RU2805433C1 - Differentiation of human embryonic stem cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: method for obtaining a population of cells and an isolated crude population of cells for obtaining pancreatic endocrine cells expressing markers characteristic of the pancreatic endoderm lineage are presented. Moreover, more than 70% of cells in the population simultaneously express PDX1 and NKX6.1.

EFFECT: invention allows it to be used to stimulate the differentiation of pluripotent stem cells into insulin-producing cells.

13 cl, 2 tbl, 5 dwg, 4 ex

Description

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent Application Serial No. 61/333831, filed May 12, 2010, which is incorporated herein by reference in its entirety for all purposes.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

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 obtaining a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 50% of the cells in the population simultaneously expressing PDX-1 and NKX6.1.

PREREQUISITES FOR CREATION OF THE INVENTION

Recent advances in cell replacement therapy for the treatment of type 1 diabetes and the shortage of islets of Langerhans available for transplantation have stimulated interest in developing sources of insulin-producing cells, or β-cells, suitable for transplantation. One approach is to generate functional β cells from pluripotent stem cells, such as embryonic stem cells.

During vertebrate embryonic development, pluripotent cells give rise to a group of cells containing three germ layers (ectoderm, mesoderm, and endoderm) through a process called gastrulation. Tissues such as the thyroid, thymus, pancreas, intestines and liver, for example, will develop from the endoderm through an intermediate stage. An intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers such as HNF3 beta, GATA4, MIXL1, CXCR4 and SOX 17.

The formation of the pancreas occurs when the definitive endoderm differentiates into pancreatic endoderm. Pancreatic endoderm cells express the pancreatic-duodenal homeobox gene, PDX1. In the absence of PDX1, pancreatic development does not proceed beyond the formation of the ventral and dorsal buds. Thus, PDX1 expression characterizes a critical stage of pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues are formed during the differentiation of pancreatic endoderm.

Cells with the properties of islet cells were reportedly obtained from mouse embryonic cells. For example, in the work of Lumelsky et al. (Science 292:1389, 2001) reported the differentiation of mouse embryonic stem cells into insulin-secreting structures similar to pancreatic islets. The work of Soria et al. (Diabetes 49:157, 2000) reported that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in mice with streptozotocin-induced diabetes.

In one example, Hori et al. (PNAS 99: 16105, 2002) discloses that treatment of mouse embryonic stem cells with phosphoinositide 3-kinase inhibitors (LY294002) resulted in β-cell-like cells.

In another example, Blyszczuk et al. (PNAS 100:998, 2003) reported the derivation of insulin-producing cells from mouse embryonic stem cells with constitutive expression of Pax4.

The work of Micallef et al. reported that retinoic acid can regulate the ability of embryonic stem cells to form PDX1-positive pancreatic endoderm. Retinoic acid induces Pdx1 expression most effectively when added to cultures at day 4 of embryonic stem cell differentiation during the period corresponding to the end of embryo gastrulation (Diabetes 54:301, 2005).

The work of Miyazaki et al. reported a mouse embryonic stem cell line overexpressing PDX1. The authors' results show that expression of exogenous PDX1 clearly increases the expression of insulin, somatostatin, glucokinase, neurogenin 3, p48, Pax6 and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).

The work of Skoudy et al. Activin A (member of the TGF-β superfamily) has been reported to increase the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (PDX1, insulin and glucagon) in mouse embryonic stem cells. The maximum effect was observed when using activin A at a concentration of 1 nM. The authors also noted that the expression levels of insulin and PDX1 mRNA were independent of the presence of retinoic acid. However, treatment with 3 nM FGF7 resulted in increased transcript levels for PDX1 (Biochem. J. 379: 749, 2004).

The work of Shiraki et al. studied the effects of growth factors that specifically accelerate the differentiation of embryonic stem cells into PDX1-positive cells. The authors noted that TGF-β2 resulted in a reproducible increase in the proportion of PDX1-positive cells (Genes Cells Jun 2005; 10(6): 503-16).

Gordon et al. demonstrated the induction of the formation of brachyuric [positive]/HNF3 beta [positive] endodermal cells from mouse embryonic stem cells in the absence of serum and in the presence of activin in combination with an inhibitor of the Wnt signaling cascade (US patent No. 2006/0003446 A1).

Gordon et al. (PNAS, vol. 103, p. 16806, 2006) states: “The formation of the anterior primitive streak required simultaneous Wnt and TGF-beta/Nodal/activin signaling cascades.”

However, the mouse model of embryonic stem cell development cannot exactly mimic the developmental program in higher mammals, such as humans.

The work of Thomson et al. isolated embryonic stem cells from human blastocysts (Science 282:114, 1998). In parallel, Gearhart et al. generated human embryonic germ cell (hEG) cell lines from embryonic gonad tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95:13726, 1998). Unlike mouse embryonic stem cells, which can be prevented from differentiating by simply culturing with leukemia inhibitory factor (LIF), human embryonic stem cells must be cultured under very specific conditions (US Patent No. 6,200,806; International Patent Applications Nos. 99/20741, 01 /51616).

The work of D'Amour et al. describes the production of enriched cultures of definitive endoderm derived from human embryonic stem cells in the presence of high concentrations of activin and low concentrations of serum (Nature Biotechnology 2005). Transplantation of these cells under the renal capsule of mice led to their differentiation into more mature cells with characteristic features of some endodermal organs. Definitive endoderm cells derived from human embryonic stem cells can be further differentiated into PDX1-positive cells after the addition of FGF-10 (US Patent No. 2005/0266554 A1).

The work of D'Amour et al. (Nature Biotechnology - 24, 1392-1401 (2006)) states: “We have developed a differentiation method that converts human embryonic stem (hES) cells into endocrine cells capable of synthesizing pancreatic hormones: insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This method mimics pancreatic organogenesis 1 in vivo, taking cells through stages reminiscent of the formation of definitive endoderm, gut tube endoderm, pancreatic endoderm, and the conversion of endocrine cell precursors to cells expressing endocrine hormones."

In another example, Fisk et al. a system for the production of pancreatic islet cells from human embryonic stem cells is reported (US Patent No. 2006/0040387 A1). In this case, the differentiation process was divided into three stages. First, human embryonic stem cells were differentiated into endoderm using a combination of sodium butyrate and activin A. The cells were then cultured with TGF-β antagonists such as Noggin in combination with EGF or betacellulin to produce PDX1-positive cells. Final differentiation was induced by nicotinamide.

In one example, Benvenistry et al. reported: “We conclude that overexpression of PDX1 increased the expression of pancreatic enriched genes, and induction of insulin expression may require additional signals present only in vivo” (Benvenistry et al., Stem Cells 2006; 24:1923–1930).

In another example, Grapin-Botton et al. reported: “Early activation of Ngn3 induced glucagon-positive cells in almost all cases, while reducing the number of pancreatic progenitor cells. As with E11.5, PDX-1 precursors can differentiate into insulin-[positive] and PP-[positive] cells" (Johansson KA et al., Developmental Cell 12, 457-465, March 2007).

For example, Diez et al. reported: “After 9 and 10 weeks, the majority of glucagon-positive cells simultaneously expressed insulin, although at these stages there were clearly some cells expressing only insulin. Cells simultaneously expressing insulin and glucagon were observed throughout the study period (weeks 9 to 21), but they represented only a small proportion of the total number of insulin and glucagon expressing cells" (J Histochem Cytochem., September 2009; 57( 9):811-24, April 13, 2009).

In one example, Chen et al. reported: “(-)-Indolactam V [(ILV)] activates the protein kinase C signaling cascade and sets the pancreatic specification of human embryonic stem cells that have already been determined to differentiate into the endoderm lineage...ILV and retinoic acid act through a related mechanism...ILV demonstrates more stronger induction of PDX-1 expressing cells (a greater percentage of PDX-1 expressing cells) than retinoic acid" (Nature Chemical Biology 5, 195-196 (April 2009) doi:10.1038/nchembio0409-195).

The work of Lyttle et al. reported: “NKX6-1 co-localizes only with insulin cells, indicating that NKX6-1 is involved only in human beta cell development” (Diabetologia, July 2008: 51(7):1169-80, 2008 ).

Thus, there remains a significant need to develop laboratory methods to create a functional insulin-expressing cell more closely related to the β-cell. The present invention provides an alternative approach to increasing the efficiency of differentiation of human embryonic stem cells into insulin-expressing cells by generating a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 50% of the cells in the population simultaneously expressing PDX-1 and NKX6.1.

SHORT DESCRIPTION

In one embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 50% of the cells in the population simultaneously expressing PDX-1 and NKX6.1.

In one embodiment, the present invention provides a method of differentiating a population of pluripotent stem cells into a population of cells expressing markers characteristic of the pancreatic endoderm lineage, comprising the following steps:

a. culturing a population of pluripotent stem cells;

b. differentiation of a population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage; And

c. differentiation of a population of cells expressing markers characteristic of the definitive endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage by treating the population of cells expressing markers characteristic of the definitive endoderm lineage with a medium to which a protein kinase C activator has been added.

In one embodiment, more than 50% of the cells in a population of cells expressing markers characteristic of the pancreatic endoderm lineage, which are obtained by the methods of the present invention, simultaneously express PDX-1 and NKX6.1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and 1B show expression data for PDX1, NKX6.1, and ISL-1 at stage 4, day 4, of the differentiation protocol described in Example 1. FIG. Figure 1A shows data on the expression of PDX1 and NKX6.1. In fig. Figure 1B shows data on the expression of NKX6.1 and ISL-1.

Figures 2A and 2B show the effect of protein kinase C (PKC) activator treatment on the proportion of cells expressing PDX1, NKX6.1 and CDX2, analyzed using the IN Cell Analyzer 1000 (Fig. 2A), as well as a comparison of different PKC activators and their effects effects on the proportion of cells expressing PDX1 and NKX6.1, as measured by the IN Cell Analyzer (Fig. 2B).

Figures 3A, 3B and 3C show the levels of circulating C-peptide in SCID mice with congenital killer cell absence that were injected with the cells of the present invention subcutaneously (Figure 3A) and through a subcutaneously implanted Theracyte device (Figure 3A). .3B). C-peptide levels were recorded at the indicated time points. In fig. Figure 3C shows a comparison of C-peptide levels observed in the group receiving cells under the renal capsule and the group receiving cells through the subcutaneous Theracyte device at 12 weeks post-transplantation.

Figures 4A, 4B, 4C and 4D show the effect of PKC activator treatment on the expression of PDX1, NKX6.1, NGN3 and PTF1 alpha in cells treated according to the methods described in Example 3.

Figures 5A, 5B, 5C and 5D show the effect of FGF7 on the expression of NKX6.1, PDX1, PTF1 alpha and CDX2 in cells treated according to the methods described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

For clarity of description and not to limit the invention, the detailed description of the present invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the present invention.

Definitions

Stem cells are undifferentiated cells defined by their ability, at the single cell level, to both self-renew and differentiate to form progeny cells, such as self-renewing progenitor cells, non-renewing progenitor cells, and terminally differentiated cells. Stem cells are also characterized by the ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm) and, upon transplantation, to give rise to tissues derived from multiple germ layers, and essentially contribute to the formation of most, if not all tissues after injection into blastocysts.

Stem cells are classified according to developmental potential as follows: (1) totipotent, capable of transforming into any of the embryonic and extraembryonic cell types; (2) pluripotent, capable of transforming into all types of embryonic cells; (3) multipotent, capable of converting into multiple cell lineages within a single tissue, organ, or physiological system (e.g., hematopoietic stem cells (HSCs) can give rise to HSCs (self-renewal), oligopotent limited blood progenitor cells, and all cell types and elements (for example, platelets), which are standard components of blood); (4) oligopotent, capable of converting into a more limited subset of cell lineages than multipotent stem cells; and (5) unipotent, capable of developing into a single cell lineage (eg, spermatogenic stem cells).

Differentiation is the process by which an unspecialized (“uncommitted”) or less specialized cell acquires the properties of a specialized cell, such as a nerve or muscle cell. A differentiated cell or a cell with induced differentiation is a cell that has taken a more specialized (“committed”) position in the lineage of the cell. The term "committed" in relation to the process of differentiation means a cell that has reached a stage during the process of differentiation from which, under normal conditions, it will continue to differentiate into a specific cell type or multiple cell types and will not normally be able to differentiate into another cell type or return back to less differentiated type. Dedifferentiation is the process by which a cell returns to a less specialized (or committed) position in the lineage. As used herein, the term “cell lineage” defines the heredity of a cell, that is, it determines which cell the cell came from and what cells it can give rise to. In the lineage of differentiation, the cell is placed into an ancestral pattern of development and differentiation. A lineage-specific marker is a characteristic feature specifically associated with the phenotype of cells of a particular lineage that can be used to assess the differentiation of an uncommitted cell to that lineage.

As used herein, the terms “cells expressing markers characteristic of the definitive endoderm lineage,” “stage 1 cells,” or “stage 1” 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, eomesodermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99 or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak cell precursor cells, primitive streak cells, mesoendoderm cells, and definitive endoderm cells.

The term “cells expressing markers characteristic of the pancreatic endoderm lineage” as used herein means 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 the pancreatic endoderm lineage include pancreatic endoderm cells, primary gut tube cells, and late foregut cells.

As used herein, the term “definitive endoderm” refers to cells having the characteristic features of cells derived from the epiblast during gastrulation and forming the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF3 beta, GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99 and MIXL1.

As used herein, the term “markers” refers to nucleic acid or polypeptide molecules differentially expressed in cells of interest. In this context, differential expression refers to an increase in the expression level for a positive marker and a decrease in the expression level for a negative marker. The detectable level of the marker nucleic acid or polypeptide in the cells of interest is significantly higher or lower compared to other cells, allowing the cell of interest to be detected and distinguished from other cells using any of a variety of methods known in the art.

As used herein, the terms “pancreatic endocrine cell” or “pancreatic hormone-expressing cell” or “cell expressing markers characteristic of a pancreatic endocrine cell line” refer to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin and pancreatic polypeptide.

Isolation, propagation and cultivation of pluripotent stem cells

Characteristics of pluripotent stem cells

Pluripotent stem cells can express one or more stage-specific embryonic antigens (SSEA) 3 and 4, as well as markers detected by antibodies designated TRa-1-60 and Tra-1-81 (Thomson et al., Science 282:1145, 1998 G.). Differentiation of pluripotent stem cells in vitro results in loss of expression of SSEA-4, Tra 1-60 and Tra 1-81 (when present) and increased expression of SSEA-1. Undifferentiated pluripotent stem cells typically have active alkaline phosphatase, which can be detected by fixing the cells with 4% paraformaldehyde followed by detection with Vector Red substrate according to the manufacturer's instructions (Vector Laboratories, Burlingame). , California State). Undifferentiated pluripotent stem cells also typically express OCT4 and TERT, detected by RT-PCR.

Another desirable phenotypic property of cultured pluripotent stem cells is the potential to differentiate into cells of all three germ layers: endodermal, mesodermal and ectodermal tissues. The pluripotency of pluripotent stem cells can be confirmed, for example, by injecting the cells into severe combined immunodeficiency (SCID) mice, fixing the resulting teratomas with 4% paraformaldehyde, and examining them histologically to provide evidence of cell types derived from the three germ layers. In an alternative embodiment, pluripotency can be determined by the formation of embryoid bodies and their analysis for the presence of markers that are associated with the three germ layers.

The cultured pluripotent stem cell lines can be karyotyped using standard Giemsa staining techniques and compared with published karyotypes of the relevant primate species. It is desirable to obtain cells that have a “normal karyotype,” that is, euploid cells in which all human chromosomes are present and have no visible changes.

Sources of pluripotent stem cells

The types of pluripotent stem cells that can be used include stable pluripotent cell lines derived from tissue formed after gestation, including preembryonic tissue (such as a blastocyst), embryonic tissue, or fetal tissue taken at any time during gestation , usually, but not necessarily, until approximately 10-12 weeks of pregnancy. Non-limiting examples are stable human embryonic stem cell lines or human embryonic germ cells, such as the H1, H7 and H9 human embryonic stem cell lines (WiCell). It is also possible to use the compositions described herein during the initial establishment or stabilization of such cells. In this case, the source cells are primary pluripotent cells taken directly from the source tissues. Also suitable for the purposes of the present invention are cells taken from a population of pluripotent stem cells already cultured in the absence of nurse cells. Mutant human embryonic stem cell lines such as BGOlv (BresaGen, Athens, Georgia) are also suitable for the purposes of the present invention.

In one embodiment, human embryonic stem cells are prepared as described in Thomson et al. (US Patent No. 5843780; Science 282:1145, 1998; Curr. Top. Dev. Biol. 38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).

Cultivation of pluripotent stem cells

In one embodiment, the pluripotent stem cells are cultured on a layer of nurse cells that support the pluripotent stem cells in various ways. In an alternative embodiment, the pluripotent stem cells are cultured in a culture system that is substantially free of nurse cells but still supports the proliferation of the pluripotent stem cells and does not allow significant differentiation. The growth of pluripotent stem cells in a culture system containing no nurse cells without differentiation is supported by the use of a medium conditioned by pre-cultivation of other cell types. In an alternative embodiment, growth of pluripotent stem cells in a nurse cell-free culture system without differentiation is supported by the use of a chemically defined medium.

In one embodiment, pluripotent stem cells can be cultured on a layer of mouse embryonic fibroblast nurse cells according to the methods set forth in Reubinoff et al (Nature Biotechnology 18: 399-404 (2000)). In an alternative embodiment, pluripotent stem cells can be cultured on a mouse embryonic fibroblast feeder cell layer according to the methods set forth in Thompson et al (Science 6, Nov. 1998: vol. 282, no. 5391, pp. 1145-1147). In an alternative embodiment, pluripotent stem cells can be cultured on any of the feeder cell layers disclosed in Richards et al (Stem Cells 21: 546-556, 2003).

In one embodiment, pluripotent stem cells can be cultured on a layer of human nurse cells in accordance with the methods set forth 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 nurse cells disclosed in Stojkovic et al (Stem Cells 2005 23: 306-314, 2005). In an alternative embodiment, pluripotent stem cells can be cultured on a layer of human nurse cells disclosed in Miyamoto et al (Stem Cells 22: 433-440, 2004). In an alternative embodiment, pluripotent stem cells can be cultured on a layer of human nurse cells disclosed in Amit et al (Biol. Reprod 68: 2150-2156, 2003). In an alternative embodiment, pluripotent stem cells can be cultured on a layer of human nurse cells disclosed in Inzunza et al (Stem Cells 23: 544-549, 2005).

In one embodiment, pluripotent stem cells can be cultured in culture medium prepared in accordance with the methods presented in US Pat. No. 20020072117. In an alternative embodiment, pluripotent stem cells can be cultured in culture medium prepared in accordance with the methods presented in US Pat. No. 6,642,048. In an alternative embodiment, pluripotent stem cells can be cultured in a culture medium prepared in accordance with the methods presented in international patent WO 2005014799. In an alternative embodiment, pluripotent stem cells can be cultured in a culture medium obtained in accordance with the methods presented in Xu et al (Stem Cells 22: 972-980, 2004). In an alternative embodiment, pluripotent stem cells can be cultured in culture medium prepared in accordance with the methods presented in US Pat. No. 20070010011. In an alternative embodiment, pluripotent stem cells can be cultured in culture medium obtained in accordance with the methods presented in US patent No. 20050233446. In an alternative embodiment, pluripotent stem cells can be cultured in culture medium prepared in accordance with the methods presented in US Pat. No. 6,800,480. In an alternative embodiment, pluripotent stem cells can be cultured in a culture medium obtained in accordance with the methods presented in international patent WO 2005065354.

In one embodiment, pluripotent stem cells can be cultured in accordance with the methods presented in Cheon et al (BioReprod DOI:10.1095/biolreprod. 105.046870, October 19, 2005). In an alternative embodiment, pluripotent stem cells can be cultured in accordance with the methods presented in the work of Levenstein et al (Stem Cells 24: 568-574, 2006). In an alternative embodiment, pluripotent stem cells can be cultured in accordance with the methods presented in US patent No. 20050148070. In an alternative embodiment, pluripotent stem cells can be cultured in accordance with the methods presented in US patent No. 20050244962. In an alternative embodiment, pluripotent stem cells can be cultured in accordance with the methods presented in international patent application No. WO 2005086845.

Pluripotent stem cells can be seeded onto an appropriate culture substrate. In one embodiment, the appropriate culture substrate is a component of the extracellular matrix, such as, for example, derived from basement membrane or one that may participate in a ligand-receptor interaction involving an adhesion layer molecule. In one embodiment, the appropriate culture substrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a soluble preparation of Engelbreth-Holm-Suorm tumor cells that gels at room temperature and forms a reconstituted basement membrane.

Alternatively, other extracellular matrix components and mixtures of components can be used. Depending on the type of proliferating cells, the latter may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, etc., individually or in various combinations.

Pluripotent stem cells can be seeded onto a substrate with an appropriate distribution over the surface and in the presence of an environment that supports survival, proliferation and maintenance of the desired cell characteristics. All of these characteristics are enhanced by careful cell distribution during seeding and can be determined by one skilled in the art.

An appropriate culture medium can be prepared, for example, from the following components: Dulbecco's modified Eagle's medium (DMEM), Gibco No. 11965-092, knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco No. 10829-018, Ham's basic medium F12/50% DMEM, 200 mM L-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma No. M7522; recombinant basic human fibroblast growth factor (bFGF), Gibco #13256-029.

Formation of cells expressing markers characteristic of the pancreatic endoderm lineage from pluripotent stem cells

In one embodiment, the present invention provides a method for producing a population of cells expressing markers characteristic of a pancreatic endoderm lineage from pluripotent stem cells, comprising the following steps:

a. culturing a population of pluripotent stem cells;

b. differentiation of a population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage; And

c. differentiation of a population of cells expressing markers characteristic of the definitive endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage by treating the population of cells expressing markers characteristic of the definitive endoderm lineage with a medium to which a protein kinase C activator has been added.

In one aspect, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 50% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 60% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 70% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 80% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 90% of the cells in the population simultaneously expressing PDX-1 and NKX6.1.

In one aspect of the present invention, a population of cells expressing markers characteristic of a pancreatic endoderm cell line can be further processed to obtain a population of cells expressing markers characteristic of a pancreatic endocrine cell line.

The efficiency of differentiation can be determined by treating a population of cells with an agent (eg, an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the desired cell type.

Methods for assessing the expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. Such methods include quantitative reverse transcription polymerase chain reaction (RT-PCR), Northern blotting, in situ hybridization (see, for example, Current Protocols in Molecular Biology (edited by Ausubel et al., 2001, add.)) , as well as immunoassay methods such as cross-sectional immunohistochemistry, Western blotting, and, for markers available in intact cells, flow cytometry (FACS) (see, for example, Harlow and Lane, Using Antibodies: A Laboratory Manual , New York: Cold Spring Harbor Laboratory Press (1998)).

The characteristics of pluripotent stem cells are well known to those skilled in the art, and additional characteristics of pluripotent stem cells continue to be identified. Markers of pluripotent stem cells include, for example, the expression of one or more of the following markers: ABCG2, CRIPTO, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA-4, Tra 1- 60, Tra 1-81.

After treatment of pluripotent stem cells using the methods of the present invention, differentiated cells can be purified by exposing the population of cells to an agent (eg, an antibody) that specifically recognizes a protein marker, for example, CXCR4, expressed by cells expressing markers characteristic of the definitive lineage endoderm.

Pluripotent stem cells suitable for use in the present invention include, for example, human embryonic stem cells line H9 (NIH code: WA09), human embryonic stem cells line H1 (NIH code: WA01), human embryonic stem cells line H7 ( NIH code: WA07) and human embryonic stem cells line SA002 (Cellartis, Sweden). Also relevant to the purposes of the present invention are cells expressing at least one of the following markers characteristic of pluripotent cells: ABCG2, CRIPTO, CD9, FOXD3, CONNEXIN43, CONNEXIN45, OCT4, SOX2, NANOG, hTERT, UTF1, ZFP42, SSEA-3, SSEA -4, Tra 1-60 and Tra 1-81.

Markers characteristic of the definitive endoderm lineage were selected from the group consisting of SOX17, GATA4, HNF3 beta, GSC, CER1, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4, CD48, eomesdermin (EOMES), DKK4, FGF17, GATA6, CXCR4, C-Kit, CD99 and OTX2. The purposes of the present invention are a cell expressing at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, the cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak progenitor cell. In another aspect of the present invention, the cell expressing markers characteristic of the definitive endoderm lineage is a mesendodermal cell. In another aspect of the present invention, the cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.

Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of PDX1, NKX6.1, HNF1 beta, PTF1 alpha, HNF6, HNF4 alpha, SOX9, HB9 and PROX1. The purposes of the present invention are a cell expressing at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, the cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.

Markers characteristic of the pancreatic endocrine cell line are selected from the group consisting of NGN3, NEUROD, NKX2.2, PDX1, NKX6.1, PAX4 and PAX6. In one embodiment, the pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin and pancreatic polypeptide. The purposes of the present invention are a cell expressing at least one of the markers characteristic of a pancreatic endocrine cell line. In one aspect of the present invention, the cell expressing markers characteristic of a pancreatic endocrine cell line is a pancreatic endocrine cell. A pancreatic endocrine cell may be a pancreatic cell that expresses hormones. In an alternative embodiment, the pancreatic endocrine cell may be a hormone-secreting pancreatic cell.

In one aspect of the present invention, the pancreatic endocrine cell is a cell expressing markers characteristic of the β-cell lineage. A cell expressing markers characteristic of the β cell lineage 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 lineage is a β cell.

Differentiation of pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage.

The production of cells expressing markers characteristic of the definitive endoderm lineage can be detected by testing for the presence of markers before and after performing a particular protocol. Pluripotent stem cells generally do not express such markers. Thus, the differentiation of pluripotent cells is determined by the onset of expression of such markers.

Pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage using any method known to those skilled in the art or using any method provided by the present invention.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by methods described in D'Amour et al., Nature Biotechnology 23, 1534-1541 (2005).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by methods described in Shinozaki et al., Development 131, 1651-1662 (2004).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by methods described in McLean et al., Stem Cells 25, 29-38 (2007).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by methods described in D'Amour et al., Nature Biotechnology 24, 1392-1401 (2006).

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 11/736908.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 11/779311.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 60/990529.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 61/076889.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 61/076900.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 61/076908.

For example, pluripotent stem cells can be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells in accordance with the methods set forth in US patent application No. 61/076915.

Differentiation of cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage

In one embodiment, the present invention provides a method for producing a population of cells expressing markers characteristic of a pancreatic endoderm lineage from pluripotent stem cells, comprising the following steps:

a. culturing a population of pluripotent stem cells;

b. differentiation of a population of pluripotent stem cells into a population of cells expressing markers characteristic of the definitive endoderm lineage; And

c. differentiation of a population of cells expressing markers characteristic of the definitive endoderm lineage into a population of cells expressing markers characteristic of the pancreatic endoderm lineage by treating the population of cells expressing markers characteristic of the definitive endoderm lineage with a medium to which a protein kinase C activator has been added.

In one aspect, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 50% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 60% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 70% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 80% of the cells in the population simultaneously expressing PDX-1 and NKX6.1. In an alternative embodiment, the present invention provides a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 90% of the cells in the population simultaneously expressing PDX-1 and NKX6.1.

In one embodiment, the protein kinase C activator is selected from the group consisting of (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadiemoylamino)benzenolactam, indolactam V ( ILV), phorbol 12-myristate 13-acetate (PMA) and phorbol 12,13-dibutyrate (PDBu). In one embodiment, the protein kinase C activator is (2S,5S)-(E,E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadiemoylamino)benzenolactam. (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino)benzenolactam can be used at a concentration of from about 20 nM to about 500 nM. (2S,5S)-(E,E)-8-(5-(4-(Trifluoromethyl)phenyl)-2,4-pentadiemoylamino)benzenolactam is referred to herein as “TPB.”

In one embodiment, the medium to which the protein kinase C activator is added is further supplemented with at least one factor selected from the group consisting of a factor capable of inhibiting BMP, a TGFβ receptor signaling cascade inhibitor, and fibroblast growth factor.

In one embodiment, the factor capable of inhibiting BMP is Noggin. Noggin can be used at concentrations ranging from about 50 ng/ml to about 500 ng/ml. In one embodiment, Noggin is used at a concentration of 100 ng/ml.

In one embodiment, the TGFβ receptor signaling cascade inhibitor is an ALK5 inhibitor. In one embodiment, the ALK5 inhibitor is an ALK5 inhibitor II. The ALK5 inhibitor II can be used at a concentration of from about 0.1 μM to about 10 μM. In one embodiment, ALK5 inhibitor II is used at a concentration of 1 μM.

In one embodiment, the fibroblast growth factor is FGF7. In an alternative embodiment, the fibroblast growth factor is FGF10.

In one embodiment, fibroblast growth factor can be used at a concentration of from about 50 pg/ml to about 50 μg/ml. In one embodiment, fibroblast growth factor is used at a concentration of 50 ng/ml.

Differentiation of cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine cell lineage

In one embodiment, populations of cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage using any method known to those skilled in the art.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in the work of D' Amour et al., Nature Biotechnology, 2006.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in the work of D' Amour et al., Nature Biotechnology, 2006.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in US patent application No. 11/736908.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in US patent application No. 11/779311.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in US patent application No. 60/953178.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in US patent application No. 60/990529.

For example, populations of cells expressing markers characteristic of the pancreatic endoderm cell lineage can be further differentiated into populations of cells expressing markers characteristic of the pancreatic endocrine cell lineage by treating the population of cells expressing markers characteristic of the pancreatic endoderm cell lineage in accordance with the methods , presented in US patent application No. 61/289671.

The present invention is further illustrated without limitation by the following examples.

EXAMPLES

Example 1

Formation of a population of cells constituting the subject of the present invention

Human embryonic stem cell line HI cells were cultured in MATRIGEL® coated (1:30 dilution) (BD Biosciences; cat. no. 356231) plates with RPMI medium (Invitrogen, cat. no. 22400) + 0.2% FBS + 100 ng /ml Activin A (PeproTech; Cat. No. 120-14) + 20 ng/ml WNT-3a (R&D Systems; Cat. No. 1324-WN/CF) for one day, followed by RPMI medium supplemented with 0 .5% FBS + 100 ng/ml activin A for two more days (stage 1), then

a. DMEM/F12 + 2% FBS + 50 ng/ml FGF7 for three days (stage 2), then

b. High glucose DMEM + 1% B27 + 0.25 µM cyclopamine-KAAD + 2 µM retinoic acid (RA) + 100 ng/ml Noggin for four days (stage 3), then either

c. treatment 1: DMEM high glucose + 1% B27 for four days (stage 4 - basal medium - BM), or

d. treatment 2: high glucose DMEM + 1% B27 + 100 ng/ml Nogin + 1 µM ALK5 inhibitor II for four days (stage 4), or

e. treatment 3: high glucose DMEM + 1% B27 + 100 ng/ml Noggin + 1 µM ALK5 inhibitor II + 20 nM phorbol 12,13-dibutyrate (PDBu) (Calbiochem, cat. no. 524390) for four days (stage 4).

Cultures were sampled in duplicate at stage 4, day 4, and imaged using an IN Cell Analyzer 1000 (GE Healthcare). To compensate for possible cell losses during the analysis and subsequent staining procedures, 100 projections were taken for each well. For each well, the total number of cells, total number of PDX1-expressing cells, total number of NKX6.1-expressing cells, and total number of CDX-2-expressing cells were measured using IN Cell Developer Toolbox 1.7 software (GE Healthcare). For each replicate data set, means and standard deviations were calculated. The total numbers of cells expressing PDX 1, NKX6.1 and CDX-2 are given as a percentage of the total cell population.

As shown in FIG. 2A, in all experimental populations at the end of stage 4, day 4, approximately 92% + 4% of cells in the population expressed PDX 1. However, treatment with PDBu (protein kinase C activator) caused a significant increase in the proportion of cells expressing NKX6.1 in the population, expressing PDX1 (Fig. 2A), compared with cell populations treated with either basal medium (treatment 1) or ALK5 inhibitor II in the presence of Noggin (treatment 2). In the PDBu treated group, 88%±4.2% of the entire population expressed NKX6.1, while in treatment 2 62%±8% of cells expressed NKX6.1, and in treatment 1 46.7%±expressed NKX6.1 0.2% of cells. Most NKX6.1-expressing cells at stage 4 also expressed PDX1. These observations are supported by an overlay of PDX1 and NKX6.1 expression images obtained for a specific cell population ( Fig. 1A ). These data show that treatment of cells with media supplemented with protein kinase C activator increased the percentage of cells simultaneously expressing PDX1 and NKX6.1 in a population of cells that express markers characteristic of the pancreatic endoderm lineage.

In the cell population treated with treatment 1, 10% of cells expressed CDX2 (a marker of intestinal tissue) (Fig. 2A). In cell populations treated with treatment 2 or 3, less than 5% of cells expressed CDX2. In all cases, the majority of cells that expressed CDX2 did not simultaneously express PDX1 and NKX6.1.

Parallel populations of stage 3 cells were also treated with the following protein kinase C activators: 20 nM phorbol 12-myristate 13-acetate (PMA) (Calbiochem #524400) or [(2S,5S)-(E,E)-8-( 5-(4-(trifluoromethyl)phenyl)-2,4-pentadienoylamino)benzenolactam] (TPB) at a concentration of 50 nM (Calbiochem #565740), which were used instead of PDBu in the above treatment 3. At the end of stage 4, day 4 , 91% of cells in the PMA-treated population and 90% of cells in the TPB-treated population expressed NKX6.1. No significant differences were observed in the total number of cells expressing PDX1 across all treatments. See fig. 2B.

This example demonstrates that protein kinase C activators can be used at relatively low concentrations to stimulate increased expression of NKX6.1 and increase the percentage of cells co-expressing PDX1 and NKX6.1 in a population of cells expressing markers characteristic of the pancreatic endoderm lineage.

Example 2

Implantation of cells of the present invention into mice with congenital absence of natural killer cells with severe combined immunodeficiency (SCID-Bg)

Human embryonic stem cell line HI cells were cultured in MATRIGEL® coated (1:30 dilution) dishes with RPMI + 0.2% FBS + 100 ng/ml Activin A + 20 ng/ml WNT-3a for one day followed by treatment RPMI medium + 0.5% FBS + 100 ng/ml activin A for another two days (stage 1), then

a. DMEM/F12 + 2% FBS + 50 ng/ml FGF7 for three days (stage 2), then

b. High glucose DMEM + 1% B27 + 0.25 µM cyclopamine-KAAD + 2 µM retinoic acid (RA) + 100 ng/ml Noggin for four days (stage 3), then

c. High glucose DMEM + 1% B27 + 100 ng/ml Nogin + 1 µM ALK5 inhibitor II + 50 nM TPB for four days (stage 4).

Five to six week old SCID-Bg mice (CB-Igh-1b/GbmsTac-Prkdc ^scid -Lyst ^bd N7) were purchased from laconic Farms. Mice were kept in microisolator cages with free access to sterilized food and water. In preparation for surgery, mice were tagged with an ear tag and their body weight and blood glucose were measured using a hand-held glucometer (LifeScan, One Touch).

Mice were anesthetized with a mixture of isoflurane and oxygen, and hair was trimmed at the surgical site using small animal scissors. Before surgery, mice were injected subcutaneously with 0.1 mg/kg Buprenex. The surgical field was prepared by sequential rinsing with 70% isopropyl alcohol and 10% povidone iodide.

Cells at the end of the fourth stage were mechanically collected with a 1 ml glass pipette and then transferred to non-attachment plates and cultured overnight. During the preoperative preparation of mice, cells were centrifuged in a 1.5 ml microcentrifuge tube, and most of the supernatant was removed, leaving only enough medium to collect the pelleted cells. Cells were collected using a Rainin Pos-D positive displacement pipette and the pipette was inverted to allow the cells to settle under their own weight. Excess medium was forced out, leaving a compact preparation of cells for transplantation.

During transplantation, a 24G × 1.9 cm (3/4 inch) intravenous catheter was used to penetrate the renal capsule and the needle was removed. The catheter was then advanced under the renal capsule to the distal edge of the kidney. The Pos-D pipette tip was firmly inserted into the catheter hub and 5 million cells were injected through the catheter under the renal capsule, to the distal edge of the kidney. The renal capsule was sealed using low-temperature cautery, after which the kidney was returned to its original anatomical position. Simultaneously, aggregates of 5 million cells were loaded into a 50 μL implantation device using a Post-D pipette tip. 50 μL devices were purchased from TheraCyte, Inc. (Irvine, CA). After cell loading, the device was sealed with medical adhesive silicone type A (Dow Corning, catalog no. 129109) and implanted under the skin of SICD-Bg mice (animals nos. 3 and 4). The muscles were sutured with a running suture using 5-0 Vicryl suture, and the skin was closed with wound staples. After surgery, mice were injected subcutaneously with 1.0 mg/kg Metacam. The mice were taken out of anesthesia and given the opportunity to fully recover.

After transplantation, mice were weighed once a week and blood samples were taken to determine glucose levels twice a week. At different times after transplantation, mice were intraperitoneally injected with 3 g/kg glucose. 60 minutes after glucose administration, blood was collected through the retroorbital sinus and placed in microcentrifuge tubes containing a small amount of heparin. The blood was centrifuged, and the plasma was collected in a second microcentrifuge tube, frozen on dry ice, and stored at −80°C until analysis for human C-peptide. Human C-peptide levels were measured using the Mercodia/ALPCO Diagnotics Ultrasensitive C-peptide ELISA kit (cat. no. 80-CPTHU-E01, Alpco Diagnostics, New Hampshire) according to the manufacturer's instructions.

Human C-peptide was detected in the serum of animals in both the renal capsule group and the Theracyte device group as early as 4-6 weeks after transplantation, and its concentration increased over time (Figs. 3A and 3B). After three months, significant amounts of circulating human C-peptide were recorded in response to glucose administration in both 100% of the animals with a renal capsule and in the group implanted with the Theracyte device. After three months, the serum glucose-stimulated human C-peptide level in the renal capsule group was 1.7 ± 0.5 ng/ml (n = 4), and the human C-peptide concentration in mice implanted with the Theracyte device was 1 ± 0. 5 ng/ml (n=2) (Fig. 3C).

This example demonstrates that a population of cells co-expressing PDX1 and NKX6.1, produced by protein kinase C activators, is capable of further differentiation into insulin-secreting cells in vivo. The ability to further differentiate into insulin-secreting cells is independent of the local environment. The authors showed that cells simultaneously expressing PDX1 and NKX6.1 are capable of further differentiation into insulin-secreting cells both in the renal capsule and in the immunoprotective device when implanted subcutaneously.

Example 3

An Alternative Method for Forming a Population of Cells Constituting the Subject of the Present Invention

Human embryonic stem cell line HI cells were cultured in MATRIGEL® coated (1:30 dilution) (BD Biosciences; cat. no. 356231) plates with RPMI medium (Invitrogen, cat. no. 22400) + 0.2% FBS + 100 ng /ml Activin A (PeproTech; Cat. No. 120-14) + 20 ng/ml WNT-3a (R&D Systems; Cat. No. 1324-WN/CF) for one day followed by RPMI medium supplemented with 0. 5% FBS + 100 ng/ml activin A for two more days (stage 1), then

a. DMEM/F12 + 2% FBS + 50 ng/ml FGF7 for three days (stage 2), then

b. High glucose DMEM + 1% B27 + 0.25 µM cyclopamine-KAAD + 2 µM retinoic acid (RA) + 100 ng/ml Noggin for four days (stage 3), then either

c. treatment 4: high glucose DMEM + 1% B27 + 20 nM PDBu + 100 ng/ml Noggin for four days (stage 4), or

d. treatment 5: high glucose DMEM + 1% B27 + 100 ng/ml Nogin + 1 µM ALK5 inhibitor II + 20 nM PDBu for four days (step 4), or

e. treatment 6: high glucose DMEM + 1% B27 + 50 ng/ml FGF10 + 20 nM PDBu for four days (stage 4).

The effect of additional factors on the protein kinase C activator-mediated increase in the percentage of cells simultaneously expressing PDX1 and NKX6.1 was examined. Cultures were sampled in duplicate at stage 4, day 4, and image analysis was performed as described in Example 1 above. The expression of ISL1 and NEUROD1 was also recorded.

In this study, the majority of cells from a population expressing markers characteristic of the pancreatic endoderm lineage showed positive expression of PDX 1. The majority of cells expressing PDX1 also simultaneously showed positive expression of NKX6.1. As shown in Table 1, addition of PKC activator alone promoted the appearance of NKX6.1-expressing cells within a population of cells expressing markers characteristic of the pancreatic endoderm lineage (treatment 4). At day 4 of stage 4, 93% of the entire population was positive for NKX6.1, and almost all cells expressing NKX6.1 showed positive PDX1 expression.

Addition of ALK5 inhibitor II to protein kinase C activator-containing medium (treatment 5) had no effect on the observed increase in NKX6.1 expression. 57.1% of the population cells expressed NEUR0D1, and 52.4% of the population cells expressed ISL1, indicating an increase in the proportion of endocrine progenitor cells in the population after this treatment. See Table 1.

PCR analysis of the samples in this example showed that the expression of PDX 1, NKX6.1 and PTF1 alpha was increased in the cell population treated with treatment 4 compared to cells treated with treatment 5. See FIG. 4A-4D. On the other hand, a significant increase in NGN3 expression was observed in cells treated with ALK5 inhibitor II and PDBu (treatment 5). See fig. 4A-4D.

The effect of adding FGF10 to the medium containing protein kinase C activator (treatment 6) was also examined. Addition of FGF10 at a concentration of 50 ng/ml in combination with PDBu (treatment 6) resulted in the formation of a population of cells expressing a marker characteristic of the pancreatic endoderm lineage, where 90% of the cells in the population express NKX6.1, but many of the cells expressing NKX6.1 also showed positive CDX2 expression. See Table 1. The mRNA levels for PDX1, NKX6.1 and PTF1 alpha did not increase compared to the levels observed for cells treated with PDBu and Noggin. See fig. 4A-4D.

This example demonstrates that a protein kinase C activator at a relatively low concentration (approximately 20 nM) in combination with a BMP inhibitor can be used to generate a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with more than 90% of the cells simultaneously expressing PDX1 and NKX6. 1.

Example 4

An Alternative Method for Forming a Population of Cells Constituting the Subject of the Present Invention

Human embryonic stem cell line H1 cells were cultured in MATRIGEL® (1:30 dilution) (BD Biosciences; Cat. No. 356231) coated dishes with RPMI medium (Invitrogen, Cat. No. 22400) + 0.2% FBS + 100 ng /ml Activin A (PeproTech; Cat. No. 120-14) + 20 ng/ml WNT-3a (R&D Systems; Cat. No. 1324-WN/CF) for one day followed by RPMI medium supplemented with 0. 5% FBS + 100 ng/ml activin A for two more days (stage 1), then

a. DMEM/F12 + 2% FBS + 50 ng/ml FGF7 for three days (stage 2), then either

b. treatment 7 (T7): DMEM high glucose + 1% B27 + 0.25 µM cyclopamine-KAAD + 2 µM retinoic acid (RA) + 100 ng/ml Noggin for four days (stage 3), or

c. treatment 8 (T8): DMEM high glucose + 1% B27 + 0.25 µM cyclopamine-KAAD + 2 µM retinoic acid (RA) + 100 ng/ml Nogin + 50 ng/ml FGF7 for four days (stage 3 ), then either

d. treatment 9 (T9): DMEM high glucose + 1% B27 + 100 ng/ml Noggin + 1 µM ALK5 inhibitor II + 20 nM PDBu for four days (stage 4), or

e. treatment 10 (T10): DMEM high glucose + 1% B27 + 50 ng/ml FGF10 + 20 nM PDBu for four days (stage 4), or

f. treatment 11 (T11): DMEM high glucose + 1% B27 + 20 nM PDBu + 100 ng/ml Noggin for four days (stage 4).

Cultures were sampled in duplicate at stage 4, day 4, and imaged using an IN Cell Analyzer 1000 (GE Healthcare). To compensate for possible cell losses during the analysis and subsequent staining procedures, 100 projections were taken for each well. For each well, the total number of cells, total number of PDX1-expressing cells, total number of NKX6.1-expressing cells, and total number of CDX2-expressing cells were measured using IN Cell Developer Toolbox 1.7 software (GE Healthcare). For each replicate data set, means and standard deviations were calculated. The total numbers of cells expressing PDX 1, NKX6.1 and CDX-2 are given as a percentage of the total cell population.

In cell populations treated with T7 followed by the addition of T9 medium, approximately 80% of the cells in the population expressed NKX6.1. See Table 2. In cell populations treated with T7 followed by T10 medium, approximately 90% of the cells expressed NKX6.1, with more cells expressing CDX2 observed in this treatment. See Table 2. Treatment of populations of T7 cells followed by treatment with T11 medium resulted in a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with 93% of the cells in the population expressing NKX6.1. The majority of cells in populations expressing NKX6.1 also expressed PDX1.

Cultures treated with T8 followed by treatment with T9 medium formed a population of cells expressing markers characteristic of the pancreatic endoderm lineage, where 56.7% of the cells in the population expressed NKX6.1. See Table 2. Cultures treated with T8 followed by treatment with T10 medium formed a population of cells expressing markers characteristic of the pancreatic endoderm lineage, where 63.5% of the population cells expressed NKX6.1; after treatment, an increase in the number of cells expressing CDX2 was noted. See Table 2. Cultures treated with T8 followed by T11 medium produced a population of cells expressing markers characteristic of the pancreatic endoderm lineage, with 74% of the cells in the population expressing NKX6.1. See Table 2. Most NKX6.1-expressing cells also expressed PDX 1.

PCR analysis also confirmed the IN Cell results in that treatment with retinoic acid, cyclopamine and Noggin at stage 3 followed by the addition of PKC activator at stage 4 on day 4 of stage 4 resulted in increased mRNA levels for NKX6.1 and PTF1 alpha (Fig. 5A-5D).

All publications cited herein are incorporated herein by reference in their entirety. Although various aspects of the present invention have been illustrated above by reference to examples and preferred embodiments, it is intended that the spirit of the present invention be limited in scope not by the foregoing description, but by the following claims, drawn up in accordance with the principles of patent law.

Claims (15)

1. A method for obtaining a population of cells, including cells of the pancreatic endoderm line, including: differentiation of a cell population, including cells of the definitive endoderm lineage, which are not human embryonic cells, in a medium supplemented with a protein kinase C activator and a factor capable of inhibiting bone morphogenetic protein (BMP), thereby producing a population of cells including cells of the pancreatic endoderm lineage. 2. The method according to claim 1, wherein the population of cells, including cells of the pancreatic endoderm lineage, expresses PDX1 and NKX6.1. 3. The method according to claim 1 or 2, where more than 60% of the population co-express PDX1 and NKX6.1. 4. Method according to any one of paragraphs. 1-3, wherein the medium is further added to at least one factor selected from the group consisting of an inhibitor of transforming growth factor (TGF)-β receptor signaling and fibroblast growth factor. 5. The method according to claim 4, where the factor capable of inhibiting BMP is noggin. 6. The method of claim 4 or 5, wherein the TGF-β receptor signaling inhibitor is an ALK5 inhibitor. 7. The method of claim 6, wherein the ALK5 inhibitor is an ALK5 inhibitor II. 8. The method according to claim 7, where the ALK II inhibitor is used in a concentration of 1 µmol to 10 µmol. 9. The method according to claim 7, in which the ALK II inhibitor is used at a concentration of 1 μmol. 10. Method according to any one of paragraphs. 1-9, characterized in that the protein kinase C activator is (2S, 5S)-(E, E)-8-(5-(4-(trifluoromethyl)phenyl)-2,4-pentadiemoylamino) benzolactam, indolactam V ( ILV), phorbol 12-myristate 13-acetate (PMA) or phorbol 12, 13-dibutyrate (PDBu). 11. An isolated, crude population of cells for obtaining pancreatic endocrine cells, containing cells of the pancreatic endoderm line obtained by the method according to any one of paragraphs. 1-10, where more than 70% of the cells in the population co-express PDX1 and NKX6.1, where the cell population is obtained without purification after isolation. 12. The cell population of claim 11, wherein the cell population expresses one or more of HNF1 beta, PTF1 alpha, HNF6, HNF4 alpha, SOX9, HB9 and PROX1. 13. The cell population of claim 11 or 12, wherein the cells in the population can further differentiate into insulin-secreting cells in vivo.