or IGF dose in a dependent manner inhibits markers associated with fully developed endoderm such as SOX17 and CXCR4. See figure 9. Expression of the NANOG pluripotency marker was also stimulated.

Janssen Biotech, Inc., United States of America Representative:

EP 2 853 589 B1

Z-16736