WO2010051223A1 - Differentiation of human embryonic stem cells to the pancreatic endocrine lineage - Google Patents

Differentiation of human embryonic stem cells to the pancreatic endocrine lineage Download PDF

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WO2010051223A1
WO2010051223A1 PCT/US2009/061774 US2009061774W WO2010051223A1 WO 2010051223 A1 WO2010051223 A1 WO 2010051223A1 US 2009061774 W US2009061774 W US 2009061774W WO 2010051223 A1 WO2010051223 A1 WO 2010051223A1
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cells
cell
inhibitor
markers characteristic
expressing markers
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PCT/US2009/061774
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French (fr)
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Alireza Rezania
Benjamin Fryer
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Centocor Ortho Biotech Inc.
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Priority to MX2011004565A priority Critical patent/MX2011004565A/en
Priority to JP2011534638A priority patent/JP5785088B2/en
Priority to AU2009308967A priority patent/AU2009308967C1/en
Priority to RU2011121842/10A priority patent/RU2522001C2/en
Application filed by Centocor Ortho Biotech Inc. filed Critical Centocor Ortho Biotech Inc.
Priority to ES09752567.9T priority patent/ES2634445T3/en
Priority to EP09752567.9A priority patent/EP2346988B1/en
Priority to CA2742268A priority patent/CA2742268C/en
Priority to KR1020117011905A priority patent/KR101712085B1/en
Priority to BRPI0919885-7A priority patent/BRPI0919885A2/en
Priority to PL09752567T priority patent/PL2346988T3/en
Priority to CN200980153865.9A priority patent/CN102272291B/en
Publication of WO2010051223A1 publication Critical patent/WO2010051223A1/en
Priority to ZA2011/03985A priority patent/ZA201103985B/en
Priority to AU2016204685A priority patent/AU2016204685B2/en
Priority to AU2018200452A priority patent/AU2018200452A1/en

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Definitions

  • the present invention provides methods to promote the differentiation of pluripotent stem cells.
  • the present invention provides a method to increase the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage.
  • a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm.
  • Definitive endoderm cells express a number of markers, such as, HNF-3 beta, GAT A4, MIXLl, CXCR4 and SOX 17.
  • pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm.
  • Cells of the pancreatic endoderm express the pancreatic- duodenal homeobox gene, PDXl.
  • PDXl pancreatic- duodenal homeobox gene
  • the mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm.
  • Cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse.
  • Lumelsky et al. (Science 292: 1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets.
  • Soria et al. (Diabetes 49: 157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin- induced diabetic mice.
  • Hori et al. (PNAS 99: 16105, 2002) disclose that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled ⁇ cells.
  • retinoic acid can regulate the commitment of embryonic stem cells to form Pdxl positive pancreatic endoderm. Retinoic acid is most effective at inducing Pdxl expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005).
  • Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdxl. Their results show that exogenous Pdxl expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenic, P48, Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).
  • the mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans.
  • D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into Pdxl positive cells after addition of FGF-10 (US 2005/0266554A1).
  • D'Amour et al. (Nature Biotechnology - 24, 1392 - 1401 (2006)) states: "We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones".
  • hES human embryonic stem
  • Fisk et al. reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006/0040387A1).
  • the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to endoderm using a combination of sodium butyrate and activin A. The cells were then cultured with TGF- ⁇ antagonists such as Noggin in combination with EGF or betacellulin to generate Pdxl positive cells. The terminal differentiation was induced by nicotinamide.
  • Benvenistry et al. states: "We conclude that over-expression of Pdxl enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo" (Benvenistry et al, Stem Cells 2006; 24: 1923-1930).
  • Cyclins have been implicated in beta cell function.
  • Lilja et al report that Cdk5 is present in the insulin-secreting pancreatic ⁇ -cell (J. Biol. Chem., Vol. 276, Issue 36, 34199-34205, September 7, 2001). Lilja et al states "Cdk5 is present in ⁇ - cells and acts as a positive regulator of insulin exocytosis.”
  • Marzo et al states "Cdk4 knockin mice have significantly increased beta cell mass and are physiologically functional, indicating that Cdk4 is a potential target for pancreatic beta cell mass regeneration in Type 1 diabetes" (Diabetalogia, Vol. 47, Number 4, 686-694, April 1, 2004.)
  • MafA is a basic leucine zipper transcription factor expressed within the beta cells of the pancreas and is required to maintain normal glucose homeostasis as it is involved in various aspects of beta cell biology. MafA protein levels are known to increase in response to high glucose through mechanisms that have yet to be fully characterized. We investigated whether discrete intracellular signaling events control mafA expression. We found that the general kinase inhibitor staurosporine induces mafA expression without altering the stability of the protein. Inhibition of the MAP -kinase JNK mimics the effects of staurosporine on the expression of mafA.
  • Calmodulin kinase and calcium signaling are also important in stimulating mafA expression by high glucose.
  • staurosporine, JNK, and calmodulin kinase have different effects on the induction of insulin expression.
  • pancreatic hormone expressing cells pancreatic hormone secreting cells.
  • the present invention provides methods to increase the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage.
  • the present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of MAFA.
  • FIG. 1 panel a shows the effect of compounds from the EMD Calbiochem kinase inhibitor library on the ratio of insulin to glucagon expression in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR.
  • the alphanumeric label corresponds to the compound identity as shown in Table 1.
  • Panel b shows the effect of compounds from the EMD Calbiochem kinase inhibitor library on the ratio of MAFA to ARX4 expression in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR.
  • the alphanumeric label corresponds to the compound identity as shown in Table 1.
  • Figure 2 A) shows a 4x micrograph of cells treated according to the methods described in Example 1, at day 4 of the stage 6 treatment.
  • B) shows a 4x micrograph of cells treated with 0.5 ⁇ M of the compound PubChemID# 5330812 at day 4 of treatment.
  • C) shows a 4x micrograph of cells treated with 1 ⁇ M of the compound PubChemID# 5330812 at day 4 of treatment.
  • D) shows a 2Ox micrograph of cells treated according to the methods described in Example 1, at day 6 of the stage 6 treatment.
  • E) shows a 2Ox micrograph of cells treated with 0.5 ⁇ M of the compound PubChemID# 5330812 at day 6 of treatment.
  • F) shows a 2Ox micrograph of cells treated with l ⁇ M of the compound PubChemID# 5330812 at day 6 of treatment.
  • Figure 3 shows the expression of the 23 genes indicated, in cells expressing markers characteristic of the pancreatic endocrine lineage following a five-day treatment of 0.5 ⁇ M (dark bars) or l.O ⁇ M (light bars) of the compound PubChem ID#5330812. Expression levels were determined at day 0, day 2 and day 5.
  • Figure 4 shows the effect of CDK inhibitor III treatment on the expression of markers characteristic of the pancreatic endocrine lineage in cells treated with Stage 7 of the differentiation protocol described in Example 4.
  • Figure 5 shows the effect of CDK inhibitor III treatment on the dithazone staining of islet-like clusters.
  • Figure 6 shows the expression of insulin, synaptophysin and glucagon in insulin- producing cells produced according to the methods described in Example 5. Expression of the proteins indicated was determined by FACS.
  • Figure 7 shows the expression of insulin, synaptophysin and glucagon in insulin- producing cells produced according to the methods described in Example 5. Expression of the proteins indicated was determined by FACS.
  • Figure 8 shows the expression of MAFA (Panel a) and insulin (Panel b), in insulin- producing cells, produced by the methods of the present invention.
  • Samples of cells were taken for PCR analysis at days 1, 2, 3, and 4. Following 4 days of treatment with CDK inhibitor, the CDK inhibitor was removed from culture and the cells were cultured additional 4 days in DMEM-F12 + 1% B27 + 20 ng/ml of activin A. At the end of the four days, samples were collected in triplicate for PCR analysis.
  • Figure 9 shows the effect of compounds from the EMD Calbiochem kinase inhibitor library I on the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR.
  • Figure 10 shows the effect of genestein on the mRNA expression of insulin, glucagon, somatostatin and MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR.
  • Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
  • Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self- renewal), blood cell restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g. , spermatogenic stem cells).
  • HSC hematopoietic stem cells
  • Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell.
  • a differentiated or differentiation-induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell.
  • the term "committed”, when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type.
  • De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell.
  • the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to.
  • the lineage of a cell places the cell within a hereditary scheme of development and differentiation.
  • a lineage- specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
  • ⁇ -cell lineage refers to cells with positive gene expression for the transcription factor PDX-I and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISLl, HNF3 beta, MAFA, PAX4, or PAX6.
  • Cells expressing markers characteristic of the ⁇ cell lineage include ⁇ cells.
  • Cells expressing markers characteristic of the definitive endoderm lineage refers to cells expressing at least one of the following markers: SOX17, GATA4, HNF3 beta, GSC, CERl, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF 17, GATA6, CXCR4, C- Kit, CD99, or OTX2.
  • Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage refers to cells expressing at least one of the following markers: PDXl, HNFl beta, PTFl alpha, FTNF-, or HB9.
  • Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells, primitive gut tube cells, and posterior foregut cells.
  • Cells expressing markers characteristic of the pancreatic endocrine lineage refers to cells expressing at least one of the following markers: NGN3, NEUROD, ISLl, PDXl, NKX6.1, PAX4, NGN3, or PTFl alpha.
  • Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the ⁇ -cell lineage.
  • Definitive endoderm refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF3 beta, GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, or MIXLl.
  • Extraembryonic endoderm refers to a population of cells expressing at least one of the following markers: SOX7, AFP, or SPARC.
  • Markers are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest.
  • differential expression means an increased level for a positive marker and a decreased level for a negative marker.
  • 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 cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
  • Mesendoderm cell refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX 17, DKK4, HNF3 beta, GSC, FGF 17, or GATA ⁇ .
  • EOMES eomesodermin
  • SOX 17, DKK4, HNF3 beta a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX 17, DKK4, HNF3 beta, GSC, FGF 17, or GATA ⁇ .
  • Pancreatic endocrine cell or “pancreatic hormone expressing cell”, as used herein, refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.
  • Pancreatic endoderm cell refers to a cell capable of expressing at least one of the following markers: NGN3, NEUROD, ISLl, PDXl, PAX4, or
  • Pantix hormone producing cell refers to a cell capable of producing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.
  • Pantix hormone secreting cell refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Posterior foregut cell refers to a cell capable of secreting at least one of the following markers: PDXl, HNFl, PTFl alpha, HNF6, HB9, or PROXl.
  • Pre-primitive streak cell refers to a cell expressing at least one of the following markers: Nodal, or FGF8.
  • Primary gut tube cell refers to a cell capable of secreting at least one of the following markers: FTNFl, or HNF 4A.
  • Primary streak cell refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
  • Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1- 60 and Tra-1-81 (Thomson et ah, Science 282: 1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra- 1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-I.
  • SSEA stage-specific embryonic antigens
  • Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the 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.
  • pluripotent stem cells Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues.
  • Pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers.
  • SCID severe combined immunodeficient
  • pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
  • Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a "normal karyotype,” which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
  • pluripotent stem cells include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • pre-embryonic tissue such as, for example, a blastocyst
  • embryonic tissue or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation.
  • Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines Hl, H7, and H9 (WiCeIl).
  • Hl, H7, and H9 WiCeIl
  • Also contemplated is use of the compositions of this disclosure 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.
  • human embryonic stem cells are prepared as described by
  • pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways.
  • pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously with another cell type.
  • the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
  • Richards et al (Stem Cells 21 : 546-556, 2003) evaluated a panel of 11 different human adult, fetal and neonatal feeder cell layers for their ability to support human pluripotent stem cell culture.
  • Richards et al states: "human embryonic stem cell lines cultured on adult skin fibroblast feeders retain human embryonic stem cell morphology and remain pluripotent”.
  • US20020072117 discloses cell lines that produce media that support the growth of primate pluripotent stem cells in feeder-free culture.
  • the cell lines employed are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells.
  • US20020072117 also discloses the use of the cell lines as a primary feeder cell layer.
  • Miyamoto et al (Stem Cells 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta.
  • Amit et al discloses a feeder cell layer derived from human foreskin.
  • Inzunza et al (Stem Cells 23 : 544-549, 2005) disclose a feeder cell layer from human postnatal foreskin fibroblasts.
  • US6642048 discloses media that support the growth of primate pluripotent stem (pPS) cells in feeder- free culture, and cell lines useful for production of such media.
  • pPS pluripotent stem
  • US6642048 states: "This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure.”
  • WO2005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells.
  • WO2005014799 states: "The culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells, in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte)."
  • Xu et al Ste Cells 22: 972-980, 2004 discloses conditioned medium obtained from human embryonic stem cell derivatives that have been genetically modified to over express human telomerase reverse transcriptase.
  • US20070010011 discloses a chemically defined culture medium for the maintenance of pluripotent stem cells.
  • An alternative culture system employs serum- free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells.
  • serum-free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells.
  • Cheon et al disclose a feeder- free, serum- free culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell self-renewal.
  • SR serum replacement
  • Levenstein et al (Stem Cells 24: 568-574, 2006) disclose methods for the long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned medium, using media supplemented with bFGF.
  • US20050148070 discloses a method of culturing human embryonic stem cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.
  • US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells.
  • the media is substantially isotonic as compared to the stem cells being cultured.
  • the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
  • a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells.
  • the basic medium is combined with a nutrient serum effective to support the growth of primate- derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells.
  • the medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt.”
  • US20050244962 states: "In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor.”
  • WO2005065354 discloses a defined, isotonic culture medium that is essentially feeder-free and serum-free, comprising: a. a basal medium; b. an amount of bFGF sufficient to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic acid sufficient to support growth of substantially undifferentiated mammalian stem cells.
  • WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGF- ⁇ ) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
  • TGF- ⁇ transforming growth factor-beta
  • FGF fibroblast growth factor
  • NIC nicotinamide
  • the pluripotent stem cells may be plated onto a suitable culture substrate.
  • the suitable culture substrate is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings.
  • a the suitable culture substrate is MATRIGEL® (Becton Dickenson).
  • MATRIGEL® is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.
  • extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations.
  • the pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
  • Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco # 11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; ⁇ -mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256- 029.
  • DMEM Dulbecco's modified Eagle's medium
  • KO DMEM Knockout Dulbecco's modified Eagle's medium
  • Ham's F12/50% DMEM basal medium 200 mM L-glutamine, Gibco # 15039-027
  • non-essential amino acid solution Gibco 11140-050
  • ⁇ -mercaptoethanol
  • the present invention provides a method for producing pancreatic hormone producing cells from pluripotent stem cells, comprising the steps of:
  • Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line Hl (NIH code: WAOl), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line 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, Connexin43, Connexin45, OCT4, SOX2, NANOG, hTERT, UTFl, ZFP42, SSEA3, SSEA4, Tral-60, or Tral-81.
  • markers characteristic of pluripotent cells ABCG2, cripto, CD9, FOXD3, Connexin43, Connexin45, OCT4, SOX2, NANOG, hTERT, UTFl, ZFP42, SSEA3, SSEA4, Tral-60, or Tral-
  • Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, HNF3 beta, GSC, CERl, NODAL, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF 17, GATA6, CXCR4, C-Kit, CD99, and OTX2.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell.
  • a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell.
  • a 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 PDXl, HNFl beta, PTFl alpha, HNF6, HB9 and PROXl.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage.
  • a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.
  • Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN3, NEUROD, ISLl, PDXl, NKX6.1, PAX4, NGN3, and PTFl alpha.
  • a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
  • Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage.
  • a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell.
  • the pancreatic endocrine cell may be a pancreatic hormone expressing cell.
  • the pancreatic endocrine cell may be a pancreatic hormone secreting cell.
  • the pancreatic endocrine cell is a cell expressing markers characteristic of the ⁇ cell lineage.
  • a cell expressing markers characteristic of the ⁇ cell lineage expresses Pdxl and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISLl, HNF3 beta, MAFA, PAX4, or PAX6.
  • a cell expressing markers characteristic of the ⁇ cell lineage is a ⁇ cell.
  • Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method proposed in this invention.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 23, 1534 - 1541 (2005).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651 - 1662 (2004).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25, 29 - 38 (2007).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration.
  • An example of this method is disclosed in Nature Biotechnology 23, 1534 - 1541 (2005).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration.
  • An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2005.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum.
  • An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 60/990,529.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,889.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,900.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,908.
  • pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,915.
  • Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.
  • an agent such as an antibody
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • Northern blots in situ hybridization
  • immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FOXD3, Connexin43, Connexin45, OCT4, SOX2, NANOG, hTERT, UTFl, ZFP42, SSEA3, SSEA4, Tral-60, or Tral-81.
  • the differentiated cells may be purified by exposing a 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 the definitive endoderm lineage.
  • an agent such as an antibody
  • a protein marker such as CXCR4
  • Cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art or by any method proposed in this invention.
  • cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine.
  • a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine an example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser. No. 60/990,529.
  • pancreatic endoderm lineage Markers characteristic of the pancreatic endoderm lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endoderm lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endoderm lineage.
  • Pancreatic endoderm lineage specific markers include the expression of one or more transcription factors such as, for example, HLXB9, PTFl alpha, PDXl, HNF6, or HNFl beta.
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.
  • an agent such as an antibody
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • Northern blots in situ hybridization
  • immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art or by any method disclosed in this invention.
  • cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-I and HGF.
  • An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-I and HGF.
  • An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4.
  • An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4.
  • An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 60/953,178, assigned to LifeScan, Inc.
  • cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in US patent application Ser. No. 60/990,529.
  • the present invention provides a method for increasing the expression of markers associated with the pancreatic endocrine lineage comprising treating cells expressing markers characteristic of the pancreatic endocrine lineage with medium comprising a sufficient amount of a TGF- ⁇ receptor agonist to cause an increase in expression of markers associated with the pancreatic endocrine lineage according to the methods disclosed in US patent application Ser. No. 61/110,278.
  • pancreatic endocrine lineage Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endocrine lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage.
  • Pancreatic endocrine lineage specific markers include the expression of one or more transcription factors such as, for example, NGN3, NEUROD, or ISLl.
  • Markers characteristic of cells of the ⁇ cell lineage are well known to those skilled in the art, and additional markers characteristic of the ⁇ cell lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the ⁇ -cell lineage, ⁇ cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, PDXl, NKX2.2, NKX6.1, ISLl, PAX6, PAX4, NEUROD, HNFl beta, HNF6, HNF3 beta, or MAFA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage.
  • an agent such as an antibody
  • the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the ⁇ cell lineage.
  • RT-PCR quantitative reverse transcriptase polymerase chain reaction
  • Northern blots in situ hybridization
  • immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
  • the efficiency of differentiation is determined by measuring the percentage of insulin positive cells in a given cell culture following treatment.
  • the methods of the present invention produce about 100% insulin positive cells in a given culture.
  • the methods of the present invention produce about 90% insulin positive cells in a given culture.
  • the methods of the present invention produce about 80% insulin positive cells in a given culture.
  • the methods of the present invention produce about 70% insulin positive cells in a given culture.
  • the methods of the present invention produce about 60% insulin positive cells in a given culture.
  • the methods of the present invention produce about 50% insulin positive cells in a given culture.
  • the methods of the present invention produce about 40% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 30% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 20% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 10% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 5% insulin positive cells in a given culture.
  • the efficiency of differentiation is determined by measuring glucose-stimulated insulin secretion, as detected by measuring the amount of C-peptide released by the cells.
  • cells produced by the methods of the present invention produce about lOOOng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 900ng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 800ng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 700ng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 600ng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 500ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 400ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 500ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 400ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 300ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 200ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about lOOng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 90ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 80ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 70ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 60ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 50ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 40ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 30ng C-peptide/pg DNA.
  • cells produced by the methods of the present invention produce about 20ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about IOng C-peptide/pg DNA.
  • the present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of MAFA.
  • the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 1.
  • the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 2.
  • the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 4.
  • the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 5.
  • the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 9.
  • the cyclin dependant kinase inhibitor may inhibit multiple isoforms of cyclin dependant kinase, in any combination thereof.
  • the cyclin dependant kinase inhibitor may be a protein. Alternatively, the cyclin dependant kinase inhibitor may be a peptide. Alternatively, the cyclin dependant kinase inhibitor may be a small molecule.
  • the small molecule cyclin dependant kinase inhibitor is selected from the group consisting of 7-n-Butyl- 6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine, 9-Nitro-7,12-dihydroindolo[3,2- d][l]benzazepin-6(5H)-one, 3-(6-Oxo-9-nitro-5,6,7,12-tetrahydroindolo[3,2- d][l]benzazepin-2-yl)propionitrile, (2R)-2-((6-((3-Amino-5-chlorophenyl)amino)-9- (l-methylethyl)-9H-purin-2-yl)amino)-3-methyl-l-butanol, Arcyriaflavin A, [6- Benzylamino-2-(3-hydroxypropylamino)-9-isopropy
  • the cyclin dependant kinase is ethyl-(6-hydroxy-4- phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate.
  • ethyl-(6- hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate is added to cells expressing markers characteristic of the endocrine lineage at a concentration from about 0.1 ⁇ M to about lO ⁇ M for about one to seven days.
  • cells expressing markers characteristic of the endocrine lineage are treated with ethyl-(6-hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-3- carboxylate for about one to about seven days.
  • RPMI medium supplemented with 2% BSA (Catalog# 152401 , MP Biomedical, Ohio), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days (Stage 1), then
  • MATRIGELTM coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 1 up to stage 5. Following this, the cells were treated for four days in DMEM/F12 + 1% B27 containing a compound from an EMD Calbiochem compound library (Catalog# 539745, Calbiochem, San Diego, Ca) at a final concentration of 1 ⁇ M. Wells containing vehicle were included as a control. Throughout the protocol media was changed daily. All samples were treated in duplicate. At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of insulin, glucagon, MAFA, and Arx4.
  • Results are expressed as a ratio insulin/glucagon (Figure 1, panel a), or MAFA versus ARX4 ( Figure 1, panel b) of the treated samples relative to the untreated control, as measured by real-time PCR.
  • the corresponding PubChem Compound ID# for each well # is listed in Table 1.
  • cells of the human embryonic stem cell line Hl at passage 42 were cultured in 10cm 2 MATRIGEL ® -coated dishes and treated according to the methods described in Example 1 up to stage 5. After stage 5, the cells were treated with DMEM/F12 containing 1% B27 containing l ⁇ M PubChem Compound ID# 5330797 for six days. Medium was changed every other day. Samples of cells were taken for real-time PCR prior to treatment with the compound, and at days two and five of compound treatment.
  • stage 6 Cells of the human embryonic stem cell line Hl at passage 52 were cultured on MATRIGEL ® coated dishes (1:30 dilution) and differentiated according to the methods described in Example 1. An additional stage (Stage 6) was added, in order to further mature the cells expressing markers characteristic of the pancreatic endocrine lineage. Stage 6 in this example consisted of a seven day treatment in DMEM/F12 + 1% B27 (Invitrogen, CA). The medium was changed daily. [0159] After stage 6, the cells were treated for 5 mins at room temperature with IX accutase (Sigma, MO). The accutase was removed, and DMEM/12 + 1% B27 was added to the cells.
  • IX accutase IX accutase
  • the attached cells were removed using a cell scarper and gently resuspended and passed through a 40 ⁇ m cell strainer.
  • the cells retained on the strainer were removed by rinsing in basal media and cultured in suspension on Ultra- Low culture plates (Catalogue# 3471, Corning, Ma).
  • the cells were then treated as follows: The cells were cultured in DMEM/F12 + 1 % B27, containing 20 ng/ml of activin A (AA), 1 ⁇ m of CDK inhibitor III (Catalog# 217714, Calbiochem, Ca) for 10 days (Stage 7). Cells treated with vehicle were included as controls. Samples were collected at days 7 through 10 for PCR analysis and dithizone staining.
  • the cells cultured in suspension according to the methods outlined in this example assumed a morphology similar to pancreatic islet clusters. Treatment with CDK inhibitor III did not appear to affect the morphology of the islet like clusters.
  • FIG 4, panels a-i shows the effect of CDK inhibitor III treatment on gene expression profile of the cell clusters.
  • Treatment with of CDK inhibitor III increased the expression of markers associated with the pancreatic endocrine lineage and in particular increased the expression of the pro-insulin transcription factor, MAFA.
  • FIG. 5 panels a-b shows the effect of CDK inhibitor III on dithazone (DTZ) staining of clusters.
  • DTZ dithazone
  • MATRIGEL ® -coated plates and differentiated into insulin producing cells using the following protocol:
  • Islet-like clusters were dispersed into single cells using TrypLE Express (Invitrogen, Carlsbad, CA) and washed in cold PBS. For fixation, the cells were resuspended in 200-300 ⁇ l Cytofix/Cytoperm Buffer (BD 554722, BD, Ca) and incubated for 30 min at 4°C. Cells were washed two times in 1 ml Perm/Wash Buffer Solution (BD 554723) and resuspended in 100 ⁇ l staining/blocking solution containing 2% normal goat serum in Perm/Wash buffer. For flow cytometric analysis, cells were stained with the following primary antibodies: Anti-Insulin (Rabbit mAb, Cell Signaling No.
  • Rabbit mAb Anti-Insulin
  • FIG. 6 panels a-c show the percentage insulin positive, synapthophysin positive, and glucagon positive cells in cells following treatment with Stage 7, in medium containing vehicle.
  • Figure 7, panels a-c shows the percentage insulin positive, synapthophysin positive, and glucagon positive cells following treatment with Stage 7 in medium containing 1 ⁇ M CDK inhibitor III for 5 days.
  • the number of single hormonal insulin positive cells increased from 3% to 8% following treatment with the CDK inhibitor.
  • the percentage of poly hormonal (insulin and glucagon positive) cells decreased following treatment with the CDK inhibitor.
  • MATRIGEL ® -coated plates and differentiated into insulin producing cells using the following protocol:
  • Samples were collected for PCR analysis at days 1, 2, 3, and 4. Following 4 days of treatment with CDK inhibitor, the CDK inhibitor was removed from culture and the cells were cultured additional 4 days in DMEM-F 12 + 1% B27 + 20 ng/ml of activin A. At the end of the four days, samples were collected in triplicate for PCR analysis.
  • FIG. 8 panels a-b show expression pattern of MAFA and insulin at various time points of stage 7.
  • CDK inhibitor treatment resulted in significant increase in MAFA and insulin expression which increased as a function of time.
  • removal of CDK inhibitor resulted in a significant drop to both MAFA and insulin expression, in samples obtained four days after removal of the compound.
  • MATRIGEL ® -coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 1 up to stage 5. Following this, the cells were grown for one day in DMEM/F12 + 1% B27 and then treated for six days in DMEM/F12 + 1% B27 containing a compound from a BIOMOLTM compound library (Catalog# 2832, BIOMOL, Madison Meeting, Pa) at a final concentration of 4 ⁇ M. Wells containing vehicle were included as a control. Throughout the treatment protocol media containing vehicle or compound was changed every other day. All samples were treated in duplicate. At the completion of this treatment RNA was collected for PCR analysis.
  • BIOMOLTM compound library Catalog# 2832, BIOMOL, Plymouth Meeting, Pa
  • MATRIGELTM coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 1 up to stage 5. Following this, the cells were grown for eight days in DMEM/F12 + 1% B27 and then treated for four days in DMEM/F12 + 1% B27 containing a cyclin dependent kinase inhibitor at a final concentration of 0.6125, 1.25, or 5.0 ⁇ M.
  • PubChem ID# 5330812 (EMD cat# 217714), PubChem ID# 4566 (EMD cat#217713), PubChem ID# 5330797 (EMD cat# 219476), PubChem ID# 73292 (EMD cat#341251), PubChem ID# 4592 (EMD cat#495620), and PubChem ID# 160355 (EMD cat #557360).
  • Wells containing vehicle were included as a control. Throughout the treatment protocol media containing vehicle or compound was changed every other day. All samples were treated in duplicate. At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of insulin, glucagon, MAFA, and ARX4. Results are expressed as the fold change relative to the vehicle treated control, as measured by real-time PCR.
  • PubChem ID# 5330797, and PubChem ID# 73292 all stimulated MAFA expression at the concentrations tested (Table 4).
  • PubChem ID# 4592 and PubChem ID# 160355 did not stimulate MAFA at the concentrations tested (Table 4).
  • the compounds PubChem ID# 5330812, PubChem ID# 4566, PubChem ID# 5330797, PubChem ID# 4592 and PubChem ID# 160355 all appeared to stimulate insulin expression (Table 4).
  • the compound PubChem ID# 5330797 reduced both glucagon and Arx4 expression (Table 4) while stimulating MAFA expression.
  • RPMI medium supplemented with 2% BSA (Catalog# 152401 , MP Biomedical, Ohio), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days (Stage 1), then
  • MATRIGEL ® -coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 9 up to stage 5.
  • the cells were fed and treated on day 1, 3, and 5 of stage 5 with media comprising DMEM-HG, 1% B27 (Invitrogen, CA), 1 ⁇ M ALK5 inhibitor II (Calbiochem, Ca) and a compound from an EMD Calbiochem compound library 1 solubilized in DMSO (Catalog# 539744, Calbiochem, San Diego, Ca) and treated at a final concentration of 2.5 ⁇ M.
  • Wells containing vehicle were included as a control.
  • media was changed daily except at stage 5 when media was changed every other day. All samples were treated in duplicate.
  • MATRIGEL ® -coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 9 up to stage 5.
  • the cells were fed and treated on day 1, 3, and 5 of stage 5 with media comprising DMEM-HG, 1% B27 (Invitrogen, CA), 1 ⁇ M ALK5 inhibitor II (Calbiochem, Ca) (Stage 5) and a compound from an EMD Calbiochem compound library II solubilized in DMSO (Tables 1 and 6, Calbiochem, San Diego, Ca) and treated at a final concentration of 2.5 ⁇ M.
  • Wells containing vehicle were included as a control.
  • media was changed daily except at stage 5 when media was changed every other day. All samples were treated in duplicate.
  • Cell growth resulting from cell cycle progression can be activated and maintained by stimulating cells with extracellular growth factors.
  • Growth factors bind to the extracellular domains of growth factor receptors, inducing a conformational switch in the receptor's intracellular domain. This shift initiates receptor dimerization and activation of tyrosine kinases located on the intracellular domain of the receptor leading to phosphorylation and activation of multiple serine/threonine kinases downstream, ultimately resulting in cell cycle progression and cell proliferation.
  • pancreatic beta cells Under normal physiologic conditions mature pancreatic beta cells, characterized by expression of insulin and the transcription factor MAFA, are quiescent and tend to remain in GO of the cell cycle. Yet, in order to generate enough cells to form a functional organ and meet the needs of a mature animal, the cells expressing markers characteristic of the pancreatic endocrine lineage of the present invention must be cell cycling. Consequently, at some point in embryonic development, the cells expressing markers characteristic of the pancreatic endocrine lineage of the present invention differentiate to beta cells and transition from an actively cell-cycling proliferating cell, to a quiescent cell.
  • a broad spectrum inhibitor like staurosporine can effectively induce MAFA, however it is also cytotoxic at effective concentrations.
  • More directed compounds like cyclin dependent kinase inhibitors (Alsterpaullone, 2-Cyanoethyl; SU9516; Alsterpaullone; or Cdkl/2 Inhibitor III) induce MAFA with less toxicity than a broad spectrum inhibitor like staurosporine.

Abstract

The present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of MAFA.

Description

DIFFERENTIATION OF HUMAN EMBRYONIC STEM CELLS TO THE PANCREATIC ENDOCRINE LINEAGE
[0001] The present invention claims priority to application serial number 61/110,287, filed October 31, 2008.
FIELD OF THE INVENTION
[0002] The present invention provides methods to promote the differentiation of pluripotent stem cells. In particular, the present invention provides a method to increase the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage.
BACKGROUND
[0003] Advances in cell-replacement therapy for Type I diabetes mellitus and a shortage of transplantable islets of Langerhans have focused interest on developing sources of insulin-producing cells, or β cells, appropriate for engraftment. One approach is the generation of functional β cells from pluripotent stem cells, such as, for example, embryonic stem cells.
[0004] In vertebrate embryonic development, a pluripotent cell gives rise to a group of cells comprising three germ layers (ectoderm, mesoderm, and endoderm) in a process known as gastrulation. Tissues such as, for example, thyroid, thymus, pancreas, gut, and liver, will develop from the endoderm, via an intermediate stage. The intermediate stage in this process is the formation of definitive endoderm. Definitive endoderm cells express a number of markers, such as, HNF-3 beta, GAT A4, MIXLl, CXCR4 and SOX 17.
[0005] Formation of the pancreas arises from the differentiation of definitive endoderm into pancreatic endoderm. Cells of the pancreatic endoderm express the pancreatic- duodenal homeobox gene, PDXl. In the absence of PDXl, the pancreas fails to develop beyond the formation of ventral and dorsal buds. Thus, PDXl expression marks a critical step in pancreatic organogenesis. The mature pancreas contains, among other cell types, exocrine tissue and endocrine tissue. Exocrine and endocrine tissues arise from the differentiation of pancreatic endoderm. [0006] Cells bearing the features of islet cells have reportedly been derived from embryonic cells of the mouse. For example, Lumelsky et al. (Science 292: 1389, 2001) report differentiation of mouse embryonic stem cells to insulin-secreting structures similar to pancreatic islets. Soria et al. (Diabetes 49: 157, 2000) report that insulin-secreting cells derived from mouse embryonic stem cells normalize glycemia in streptozotocin- induced diabetic mice.
[0007] In one example, Hori et al. (PNAS 99: 16105, 2002) disclose that treatment of mouse embryonic stem cells with inhibitors of phosphoinositide 3-kinase (LY294002) produced cells that resembled β cells.
[0008] In another example, Blyszczuk et al. (PNAS 100:998, 2003) reports the generation of insulin-producing cells from mouse embryonic stem cells constitutively expressing Pax4.
[0009] Micallef et al. reports that retinoic acid can regulate the commitment of embryonic stem cells to form Pdxl positive pancreatic endoderm. Retinoic acid is most effective at inducing Pdxl expression when added to cultures at day 4 of embryonic stem cell differentiation during a period corresponding to the end of gastrulation in the embryo (Diabetes 54:301, 2005).
[0010] Miyazaki et al. reports a mouse embryonic stem cell line over-expressing Pdxl. Their results show that exogenous Pdxl expression clearly enhanced the expression of insulin, somatostatin, glucokinase, neurogenic, P48, Pax6, and HNF6 genes in the resulting differentiated cells (Diabetes 53: 1030, 2004).
[0011] Skoudy et al. reports that activin A (a member of the TGF-β superfamily) upregulates the expression of exocrine pancreatic genes (p48 and amylase) and endocrine genes (Pdxl, insulin, and glucagon) in mouse embryonic stem cells. The maximal effect was observed using InM activin A. They also observed that the expression level of insulin and Pdxl mRNA was not affected by retinoic acid; however, 3nM FGF7 treatment resulted in an increased level of the transcript for Pdxl (Biochem. J. 379: 749, 2004).
[0012] Shiraki et al. studied the effects of growth factors that specifically enhance differentiation of embryonic stem cells into Pdxl positive cells. They observed that TGF-β2 reproducibly yielded a higher proportion of Pdxl positive cells (Genes Cells. 2005 Jun; 10(6): 503-16.).
[0013] Gordon et al. demonstrated the induction of brachyury+/HNF-3beta+ endoderm cells from mouse embryonic stem cells in the absence of serum and in the presence of activin along with an inhibitor of Wnt signaling (US 2006/0003446A1).
[0014] Gordon et al (PNAS, VoI 103, page 16806, 2006) states "Wnt and TGF -beta/ nodal/ activin signaling simultaneously were required for the generation of the anterior primitive streak".
[0015] However, the mouse model of embryonic stem cell development may not exactly mimic the developmental program in higher mammals, such as, for example, humans.
[0016] Thomson et al. isolated embryonic stem cells from human blastocysts (Science
282: 114, 1998). Concurrently, Gearhart and coworkers derived human embryonic germ (hEG) cell lines from fetal gonadal tissue (Shamblott et al., Proc. Natl. Acad. Sci. USA 95: 13726, 1998). Unlike mouse embryonic stem cells, which can be prevented from differentiating simply by culturing with Leukemia Inhibitory Factor (LIF), human embryonic stem cells must be maintained under very special conditions (U.S. Pat. No. 6,200,806; WO 99/20741; WO 01/51616).
[0017] D'Amour et al. describes the production of enriched cultures of human embryonic stem cell-derived definitive endoderm in the presence of a high concentration of activin and low serum (Nature Biotechnology 2005). Transplanting these cells under the kidney capsule of mice resulted in differentiation into more mature cells with characteristics of some endodermal organs. Human embryonic stem cell-derived definitive endoderm cells can be further differentiated into Pdxl positive cells after addition of FGF-10 (US 2005/0266554A1).
[0018] D'Amour et al. (Nature Biotechnology - 24, 1392 - 1401 (2006)) states: "We have developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursor en route to cells that express endocrine hormones".
[0019] In another example, Fisk et al. reports a system for producing pancreatic islet cells from human embryonic stem cells (US2006/0040387A1). In this case, the differentiation pathway was divided into three stages. Human embryonic stem cells were first differentiated to 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 generate Pdxl positive cells. The terminal differentiation was induced by nicotinamide.
[0020] In one example, Benvenistry et al. states: "We conclude that over-expression of Pdxl enhanced expression of pancreatic enriched genes, induction of insulin expression may require additional signals that are only present in vivo" (Benvenistry et al, Stem Cells 2006; 24: 1923-1930).
[0021] Cyclins have been implicated in beta cell function. For example, Lilja et al report that Cdk5 is present in the insulin-secreting pancreatic β-cell (J. Biol. Chem., Vol. 276, Issue 36, 34199-34205, September 7, 2001). Lilja et al states "Cdk5 is present in β- cells and acts as a positive regulator of insulin exocytosis."
[0022] In another example, Marzo et al states "Cdk4 knockin mice have significantly increased beta cell mass and are physiologically functional, indicating that Cdk4 is a potential target for pancreatic beta cell mass regeneration in Type 1 diabetes" (Diabetalogia, Vol. 47, Number 4, 686-694, April 1, 2004.)
[0023] In another example, Ubeda et al report that inhibition of cyclin-dependant kinase 5 activity protects pancreatic beta cells from glucotoxicity (J. Biol. Chem., Vol. 281, Issue 39, 28858-28864, September 29, 2006).
[0024] In another example, Wei et al report Cdk5-dependent regulation of glucose-stimulated insulin secretion (Nature Medicine 11, 1104-1108 (1 October 2005.)
[0025] In another example, Vanderford et al state "MafA is a basic leucine zipper transcription factor expressed within the beta cells of the pancreas and is required to maintain normal glucose homeostasis as it is involved in various aspects of beta cell biology. MafA protein levels are known to increase in response to high glucose through mechanisms that have yet to be fully characterized. We investigated whether discrete intracellular signaling events control mafA expression. We found that the general kinase inhibitor staurosporine induces mafA expression without altering the stability of the protein. Inhibition of the MAP -kinase JNK mimics the effects of staurosporine on the expression of mafA. Calmodulin kinase and calcium signaling are also important in stimulating mafA expression by high glucose. However, staurosporine, JNK, and calmodulin kinase have different effects on the induction of insulin expression. These data reveal that MafA levels are tightly controlled by the coordinated action of multiple kinase pathways." (Archives of Biochemistry and Biophysics (2008), doi: 10.1016/j.abb.2008.10.001).
[0026] Therefore, there still remains a significant need to develop methods for differentiating pluripotent stem cells into pancreatic endocrine cells, pancreatic hormone expressing cells, or pancreatic hormone secreting cells. The present invention provides methods to increase the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage.
SUMMARY
[0027] In one embodiment, the present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of MAFA.
BRIEF DESCRIPTION OF THE DRAWINGS
[0028] Figure 1 , panel a shows the effect of compounds from the EMD Calbiochem kinase inhibitor library on the ratio of insulin to glucagon expression in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR. The alphanumeric label corresponds to the compound identity as shown in Table 1. Panel b shows the effect of compounds from the EMD Calbiochem kinase inhibitor library on the ratio of MAFA to ARX4 expression in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR. The alphanumeric label corresponds to the compound identity as shown in Table 1.
[0029] Figure 2 A) shows a 4x micrograph of cells treated according to the methods described in Example 1, at day 4 of the stage 6 treatment. B) shows a 4x micrograph of cells treated with 0.5μM of the compound PubChemID# 5330812 at day 4 of treatment. C) shows a 4x micrograph of cells treated with 1 μM of the compound PubChemID# 5330812 at day 4 of treatment. D) shows a 2Ox micrograph of cells treated according to the methods described in Example 1, at day 6 of the stage 6 treatment. E) shows a 2Ox micrograph of cells treated with 0.5μM of the compound PubChemID# 5330812 at day 6 of treatment. F) shows a 2Ox micrograph of cells treated with lμM of the compound PubChemID# 5330812 at day 6 of treatment.
[0030] Figure 3 shows the expression of the 23 genes indicated, in cells expressing markers characteristic of the pancreatic endocrine lineage following a five-day treatment of 0.5μM (dark bars) or l.OμM (light bars) of the compound PubChem ID#5330812. Expression levels were determined at day 0, day 2 and day 5.
[0031] Figure 4 shows the effect of CDK inhibitor III treatment on the expression of markers characteristic of the pancreatic endocrine lineage in cells treated with Stage 7 of the differentiation protocol described in Example 4.
[0032] Figure 5 shows the effect of CDK inhibitor III treatment on the dithazone staining of islet-like clusters.
[0033] Figure 6 shows the expression of insulin, synaptophysin and glucagon in insulin- producing cells produced according to the methods described in Example 5. Expression of the proteins indicated was determined by FACS.
[0034] Figure 7 shows the expression of insulin, synaptophysin and glucagon in insulin- producing cells produced according to the methods described in Example 5. Expression of the proteins indicated was determined by FACS.
[0035] Figure 8 shows the expression of MAFA (Panel a) and insulin (Panel b), in insulin- producing cells, produced by the methods of the present invention. Samples of cells were taken for PCR analysis at days 1, 2, 3, and 4. Following 4 days of treatment with CDK inhibitor, the CDK inhibitor was removed from culture and the cells were cultured additional 4 days in DMEM-F12 + 1% B27 + 20 ng/ml of activin A. At the end of the four days, samples were collected in triplicate for PCR analysis.
[0036] Figure 9 shows the effect of compounds from the EMD Calbiochem kinase inhibitor library I on the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR.
[0037] Figure 10 shows the effect of genestein on the mRNA expression of insulin, glucagon, somatostatin and MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage, as determined by real-time PCR.
DETAILED DESCRIPTION
[0038] For clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the following subsections that describe or illustrate certain features, embodiments or applications of the present invention.
Definitions
[0039] Stem cells are undifferentiated cells defined by their ability at the single cell level to both self-renew and differentiate to produce progeny cells, including self-renewing progenitors, non-renewing progenitors, and terminally differentiated cells. Stem cells are also characterized by their ability to differentiate in vitro into functional cells of various cell lineages from multiple germ layers (endoderm, mesoderm and ectoderm), as well as to give rise to tissues of multiple germ layers following transplantation and to contribute substantially to most, if not all, tissues following injection into blastocysts.
[0040] Stem cells are classified by their developmental potential as: (1) totipotent, meaning able to give rise to all embryonic and extraembryonic cell types; (2) pluripotent, meaning able to give rise to all embryonic cell types; (3) multipotent, meaning able to give rise to a subset of cell lineages but all within a particular tissue, organ, or physiological system (for example, hematopoietic stem cells (HSC) can produce progeny that include HSC (self- renewal), blood cell restricted oligopotent progenitors, and all cell types and elements (e.g., platelets) that are normal components of the blood); (4) oligopotent, meaning able to give rise to a more restricted subset of cell lineages than multipotent stem cells; and (5) unipotent, meaning able to give rise to a single cell lineage (e.g. , spermatogenic stem cells).
[0041] Differentiation is the process by which an unspecialized ("uncommitted") or less specialized cell acquires the features of a specialized cell such as, for example, a nerve cell or a muscle cell. A differentiated or differentiation-induced cell is one that has taken on a more specialized ("committed") position within the lineage of a cell. The term "committed", when applied to the process of differentiation, refers to a cell that has proceeded in the differentiation pathway to a point where, under normal circumstances, it will continue to differentiate into a specific cell type or subset of cell types, and cannot, under normal circumstances, differentiate into a different cell type or revert to a less differentiated cell type. De-differentiation refers to the process by which a cell reverts to a less specialized (or committed) position within the lineage of a cell. As used herein, the lineage of a cell defines the heredity of the cell, i.e., which cells it came from and what cells it can give rise to. The lineage of a cell places the cell within a hereditary scheme of development and differentiation. A lineage- specific marker refers to a characteristic specifically associated with the phenotype of cells of a lineage of interest and can be used to assess the differentiation of an uncommitted cell to the lineage of interest.
[0042] "β-cell lineage" refers to cells with positive gene expression for the transcription factor PDX-I and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISLl, HNF3 beta, MAFA, PAX4, or PAX6. Cells expressing markers characteristic of the β cell lineage include β cells.
[0043] "Cells expressing markers characteristic of the definitive endoderm lineage", as used herein, refers to cells expressing at least one of the following markers: SOX17, GATA4, HNF3 beta, GSC, CERl, Nodal, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF 17, GATA6, CXCR4, C- Kit, CD99, or OTX2. Cells expressing markers characteristic of the definitive endoderm lineage include primitive streak precursor cells, primitive streak cells, mesendoderm cells and definitive endoderm cells. [0044] "Cells expressing markers characteristic of the pancreatic endoderm lineage", as used herein, refers to cells expressing at least one of the following markers: PDXl, HNFl beta, PTFl alpha, FTNF-, or HB9. Cells expressing markers characteristic of the pancreatic endoderm lineage include pancreatic endoderm cells, primitive gut tube cells, and posterior foregut cells.
[0045] "Cells expressing markers characteristic of the pancreatic endocrine lineage", as used herein, refers to cells expressing at least one of the following markers: NGN3, NEUROD, ISLl, PDXl, NKX6.1, PAX4, NGN3, or PTFl alpha. Cells expressing markers characteristic of the pancreatic endocrine lineage include pancreatic endocrine cells, pancreatic hormone expressing cells, and pancreatic hormone secreting cells, and cells of the β-cell lineage.
[0046] "Definitive endoderm", as used herein, refers to cells which bear the characteristics of cells arising from the epiblast during gastrulation and which form the gastrointestinal tract and its derivatives. Definitive endoderm cells express the following markers: HNF3 beta, GATA4, SOX17, Cerberus, OTX2, goosecoid, C-Kit, CD99, or MIXLl.
[0047] "Extraembryonic endoderm", as used herein, refers to a population of cells expressing at least one of the following markers: SOX7, AFP, or SPARC.
[0048] "Markers", as used herein, are nucleic acid or polypeptide molecules that are differentially expressed in a cell of interest. In this context, differential expression means an increased level for a positive marker and a decreased level for a negative marker. 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 cell of interest can be identified and distinguished from other cells using any of a variety of methods known in the art.
[0049] "Mesendoderm cell", as used herein, refers to a cell expressing at least one of the following markers: CD48, eomesodermin (EOMES), SOX 17, DKK4, HNF3 beta, GSC, FGF 17, or GATAό.
[0050] "Pancreatic endocrine cell", or "pancreatic hormone expressing cell", as used herein, refers to a cell capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide. [0051] "Pancreatic endoderm cell", as used herein, refers to a cell capable of expressing at least one of the following markers: NGN3, NEUROD, ISLl, PDXl, PAX4, or
NKX2.2.
[0052] "Pancreatic hormone producing cell", as used herein, refers to a cell capable of producing at least one of the following hormones: insulin, glucagon, somatostatin, or pancreatic polypeptide.
[0053] "Pancreatic hormone secreting cell" as used herein, refers to a cell capable of secreting at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide.
[0054] "Posterior foregut cell", as used herein, refers to a cell capable of secreting at least one of the following markers: PDXl, HNFl, PTFl alpha, HNF6, HB9, or PROXl.
[0055] "Pre-primitive streak cell", as used herein, refers to a cell expressing at least one of the following markers: Nodal, or FGF8.
[0056] "Primitive gut tube cell", as used herein, refers to a cell capable of secreting at least one of the following markers: FTNFl, or HNF 4A.
[0057] "Primitive streak cell", as used herein, refers to a cell expressing at least one of the following markers: Brachyury, Mix-like homeobox protein, or FGF4.
Isolation, Expansion and Culture of Pluripotent Stem Cells
Characterization of Pluripotent Stem Cells
[0058] Pluripotent stem cells may express one or more of the stage-specific embryonic antigens (SSEA) 3 and 4, and markers detectable using antibodies designated Tra-1- 60 and Tra-1-81 (Thomson et ah, Science 282: 1145, 1998). Differentiation of pluripotent stem cells in vitro results in the loss of SSEA-4, Tra- 1-60, and Tra-1-81 expression (if present) and increased expression of SSEA-I. Undifferentiated pluripotent stem cells typically have alkaline phosphatase activity, which can be detected by fixing the 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.
[0059] Another desirable phenotype of propagated pluripotent stem cells is a potential to differentiate into cells of all three germinal layers: endoderm, mesoderm, and ectoderm tissues. Pluripotency of pluripotent stem cells can be confirmed, for example, by injecting cells into severe combined immunodeficient (SCID) mice, fixing the teratomas that form using 4% paraformaldehyde, and then examining them histologically for evidence of cell types from the three germ layers. Alternatively, pluripotency may be determined by the creation of embryoid bodies and assessing the embryoid bodies for the presence of markers associated with the three germinal layers.
[0060] Propagated pluripotent stem cell lines may be karyotyped using a standard G-banding technique and compared to published karyotypes of the corresponding primate species. It is desirable to obtain cells that have a "normal karyotype," which means that the cells are euploid, wherein all human chromosomes are present and not noticeably altered.
Sources of Pluripotent Stem Cells
[0061] The types of pluripotent stem cells that may be used include established lines of pluripotent cells derived from tissue formed after gestation, including pre-embryonic tissue (such as, for example, a blastocyst), embryonic tissue, or fetal tissue taken any time during gestation, typically but not necessarily before approximately 10-12 weeks gestation. Non-limiting examples are established lines of human embryonic stem cells or human embryonic germ cells, such as, for example the human embryonic stem cell lines Hl, H7, and H9 (WiCeIl). Also contemplated is use of the compositions of this disclosure 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. Also suitable are cells taken from a pluripotent stem cell population already cultured in the absence of feeder cells. Also suitable are mutant human embryonic stem cell lines, such as, for example, BGOIv (BresaGen, Athens, GA). [0062] In one embodiment, human embryonic stem cells are prepared as described by
Thomson et al. (U.S. Pat. No. 5,843,780; Science 282: 1145, 1998; Curr. Top. Dev. Biol. 38: 133 ff., 1998; Proc. Natl. Acad. ScL U.S.A. 92:7844, 1995).
Culture of Pluripotent Stem Cells
[0063] In one embodiment, pluripotent stem cells are typically cultured on a layer of feeder cells that support the pluripotent stem cells in various ways. Alternatively, pluripotent stem cells are cultured in a culture system that is essentially free of feeder cells, but nonetheless supports proliferation of pluripotent stem cells without undergoing substantial differentiation. The growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a medium conditioned by culturing previously with another cell type. Alternatively, the growth of pluripotent stem cells in feeder-free culture without differentiation is supported using a chemically defined medium.
[0064] For example, Reubinoff et al (Nature Biotechnology 18: 399 - 404 (2000)) and
Thompson et al (Science 6 November 1998: Vol. 282. no. 5391, pp. 1145 - 1147) disclose the culture of pluripotent stem cell lines from human blastocysts using a mouse embryonic fibroblast feeder cell layer.
[0065] Richards et al, (Stem Cells 21 : 546-556, 2003) evaluated a panel of 11 different human adult, fetal and neonatal feeder cell layers for their ability to support human pluripotent stem cell culture. Richards et al, states: "human embryonic stem cell lines cultured on adult skin fibroblast feeders retain human embryonic stem cell morphology and remain pluripotent".
[0066] US20020072117 discloses cell lines that produce media that support the growth of primate pluripotent stem cells in feeder-free culture. The cell lines employed are mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. US20020072117 also discloses the use of the cell lines as a primary feeder cell layer.
[0067] In another example, Wang et al (Stem Cells 23: 1221-1227, 2005) discloses methods for the long-term growth of human pluripotent stem cells on feeder cell layers derived from human embryonic stem cells. [0068] In another example, Stojkovic et al (Stem Cells 2005 23 : 306-314, 2005) disclose a feeder cell system derived from the spontaneous differentiation of human embryonic stem cells.
[0069] In a further example, Miyamoto et al (Stem Cells 22: 433-440, 2004) disclose a source of feeder cells obtained from human placenta.
[0070] Amit et al (Biol. Reprod 68: 2150-2156, 2003) discloses a feeder cell layer derived from human foreskin.
[0071] In another example, Inzunza et al (Stem Cells 23 : 544-549, 2005) disclose a feeder cell layer from human postnatal foreskin fibroblasts.
[0072] US6642048 discloses media that support the growth of primate pluripotent stem (pPS) cells in feeder- free culture, and cell lines useful for production of such media. US6642048 states: "This invention includes mesenchymal and fibroblast-like cell lines obtained from embryonic tissue or differentiated from embryonic stem cells. Methods for deriving such cell lines, processing media, and growing stem cells using the conditioned media are described and illustrated in this disclosure."
[0073] In another example, WO2005014799 discloses conditioned medium for the maintenance, proliferation and differentiation of mammalian cells. WO2005014799 states: "The culture medium produced in accordance with the present invention is conditioned by the cell secretion activity of murine cells, in particular, those differentiated and immortalized transgenic hepatocytes, named MMH (Met Murine Hepatocyte)."
[0074] In another example, Xu et al (Stem Cells 22: 972-980, 2004) discloses conditioned medium obtained from human embryonic stem cell derivatives that have been genetically modified to over express human telomerase reverse transcriptase.
[0075] In another example, US20070010011 discloses a chemically defined culture medium for the maintenance of pluripotent stem cells.
[0076] An alternative culture system employs serum- free medium supplemented with growth factors capable of promoting the proliferation of embryonic stem cells. For example, Cheon et al (BioReprod DOI: 10.1095/biolreprod.105.046870, October 19, 2005) disclose a feeder- free, serum- free culture system in which embryonic stem cells are maintained in unconditioned serum replacement (SR) medium supplemented with different growth factors capable of triggering embryonic stem cell self-renewal.
[0077] In another example, Levenstein et al (Stem Cells 24: 568-574, 2006) disclose methods for the long-term culture of human embryonic stem cells in the absence of fibroblasts or conditioned medium, using media supplemented with bFGF.
[0078] In another example, US20050148070 discloses a method of culturing human embryonic stem cells in defined media without serum and without fibroblast feeder cells, the method comprising: culturing the stem cells in a culture medium containing albumin, amino acids, vitamins, minerals, at least one transferrin or transferrin substitute, at least one insulin or insulin substitute, the culture medium essentially free of mammalian fetal serum and containing at least about 100 ng/ml of a fibroblast growth factor capable of activating a fibroblast growth factor signaling receptor, wherein the growth factor is supplied from a source other than just a fibroblast feeder layer, the medium supported the proliferation of stem cells in an undifferentiated state without feeder cells or conditioned medium.
[0079] In another example, US20050233446 discloses a defined media useful in culturing stem cells, including undifferentiated primate primordial stem cells. In solution, the media is substantially isotonic as compared to the stem cells being cultured. In a given culture, the particular medium comprises a base medium and an amount of each of bFGF, insulin, and ascorbic acid necessary to support substantially undifferentiated growth of the primordial stem cells.
[0080] In another example, US6800480 states "In one embodiment, a cell culture medium for growing primate-derived primordial stem cells in a substantially undifferentiated state is provided which includes a low osmotic pressure, low endotoxin basic medium that is effective to support the growth of primate-derived primordial stem cells. The basic medium is combined with a nutrient serum effective to support the growth of primate- derived primordial stem cells and a substrate selected from the group consisting of feeder cells and an extracellular matrix component derived from feeder cells. The medium further includes non-essential amino acids, an anti-oxidant, and a first growth factor selected from the group consisting of nucleosides and a pyruvate salt." [0081] In another example, US20050244962 states: "In one aspect the invention provides a method of culturing primate embryonic stem cells. One cultures the stem cells in a culture essentially free of mammalian fetal serum (preferably also essentially free of any animal serum) and in the presence of fibroblast growth factor that is supplied from a source other than just a fibroblast feeder layer. In a preferred form, the fibroblast feeder layer, previously required to sustain a stem cell culture, is rendered unnecessary by the addition of sufficient fibroblast growth factor."
[0082] In a further example, WO2005065354 discloses a defined, isotonic culture medium that is essentially feeder-free and serum-free, comprising: a. a basal medium; b. an amount of bFGF sufficient to support growth of substantially undifferentiated mammalian stem cells; c. an amount of insulin sufficient to support growth of substantially undifferentiated mammalian stem cells; and d. an amount of ascorbic acid sufficient to support growth of substantially undifferentiated mammalian stem cells.
[0083] In another example, WO2005086845 discloses a method for maintenance of an undifferentiated stem cell, said method comprising exposing a stem cell to a member of the transforming growth factor-beta (TGF-β) family of proteins, a member of the fibroblast growth factor (FGF) family of proteins, or nicotinamide (NIC) in an amount sufficient to maintain the cell in an undifferentiated state for a sufficient amount of time to achieve a desired result.
[0084] The pluripotent stem cells may be plated onto a suitable culture substrate. In one embodiment, the suitable culture substrate is an extracellular matrix component, such as, for example, those derived from basement membrane or that may form part of adhesion molecule receptor-ligand couplings. In one embodiment, a the suitable culture substrate is MATRIGEL® (Becton Dickenson). MATRIGEL® is a soluble preparation from Engelbreth-Holm Swarm tumor cells that gels at room temperature to form a reconstituted basement membrane.
[0085] Other extracellular matrix components and component mixtures are suitable as an alternative. Depending on the cell type being proliferated, this may include laminin, fibronectin, proteoglycan, entactin, heparan sulfate, and the like, alone or in various combinations. [0086] The pluripotent stem cells may be plated onto the substrate in a suitable distribution and in the presence of a medium that promotes cell survival, propagation, and retention of the desirable characteristics. All these characteristics benefit from careful attention to the seeding distribution and can readily be determined by one of skill in the art.
[0087] Suitable culture media may be made from the following components, such as, for example, Dulbecco's modified Eagle's medium (DMEM), Gibco # 11965-092; Knockout Dulbecco's modified Eagle's medium (KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mM L-glutamine, Gibco # 15039-027; non-essential amino acid solution, Gibco 11140-050; β-mercaptoethanol, Sigma # M7522; human recombinant basic fibroblast growth factor (bFGF), Gibco # 13256- 029.
Formation of Pancreatic Hormone Producing Cells from Pluripotent Stem Cells
[0088] In one embodiment, the present invention provides a method for producing pancreatic hormone producing cells from pluripotent stem cells, comprising the steps of:
a. Culturing pluripotent stem cells,
b. Differentiating the pluripotent stem cells into cells expressing markers characteristic of the definitive endoderm lineage,
c. Differentiating the cells expressing markers characteristic of the definitive endoderm lineage into cells expressing markers characteristic of the pancreatic endoderm lineage, and
d. Differentiating the cells expressing markers characteristic of the pancreatic endoderm lineage into cells expressing markers characteristic of the pancreatic endocrine lineage.
[0089] Pluripotent stem cells suitable for use in the present invention include, for example, the human embryonic stem cell line H9 (NIH code: WA09), the human embryonic stem cell line Hl (NIH code: WAOl), the human embryonic stem cell line H7 (NIH code: WA07), and the human embryonic stem cell line 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, Connexin43, Connexin45, OCT4, SOX2, NANOG, hTERT, UTFl, ZFP42, SSEA3, SSEA4, Tral-60, or Tral-81.
[0090] Markers characteristic of the definitive endoderm lineage are selected from the group consisting of SOX17, GATA4, HNF3 beta, GSC, CERl, NODAL, FGF8, Brachyury, Mix-like homeobox protein, FGF4 CD48, eomesodermin (EOMES), DKK4, FGF 17, GATA6, CXCR4, C-Kit, CD99, and OTX2. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the definitive endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the definitive endoderm lineage is a primitive streak precursor cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a mesendoderm cell. In an alternate aspect, a cell expressing markers characteristic of the definitive endoderm lineage is a definitive endoderm cell.
[0091] Markers characteristic of the pancreatic endoderm lineage are selected from the group consisting of PDXl, HNFl beta, PTFl alpha, HNF6, HB9 and PROXl. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endoderm lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endoderm lineage is a pancreatic endoderm cell.
[0092] Markers characteristic of the pancreatic endocrine lineage are selected from the group consisting of NGN3, NEUROD, ISLl, PDXl, NKX6.1, PAX4, NGN3, and PTFl alpha. In one embodiment, a pancreatic endocrine cell is capable of expressing at least one of the following hormones: insulin, glucagon, somatostatin, and pancreatic polypeptide. Suitable for use in the present invention is a cell that expresses at least one of the markers characteristic of the pancreatic endocrine lineage. In one aspect of the present invention, a cell expressing markers characteristic of the pancreatic endocrine lineage is a pancreatic endocrine cell. The pancreatic endocrine cell may be a pancreatic hormone expressing cell. Alternatively, the pancreatic endocrine cell may be a pancreatic hormone secreting cell. [0093] 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 Pdxl and at least one of the following transcription factors: NGN3, NKX2.2, NKX6.1, NEUROD, ISLl, HNF3 beta, MAFA, PAX4, or PAX6. In one aspect of the present invention, a cell expressing markers characteristic of the β cell lineage is a β cell.
Formation of Cells Expressing Markers Characteristic of the Definitive
Endoderm Lineage
[0094] Pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by any method in the art or by any method proposed in this invention.
[0095] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 23, 1534 - 1541 (2005).
[0096] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in Shinozaki et al, Development 131, 1651 - 1662 (2004).
[0097] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in McLean et al, Stem Cells 25, 29 - 38 (2007).
[0098] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
[0099] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A and serum, and then culturing the cells with activin A and serum of a different concentration. An example of this method is disclosed in Nature Biotechnology 23, 1534 - 1541 (2005). [0100] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A in the absence of serum, then culturing the cells with activin A with serum of another concentration. An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2005.
[0101] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by culturing the pluripotent stem cells in medium containing activin A and a Wnt ligand in the absence of serum, then removing the Wnt ligand and culturing the cells with activin A with serum. An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
[0102] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
[0103] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
[0104] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 60/990,529.
[0105] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,889.
[0106] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,900. [0107] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,908.
[0108] For example, pluripotent stem cells may be differentiated into cells expressing markers characteristic of the definitive endoderm lineage by treating the pluripotent stem cells according to the methods disclosed in US patent application Ser. No. 61/076,915.
Differentiation of Cells Expressing Markers Characteristic of the Definitive
Endoderm Lineage
[0109] Formation of cells expressing markers characteristic of the definitive endoderm lineage may be determined by testing for the presence of the markers before and after following a particular protocol. Pluripotent stem cells typically do not express such markers. Thus, differentiation of pluripotent cells is detected when cells begin to express them.
[0110] The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the definitive endoderm lineage.
[0111] Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et ah, eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
[0112] 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. Pluripotent stem cell markers include, for example, the expression of one or more of the following: ABCG2, cripto, FOXD3, Connexin43, Connexin45, OCT4, SOX2, NANOG, hTERT, UTFl, ZFP42, SSEA3, SSEA4, Tral-60, or Tral-81.
[0113] After treating pluripotent stem cells with the methods of the present invention, the differentiated cells may be purified by exposing a 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 the definitive endoderm lineage.
Formation of Cells Expressing Markers Characteristic of the Pancreatic
Endoderm Lineage
[0114] Cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage by any method in the art or by any method proposed in this invention.
[0115] For example, cells expressing markers characteristic of the definitive endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
[0116] For example, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with a fibroblast growth factor and the hedgehog signaling pathway inhibitor KAAD-cyclopamine, then removing the medium containing the fibroblast growth factor and KAAD-cyclopamine and subsequently culturing the cells in medium containing retinoic acid, a fibroblast growth factor and KAAD-cyclopamine. An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
[0117] In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
[0118] In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage with retinoic acid and at least one fibroblast growth factor for a period of time, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
[0119] In one aspect of the present invention, cells expressing markers characteristic of the definitive endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endoderm lineage, by treating the cells expressing markers characteristic of the definitive endoderm lineage according to the methods disclosed in US patent application Ser. No. 60/990,529.
Detection of Cells Expressing Markers Characteristic of the Pancreatic Endoderm
Lineage
[0120] Markers characteristic of the pancreatic endoderm lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endoderm lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endoderm lineage. Pancreatic endoderm lineage specific markers include the expression of one or more transcription factors such as, for example, HLXB9, PTFl alpha, PDXl, HNF6, or HNFl beta.
[0121] The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endoderm lineage.
[0122] Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et ah, eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
Formation of Cells Expressing Markers Characteristic of the Pancreatic
Endocrine Lineage
[0123] Cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage by any method in the art or by any method disclosed in this invention.
[0124] For example, cells expressing markers characteristic of the pancreatic endoderm lineage may be differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage according to the methods disclosed in D'Amour et al, Nature Biotechnology 24, 1392 - 1401 (2006).
[0125] For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4, then removing the medium containing DAPT and exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-I and HGF. An example of this method is disclosed in Nature Biotechnology 24, 1392 - 1401 (2006).
[0126] For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4, then removing the medium containing exendin 4 and subsequently culturing the cells in medium containing exendin 1, IGF-I and HGF. An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
[0127] For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing DAPT and exendin 4. An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
[0128] For example, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by culturing the cells expressing markers characteristic of the pancreatic endoderm lineage in medium containing exendin 4. An example of this method is disclosed in D' Amour et al, Nature Biotechnology, 2006.
[0129] In one aspect of the present invention, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/736,908, assigned to LifeScan, Inc.
[0130] In one aspect of the present invention, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 11/779,311, assigned to LifeScan, Inc.
[0131] In one aspect of the present invention, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage with a factor that inhibits the Notch signaling pathway, according to the methods disclosed in US patent application Ser. No. 60/953,178, assigned to LifeScan, Inc.
[0132] In one aspect of the present invention, cells expressing markers characteristic of the pancreatic endoderm lineage are further differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage, by treating the cells expressing markers characteristic of the pancreatic endoderm lineage according to the methods disclosed in US patent application Ser. No. 60/990,529. [0133] In one aspect of the present invention, the present invention provides a method for increasing the expression of markers associated with the pancreatic endocrine lineage comprising treating cells expressing markers characteristic of the pancreatic endocrine lineage with medium comprising a sufficient amount of a TGF-β receptor agonist to cause an increase in expression of markers associated with the pancreatic endocrine lineage according to the methods disclosed in US patent application Ser. No. 61/110,278.
Detection of Cells Expressing Markers Characteristic of the Pancreatic Endocrine
Lineage
[0134] Markers characteristic of cells of the pancreatic endocrine lineage are well known to those skilled in the art, and additional markers characteristic of the pancreatic endocrine lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the pancreatic endocrine lineage. Pancreatic endocrine lineage specific markers include the expression of one or more transcription factors such as, for example, NGN3, NEUROD, or ISLl.
[0135] Markers characteristic of cells of the β cell lineage are well known to those skilled in the art, and additional markers characteristic of the β cell lineage continue to be identified. These markers can be used to confirm that the cells treated in accordance with the present invention have differentiated to acquire the properties characteristic of the β-cell lineage, β cell lineage specific characteristics include the expression of one or more transcription factors such as, for example, PDXl, NKX2.2, NKX6.1, ISLl, PAX6, PAX4, NEUROD, HNFl beta, HNF6, HNF3 beta, or MAFA, among others. These transcription factors are well established in the art for identification of endocrine cells. See, e.g., Edlund (Nature Reviews Genetics 3: 524-632 (2002)).
[0136] The efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the pancreatic endocrine lineage. Alternatively, the efficiency of differentiation may be determined by exposing a treated cell population to an agent (such as an antibody) that specifically recognizes a protein marker expressed by cells expressing markers characteristic of the β cell lineage.
[0137] Methods for assessing expression of protein and nucleic acid markers in cultured or isolated cells are standard in the art. These include quantitative reverse transcriptase polymerase chain reaction (RT-PCR), Northern blots, in situ hybridization (see, e.g., Current Protocols in Molecular Biology (Ausubel et ah, eds. 2001 supplement)), and immunoassays such as immunohistochemical analysis of sectioned material, Western blotting, and for markers that are accessible in intact cells, flow cytometry analysis (FACS) (see, e.g., Harlow and Lane, Using Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press (1998)).
[0138] In one aspect of the present invention, the efficiency of differentiation is determined by measuring the percentage of insulin positive cells in a given cell culture following treatment. In one embodiment, the methods of the present invention produce about 100% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 90% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 80% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 70% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 60% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 50% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 40% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 30% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 20% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 10% insulin positive cells in a given culture. In an alternate embodiment, the methods of the present invention produce about 5% insulin positive cells in a given culture.
[0139] In one aspect of the present invention, the efficiency of differentiation is determined by measuring glucose-stimulated insulin secretion, as detected by measuring the amount of C-peptide released by the cells. In one embodiment, cells produced by the methods of the present invention produce about lOOOng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 900ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 800ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 700ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 600ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 500ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 400ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 500ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 400ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 300ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 200ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about lOOng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 90ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 80ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 70ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 60ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 50ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 40ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 30ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about 20ng C-peptide/pg DNA. In an alternate embodiment, cells produced by the methods of the present invention produce about IOng C-peptide/pg DNA. Increasing Expression of MAFA in Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
[0140] In one embodiment, the present invention provides a method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin-dependant kinase inhibitor to cause an increase in expression of MAFA.
[0141] The cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 1.
Alternatively, the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 2. Alternatively, the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 4. Alternatively, the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 5. Alternatively, the cyclin dependant kinase inhibitor may inhibit cyclin dependant kinase 9. Alternatively, the cyclin dependant kinase inhibitor may inhibit multiple isoforms of cyclin dependant kinase, in any combination thereof.
[0142] The cyclin dependant kinase inhibitor may be a protein. Alternatively, the cyclin dependant kinase inhibitor may be a peptide. Alternatively, the cyclin dependant kinase inhibitor may be a small molecule. In one embodiment, the small molecule cyclin dependant kinase inhibitor is selected from the group consisting of 7-n-Butyl- 6-(4-hydroxyphenyl)[5H]pyrrolo[2,3-b]pyrazine, 9-Nitro-7,12-dihydroindolo[3,2- d][l]benzazepin-6(5H)-one, 3-(6-Oxo-9-nitro-5,6,7,12-tetrahydroindolo[3,2- d][l]benzazepin-2-yl)propionitrile, (2R)-2-((6-((3-Amino-5-chlorophenyl)amino)-9- (l-methylethyl)-9H-purin-2-yl)amino)-3-methyl-l-butanol, Arcyriaflavin A, [6- Benzylamino-2-(3-hydroxypropylamino)-9-isopropylpurine, Butyrolactone I, (Z)-I- (3-Ethyl-5-methoxy-2,3-dihydrobenzothiazol-2-ylidene)propan-2-one, 2-(3- Hydroxypropylamino)-6-(o-hydroxybenzylamino)-9-isopropylpurine, l-(2,6- Dichlorophenyl)-l,5-dihydro-6-((4-(2 -hydroxy ethoxy )phenyl)methyl)-3-(l- methylethyl)-4H-pyrazolo[3,4-d]pyrimidin-4-one, Cdk/ Cyclin Inhibitory Peptide III, 3-(2-Chloro-3-indolylmethylene)-l,3-dihydroindol-2-one, Ethyl-(6-hydroxy-4- phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate, RO-3306, N-(cis-2- Aminocyclohexyl)-N-(3-chlorophenyl)-9-ethyl-9H-purine-2,6-diamine, 6- Cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine, 5-Amino-3-((4- (aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)- IH- 1 ,2,4-triazole- 1 - carbothioamide, 3-Amino-lH-pyrazolo[3,4-b]quinoxaline, Cdk2 Inhibitor I, Cdk2 Inhibitor II, 2(bis-(Hydroxyethyl)amino)-6-(4-methoxybenzylamino)-9- isopropylpurine, 4-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)-N,N- diethylbenzamide, N4-(6-Aminopyrimidin-4-yl)-sulfanilamide, (4-(2-Amino-4- methylthiazol-5-yl)pyrimidin-2-yl)-(3-nitrophenyl)amine, 2-Bromo-12,13-dihydro- 5H-indolo[2,3-a]pyrrolo[3,4-c]carbazole-5,7(6H)-dione, 1,4-Dimethoxyacridine- 9(10H)-thione, 5-(N-(4-Methylphenyl)amino)-2-methyl-4,7-dioxobenzothiazole, A- (3,5-Diamino-lHpyrazol-4-ylazo)-phenol, 2-(2-Hydroxyethylamino)-6-(3- chloroanilino)-9-isopropylpurine, Fascaplysin, Indirubin-3'-monoxime, Indirubin-3 '- monoxime, 5-Iodo-, Indirubin-3 '-monoxime-5-sulphonic Acid, Isogranulatimide, 2- (2-Hydroxyethylamino)-6-benzylamino-9-methylpurine, 6-(2-Hydroxybenzylamino)- 2-((lR)-(hydroxymethyl)propyl)amino)-9-isopropylpurine, 5-Bromo-3-(2-(4- Fluorophenyl)-2-oxoethylidine)-l,3-dihydroindol-2-one, N6,N6-Dimethyladenine, 2- (lR-Isopropyl-2-hydroxyethylamino)-6-(3-chloroanilino)-9-isopropyl-purine, rapamycin, 2-(R)-( 1 -Ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine, Scytonemin, 3 - [ 1 -(3 H-Imidazol-4-yl)-meth-(Z)-ylidene] -5-methoxy- 1,3- dihydroindol-2-one, and 4-(3 '-Hydroxyphenyl)amino-6,7-dimethoxyquinazoline.
[0143] In one embodiment, the cyclin dependant kinase is ethyl-(6-hydroxy-4- phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate. In one embodiment, ethyl-(6- hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-3-carboxylate is added to cells expressing markers characteristic of the endocrine lineage at a concentration from about 0.1 μM to about lOμM for about one to seven days.
[0144] In one embodiment, cells expressing markers characteristic of the endocrine lineage are treated with ethyl-(6-hydroxy-4-phenylbenzo[4,5]furo[2,3-b])pyridine-3- carboxylate for about one to about seven days.
[0145] The present invention is further illustrated, but not limited by, the following examples.
EXAMPLES
Example 1 Differentiation of Human Embryonic Stem Cells of the Cell Line Hl to Pancreatic Endocrine Cells in the Absence of Fetal Bovine Serum
[0146] Cells of the human embryonic stem cells line Hl at passage 52 were cultured on MATRIGEL®-coated dishes (1 :30 dilution) and exposed to the following differentiation protocol, in order to differentiate the cells to cells expressing markers characteristic of the pancreatic endocrine lineage.
a. RPMI medium supplemented with 2% BSA (Catalog# 152401 , MP Biomedical, Ohio), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days (Stage 1), then
b. DMEM/F12 + 2% BSA + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD (#239804, Calbiochem, CA) for two days (Stage T), then
c. DMEM/F12 + 1% B27 (Invitrogen, CA) + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD + 2 μM Retinoic acid (RA) (Sigma, MO) + 100 ng/ml of Noggin (R & D Systems, MN) for four days (Stage 3), then
d. DMEM/F12 + 1% B27 (Invitrogen, CA) + 100 ng/ml Noggin + 1 μM DAPT (a gamma-secretase inhibitor) (Catalog# 565784, Calbiochem, CA) + 1 μM ALK5 inhibitor II (Catalog# 616452, Calbiochem, Ca) + 100 ng/ml of Netrin- 4 (R&D Systems, MN) for three days (Stage 4), then
e. DMEM/F12 + 1% B27 (Invitrogen, CA) + 1 μM ALK5 inhibitor II (Calbiochem, Ca) for seven days (Stage 5).
[0147] Medium was changed daily. At each stage the cell number was calculated using a hemocytometer and RNA was collected for PCR analysis. All samples were collected in triplicate. Example 2
Screening of the Effects of Compounds from the EMD Kinase Inhibitor Library II on Cells that have been Treated according to the Differentiation Protocol
Outlined in Example 1
[0148] Cells of the human embryonic stem cell line Hl at passage 44 were seeded onto
MATRIGEL™ coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 1 up to stage 5. Following this, the cells were treated for four days in DMEM/F12 + 1% B27 containing a compound from an EMD Calbiochem compound library (Catalog# 539745, Calbiochem, San Diego, Ca) at a final concentration of 1 μM. Wells containing vehicle were included as a control. Throughout the protocol media was changed daily. All samples were treated in duplicate. At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of insulin, glucagon, MAFA, and Arx4. Results are expressed as a ratio insulin/glucagon (Figure 1, panel a), or MAFA versus ARX4 (Figure 1, panel b) of the treated samples relative to the untreated control, as measured by real-time PCR. The corresponding PubChem Compound ID# for each well # is listed in Table 1.
[0149] Treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with compounds A6, B7, B8, or C2 at a lμM concentration resulted in an insulin/ glucagon expression ratio of approximately 3.0 or higher (see Figure 1, panel a).
[0150] We next examined the effect of these compounds on the ratio of MAFA/ARX4, and we observed that treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with several of the compounds resulted in a much greater change in the ratio of MAFA to ARX4 than other compounds tested in the library: Cells treated with compound C2 showed a ratio of MAFA/ARX4 of approximately 1000. Treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with compound C2 resulted in a MAFA/ARX4 ratio of approximately 100. (See Figure 1, panel b). Example 3
The Effects of Cyclin Dependant Kinase Inhibitor Treatment on Insulin and
MAFA Expression in Cells that have been Treated according to the
Differentiation Protocol Outlined in Example 1
[0151] Several of the compounds that increased the ratio of insulin to glucagon expression, or MAFA to ARX4 expression in Example 2 were cyclin dependant kinase inhibitors. One such compound was PubChem Compound ID# 5330797 (5-Amino-3-((4- (aminosulfonyl)phenyl)amino)-N-(2,6-difluorophenyl)-lH-l,2,4-triazole-l- carbothioamide) (Catalogue # 217714; Calbiochem, San Diego, Ca). To confirm these observations, cells of the human embryonic stem cell line Hl at passage 42 were cultured in 10cm2 MATRIGEL®-coated dishes and treated according to the methods described in Example 1 up to stage 5. After stage 5, the cells were treated with DMEM/F12 containing 1% B27 containing lμM PubChem Compound ID# 5330797 for six days. Medium was changed every other day. Samples of cells were taken for real-time PCR prior to treatment with the compound, and at days two and five of compound treatment.
[0152] Characteristic micrographs of the cells at day 4 or day 6 of compound treatment versus untreated controls are shown in Figure 2. Untreated cells are highly packed (Figure 3, panels a and d) and it is difficult to distinguish individual cells. However, after treatment with 0.5 μM or lμM of PubChem Compound ID#5330797 for six days, individual nuclei became visible (Figures 2, panels e and f) as compared to the untreated control (Figure 2, panel d), indicating that there was differentiation occurring in the cell population. This was also accompanied by some cell death, which can be seen by gaps in the layer of cells as shown in Figure 2, panels b and c.
[0153] Treatment of cells with PubChem Compound ID# 5330797 resulted in the increase in expression of insulin, glucagon, MAFA, MAFB and somatostatin, albeit to differing degrees. The relative induction of gene expression per treatment as compared to day 0 (pretreatment) cultures is shown in Figures 3, panels a-v. Cells expressing markers characteristic of the pancreatic endocrine lineage that were treated with 1 μM PubChem Compound ID# 5330797 resulted in an approximately 1.5 fold increase in glucagon expression at 48 hrs of treatment. This expression declined to below pretreatment levels after 5 days of treatment. No increase in glucagon expression was observed with treatment of 0.5μM PubChem Compound ID# 5330797. (See Figure 3, panel a).
[0154] Cells expressing markers characteristic of the pancreatic endocrine lineage that were treated with lμM PubChem Compound ID# 5330797 for five days resulted in an approximately 1.5 fold increase in insulin expression. (See Figure 3, panel b).
[0155] Cells expressing markers characteristic of the pancreatic endocrine lineage that were treated with lμM PubChem Compound ID# 5330797 for five days resulted in an approximately 200 fold increase in MAFA expression. (See Figure 3, panel d).
[0156] Cells expressing markers characteristic of the pancreatic endocrine lineage that were treated with 0.5μM PubChem Compound ID# 5330797 for five days resulted in an approximately 1.5 fold increase in MAFB expression. (See Figure 3, panel c). A dose-dependent increase in the expression of somatostatin was observed (Figure 3, panel e).
[0157] No change in the expression of amylase was observed in cells expressing markers characteristic of the pancreatic endocrine lineage that were treated with PubChem Compound ID# 5330797 for five days. (See Figure 3, panel f). However, decreases in the level of expression of PAX4 (Figure 3, panel h), NKX6.1 (Figure 3, panel k), PDXl (Figure 3, panel 1), NEUROD (Figure 3, panel o), and BRN4 (Figure 3, panel q) was observed.
Example 4
Cyclin Dependant Kinase Inhibitor Treatment Increased the Expression of
MAFA in Islet-Like Clusters.
[0158] Cells of the human embryonic stem cell line Hl at passage 52 were cultured on MATRIGEL® coated dishes (1:30 dilution) and differentiated according to the methods described in Example 1. An additional stage (Stage 6) was added, in order to further mature the cells expressing markers characteristic of the pancreatic endocrine lineage. Stage 6 in this example consisted of a seven day treatment in DMEM/F12 + 1% B27 (Invitrogen, CA). The medium was changed daily. [0159] After stage 6, the cells were treated for 5 mins at room temperature with IX accutase (Sigma, MO). The accutase was removed, and DMEM/12 + 1% B27 was added to the cells. The attached cells were removed using a cell scarper and gently resuspended and passed through a 40 μm cell strainer. The cells retained on the strainer were removed by rinsing in basal media and cultured in suspension on Ultra- Low culture plates (Catalogue# 3471, Corning, Ma). The cells were then treated as follows: The cells were cultured in DMEM/F12 + 1 % B27, containing 20 ng/ml of activin A (AA), 1 μm of CDK inhibitor III (Catalog# 217714, Calbiochem, Ca) for 10 days (Stage 7). Cells treated with vehicle were included as controls. Samples were collected at days 7 through 10 for PCR analysis and dithizone staining. The cells cultured in suspension according to the methods outlined in this example assumed a morphology similar to pancreatic islet clusters. Treatment with CDK inhibitor III did not appear to affect the morphology of the islet like clusters.
[0160] Figure 4, panels a-i shows the effect of CDK inhibitor III treatment on gene expression profile of the cell clusters. Treatment with of CDK inhibitor III increased the expression of markers associated with the pancreatic endocrine lineage and in particular increased the expression of the pro-insulin transcription factor, MAFA.
[0161] Figure 5, panels a-b shows the effect of CDK inhibitor III on dithazone (DTZ) staining of clusters. Cell clusters treated with CDK inhibitor and stained with DTZ, showed a more reddish staining pattern as compared to clusters not treated with the CDK inhibitor III.
Example 5
FACS Analysis of Insulin Producing Cells Produced by the Methods of the
Present Invention.
[0162] Cells of the human embryonic stem cell line Hl at passage 42 were cultured on
MATRIGEL®-coated plates, and differentiated into insulin producing cells using the following protocol:
a. RPMI medium supplemented with 2% BSA (Catalog# 152401 , MP
Biomedical, Ohio), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days (Stage 1), then
b. DMEM/F12 + 2% BSA + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD (#239804, Calbiochem, CA) for two days (Stage 2), then
c. DMEM/F12 + 1% B27 (Invitrogen, CA) + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD + 2 μM Retinoic acid (RA) (Sigma, MO) + 100 ng/ml of Noggin (R & D Systems, MN) for four days (Stage 3), then
d. DMEM/F12 + 1% B27 (Invitrogen, CA) + 100 ng/ml Noggin + 1 μM DAPT (a gamma-secretase inhibitor) (Catalog# 565784, Calbiochem, CA) + 1 μM ALK5 inhibitor II (Catalog# 616452, Calbiochem, Ca) + 100 ng/ml of Netrin- 4 (R&D Systems, MN) for three days (Stage 4), then
e. DMEM/F12 + 1% B27 (Invitrogen, CA) + 1 μM ALK5 inhibitor II (Calbiochem, Ca) for seven days (Stage 5), then
f. DMEM/F 12 + 1 % B27 for seven days (Stage 6), then
g. Treatment with Accutase for 5 minutes, followed by scraping to remove any remaining attached cells. The cell suspension was then passed through a 40μm cell strainer. The cells retained on the strainer were removed by rinsing in basal media and cultured in suspension on Ultra-Low culture plates in DMEM-High Glucose (Catalogue# 11995-073, Invitrogen, Ca) + 1 % B27 + 20 ng/ml of activin A (AA) 1 μm of CDK inhibitor III (Catalog# 217714, Calbiochem, Ca) for 5 days (Stage 7).
Islet-like clusters were dispersed into single cells using TrypLE Express (Invitrogen, Carlsbad, CA) and washed in cold PBS. For fixation, the cells were resuspended in 200-300 μl Cytofix/Cytoperm Buffer (BD 554722, BD, Ca) and incubated for 30 min at 4°C. Cells were washed two times in 1 ml Perm/Wash Buffer Solution (BD 554723) and resuspended in 100 μl staining/blocking solution containing 2% normal goat serum in Perm/Wash buffer. For flow cytometric analysis, cells were stained with the following primary antibodies: Anti-Insulin (Rabbit mAb, Cell Signaling No. C27C9; 1: 100 dilution); Anti-Glucagon (Mouse Mab, Sigma No. G2654, 1 : 100); Anti-Synaptophysin (Rabbit Polyclonal antibody, DakoCytomation No AOOlO, 1 :50). Cells were incubated for 30 min at 4°C followed by two washes in Perm/Wash buffer and a further 30 min incubation in appropriate secondary antibodies as follows: Goat anti-Rabbit Alexa 647 (Invitrogen No. A21246) or Goat anti-Mouse 647 (Invitrogen No. A21235); Goat anti-Rabbit R-PE (BioSource No. ALI4407). All secondary antibodies were used at a 1:200 dilution. Cells were washed at least once in Perm/Wash buffer and analyzed using BD FACSArray. At least 10,000 events were acquired for analysis. Controls included undifferentiated Hl cells and the β-TC (CRL-11506™ ATCC, VA) cell line.
[0164] Figure 6, panels a-c show the percentage insulin positive, synapthophysin positive, and glucagon positive cells in cells following treatment with Stage 7, in medium containing vehicle. Figure 7, panels a-c shows the percentage insulin positive, synapthophysin positive, and glucagon positive cells following treatment with Stage 7 in medium containing 1 μM CDK inhibitor III for 5 days. The number of single hormonal insulin positive cells increased from 3% to 8% following treatment with the CDK inhibitor. Additionally, the percentage of poly hormonal (insulin and glucagon positive) cells decreased following treatment with the CDK inhibitor.
Example 6
Kinetics of CDK Inhibitor-Induced MAFA Expression.
[0165] Cells of the human embryonic stem cell line Hl at passage 42 were cultured on
MATRIGEL®-coated plates, and differentiated into insulin producing cells using the following protocol:
a. RPMI medium supplemented with 2% BSA (Catalog# 152401 , MP
Biomedical, Ohio), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days (Stage 1), then b. DMEM/F12 + 2% BSA + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD (#239804, Calbiochem, CA) for two days (Stage 2), then
c. DMEM/F12 + 1% B27 (Invitrogen, CA) + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD + 2 μM Retinoic acid (RA) (Sigma, MO) + 100 ng/ml of Noggin (R & D Systems, MN) for four days (Stage 3), then
d. DMEM/F12 + 1% B27 (Invitrogen, CA) + 100 ng/ml Noggin + 1 μM DAPT (a gamma-secretase inhibitor) (Catalog# 565784, Calbiochem, CA) + 1 μM ALK5 inhibitor II (Catalog# 616452, Calbiochem, Ca) + 100 ng/ml of Netrin- 4 (R&D Systems, MN) for three days (Stage 4), then
e. DMEM/F12 + 1% B27 (Invitrogen, CA) + 1 μM ALK5 inhibitor II (Calbiochem, Ca) for seven days (Stage 5), then
f. DMEM/F12 + 1% B27 for seven days (Stage 6), then
g. Treatment with Accutase for 5 minutes, followed by scraping to remove any remaining attached cells. The cell suspension was then passed through a 40μm cell strainer. The cells retained on the strainer were removed by rinsing in basal media and cultured in suspension on Ultra-Low culture plates in DMEM-High Glucose (Catalogue# 11995-073, Invitrogen, Ca) + 1 % B27 + 20 ng/ml of activin A (AA) 2 μm of CDK inhibitor III (Catalog# 217714, Calbiochem, Ca) for 1-8 days (Stage 7).
[0166] Samples were collected for PCR analysis at days 1, 2, 3, and 4. Following 4 days of treatment with CDK inhibitor, the CDK inhibitor was removed from culture and the cells were cultured additional 4 days in DMEM-F 12 + 1% B27 + 20 ng/ml of activin A. At the end of the four days, samples were collected in triplicate for PCR analysis.
[0167] Figure 8, panels a-b show expression pattern of MAFA and insulin at various time points of stage 7. CDK inhibitor treatment resulted in significant increase in MAFA and insulin expression which increased as a function of time. However, removal of CDK inhibitor resulted in a significant drop to both MAFA and insulin expression, in samples obtained four days after removal of the compound. Example 7
Screening of the Effects of Compounds from the BIOMOL™ Kinase Inhibitor
Library on Cells that have been Treated According to the Differentiation
Protocol Outlined in Example 1.
[0168] Cells of the human embryonic stem cell line Hl at passage 51 were seeded onto
MATRIGEL®-coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 1 up to stage 5. Following this, the cells were grown for one day in DMEM/F12 + 1% B27 and then treated for six days in DMEM/F12 + 1% B27 containing a compound from a BIOMOL™ compound library (Catalog# 2832, BIOMOL, Plymouth Meeting, Pa) at a final concentration of 4μM. Wells containing vehicle were included as a control. Throughout the treatment protocol media containing vehicle or compound was changed every other day. All samples were treated in duplicate. At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of insulin, glucagon, MAFA, and ARX4. Results are expressed as a ratio insulin/glucagon (Table 2), or MAFA versus Arx4 (Table 2) of the treated samples relative to the untreated control, as measured by real-time PCR. The corresponding catalog#, CAS#, and compound name or ID number for each alpha numeric well# is listed in Table 3.
[0169] Treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with compounds C8 or Fl at a 4μM concentration resulted in an insulin/ glucagon expression ratio of approximately 10.0 or higher. Cells treated with D9 had an insulin/ glucagon expression ratio of approximately 1840.0 (Table T).
[0170] We next examined the effect of these compounds on the ratio of MAFA/ARX4, and we observed that treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with several of the compounds resulted in a much greater change in the ratio of MAFA to ARX4 than other compounds tested in the library: Cells treated with compound B6 or Fl showed a ratio of MAFA/ARX4 of approximately greater than 10. Treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with compound C8 resulted in a MAFA/ ARX4 ratio of approximately 84, while cells treated with D9 had a MAFA/ ARX4 ratio of approximately 212. (Table 2).
Example 8
The Effect of Cyclin Dependant Kinase Inhibitors on Insulin and MAFA Expression in Cells Treated according to the Differentiation Protocol Outlined in
Example 1.
[0171] Cells of the human embryonic stem cell line Hl at passage 51 were seeded onto
MATRIGEL™ coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 1 up to stage 5. Following this, the cells were grown for eight days in DMEM/F12 + 1% B27 and then treated for four days in DMEM/F12 + 1% B27 containing a cyclin dependent kinase inhibitor at a final concentration of 0.6125, 1.25, or 5.0 μM. We tested 6 inhibitors: PubChem ID# 5330812 (EMD cat# 217714), PubChem ID# 4566 (EMD cat#217713), PubChem ID# 5330797 (EMD cat# 219476), PubChem ID# 73292 (EMD cat#341251), PubChem ID# 4592 (EMD cat#495620), and PubChem ID# 160355 (EMD cat #557360). Wells containing vehicle were included as a control. Throughout the treatment protocol media containing vehicle or compound was changed every other day. All samples were treated in duplicate. At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of insulin, glucagon, MAFA, and ARX4. Results are expressed as the fold change relative to the vehicle treated control, as measured by real-time PCR.
[0172] We observed that the compounds PubChem ID# 5330812, PubChem ID# 4566,
PubChem ID# 5330797, and PubChem ID# 73292 all stimulated MAFA expression at the concentrations tested (Table 4). PubChem ID# 4592 and PubChem ID# 160355 did not stimulate MAFA at the concentrations tested (Table 4). The compounds PubChem ID# 5330812, PubChem ID# 4566, PubChem ID# 5330797, PubChem ID# 4592 and PubChem ID# 160355 all appeared to stimulate insulin expression (Table 4). The compound PubChem ID# 5330797 reduced both glucagon and Arx4 expression (Table 4) while stimulating MAFA expression. Example 9
Differentiation of Human Embryonic Stem Cells of the Cell Line Hl to
Pancreatic Endocrine Cells with DMEM containing 25mM glucose (DMEM-
HG), Lacking Fetal Bovine Serum
[0173] Cells of the human embryonic stem cells line Hl were cultured on MATRIGEL®- coated dishes (1:30 dilution) and differentiated into cells expressing markers characteristic of the pancreatic endocrine lineage using the following protocol:
a. RPMI medium supplemented with 2% BSA (Catalog# 152401 , MP Biomedical, Ohio), and 100 ng/ml activin A (R&D Systems, MN) plus 20 ng/ml WNT-3a (Catalog# 1324-WN-002, R&D Systems, MN) plus 8 ng/ml of bFGF (Catalog# 100-18B, PeproTech, NJ), for one day followed by treatment with RPMI media supplemented with 2% BSA and 100 ng/ml activin A plus 8 ng/ml of bFGF for an additional two days (Stage 1), then
b. RPMI medium supplemented with 2% BSA + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD (#239804, Calbiochem, CA) for two days (Stage 2), then
c. DMEM-HG + 1% B27 (Invitrogen, CA) + 50 ng/ml FGF7 + 0.25 μM Cyclopamine- KAAD + 2 μM Retinoic acid (RA) (Sigma, MO) + 100 ng/ml of Noggin (R & D Systems, MN) for six days (Stage 3), then
d. DMEM-HG + 1% B27 (Invitrogen, CA) + 100 ng/ml Noggin + 1 μM ALK5 inhibitor II (Catalog# 616452, Calbiochem, Ca) for three days (Stage 4), then
e. DMEM-HG + 1% B27 (Invitrogen, CA) + 1 μM ALK5 inhibitor II (Calbiochem, Ca) for seven days (Stage 5).
[0174] Medium was changed daily. At each stage the cell number was calculated using a hemocytometer and RNA was collected for PCR analysis. All samples were collected in triplicate. Example 10
Screening of the Effects of Compounds from the EMD Kinase Inhibitor Library I on Cells Treated according to the Differentiation Protocol Outlined in Example
9
[0175] Cells of the human embryonic stem cell line Hl at passage 45 were seeded onto
MATRIGEL®-coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 9 up to stage 5. Following this, the cells were fed and treated on day 1, 3, and 5 of stage 5 with media comprising DMEM-HG, 1% B27 (Invitrogen, CA), 1 μM ALK5 inhibitor II (Calbiochem, Ca) and a compound from an EMD Calbiochem compound library 1 solubilized in DMSO (Catalog# 539744, Calbiochem, San Diego, Ca) and treated at a final concentration of 2.5μM. Wells containing vehicle were included as a control. Throughout the protocol media was changed daily except at stage 5 when media was changed every other day. All samples were treated in duplicate.
[0176] At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of MAFA. Results are expressed as the fold increase in MAFA expression versus untreated Hl human embryonic stem cells (Table 5), as measured by real-time PCR.
[0177] Treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with compounds A4 (Cat#, 124001, Akt Inhibitor IV), E8 (Cat# 527450, PKR Inhibitor), and F9 (Cat#539648, Staurosporine, N-benzoyl-) at a 2.5μM concentration resulted in an increase in MAFA expression at least 4 fold higher than vehicle treated controls (Table 5). Treatment with the compound E6 (Cat# 521233, PDGF Receptor Tyrosine Kinase Inhibitor IV) at a 2.5μM concentration resulted in an increase in MAFA expression at least 2.5 fold higher than vehicle treated controls (Table 5). Example 11
Screening of the Effects of Compounds from the EMD Kinase Inhibitor Library II on Cells Treated according to the Differentiation Protocol Outlined in
Example 9
[0178] Cells of the human embryonic stem cell line Hl at passage 46 were seeded onto
MATRIGEL®-coated 24-well dishes (1 :30 dilution), and differentiated according to the methods described in Example 9 up to stage 5. Following this, the cells were fed and treated on day 1, 3, and 5 of stage 5 with media comprising DMEM-HG, 1% B27 (Invitrogen, CA), 1 μM ALK5 inhibitor II (Calbiochem, Ca) (Stage 5) and a compound from an EMD Calbiochem compound library II solubilized in DMSO (Tables 1 and 6, Calbiochem, San Diego, Ca) and treated at a final concentration of 2.5μM. Wells containing vehicle were included as a control. Throughout the protocol media was changed daily except at stage 5 when media was changed every other day. All samples were treated in duplicate.
[0179] At the completion of this treatment RNA was collected for PCR analysis. Samples were analyzed by real-time PCR for expression of MAFA. Results for compounds that stimulated the expression of MAFA are shown and expressed as the fold increase in MAFA expression versus control samples (Figure 9), as measured by real-time PCR.
[0180] Treatment of cells expressing markers characteristic of the pancreatic endocrine lineage with either: Alsterpaullone, 2-Cyanoethyl; SU9516; Alsterpaullone; Cdkl/2 Inhibitor III; Casein Kinase I Inhibitor, D4476; or MEK1/2 Inhibitor at a 2.5μM concentration resulted in a 4.5 fold increase in MAFA expression versus untreated controls (Table 7).
Example 12
Inhibiting Cell Cycle Progression in Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage with Small Molecule Inhibitors Promotes MAFA Expression in Cells Expressing Markers Characteristic of the Pancreatic
Endocrine Lineage
[0181] Cell growth resulting from cell cycle progression can be activated and maintained by stimulating cells with extracellular growth factors. Growth factors bind to the extracellular domains of growth factor receptors, inducing a conformational switch in the receptor's intracellular domain. This shift initiates receptor dimerization and activation of tyrosine kinases located on the intracellular domain of the receptor leading to phosphorylation and activation of multiple serine/threonine kinases downstream, ultimately resulting in cell cycle progression and cell proliferation.
[0182] Under normal physiologic conditions mature pancreatic beta cells, characterized by expression of insulin and the transcription factor MAFA, are quiescent and tend to remain in GO of the cell cycle. Yet, in order to generate enough cells to form a functional organ and meet the needs of a mature animal, the cells expressing markers characteristic of the pancreatic endocrine lineage of the present invention must be cell cycling. Consequently, at some point in embryonic development, the cells expressing markers characteristic of the pancreatic endocrine lineage of the present invention differentiate to beta cells and transition from an actively cell-cycling proliferating cell, to a quiescent cell.
[0183] Our data indicate that by inhibiting cell cycle progression by blocking signaling cascades with small molecule kinase inhibitors, we can induce the cells expressing markers characteristic of the pancreatic endocrine lineage to express MAFA, a marker of mature pancreatic beta cells. Kinase inhibitors targeted to a growth factor receptor, (PDGF Receptor Tyrosine Kinase Inhibitor IV), or inhibitors which disrupt kinases downstream of tyrosine kinase receptors (MEK 1/2 Inhibitor, PKR Inhibitor, or Akt Inhibitor IV) disrupt proliferative growth factor/kinase based signaling resulting in cell cycle arrest and induction of MAFA expression. Use of a broad spectrum inhibitor like staurosporine, can effectively induce MAFA, however it is also cytotoxic at effective concentrations. More directed compounds like cyclin dependent kinase inhibitors (Alsterpaullone, 2-Cyanoethyl; SU9516; Alsterpaullone; or Cdkl/2 Inhibitor III) induce MAFA with less toxicity than a broad spectrum inhibitor like staurosporine.
[0184] In order to determine if a broad spectrum kinase inhibitor could induce MAFA expression and a more mature phenotype in the cells expressing markers characteristic of the pancreatic endocrine lineage of the present invention we differentiated Hl human ES cells according to the methods described in example 9, and treated them on days 1, 3, and 5 of stage 5 with the protein-tyrosine kinase inhibitor, Genistein, which has been shown to induce G2 phase arrest in human and murine cell lines and inhibit multiple kinases. At doses of 10 and 30ng/ml the endocrine hormones insulin, somatostatin, and the transcription factor MAFA, all showed increased expression versus untreated controls, while at 10ng/ml the endocrine hormone glucagon had increased expression (Figure 10). We observed significant toxicity at a 100ng/ml dose of genistein that correlated with loss of insulin, glucagon, and somatostatin expression.
[0185] These data indicate that by inhibiting cell cycle progression by blocking signaling cascades with small molecule kinase inhibitors targeted to inhibit signal transduction from a growth factor receptor tyrosine kinase through intracellular signaling kinases to the nucleus and cyclin dependent kinases, we can induce the cells expressing markers characteristic of the pancreatic endocrine lineage of the present invention to express MAFA, a marker of mature pancreatic beta cells.
[0186] Publications cited throughout this document are hereby incorporated by reference in their entirety. Although the various aspects of the invention have been illustrated above by reference to examples and preferred embodiments, it will be appreciated that the scope of the invention is defined not by the foregoing description but by the following claims properly construed under principles of patent law. Well PubChemlD# Well PubChemlD# Well PubChemlD# Well PubChemlD#
A2 16760529 B2 16760303 C2 5330797 D2 9797929
A3 5278396 B3 5326739 C3 3004085 D3 11493598
A4 6605258 B4 5353431 C4 481747 D4 16760417
A5 5005498 B5 2422 C5 1893668 D5 73292
A6 16760286 B6 5472558 C6 9969021 D6 4124851
A7 5326843 B7 2794188 C7 2856 D7 6539732
A8 3641059 B8 5330812 C8 6918386 D8 448014
A9 6604931 B9 438981 C9 10202471 D9 5287844
A10 11524144 B10 6419753 C10 9549301 D10 6538818
A11 9549303 B11 16760346 C11 5339183 D11 10020713
E2 5312137 F2 11382492 G2 6419739 H2 176155
E3 6419766 F3 11624601 G3 4665 H3 16219471
Table 1. AlphaNumeric Well
E4 6419741 F4 490561 G4 4713 H4 3387354 Label and the Corresponding
E5 3674 F5 3820 G5 4712 H5 5174
E6 9903786 F6 5312122 G6 5164 H6 5228 PubChem ID#forEMD
E7 6419764 F7 9951490 G7 4987 H7 16760659 Calbiochem Kinase Inhibitor
E8 8515 F8 389898 G8 9549289 H8 451705 II compound library
E9 11665831 F9 9549284 G9 5702541 H9 16760660
E10 11422035 F10 116^4425 G10 5162 H10 5289419
E11 16760525 F11 509554 G11 5353940 H11 9549300
Table 2. The Effect of compounds of the BIOMOL Inhibitor compound library on the Ratio of Insulin/glucagon and MAFA/Arx4 expression as determined by realtime PCR in Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage. The AlphaNumeric well# corresponds to the compound identity in Table 3.
Ratio vs . Control
Well # Insulin to Glucag on MAFA to Arx4
Bl 1.6 0.9
B2 2.4 1.3
B3 2.9 2.9
B4 1.1 2.0
B5 1.3 1.3
B6 1.6 16.3
B7 1.3 0.5
B8 1.6 0.5
B9 1.1 1.5
BlO 1.2 1.5
BI l 1.1 2.1
B12 1.0 2.0
Cl 0.7 0.8
C2 0.9 1.0
C3 1.3 0.9
C4 1.2 1.7
C5 1.0 1.1
C6 1.6 1.2
C7 4.3 0.2
C8 40.2 84.3
C9 0.8 0.5
ClO 2.3 1.9
CI l 1.1 0.4
C12 1.0 0.4
Dl 2.7 1.2 D2 3.4 1.3
D3 1.7 2.1
D4 5.8 6.4
D5 1.4 1.1
D6 1.8 3.9
D7 1.7 0.6
D8 2.8 5.1
D9 1842.5 212.6
DlO 0.9 1.3
DI l 1.0 0.7
D12 1.1 2.5
El 1.1 0.9
E2 0.8 0.8
E3 1.2 1.0
E4 2.1 1.3
E5 1.3 1.1
E6 2.0 1.5
E7 4.8 0.2
E8 3.7 0.0
E9 1.0 0.8
ElO 0.6 0.2
El l 1.0 0.3
E12 0.8 0.2
Fl 10.3 9.5
F2 2.9 1.9
F3 2.6 2.5
F4 1.5 2.7
F5 1.9 1.4
F6 1.6 1.2
F7 1.9 0.6
F8 1.5 0.6
F9 1.0 1.4
FlO 5.4 3.4 FI l 0.8 1.9
F12 1.0 1.4
Gl 0.8 1.2
G2 0.6 1.1
G3 2.0 1.6
G4 1.3 1.6
G5 1.7 1.5
G6 1.5 1.3
G7 4.6 0.2
G8 3.9 0.4
G9 1.0 0.7
GlO 1.3 0.7
GI l 1.9 0.6
G12 1.4 0.8
Hl 3.1 0.6
H2 1.8 3.5
H3 1.8 3.9
H4 1.2 4.0
H5 1.8 2.0
H6 1.4 2.9
H7 1.5 0.6
H8 2.1 0.8 vehicle control 1.0 1.0
Table 3. AlphaNumeric Well Label and the Corresponding Catalog#, CAS#, and Compound Name or ID Number for the BIOMOL Kinase Inhibitor compound library
PLATE LOCATION CATALOG # CAS # COMPOUND NAME OR ID
NUMBER
Bl EI-360 167869-21-8 PD-98059
B2 EI-282 109511-58-2 U-0126
B3 EI-286 152121-47-6 SB-203580
B4 EI- 148 84477-87-2 H-7
B5 EI- 195 84468-17-7 H-9
B6 EI- 156 62996-74-1 Staurosporine
B7 EI-228 133550-35-5 AG-494
B8 EI-267 AG-825
B9 EI- 185 125697-92-9 Lavendustin A
BlO EI-253 136831-49-7 RG- 14620
BI l EI-191 118409-57-7 Tyrphostin 23
B12 EI- 187 118409-58-8 Tyrphostin 25
Cl EI-257 122520-85-8 Tyrphostin 46
C2 EI- 188 122520-86-9 Tyrphostin 47
C3 EI- 189 122520-90-5 Tyrphostin 51
C4 EI- 190 2826-26-8 Tyrphostin 1
C5 EI-335 116313-73-6 Tyrphostin AG 1288
C6 EI-277 63177-57-1 Tyrphostin AG 1478
C7 AC-1133 71897-07-9 Tyrphostin AG 1295
C8 EI-215 10537-47-0 Tyrphostin 9
C9 EI-247 HNMPA (Hydroxy-2- naphthalenylmethylphosphonic acid)
ClO EI-370 120685-11-2 PKC-412
CI l EI-271 10083-24-6 Piceatannol
C12 EI-275 172889-26-8 PPl
Dl EI-272 133550-35-3 AG-490
D2 EI-263 AG-126 D3 EI-229 AG-370
D4 EI-258 AG-879
D5 ST-420 154447-36-6 LY 294002
D6 ST-415 19545-26-7 Wortmannin
D7 EI-246 133052-90-1 GF 109203X
D8 EI-226 548-04-9 Hypericin
D9 EI-283 138489-18-6 Ro 31-8220
DlO EI-155 123-78-4 Sphingosine
DI l EI- 196 127243-85-0 H-89
D12 EI-158 84478-11-5 H-8
El EI- 184 91742-10-8 HA- 1004
E2 EI-233 103745-39-7 HA- 1077
E3 EI-232 HDBA (2-Hydroxy-5-(2,5- dihydroxybenzylamino)benzoic acid)
E4 EI-230 127191-97-3 KN-62
E5 EI-268 KN-93
E6 EI- 197 109376-83-2 ML-7
E7 EI-153 105637-50-1 ML-9
E8 CC-100 452-06-2 2-Aminopurine
E9 CC-202 158982-15-1 N9-Isopropyl-olomoucine
ElO CC-200 101622-51-9 Olomoucine
El l CC-201 101622-50-8 iso-Olomoucine
E12 CC-205 186692-46-6 Roscovitine
Fl EI-293 24386-93-4 5-Iodotubercidin
F2 EI-295 62004-35-7 LFM-A 13
F3 EI-294 152121-30-7 SB-202190
F4 EI-297 172889-27-9 PP2
F5 EI-298 208260-29-1 ZM 336372
F6 EI-306 5812-07-7 SU 4312
F7 EI-303 146535-11-7 AG-1296
F8 EI-307 220904-83-6 GW 5074
F9 AC-1121 6865-14-1 Palmitoyl-DL-carnitine Cl
FlO EI-270 82-08-6 Rottlerin FI l EI- 147 446-72-0 Genistein
F12 ST-110 486-66-8 Daidzein
Gl EI- 146 63177-57-1 Erbstatin analog
G2 AC-1142 6151-25-3 Quercetin dihydrate
G3 AC-1293 SU1498
G4 EI-357 4452-06-6 ZM 449829
G5 EI-278 195462-67-7 BAY 11-7082
G6 EI-231 53-85-0 DRB (5,6-Dichloro-l-β-D- ribofuranosylbenzimidazole)
G7 EI-273 HBDDE (2,2',3,3',4,4'-Hexahydroxy-
1 , 1 '-biphenyl-6,6'-dimethanol dimethyl ether)
G8 EI-305 129-56-6 SP 600125
G9 CC-206 479-41-4 Indirubin
GlO CC-207 160807-49-8 Indirubin-3 '-monoxime
GI l EI-299 146986-50-7 Y-27632
G12 EI-310 142273-20-9 Kenpaullone
Hl EI-328 121-40-4 Terreic acid
H2 EI-332 35943-35-2 Triciribine
H3 EI-336 BML-257
H4 EI-343 SC-514
H5 EI-344 BML-259
H6 EI-345 520-36-5 Apigenin
H7 EI-346 BML-265 (Erlotinib analog)
H8 A-275 53123-88-9 Rapamycin
Table 4. The Effect of compounds of the BIOMOL Inhibitor compound library on the Expression of Insulin, glucagon, MAFA andArx4 in Cells Expressing Markers Characteristic of the Pancreatic Endocrine Lineage
Concentration and
PubChem ID# MAFA Insulin Glucagon Arx4
O.όlμM 5330812 46.3 0.9 0.26 0.68
1.25μM 5330812 209.2 1.3 0.31 0.66
5.0μM 5330812 2909.9 66.3 4.71 0.92
O.όlμM 4566 1.0 1.0 0.77 0.78
1.25μM 4566 0.8 1.1 0.90 0.78
5.0μM 4566 1.0 1.1 0.96 0.69
O.όlμM 5330797 0.7 0.6 0.34 0.36
1.25 μM 5330797 1.5 0.8 0.25 0.37
5.0μM 5330797 6.3 1.3 0.04 0.16
O.όlμM 73292 0.7 0.7 0.29 0.38
1.25μM 73292 1.3 1.0 0.25 0.42
5.0μM 73292 3.1 0.8 0.13 0.33
O.όlμM 4592 0.9 0.9 0.81 0.61
1.25 μM 4592 1.0 1.0 0.70 0.54
5.0μM 4592 0.6 1.3 1.08 0.77
O.όlμM 160355 0.9 0.9 0.76 0.77
1.25μM 160355 0.7 1.0 0.61 0.65
5.0μM 160355 0.8 1.1 0.59 0.86
Vehicle Treated 1.0 1.0 1.00 1.00 Table 5. AlphaNumeric Well Label and the Corresponding Catalog^, and Compound Name or ID Number for the EMD Calbiochem Kinase Inhibitor compound library I
Gene
Plate Induction
Location Catalog# Compound Name vs. H1 :
MAFa
A10 197221 Bcr-abl Inhibitor 1 .5
A1 1 203290 Bisindolylmaleimide I 0.8
A12 DMSO Control 1 .5
A2 121767 AG 1024 0.8
A3 121790 AGL 2043 0.8
A4 124011 Akt Inhibitor IV 45.7
A5 124012 Akt Inhibitor V, Triciribine 0.9
A6 124018 Akt Inhibitor VIII, Isozyme-Selective, Akti-1/2 1 .6
A7 124020 Akt Inhibitor X 1 .4
A8 521275 PDK1/Akt/Flt Dual Pathway Inhibitor 2.1
A9 189404 Aurora Kinase Inhibitor Il 1 .3
B10 317200 DMBI 1 .9
B1 1 324673 EGFR/ErbB-2 Inhibitor 2.4
B12 DMSO Control 1 .9
B2 203297 Bisindolylmaleimide IV 1 .9
B3 203696 BPIQ-I 1 .6
B4 220285 Chelerythrine Chloride 2.3
B5 234505 Compound 56 1 .8
B6 260961 DNA-PK Inhibitor Il 2.0
B7 260962 DNA-PK Inhibitor III 2.2
B8 528100 PM 03 1 .9
B9 266788 Diacylglycerol Kinase Inhibitor Il 1 .5
C10 375670 Herbimycin A, Streptomyces sp. 1 .3
C1 1 343022 Flt-3 Inhibitor III 1 .1
C12 DMSO Control 1 .1
C2 324674 EGFR Inhibitor 3.5
C3 324840 EGFR/ErbB-2/ErbB-4 Inhibitor 0.9
C4 343020 Flt-3 Inhibitor 0.6
C5 343021 Flt-3 Inhibitor Il 0.5
C6 344036 cFMS Receptor Tyrosine Kinase Inhibitor 2.2
C7 365250 Go 6976 1 .9
C8 365251 Go 6983 1 .0
C9 371806 GTP-14564 0.7
D10 440203 LY 30351 1 1 .7
D1 1 448101 Met Kinase Inhibitor 2.1
D12 BLANK 1 .7 D2 407248 IGF-1R Inhibitor Il 1.6
D3 407601 IRAK-1/4 Inhibitor 2.2
D4 420099 JAK Inhibitor I 1.4
D5 420104 JAK3 Inhibitor Il 2.0
D6 420121 JAK3 Inhibitor IV 1.7
D7 420126 JAK3 Inhibitor Vl 1.9
D8 428205 Lck Inhibitor 1.9
D9 440202 LY 294002 2.3
E10 528106 Pl 3-Kg Inhibitor 1.6
E11 528108 Pl 3-Kblnhibitor Il 1.4
E12 BLANK 1.6
E2 513035 PD 158780 0.8
E3 513040 PD 174265 1.0
E4 521231 PDGF Receptor Tyrosine Kinase Inhibitor Il 0.8
E5 521232 PDGF Receptor Tyrosine Kinase Inhibitor III 1.7
E6 521233 PDGF Receptor Tyrosine Kinase Inhibitor IV 5.5
E7 521234 PDGF RTK Inhibitor 1.9
E8 527450 PKR Inhibitor 24.6
E9 527455 PKR Inhibitor, Negative Control 1.5
F10 567805 Src Kinase Inhibitor I 2.3
F11 572660 SU11652 1.7
F12 DMSO Control 2.1
F2 529574 PP3 1.3
F3 529581 PP1 Analog II, 1NM-PP1 2.3
F4 539652 PKCbll/EGFR Inhibitor 1.8
F5 539654 PKCb Inhibitor 1.6
F6 553210 Rapamycin 1.2
F7 555553 Rho Kinase Inhibitor III, Rockout 1.7
F8 555554 Rho Kinase Inhibitor IV 2.3
F9 539648 Staurosporine, N-benzoyl- 11.7
G10 658550 AG 1295 1.4
G11 658551 AG 1296 1.1
G12 DMSO Control 1.2
G2 574711 Syk Inhibitor 1.2
G3 574712 Syk Inhibitor Il 0.8
G4 574713 Syk Inhibitor III 1.1
G5 616451 TGF-b Rl Kinase Inhibitor 1.1
G6 616453 TGF-b Rl Inhibitor III 1.6
G7 658390 AG 9 1.5
G8 658401 AG 490 1.4
G9 658440 AG 112 1.4
H10 189405 Aurora Kinase Inhibitor III 2.0
H11 569397 Staurosporine, Streptomyces sp. 0.0*
H12 DMSO Control 1.5
H2 658552 AG 1478 2.7
H3 676480 VEGF Receptor 2 Kinase Inhibitor I 1.7
H4 676481 VEGF Receptor Tyrosine Kinase Inhibitor Il 1.0
H5 676482 VEGF Receptor Tyrosine Kinase Inhibitor III,
KRN633 1.6
H6 676485 VEGF Receptor 2 Kinase Inhibitor Il 1.0
H7 676487 VEGF Receptor 2 Kinase Inhibitor III 1.1 H8 676489 VEGF Receptor 2 Kinase Inhibitor IV 2.1
H9 260964 DNA-PK Inhibitor V 1 .5
Table 6. AlphaNumeric Well Label, Corresponding Catalogtt, and Compound #or EMD Calbiochem Kinase Inhibitor II compound library
Well # Catalog # Compound #
A1 DMSO
A2 422706 KN-62
A3 1 18500 ATM Kinase Inhibitor
A4 1 18501 ATM/ATR Kinase Inhibitor
A5 126870 Alsterpaullone
A6 126871 Alsterpaullone, 2-Cyanoethyl
A7 128125 Aloisine A, RP107
A8 128135 Aloisine, RP106
A9 164640 Aminopurvalanol A
A10 171260 AMPK Inhibitor, Compound C
A11 189405 Aurora Kinase Inhibitor III
A12 BLANK B1 DMSO
B2 189406 Aurora Kinase/Cdk Inhibitor
B3 402085 lndirubin-3'-monoxime
B4 196870 BAY 1 1-7082
B5 203600 Bohemine
B6 217695 Cdk1 Inhibitor
B7 217696 Cdk1 Inhibitor, CGP74514A
B8 217714 Cdk1/2 Inhibitor III
B9 217720 Cdk1/5 Inhibitor
B10 218696 Casein Kinase I Inhibitor, D4476
B11 218710 Casein Kinase Il Inhibitor III, TBCA
B12 BLANK C1 DMSO
C2 219476 Cdk4 Inhibitor
C3 219477 Cdk4 Inhibitor II, NSC 625987
C4 219478 Cdk4 Inhibitor III
C5 219479 Cdc2-Like Kinase Inhibitor, TG003
C6 220486 Chk2 Inhibitor Il
C7 234503 Compound 52
C8 238803 Cdk2 Inhibitor III
C9 238804 Cdk2 Inhibitor IV, NU6140
C10 219491 Cdk/Crk Inhibitor
C1 1 328009 ERK Inhibitor III
C12 BLANK D1 DMSO
D2 688000 ROCK Inhibitor, Y-27632
D3 328007 ERK Inhibitor II, FR180204
D4 328008 ERK Inhibitor II, Negative control
D5 341251 Fascaplysin, Synthetic
D6 361540 GSK-3b Inhibitor I
D7 361541 GSK-3b Inhibitor Il
D8 361549 GSK-3b Inhibitor VIII D9 361550 GSK-3 Inhibitor IX
D10 361551 GSK-3 Inhibitor X
D1 1 361553 GSK-3b Inhibitor Xl
D12 BLANK
E1 DMSO
E2 572635 SU6656
E3 361555 GSK-3 Inhibitor XIII
E4 371957 lsogranulatimide
E5 400090 IC261
E6 401481 IKK-2 Inhibitor IV
E7 402081 lndirubin Derivative E804
E8 4201 19 JNK Inhibitor Il
E9 420123 JNK Inhibitor, Negative Control
E10 420129 JNK Inhibitor V
E11 420136 JNK Inhibitor IX
E12 BLANK
F1 DMSO
F2 475863 MK2a Inhibitor
F3 420135 JNK Inhibitor VIII
F4 420298 K-252a, Nocardiopsis sp.
F5 422000 Kenpaullone
F6 422708 KN-93
F7 444937 MEK Inhibitor I
F8 444938 MEK Inhibitor Il
F9 444939 MEK1/2 Inhibitor
F10 454861 MNK1 Inhibitor
F1 1 481406 NF-κB Activation Inhibitor
F12 BLANK
G1 DMSO
G2 506121 p38 MAP Kinase Inhibitor III
G3 506126 p38 MAP Kinase Inhibitor
G4 513000 PD 98059
G5 513030 PD 169316
G6 559396 SB220025
G7 540500 Purvalanol A
G8 361554 GSK3b Inhibitor XII, TWS1 19
G9 371963 H-89, Dihydrochloride
G10 559387 SB 202474, Negative control for p38 MAPK inhibition studies
G11 559388 SB 202190
G12 BLANK
H1 DMSO
H2 559389 SB 203580
H3 371970 HA 1077, Dihydrochloride Fasudil
H4 559402 SB 218078
H5 565625 SC-68376
H6 567305 SKF-86002
H7 567731 Sphingosine Kinase Inhibitor
H8 569397 Staurosporine, Streptomyces sp.
H9 570250 STO-609
H10 572650 SU9516
H1 1 616373 Tpl2 Kinase Inhibitor
H12 BLANK Table 7. Fold Induction of MAFA expression by several Compounds from the EMD Kinase Inhibitor Library II on Cells Treated according to the Differentiation Protocol Outlined in Example 9
fold vs. well# control cat# dru name
Figure imgf000059_0001

Claims

What is claimed is:
1. A method for increasing the expression of MAFA in cells expressing markers characteristic of the pancreatic endocrine lineage comprising the steps of culturing the cells expressing markers characteristic of the pancreatic endocrine lineage in medium comprising a sufficient amount of a cyclin- dependant kinase inhibitor to cause an increase in expression of MAFA.
PCT/US2009/061774 2008-10-31 2009-10-23 Differentiation of human embryonic stem cells to the pancreatic endocrine lineage WO2010051223A1 (en)

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RU2011121842/10A RU2522001C2 (en) 2008-10-31 2009-10-23 Differentiation of human embrionic stem cells in line of pancreatic endocrine cells
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