WO2018119155A1 - Méthodes de différenciation pancréatique de cellules souches - Google Patents

Méthodes de différenciation pancréatique de cellules souches Download PDF

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WO2018119155A1
WO2018119155A1 PCT/US2017/067743 US2017067743W WO2018119155A1 WO 2018119155 A1 WO2018119155 A1 WO 2018119155A1 US 2017067743 W US2017067743 W US 2017067743W WO 2018119155 A1 WO2018119155 A1 WO 2018119155A1
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cell
cells
days
modulated
equivalent
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Louise LAURENT
Sergio MORA-CASTILLA
Thomas Touboul
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The Regents Of The University Of California
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/385—Hormones with nuclear receptors of the family of the retinoic acid recptor, e.g. RAR, RXR; Peroxisome proliferator-activated receptor [PPAR]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
    • C12N2501/41—Hedgehog proteins; Cyclopamine (inhibitor)
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/40—Regulators of development
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/999—Small molecules not provided for elsewhere
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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    • C12N2513/00—3D culture
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
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    • C12N2533/52—Fibronectin; Laminin
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • This disclosure resides in the field of pancreatic cell differentiation. Particularly, this disclosure relates to methods of differentiating stem cells into pancreatic cells in vitro.
  • Type 1 diabetes is an autoimmune disease in which the immune system attacks and destroys the insulin-producing beta cells in the pancreas.
  • Islet transplantation is a suitable therapy for diabetes, but the scarceness of cadaveric islet donors makes this approach practically impossible in a higher scale.
  • Promising alternative cell sources for treating diabetes comes from human embryonic stem cells (hESCs), and numerous groups have generated functional insulin-producing cells in vitro using stepwise differentiation protocols that mimic pancreatic development, including from diabetic patients.
  • hESCs human embryonic stem cells
  • stepwise differentiation protocols that mimic pancreatic development, including from diabetic patients.
  • the limited transcriptomic data on these cells indicate that there remain marked differences between the most mature hESC-derived cells and primary human adult beta cells. Therefore,
  • methods for differentiating a stem cell into a pancreatic cell are provided by this disclosure.
  • this disclosure provides pancreatic cells produced according to the disclosed methods and compositions thereof.
  • this disclosure provides methods of treating diabetes comprising administering a pancreatic cell produced according to the disclosed methods.
  • a method of differentiating a stem cell into a pancreatic beta cell comprising, or consisting of, or consisting essentially of, culturing the stem cell under conditions to modulate one or more signaling pathways selected from the group of: Activin, WNT, BMP, FGF, or AKT signaling pathways to produce an endoderm cell.
  • the stem cell is an embryonic stem cell or an iPSC. Further provided is a population of the differentiated cells. In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone differentiation.
  • the stem cell has been previously adapted to a system comprising feeder free medium and a growth surface coated with vitronectin or an equivalent thereof.
  • the feeder free medium comprises E8 culture medium or an equivalent thereof.
  • the culture conditions comprise culturing in a medium comprising CDM-PVA.
  • one of the Activin, WNT, BMP, FGF, or AKT signaling pathways is modulated.
  • two of the Activin, WNT, BMP, FGF, or AKT signaling pathways are modulated.
  • three of the Activin, WNT, BMP, FGF, or AKT signaling pathways are modulated.
  • four of the Activin, WNT, BMP, FGF, or AKT signaling pathways are modulated.
  • the Activin signaling pathway is modulated by contacting the stem cell with about 50 ng/mL to about 150 ng/mL ActA or an equivalent thereof for an effective amount of time, e.g., from about 1 to 4 days or alternatively for about three days.
  • the WNT signaling pathway is modulated by contacting the stem cell with about 1 ⁇ to about 10 ⁇ CHIR99021 or an equivalent thereof for an effective amount of time, e.g., during the first about 1 hour to about 36 hours of culture.
  • the BMP signaling pathway is modulated by contacting the stem cell with about 5 ng/mL to about 50 ng/mL BMP4 or an equivalent thereof for an effective amount of time, e.g., the first 18 to 36 hours of culture followed by about 36 to about 72 hours of contacting the stem cell with about 50 to about 150 nM dorsomorphin or an equivalent thereof.
  • the FGF signaling pathway is modulated by contacting the stem cell with about 10 ng/mL to about 50 ng/mL FGF2 or an equivalent thereof for an effective amount of time, e.g., from about one to about four days, e.g., for about three days.
  • the AKT signaling pathway is modulated by contacting the stem cell with about 5 ⁇ to about 50 ⁇ LY294002 or an equivalent thereof for an effective amount of time, e.g., for about one to about four days, or for about three days.
  • the endoderm cell expresses at least one of CXCR4 and SOX17. In another aspect, the endoderm cell expresses both.
  • the method further comprises, or consists of, or consists essentially of culturing the endoderm cell under conditions to modulate one or more signaling pathways selected from the group of: BMP, TGFp, or FGF, signaling pathways to produce a posterior foregut (PF) cell.
  • PF posterior foregut
  • a population of the cells after modulation In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the culture conditions comprise culturing in the presence of an effective amount of Advanced DMEM culture medium.
  • the culture conditions comprise contacting the endoderm cell with an effective amount of retinoic acid or an equivalent thereof.
  • the endoderm cell is contacted with from about 0.5 ⁇ to about 5 ⁇ retinoic acid for about one to four days, or for about three days.
  • a population of the modulated or the differentiated cells are provided. In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%) of the cells in the population have undergone modulation and/or differentiation.
  • all of the BMP, TGFP, and FGF signaling pathways are modulated.
  • one of the BMP, TGFP, and FGF signaling pathways is modulated.
  • two of the BMP, TGFp, and FGF signaling pathways are modulated. In another aspect, all three are modulated.
  • the TGFP signaling pathway is modulated by contacting the endoderm cell with from about 1 ⁇ to about 50 ⁇ SB431542 for about one to about four days, or for about three days. Further provided is a population of the modulated or the differentiated cells. In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%) of the cells in the population have undergone modulation and/or differentiation.
  • the BMP signaling pathway is modulated by contacting the endoderm cell with from about 50 nM to about 150 nM dorsomorphin or an equivalent thereof for about one to about four days, or for about three days. Further provided is a population of the modulated or the differentiated cells. In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation and/or differentiation.
  • the FGF signaling pathway is modulated by contacting the endoderm cell with from about 10 ng/mL to about 100 ng/mL FGF10 or an equivalent thereof for one to about four days, or for about three days. Further provided is a population of the modulated or the differentiated cells. In one aspect, at least 75%, or alternatively at least 80%), or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation and/or differentiation.
  • the PF cell expresses at least one or both of UNFip and HNF4A, but does not express OCT4, NANOG, HHEX, HOXA3, nor CDX2. Further provided is a population of the cells expressing the marker(s). In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%> of the cells in the population express the marker(s).
  • the method further comprises, consists of, or consists essentially of culturing the PF cell under conditions to modulate one or more signaling pathways selected from the group of: BMP, Sonic hedgehog, or FGF signaling pathways to produce a pancreatic progenitor cell.
  • a population of the modulated cells In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the culture conditions comprise culturing the PF cell in the presence of an effective amount of Advanced DMEM culture medium.
  • the PF cell is contacted with about 0.5 ⁇ to about 5 ⁇ retinoic acid for about one to about four days, or about three days.
  • all of the BMP, Sonic Hedgehog, and FGF signaling pathways are modulated.
  • one of the BMP, Sonic Hedgehog, and FGF signaling pathways is modulated.
  • two of the BMP, Sonic Hedgehog, and FGF signaling pathways are modulated.
  • all three are modulated.
  • a population of the modulated cells In one aspect, at least 75%), or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the Sonic hedgehog signaling pathway is modulated by contacting the PF cell with an effective amount of, or alternatively from about 0.1 ⁇ to about 0.5 ⁇ cyclopamine or an equivalent thereof for about one to about four days, or for about three days.
  • the FGF signaling pathway is modulated by contacting the PF cell with about 50 ng/mL FGF 10 for one to about four days, or for about 3 days.
  • the BMP signaling pathway is modulated by contacting the PF cell with from about 50 nM to about 150 nM dorsomorphin or an equivalent thereof for one to about four days, or for about three days.
  • the pancreatic progenitor cell expresses PDX1. Further provided is a population of the cells that express PDX1. In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%> of the cells in the population that express PDX1.
  • the method further comprises, consists of, or consists essentially of culturing the pancreatic progenitor cell under conditions to modulate the FGF signaling pathway followed by modulation of the Notch signaling pathway to produce an endocrine pancreas cell. In a further embodiment, all three are modulated. Further provided is a population of the modulated cells. In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the culture conditions comprise culturing the pancreatic progenitor cell in an effective amount of Advanced DMEM culture medium.
  • the method further comprises contacting the pancreatic progenitor cell with an effective amount of B27 or an equivalent thereof.
  • the pancreatic progenitor cell is contacted with from about 0.1% to about 10% B27 without insulin for about one day to about four days. In a further embodiment, all three are modulated. Further provided is a population of the cells having been contacted with the B27 or an equivalent thereof. In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the FGF signaling pathway is modulated by contacting the pancreatic progenitor cell with from about 10 ng/mL to about 100 ng/mL FGF 10 for about one to about four days, or for three days.
  • the Notch signaling pathway is modulated by contacting the pancreatic progenitor cell with about 1 ⁇ to about 50 ⁇ DAPT for about one to about four days, or for about three days.
  • the endocrine pancreas cell expresses at least one of
  • at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%>, or alternatively at least 95%>, or alternatively at least 98%> of the cells in the population express the marker(s).
  • the method further comprises, consists of, or consists essentially of culturing the endocrine pancreas cell under conditions to modulate the TGFP signaling pathway to produce an islet precursor cell, wherein the endocrine pancreas cell is cultured in three-dimensional culture conditions. In a further embodiment, all three are modulated.
  • a population of the modulated cells In one aspect, at least 75%>, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%>, or alternatively at least 95%>, or alternatively at least 98%> of the cells in the population have undergone modulation.
  • the culture conditions comprise culturing in the presence of an effective amount of DMEM culture medium.
  • the method further comprises contacting the endocrine pancreas cell with an effective amount of dexamethasone or an equivalent thereof.
  • the endocrine pancreas cell is contacted with from about 1 ⁇ to about 100 ⁇ dexamethasone for about 2 to about 8 days, or for about 6 days.
  • all three are modulated.
  • a population of the modulated cells In one aspect, at least 75%>, or alternatively at least 80%>, or alternatively at least 85%>, or
  • the method further comprises contacting the endocrine pancreas cell with nicotinamide or an equivalent thereof.
  • the endocrine pancreas cell is contacted with from about 1 mM to about 100 mM nicotinamide for about 2 to about 8 days, or for about 6 days about 2 to about 8 days, or for about 6 days.
  • all three are modulated.
  • a population of the modulated cells In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%o, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98%o of the cells in the population have undergone modulation.
  • the method further comprises contacting the endocrine pancreas cell with an effective amount of B27 or an equivalent thereof.
  • the endocrine pancreas cell is contacted with from about 0.1%> to about 10%> B27 without insulin for about 2 to about 8 days, or for about 6 days.
  • all three are modulated. Further provided is a population of the modulated cells. In one aspect, at least 75%, or alternatively at least 80%>, or alternatively at least 85%>, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the TGFP signaling pathway is modulated by contacting the endocrine pancreas cell with from about 0.1 ⁇ to about 10 ⁇ Alk5 inhibitor II or an equivalent thereof for about 2 to about 8 days, or for about 6 days. In a further embodiment, all three are modulated. Further provided is a population of the modulated cells. In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population have undergone modulation.
  • the islet precursor cell expresses at least one of INS, PDX1, and NGN3, or alternatively at least two, or all three. In a further embodiment, all three are modulated. Further provided is a population of the cells that express the marker(s). In one aspect, at least 75%, or alternatively at least 80%, or alternatively at least 85%, or alternatively at least 90%, or alternatively at least 95%, or alternatively at least 98% of the cells in the population express the marker(s).
  • the methods further comprise monitoring the efficiency of the differentiation process and marker expression of the cells or population through immunostaining, flow cytometry, gene expression analysis, or assaying for the production of c-peptide upon glucose stimulation.
  • the stem cell is an animal stem cell or alternatively, mammalian stem cell.
  • the mammalian stem cell is a bovine, canine, feline, murine, equine, or human stem cell.
  • the stem cell is a human stem cell.
  • an islet precursor cell produced according to the methods disclosed herein is provided herein.
  • composition comprising a cell produced according to the method disclosed herein and a carrier.
  • the carrier comprises a a non-naturally occurring carrier or a pharmaceutically acceptable carrier.
  • the composition further comprises an effective amount of preservative to extend the short and long term viability of the cells.
  • pancreatic diseases and pancreas- related disorders or conditions including but not limited to juvenile onset (Type I) diabetes mellitus, pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes).
  • IDDM pediatric insulin-dependent diabetes mellitus
  • Type II diabetes Type II diabetes
  • a method of treating a subject suffering from diabetes in a subject and in need thereof by administering to the subject an effective amount of a cell produced according to the methods disclosed herein or a composition comprising a cell produced according to the methods disclosed herein.
  • the subject is an animal, or it may be a mammal.
  • the subject is a bovine, equine, canine, feline, murine, or human.
  • the subject is a human.
  • the cell or population can be allogenic or autologous to the subject being treated.
  • FIG. 1A-E show stage 1 differentiation.
  • CHIR99021 treatments and two AKT inhibitors: LY294002 and PI- 103 in two surface coated plates (Gelatin FBS and Vitronectin).
  • Undifferentiated clusters (U) were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, with or without 10 nM LY294002 and 60 nM PI- 103, and with or without 2 uM CHIR99021.
  • FIG. 1A shows the experimental scheme.
  • FIG. IB Immunostaining for OCT4 and SOX17 in DE cells in LY294002, PI-103 or control (non-AKT inh) cells. Scale bars: 250 ⁇ .
  • FIG. 1C qPCR analysis of SOX17, OCT4, MIXL1 and GOOSECOID in CHIR99021 treated or not treated cells during primitive streak (PS) commitment. *p-value>0.05.
  • FIG. ID shows the experimental scheme to test the importance of inhibiting BMP4 and AKT signaling pathways after PS formation.
  • Undifferentiated clusters were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, 10 nM LY294002, and 2 uM CHIR99021. After this treatment, cells were maintained 2 more days with 100 ng/mL Activin A and 200 ng/mL of Noggin or 10 ng/mL BMP4 in CDM-PVA media. qPCR analysis show expression of SOX17 and HHEXm ' LY294002 treated or not treated cells during DE commitment. *p-value>0.05
  • FIG. 2A-C show stage 2 differentiation.
  • Applicants treated cells with TGFbeta inhibitor SB431542 during stage 2 of differentiation in cells that the BMP pathway was active or repressed (Noggin) after PS.
  • FIG. 2A shows the experimental scheme.
  • Undifferentiated clusters were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, 10 nM LY294002 and 2 uM CHIR99021 to induce PS formation.
  • cells were maintained 2 more days with 100 ng/mL Activin A, 20 ng/mL FGF2 and 200 ng/mL of Noggin or 10 ng/mL BMP4.
  • Stage 2 cells were differentiated in the presence of 2 uM RA, 10 ng/mL FGF10, 50 ng/mL Noggin and with or without 10 uM SB431542 during 3 days.
  • FIG. 2B Immunostaining of OCT4, HNFIB and HNF4A of stage 2 cells after blockade of TGFbeta with 10 uM SB431542 (squared condition shown in A). Scale bars: 250 ⁇ .
  • FIG. 3A-F show stage 3 differentiation.
  • DM dorsomorphin
  • FIG. 3A Experimental scheme comparing Cho et al descriptions and our improved protocol. Undifferentiated clusters were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, 10 nM LY294002 and 2 uM CHIR99021 to induce PS formation.
  • cells were maintained 2 more days in CDM-PVA media with 100 ng/mL Activin A, 20 ng/mL FGF2, 10 nM LY294002 and 10 ng/mL BMP4 (control conditions) or with a gradient of 25-200 ng/mL of Noggin or 50-500 nM dorsomorphin.
  • Applicants noticed a toxic effect of 500 nM dorsomorphin conditions.
  • stages 2 and 3 cells were maintained with 50 ng/mL of Noggin for control conditions (BMP4 during Stagel) or with a gradient of 50-200 ng/mL of Noggin or 50-200 nM dorsomorphin for improved protocol.
  • FIG. 3B qPCR analysis oiMIXLl and NANOG in Stage 1 cells in a gradient of Noggin or dorsomorphin.
  • FIG. 3C Immunostaining of PDX1 at the end of Stage 3 in a gradient of Noggin or dorsomorphin. Scale bars: 250 ⁇ .
  • FIG. 3D Blockade of Sonic Hedgehog pathway with Cyclopamine or SANT1. Applicants tested the combined actions of 0.25 uM Cyclopamine or 0.25 uM SANT1 with or without 20 ng/mL Activin A. Immunostaining of PDX1 at the end of Stage 3. Scale bars: 250 ⁇ .
  • FIG. 3E qPCR analysis shows expression oiPDXl, HNF6, RFX6 and NGN3 in stage 3 cells treated combined actions of Cyclopamine or SANT1 with Activin A.
  • FIG. 3F Immunostaining of PDX1, HNF6 and CD24 at the end of Stage 3 in H9 cells treated with 0.25 uM Cyclopamine, 2 uM Retinoic acid (RA), 50 ng/mL FGF10, 100 nM DM. Scale bars: 100 ⁇ .
  • FIG. 4A-C show experimental approach for pancreatic differentiation.
  • FIG. 4B Flow cytometry analysis of cells stained with known markers for stages 1 to 4 of differentiation. Cut-off bars in the plots are obtained with corresponding isotypic controls for each antibody.
  • FIG. 4C RT-qPCR analysis during beta-cell differentiation. Gene expression profile of hESC-derived cells (U: undifferentiated, Stage 1 : Stl, Stage 2: St2, Stage 3 : St3, Stage 4: St4, Stage 5: St5), fetal pancreatic (FP) and adult islet (AI) cells.
  • FP fetal pancreatic
  • AI adult islet
  • FIG. 5A-J show the protocol for pancreatic differentiation is performed in 5 stages, with Stages 1-4 in 2D conditions, followed by formation of 3D spheroids for Stage 5.
  • FIGs. 5 A-C demonstrate the morphology of the Stage 5 spheroids, as well as their expression of mRNA and protein markers of pancreatic beta cells.
  • FIG. 5A Stage 5 spheroids were embedded in paraffin and sectioned in slides of 5 microns. Slides were stained with hematoxylin and eosin. Scale bar: 50 microns.
  • FIG. 5A-J show the protocol for pancreatic differentiation is performed in 5 stages, with Stages 1-4 in 2D conditions, followed by formation of 3D spheroids for Stage 5.
  • FIGs. 5 A-C demonstrate the morphology of the Stage 5 spheroids, as well as their expression of mRNA and protein markers of pancreatic beta cells.
  • FIG. 5A Stage 5 spheroids were embedded in paraffin and sectione
  • FIG. 5B RT-qPCR analysis of mature markers for pancreatic differentiation (NKX6.1, PDX1, NEURODl, INS) in Stage 5 (St5), Fetal Pancreas (FP), and Adult Islets (AI).
  • FIG. 5C C-peptide and NKX6.1 immunostaining of stage 5 spheroids sections in 5 micron slices. Scale bar: 50 microns.
  • FIGs. 5D-E show that the combined 2D (Stages l-4)/3D (Stage5) approach results in higher expression of pancreatic endoderm markers compared to purely 2D (Stages 1-5) or purely 3D (Stages 1-5) approaches. This data demonstrates that the purely 3D approach results in aberrant expression of hepatic markers.
  • FIG. 5D Images of 2D and 3D St5 cells.
  • 2D conditions cells were maintained in vitronectin-coated plates during the whole process of differentiation, no dissociation of St4 cells was applied.
  • 3D conditions St4 cells were dissociated and processed for spheroid formation. Scale bar: 100 microns.
  • FIG. 5E RT-qPCR analysis of mature markers for pancreatic differentiation (PDX1, NGN3 and INS. *p-value>0.05) in 2D and 3D conditions.
  • FIG. 5F Secreted C-peptide in response to low (2.5 mM) or high (25 mM) concentrations of D-glucose and 30 mM potassium chloride (KC1) was measured with an Ultra sensitive C-peptide ELISA kit from Stage 5 spheroids.
  • FIG. 5G Glucose stimulatory insulin release (GSIR) of Stage 5 cells after transplantation.
  • FIG. 5 J Gene clusters selected on heatmap of FIG. 5H.
  • FIG. 6 is a schematic representation of an embodiment of the disclosed methods.
  • undifferentiated cells are subjected to 21 days of culture, during which they differentiate through 5 distinct stages, ultimately producing islet precursor cells.
  • the cells are cultured in various media during the 21 day period including E8, CDM-PVA, Advanced DMEM, and DMEM. Further, the cytokine, nutrient, and other effectors of differentiation are altered throughout the different stages.
  • the bottom panel depicts the morphology of cells at each stage of differentiation.
  • FIG. 7 shows the result of RT-qPCR analysis of mature markers for pancreatic differentiation (PDX1, NGN3 and INS. *p-value>0.05) in 2D, 3D, and 2D followed by 3D conditions. These results demonstrate appropriate secretion of insulin by the 2D/3D Stage 5 cells in vitro and after transplantation into mice.
  • compositions and methods are intended to mean that the compositions and methods include the recited elements, but not excluding others.
  • compositions and methods shall mean excluding other elements of any essential significance to the combination for the stated purpose.
  • a composition consisting essentially of the elements as defined herein would not exclude trace contaminants from the isolation and purification method and
  • compositions of this invention are within the scope of this invention.
  • pharmaceutically acceptable carriers such as phosphate buffered saline, preservatives and the like.
  • Consisting of shall mean excluding more than trace elements of other ingredients and substantial method steps for administering the compositions of this invention or process steps to produce a composition or achieve an intended result. Embodiments defined by each of these transition terms are within the scope of this invention.
  • isolated refers to molecules or biological or cellular materials being substantially free from other materials, e.g., greater than 70%, or 80%, or 85%), or 90%), or 95%, or 98%>.
  • isolated refers to nucleic acid, such as DNA or RNA, or protein or polypeptide, or cell or cellular organelle, or tissue or organ, separated from other DNAs or RNAs, or proteins or polypeptides, or cells or cellular organelles, or tissues or organs, respectively, that are present in the natural source and which allow the manipulation of the material to achieve results not achievable where present in its native or natural state, e.g., recombinant replication or manipulation by mutation.
  • isolated also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized.
  • an "isolated nucleic acid” is meant to include nucleic acid fragments which are not naturally occurring as fragments and would not be found in the natural state.
  • isolated is also used herein to refer to polypeptides which are isolated from other cellular proteins and is meant to encompass both purified and recombinant polypeptides, e.g., with a purity greater than 70%, or 80%), or 85%>, or 90%, or 95%, or 98%.
  • isolated is also used herein to refer to cells or tissues that are isolated from other cells or tissues and is meant to encompass both cultured and engineered cells or tissues.
  • stem cell defines a cell with the ability to divide for indefinite periods in culture and give rise to specialized cells. At this time and for convenience, stem cells are categorized as somatic (adult), embryonic or induced pluripotent stem cells.
  • a somatic stem cell is an undifferentiated cell found in a differentiated tissue that can renew itself (clonal) and (with certain limitations) differentiate to yield all the specialized cell types of the tissue from which it originated.
  • An embryonic stem cell is a primitive
  • Pluripotent embryonic stem cells can be distinguished from other types of cells by the use of markers including, but not limited to, Oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, Germ cell nuclear factor, SSEA1, SSEA3, and SSEA4.
  • markers including, but not limited to, Oct-4, alkaline phosphatase, CD30, TDGF-1, GCTM-2, Genesis, Germ cell nuclear factor, SSEA1, SSEA3, and SSEA4.
  • Non-limiting examples of stem cells suitable for practicing the disclosed methods are WA09 and sHDF#l .
  • the term “culturing” refers to the in vitro propagation of cells or organisms on or in synthetic culture conditions such as culture media of various kinds. In some aspects, the medium is changed daily. It is understood that the descendants of a cell grown in culture may not be completely identical ⁇ i.e., morphologically, genetically, or phenotypically) to the parent cell. By “expanded” is meant any proliferation, growth, or division of cells.
  • culture methods that support differentiation by in inclusion of nutrients and effector molecules necessary to promote or support the differentiation of stem cells into differentiated cells. Further disclosed herein are culture methods that are two dimensional (2D) or three dimensional (3D). In 2D culture, cells are grown in a monolayer typically on a single, flat surface.
  • a 3D cell culture allows cells to grow, proliferate, or interact with their environment and the surrounding cells in all three dimensions, similar to how they would in vivo.
  • the disclosed method involves 2D culturing the stem cell on a single growth surface such as a well of a culture plate followed by a transition of the expanded cells to 3D culture.
  • “Differentiation” describes the process whereby an unspecialized cell acquires the features of a specialized cell such as a heart, liver, pancreas, or muscle cell.
  • Directed differentiation refers to the manipulation of stem cell culture conditions to induce differentiation into a particular cell type.
  • “Dedifferentiated” defines a cell that reverts to a less committed position within the lineage of a cell.
  • the term “differentiates or differentiated” defines a cell that takes on a more committed (“differentiated") position within the lineage of a cell and may also include maturation or development of the cell.
  • a cell that differentiates into pancreatic beta cell defines any cell that can become a committed pancreatic cells that produces insulin.
  • Non-limiting examples of cells that are capable of differentiating into pancreatic beta cells include embryonic stem cells, pluripotent stem cells, induced pluripotent stem cells (iPSCs), definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor cells, pancreatic endoderm, pancreatic endocrine precursors, endocrine pancreas cells, islet precursors, intermediate beta progenitor cells, and immature beta cells.
  • embryonic stem cells pluripotent stem cells, induced pluripotent stem cells (iPSCs), definitive endoderm, primitive gut tube, posterior foregut, pancreatic progenitor cells, pancreatic endoderm, pancreatic endocrine precursors, endocrine pancreas cells, islet precursors, intermediate beta progenitor cells, and immature beta cells.
  • iPSCs induced pluripotent stem cells
  • a “pluripotent cell” defines a less differentiated cell that can give rise to at least two distinct (genotypically and/or phenotypically) further differentiated progeny cells.
  • a “pluripotent cell” includes a Induced Pluripotent Stem Cell (iPSC) which is an artificially derived stem cell from a non-pluripotent cell, typically an adult somatic cell, produced by inducing expression of one or more stem cell specific genes.
  • iPSC Induced Pluripotent Stem Cell
  • compositions are intended to encompass a combination of active agent and another "carrier,” e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • carrier e.g., compound or composition, inert (for example, a detectable agent or label) or active, such as an adjuvant, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
  • compositions may include stabilizers and preservatives.
  • pharmaceutically acceptable carrier encompasses any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, and emulsions, such as an oil/water or water/oil emulsion, and various types of wetting agents.
  • Carriers also include biocompatible scaffolds, pharmaceutical excipients and additives proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
  • Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/antibody
  • components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
  • Carbohydrate excipients are also intended within the scope of this invention, examples of which include but are not limited to monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like;
  • polysaccharides such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like
  • alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol) and myoinositol.
  • a population of cells intends a collection of more than one cell that is identical (clonal) or non-identical in phenotype and/or genotype.
  • substantially homogeneous describes a population of cells in which more than about 50%, or alternatively more than about 60 %, or alternatively more than 70 %, or alternatively more than 75 %, or alternatively more than 80%, or alternatively more than 85 %, or alternatively more than 90%, or alternatively, more than 95 %, of the cells are of the same or similar phenotype. Phenotype can be determined by assaying for expression of a pre-selected cell surface marker or other marker.
  • an "effective amount” is an amount sufficient to effect beneficial or desired results.
  • the term “effective amount” as used herein refers to the amount needed to modulate the targeted signaling pathway and/or direct differentiation of the cell.
  • the term “effective amount” as used herein refers to the amount the alleviate at least one or more symptom of a pancreatic or immune disorder or condition (e.g., diabetes), and relates to a sufficient amount of the cell, population, or composition to provide the desired effect (e.g., increase insulin production).
  • an effective amount as used herein would also include an amount sufficient to delay the development of a pancreatic or immune disorder or condition symptom, alter the course of a pancreatic or immune disorder or condition symptom (for example but not limited to, slow the progression of a symptom of diabetes), or reverse a symptom of a pancreatic or immune disorder or condition. Thus, it is not possible to specify the exact "effective amount.” However, for any given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using only routine experimentation.
  • An effective amount can be administered in one or more administrations, applications or dosages. Such delivery is dependent on a number of variables including the time period for which the individual dosage unit is to be used, the bioavailability of the therapeutic agent, the route of administration, etc. It is understood, however, that specific dose levels of the therapeutic agents of the present invention for any particular subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, and diet of the subject, the time of administration, the rate of excretion, the drug combination, and the severity of the particular disorder being treated and form of administration. Treatment dosages generally may be titrated to optimize safety and efficacy. The dosage can be determined by a physician and adjusted, as necessary, to suit observed effects of the treatment.
  • RNA virus replication ex vivo is an amount sufficient to inhibit RNA virus replication ex vivo, in vitro or in vivo.
  • administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, ICV, intraci sternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration.
  • the invention is not limited by the route of administration, the formulation or dosing schedule.
  • An "enriched population" of cells intends a substantially homogenous population of cells having certain defined characteristics.
  • the cells are greater than 60%, or alternatively greater than 65%, or alternatively greater than 70 %, or alternatively greater than 75 %, or alternatively greater than 80 %, or alternatively greater than 85 %, or alternatively greater than 90 %, or alternatively greater than 95 %, or alternatively greater than 98% identical in the defined characteristics.
  • the substantially homogenous population of cells express markers that correlate with pluripotent cell identity such as expression of stem-cell specific genes like OCT4 and NANOG.
  • the substantially homogenous population of cells express markers that are correlated with definitive endoderm cell identity such SOX17, CXCR4, FOXA2, and GATA4.
  • the substantially homogenous population of cells express markers that are correlated with posterior foregut cell identity such as ⁇ , HNF4A while suppressing expression of HHEX, HOXA3, CDX2, OCT4, and NANOG.
  • the substantially homogenous population of cells express markers that are correlated with pancreatic progenitor cell identity such as PDX1 (pancreatic duodenal homeobox gene 1).
  • the substantially homogenous population of cells express markers that are correlated with endocrine pancreas cell identity such as NKX6.1, NEURO-D1, and NGN3.
  • the substantially homogenous population of cells express markers that are correlated with islet precursor cell identity such as INS. This population may further be identified by its ability to secrete C-peptide.
  • a “gene” refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular RNA, polypeptide, or protein after being transcribed and/or translated.
  • the term “express” refers to the production of a gene product.
  • expression refers to the process by which polynucleotides are transcribed into RNA and/or the process by which the transcribed RNA such as mRNA is subsequently being translated into peptides, polypeptides, or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • a “gene product” or alternatively a “gene expression product” refers to the amino acid (e.g., peptide or polypeptide) or functional RNA (e.g. a tRNA, miRNA, rRNA, or shRNA) generated when a gene is transcribed and translated.
  • amino acid e.g., peptide or polypeptide
  • functional RNA e.g. a tRNA, miRNA, rRNA, or shRNA
  • pancreatic or immune disorder or condition refers to ameliorating the effects of, or delaying, halting or reversing the progress of, or delaying or preventing the onset of, a pancreatic or immune condition such as diabetes, prediabetes, juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes).
  • Type I diabetes mellitus including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes).
  • Treatment includes preventing the disease or condition (i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease), inhibiting the disease or condition (i.e., arresting or reducing the development of the disease or its clinical symptoms), or relieving the disease or condition (i.e., causing regression of the disease or its clinical symptoms).
  • the disease or condition i.e., causing the clinical symptoms of the disease not to develop in a patient that may be predisposed to the disease but does not yet experience or display symptoms of the disease
  • inhibiting the disease or condition i.e., arresting or reducing the development of the disease or its clinical symptoms
  • relieving the disease or condition i.e., causing regression of the disease or its clinical symptoms.
  • a mammalian stem cell intends a stem cell having an origin from a mammal.
  • Non-limiting examples include, e.g., a murine, a canine, an equine, a simian and a human.
  • An animal stem cell intends a stem cell having an origin from an animal, e.g., a mammalian stem cell.
  • a "subject,” “individual” or “patient” is used interchangeably herein, and refers to a vertebrate, preferably a mammal, more preferably a human.
  • Mammals include, but are not limited to, murines, rats, rabbit, simians, bovines, ovine, porcine, canines, feline, farm animals, sport animals, pets, equine, and primate, particularly human.
  • the methods and compositions disclosed herein are also useful for veterinary treatment of companion mammals, exotic animals and domesticated animals, including mammals, rodents, and the like which is susceptible to diabetes or other immune or pancreatic diseases or conditions.
  • the mammals include horses, dogs, and cats.
  • the human is an adolescent or infant under the age of eighteen years.
  • An immature stem cell intends a phenotype wherein the cell expresses or fails to express one or more markers of a mature phenotype. Examples of such are known in the art, e.g., telomerase length or the expression of actin for mature cardiomyocytes derived or differentiated from a less mature phenotype such as an embryonic stem cell.
  • An immature beta cell intends a pancreatic cell that has insulin secretory granules but lacks GSIS. In contrast, mature beta cells typically are positive for GSIS and have low lactate dehydrogenase (LDH).
  • Vitronectin refers to a an extracellular matrix glycoprotein encoded by the VTN gene (Entrez gene: 7448, UniProt: P04004). Vitronectin is available from, for example, Sigma Aldrich (e.g. CAS#: 83380-82-9; cat.#: V8379). In some embodiments, Vitronectin is truncated human protein, corresponding to the amino acid fragment 62-478 of human vitronectin. In some embodiments, vitronectin is purified from inclusion bodies and refolded for use as a substrate for the feeder-free culture of human PSCs (1).
  • vitronectin When used with Essential 8TM Medium, VTN-N has demonstrated the ability to maintain pluripotency and normal growth characteristics in pluripotent stem cell lines.
  • Equivalents of vitronectin include chimeric and recombinant vitronectin, as well as other matricellular proteins and other extracellular matrix (ECM) molecules and compositions that are capable of supporting growth and/or adhesion of cultured stem cells such as laminin, collagen, gelatin, fibronectin, entactin, nidogen-2, elastin, perlecan, and tropoelastin, and basement membrane matrices (e.g. Matrigel).
  • ECM extracellular matrix
  • ECMs with an arginine-glycine-aspartic acid ('RGD') motif serve as ligands for cellular integrins.
  • RGD ECMs include laminin, vitronectin, and fibronectin.
  • Laminin has demonstrated binding to integrin heterodimers including ⁇ , ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1, ⁇ , ⁇ 6 ⁇ 4, ⁇ 7 ⁇ 1, ⁇ 9 ⁇ 1, and ⁇ 3.
  • Vitronectin and fibronectin mediate cell adhesion through binding integrins such as ⁇ 3 ⁇ 1, ⁇ 5 ⁇ 1, ⁇ 8 ⁇ 1, ⁇ , ⁇ 3, ⁇ 5, and ⁇ .
  • ECMs ECMs, serums, and mixtures of various ECMs have also demonstrated ability to support cultured stem cells including Matrigel, an ECM mixture containing laminin, collagen type IV, heparin sulfate, proteoglycans, entactin, and nidogen.
  • the methods herein use vitronectin or equivalents that are devoid of animal products to avoid the presence of undefined factors that may increase experimental variability.
  • Collagen is a structural protein that is generally found in connective tissue and the extracellular space of animals. Collagen is classified into several types including but not limited to type I (e.g.
  • COL1 Al Entrez gene: 1277, UniProt: P02452; COL1 A2 (Entrez gene: 1278, UniProt: P08123)), type II (e.g. COL2A1 (Entrez gene: 1280, UniProt: P02458)), type III (e.g. COL3A1 (Entrez gene: 1281, UniProt: P02461)), type IV (basement membrane collagen, e.g.
  • COL4A1 Entrez gene: 1282, UniProt: P02462
  • COL4A2 Entrez gene: 1284, UniProt: P08572
  • COL4A3 Entrez gene: 1285, UniProt: Q01955)
  • COL4A4 Entrez gene: 1286, UniProt: P53420
  • COL4A5 Entrez gene: 1287, UniProt: P29400
  • COL4A6 Entrez gene: 1288, UniProt: Q14031
  • type V e.g.
  • COL5A1 Entrez gene: 1289, UniProt: P20908
  • COL5A2 Entrez gene: 1290, UniProt: P05997
  • COL5A3 Entrez gene: 5059, UniProt: P25940
  • type VI e.g. COL6A1 (Entrez gene: 1291, UniProt: P12109), COL6A2 (Entrez gene: 1292, UniProt: P12110), COL6A3 (Entrez gene: 1293, UniProt: P12111), COL6A5 (Entrez gene: 256076, UniProt: PA8TX70, H0Y935)
  • type VII e.g.
  • COL7A1 (Entrez gene: 1294, UniProt: Q02388)), type VIII (e.g. COL8A1 (Entrez gene: 1295, UniProt: P27658), COL8A2 (Entrez gene: 1296, UniProt: P25067, Q4VAQ0)), type IX (e.g. COL9A1 (Entrez gene: 1297, UniProt: P20908), COL9A2 (Entrez gene: 1290, UniProt: P05997), COL9A3 (Entrez gene: 5059, UniProt: P25940)), type X (e.g.
  • COL 10A1 Entrez gene: 1300, UniProt: A03692
  • type XI e.g. COL11A1 (Entrez gene: 1301, UniProt: P12107), COL11A2 (Entrez gene: 1302, UniProt: P13942)
  • type XII e.g. COL 12 Al (Entrez gene: 1303, UniProt:
  • the collagen is type IV collagen.
  • Collagens are available from, for example, Sigma Aldrich, St. Louis, Missouri, U.S.A. (e.g. CAS# 9007-34-5, cat.#: C6745).
  • Laminins are heterotrimeric proteins formed by combinations of an alpha chain (e.g. LAMA1 (Entrez gene: 284217, UniProt: P25391), LAMA2 (Entrez gene: 3908, UniProt: P24043), LAMA3 (Entrez gene: 3909, UniProt: Q16787), LAMA4 (Entrez gene: 3910, UniProt: Q16363), or LAMA5 (Entrez gene: 3911, UniProt: O15230)), beta chain (e.g. LAMA1 (Entrez gene: 284217, UniProt: P25391), LAMA2 (Entrez gene: 3908, UniProt: P24043), LAMA3 (Entrez gene: 3909, UniProt: Q16787), LAMA4 (Entrez gene: 3910, UniProt: Q16363), or LAMA5 (Entrez gene: 3911, UniProt: O15230)), beta chain (e.g.
  • alpha chain e.g. LAMA1 (Entrez gene:
  • LAMB1 Entrez gene: 3912, UniProt: P07942
  • LAMB2 Entrez gene: 3913, UniProt:
  • LAMB 3 Entrez gene: 3914, UniProt: Q13751
  • LAMB4 Entrez gene: 22798
  • gamma chain e.g. LAMC1 (Entrez gene: 22798, UniProt: P25391), LAMC2 (Entrez gene: 284217, UniProt: P25391), or LAMC3 (Entrez gene: 284217, UniProt: P25391)) of laminin.
  • Laminins include but are not limited to the following trimers: laminin 111, laminin 211, laminin 121, laminin 221, laminin 332, laminin 3A32, laminin 3B32, laminin 311, laminin 3 Al l, laminin 312, laminin 3A21, laminin 411, laminin 421, laminin 511, laminin 521, laminin 213, laminin 423, laminin 522, and laminin 523.
  • Laminin is available from, for example, Sigma Aldrich (e.g. Laminin from Engelbreth-Holm- Swarm murine sarcoma basement membrane, CAS #: 114956-81-9, catJ: L2020).
  • basement membrane matrices are derived from the extracellular matrix that separates the epithelium, endothelium, muscle, or neuronal cells from adj ascent stromal tissue (known as the basement membrane).
  • the basement membrane is comprised of a thin, delicate membrane of extracellular matrix proteins and glycosaminoglycans (LeBleu, V.S. et al. Exp Biol Med 232(9): 1121-29 (2007)).
  • a solubilized basement membrane matrix can be prepared from the extracellular matrix of Engelbreth-Holm- Swarm (EHS) mouse sarcoma cells (see, e.g. Robinson, L.K. et al. J Biol. Chem. 264(9): 5141-47 (1989).
  • EHS Engelbreth-Holm- Swarm
  • Matrigel is derived from EHS and comprises extracellular matrix proteins such as laminin, collagen IV, growth factors, heparin sulfate proteoglycans, entactin, and nidogen.
  • Matrigel is available in a standard formulation (Corning cat.# 356234), high protein concentration formulation (Corning cat.# 354248), growth factor reduced formulation comprising a more highly defined basement membrane preparation (Corning cat.# 356230), and hESC-qualified formulations that have been pre-screened for compatibility with mTeSRl medium from Stem Cell Technologies (Corning cat.# 354277).
  • the standard protein concentration formulations comprise 8-12 mg/mL.
  • the high protein concentration formulations comprise 18-21 mg/mL.
  • Cultrex BME is also derived from EHS and comprises laminin, collagen IV, entactin, and heparin sulfate proteoglycans.
  • Cultrex BME is available in a reduced growth factor formulation for 3-D culture (Trevigen cat.# 3433-001-01), stem cell qualified formulation (Trevigen cat.# 3434-001-02), and organoid qualified formulations (e.g., Trevigen cat.# 3533-001-02). All Cultrex formulations range from 12-18 mg/mL of protein except the reduced growth factor culture formulation which ranges from 14-16 mg/mL of protein.
  • Gibco Geltrex Matrix is a basement membrane matrix derived from EHS and available in reduced growth factor formulations (ThermoFisher Scientific cat.# A1569601).
  • Gelatin is a heterogeneous mixture of proteins and protein fragments (e.g. peptides) produced by hydrolysis of collagen.
  • Type A gelatin is derived from acid-cured animal tissue (e.g. skin, tendons, ligaments, and bones) and type B gelatin is derived from lime-cured animal tissue.
  • Gelatin is available from, for example, Sigma Aldrich (e.g. CAS#: 9000-70-8, catJ: G1393, G9391, G1890).
  • a “signaling pathway” intends a process of signal transduction through which a chemical or physical signal is transmitted inside of a cell or between cells through a series of molecular or biochemical events, such as protein phosphorylation.
  • the output of the signal transduction process may be repression or expression of a gene or set of genes, as well as other molecular changes inside the cell that relate to cell function, location, or identity.
  • Components of a signaling pathway may include first messengers (i.e. ligands and/or extracellular and intracellular signaling molecules)), signal transducers (i.e. receptors), primary effectors (i.e. expressed genes), second messengers, and secondary effectors.
  • Non- limiting examples of signaling pathways that are relevant to the methods disclosed herein are Activin, WNT, BMP, FGF, AKT, TGFp, Notch, and Sonic Hedgehog pathways.
  • modulation of a signaling pathway means any method of altering or stabilizing the level of signaling or signaling output, including inhibition of signaling, activation of signaling, stimulation of signaling, increased signaling, and decreased signaling. Modes of modulation may involve targeting one or more specific messengers, one or more signal transducers, and/or one or more effectors in a signaling pathway to produce a desired result.
  • Specific methods modulation include use of inhibitors, activators, competitors, mutation or deletion of pathway genes, changes in transcription of pathway genes, changes in stability of mRNA encoding pathway genes, changes in translation of pathway genes, posttranslational modification of pathway gene products, and/or changes in the stability or degradation of pathway molecules.
  • Non-specific modulation of signaling pathways may involve targeting components of the signaling pathway that are involved in a plurality of different signaling pathways.
  • broad, non-specific inhibition of signaling may be achieved through inhibition of kinase activity (e.g. use of kinase inhibitors), inhibition of cyclic AMP, or inhibition of protein synthesis (e.g.
  • Activins are members of the Transforming growth factor beta (TGFP) family and participate in cell differentiation, proliferation, immune responses, apoptosis, and other biological processes.
  • Activin A (ActA) binds to Activin A receptors type II and recruits Activin A receptor, type IB which then signals through the SMAD family.
  • Activin signaling may be increased or stimulated by ActA, FAST-1/2, or PC2 (TIG1).
  • Activin signaling may be inhibited by Follistatin, FSRP, Inhibin, IGSFl, BAMBI, FKBP12, SMURFl, or SMAD7. Additional methods of modulation of Activin signaling may involve targeting any other messengers, signal transducers, or effectors in the Activin signaling pathway.
  • WNT signaling pathways include the canonical WNT pathway and two non- canonical pathways: planar cell polarity and Wnt/calcium pathways.
  • the WNT pathways are activated when a Wnt family protein ligand binds to a Frizzled family receptor, which then transduces the signal to a Disheveled intracellular protein.
  • the outputs of WNT signaling pathways include changes in gene transcription, changes in the cytoskeleton, or regulation of calcium.
  • Human WNT family ligands include WNT1, WNT2, WNT2B, WNT3, WNT3A, WNT4, WNT 5 A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9B, WNT 1 OA, WNT 1 OB, WNT11, and WNT16.
  • Murine Wnt family ligands include Wntl, Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B, WntlOA, WntlOB, Wntl 1, and Wntl6.
  • WNT3a is involved in proliferation, apoptosis, and function of pancreatic cells.
  • WNT3a/Wnt signaling may be modulated by CHIR99021, Dkkl, or wortmannin.
  • Additional methods of modulation of WNT signaling may involve targeting any other messengers, signal transducers, or effectors in the WNT signaling pathway such as Frizzled, Disheveled, ⁇ -catenin, RAC, INK, cJUN, APC, JNK2, TAK1, NLK, NFAT, Calmodulin, and RhoA.
  • any other messengers, signal transducers, or effectors in the WNT signaling pathway such as Frizzled, Disheveled, ⁇ -catenin, RAC, INK, cJUN, APC, JNK2, TAK1, NLK, NFAT, Calmodulin, and RhoA.
  • the BMP signaling pathways comprise bone morphogenic proteins (BMPs), which are a group of cytokines capable of inducing formation of bone and cartilage, in addition to other important roles in transmitting morphogenetic signals and regulating tissue
  • BMPs bone morphogenic proteins
  • BMPs belong to the TGFP superfamily of proteins.
  • BMPs include BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP 8b, BMP 10, and BMP15.
  • BMP4 pathway signaling can be modulated by BMP4, Noggin, Chordin, LDN193189 dihydrochloride, DMH-1, DMH-2, K02299, ML347, and
  • Additional methods of modulation of BMP signaling may involve targeting any other messengers, signal transducers, or effectors in the BMP signaling pathway such as the SMAD family, ALK2, ALK3, ALK6, ALK7, BMPR2, BMPR1, ERK, J K, or p38.
  • Fibroblast Growth Factor (FGF) signaling pathways are comprised of secreted proteins that interact with tyrosine kinase FGF receptors (FGFRs) to effect signaling through the RAS-MAPK, PI3K-AKT, PLCgamma, and STAT intracellular signaling pathways.
  • FGF Fibroblast Growth Factor
  • Mammalian FGFs include FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF 10, FGF11, FGF 12, FGF13, FGF 14, FGF 15, FGF 16, FGF 17, FGF 18, FGF 19, FGF20, FGF21, FGF22, and FGF23.
  • Mammalian FGFRs include FGFR1, FGFR2, FGFR3, FGFR4, and FGFRLl .
  • FGF2 is also known as Basic FGF (bFGF) and FIBGF2 (heparin-binding growth factor 2).
  • FGF2 and FGF 10 may use Heparin or Heparan sulfate as cofactors.
  • Methods of modulation of the FGF signaling pathway may involve targeting any other messengers, signal transducers, or effectors in the FGF signaling pathway.
  • AKT Protein Kinase B
  • PI3K phosphatidylinositol 3-kinase
  • AKT is activated by the phosphorylated plasma membrane docking sites and mediates downstream signaling.
  • the AKT signaling pathway can be modulated by PTEN, an antagonist of PI3K activity, reduction of PIP 3 levels, SHIP, F- ⁇ , protein phosphatase 2A (PP2A) and inhibitors of PI3K such as LY294002, A66, AS252424, AS605240, AZD6482, BAG956, CZC24832, ETP45658, GSK1059615, KU0060648, LTURM36, 3-Methyladenine, PF04691502, PF05212384, PI103hydrochloride, PI3065, PI828, PP121, Quercetin,
  • Akti-1/2, API-1, API-2, 10-DEBC hydrochloride, FPA124, GSK690639, PHT427, OSU03012, and SC66 are inhibitors of AKT.
  • AC79 is an activator of AKT. Additional methods of modulation of AKT signaling may involve targeting any other messengers, signal transducers, or effectors in the AKT signaling pathway.
  • the TGFP signaling pathway involves a ligand from the TFGP super family of ligands which binds to a TGFP type II receptor, which subsequently phosphorylates a TGFP type I receptor.
  • the phosphorylated type I receptor then activates the SMAD family which can regulate transcription in the nucleus.
  • the TGFP signaling pathway can be inhibited by SB-431542, A83-01, D4476, GW788388, LY364947, R268712, RepSox, SB-505124, SB- 525334, SD208, ITD1, and SIS3.
  • Inhibitors of ALK5 also modulate the TGFp signaling pathway. N-Acetylpuromycin promotes TFGP signaling. Additional methods of modulation of TGFP signaling may involve targeting any other messengers, signal transducers, or effectors in the TGFP signaling pathway.
  • Retinoic acid is a metabolite of vitamin A.
  • Equivalents of retinoic acid include but are not limited to other metabolites of vitamin A such as tretinoin, isotretinoin, retinol, retinal, beta-carotene, and other provitamin A carotenoids.
  • B-27 is a supplement to growth medium which may or may not contain insulin. Though the concentrations of the components are proprietary, B-27 contains Biotin, DL Alpha Tocopherol Acetate, DL Alpha-Tocopherol, Vitamin A (acetate), BSA, fatty acid free Fraction V, Catalase, Human Transferrin, Superoxide Dismutase, Corticosterone, D- Galactose, Ethanolamine HC1, Gutathione (reduced) L-Carnitine HC1, Linoleic Acid, Linolenic Acid, Progesterone, Putrescine 2HC1, Sodium Selenite, and T3 (trido-L-thyronine). B-27 is available from ThermoFisher Scientific. Equivalents of B-27 include other growth medium supplements that contain at least a majority of the above-listed components.
  • FIGs. 1 and 6 depict embodiments of the disclosed methods.
  • definitive endoderm cells can be obtained after from about one to about four days, or for about three days of differentiation with striking and precise dynamic modulation of Activin, Wnt3, BMP4, bFGF (also known as FGF2) and AKT signaling pathways. Special attention was put on the Primitive Streak (PS) commitment and transition of cells from pluripotency to the DE stage. The methods described herein obtain more efficiently anterior definitive endoderm progenitors that will give homogeneous populations of pancreatic progenitors on later stages.
  • PS Primitive Streak
  • Disclosed herein is a novel protocol for obtaining insulin-producing cells from human ESCs. This method is an improved, robust, and reliable method for in vitro pancreatic beta-cell differentiation.
  • the work described herein broadens knowledge in the stem cell field and allows new approaches for techniques in cell manipulation, as well as finding new strategies for improving differentiation protocols.
  • This research is a novel approach to an important clinical problem, which applies chemical screening to produce cells that mimic functional beta-cells for potential stem cell-based therapies in type I diabetes.
  • BMP and Wnt pathways can be targeted to specifying anterior primitive streak formation (progenitor to endoderm) during the first 24 hours of PS commitment.
  • the dynamics of activation of Wnt pathway were analyzed in a time- and dose- dependent way by varying exposure to the Wnt inhibitor CHTR99021.
  • the same strategy was used to target the BMP pathway, using dorsomorphin.
  • a protocol was developed in which cells are committed to the PS by combined actions of high doses of Activin- A, low levels of BMP4, bFGF, and the PI3K inhibitor LY294002, activating Wnt3a signaling pathway with GSK3 inhibitor CHIR99021 (2 ⁇ ) during the first 24 hours of differentiation.
  • the blockade of the BMP4 signaling pathway with 100 ⁇ dorsomorphin avoids mesoderm contamination in future steps of differentiation.
  • PI3K inhibition may be necessary to enhance DE differentiation.
  • BMP4 and AKT blockade after short stimulation of WNT during PS enhances expression of HHEX, an important factor for proper
  • the culture system for DE specification is devoid of animal products including BSA, serum, complex extracellular matrices such as Matrigel or feeder cells at all steps, thereby avoiding the presence of undefined factors that may increase experimental variability.
  • DE progenitors were transitioned to Advanced DMEM media with RA, TGF beta inhibitor SB431542, and Noggin.
  • Noggin was previously shown to specify the emerging gut tube.
  • Noggin can be replaced by low doses of dorsomorphin.
  • RA and inhibition of the BMP pathway are important for specification of stage 2 cells in vitro. However, depending on the chemistry used during DE specification, the anterior-posterior patterning of the foregut is altered. In some embodiments, inhibition of BMP after PS commitment generates a more PF with almost complete absence of the midgut and hindgut markers HOXA3 and CDX2.
  • blockade of TGF beta with SB431542 inhibitor in the presence of Noggin highly enhanced expression of HNFIB and significantly suppresses expression of HHEX, HOXA3 and CDX2.
  • Noggin or Dorsomorphin, as noted above
  • HNFIB highly enhanced expression of HNFIB and significantly suppresses expression of HHEX, HOXA3 and CDX2.
  • the absence of HHEX expression suggested a dorsal identity for these foregut cells, predisposing them to pancreatic determination, while the absence of HOXA3 and CDX2 excluded the presence of midgut and hindgut cells, respectively.
  • other cytokines used in this work for specification of the PF are implicated in pancreatic development in vivo.
  • RA and Noggin are necessary for the induction of pancreatic specification, and FGF10 is also necessary for the proliferation and survival of pancreatic endoderm in vitro.
  • TGF beta inhibition has been used previously to specify PF, but the methods disclosed herein are the first to implement this important stage of differentiation with the combined actions of RA, FGF10 and dorsomorphin.
  • BMP signaling during pancreatic commitment has been demonstrated to participate actively in later decisions of pancreatic progenitors.
  • BMP signaling from the septum transversum mesenchyme promotes hepatic fate, and at high levels, without being bound by theory, it represses pancreatic development.
  • Several groups have used chemical inhibitors of the BMP pathway for pancreatic differentiation these protocols use dorsomorphin or noggin to inhibit BMP4 pathway only after PF commitment (after St2 of differentiation).
  • the methods disclosed herein utilize low doses of dorsomorphin to mimic inhibitory actions of high levels of Noggin and are novel in utilizing this chemical inhibition immediately after PS commitment, at very early stages of DE differentiation in cells previously conditioned in E8 media and vitronectin-coated plates. Use of this chemical in later stages of DE and during St2 and St3 of differentiation results in almost entirely pure populations of PDX1 -positive cells after only nine days of
  • Pancreatic progenitors rapidly specify after Sonic hedgehog inhibition with cyclopamine, so the cells were maintained in the presence of this chemical for 3 days of pancreatic commitment.
  • Sonic hedgehog inhibitors SANT-1
  • pancreatic progenitors To facilitate the transition of obtained pancreatic progenitors to endocrine pancreas (St4), cells were maintained in the presence of FGF10 for 3 days and the Notch pathway was inhibited for another subsequent three days.
  • FGF10 is highly expressed in pancreatic mesenchyme, promoting epithelial proliferation, migration, and correct positioning of the liver, pancreas, and various hepatobiliary ducts.
  • mesenchymal FGF10 then promotes the proliferation of pancreatic progenitor).
  • the Notch-delta signaling pathway is required for segregating the endocrine lineages via NGN3, SOX9, and FIES1 transcription factor).
  • the ductal versus endocrine fate decision is controlled by graded Notch activity.
  • High Notch activity promotes the ductal fate by activating both HES1, a repressor of NGN3, and SOX9, an NGN3 activator.
  • Low Notch activity only NGN3 is activated leading to endocrine differentiation.
  • FGF10 and DAPT induce expression of important endocrine genes like NG3, NKX6.1 and NEURODl, confirming developmental precedents.
  • Endocrine progenitors obtained in stage 4 of differentiation of this protocol are highly proliferative and they support several rounds of passage and cryopreservation without losing their properties.
  • stage 5 After endocrine pancreas commitment, cells are transitioned to DMEM for maturation of islet precursor cells. Advanced DMEM was avoided in the final stage (stage 5) of differentiation due to the high level of insulin of this medium, which could interfere with c-peptide assays. In stage 5 of the differentiation protocol, a three-dimensional culture condition was employed to produce spheroids. Endocrine progenitors were then matured in the presence of Alk5 inhibitor, nicotinamide, and dexamethasone. Without being bound by theory, Nicotinamide, also known as vitamin B3, has been shown to promote endocrine differentiation from human fetal explants and hESC by specific induction of NKX6.1 expression.
  • Dexamethasone is a steroid molecule that has been shown to improve maturation of hepatocytes and pancreatic cells, but the molecular actions are unknown.
  • TGF-beta RI Kinase Inhibitor II (Alk5 inh ll) is used for pancreatic maturation and functionality experiments with excellent results. Transition from 2D to 3D conditions by centrifugation and compaction of stage 5 cells enhances expression of INS, PDX1, and NGN3 genes.
  • Stage 5 spheroids show a gene expression pattern similar to fetal pancreas and adult islet samples. In fact, cells release c-peptide after glucose stimulation, confirming their functionality in vitro. Without being bound by theory, all of these results highlight the importance of a correct protocol improved from early to late stages of differentiation to obtain the desired cell phenotype.
  • Non-limiting examples of appropriate cell lines for use in practicing this method are described herein and include the H9 (WA09) human embryonic stem cell line and the iPSC line sHDF#l .
  • This method works efficiently on homogenous or substantially homogenous populations of human pluripotent stem cells. Without being bound by theory, contamination by differentiated cells strongly interferes with endodermal differentiation. Optimal results may be achieved with a starting population of cells in which at least 80% of the cells are uniformly positive for OCT4 and NANOG.
  • this method is tested on hESC and hiPSC cultures grown on vitronectin-coated plates in E8 medium (Gibco). Vitronectin can support undifferentiated hESC cultures and is compatible with definitive endoderm (DE) specification. Use of FBS and gelatin may result in the undesired induction of mesoderm contaminants by BMP molecules present in FBS.
  • the morphology of differentiating cells should be monitored every day. Monitoring of the efficiency of the differentiation process should include immunostaining, FACS and gene expression analysis.
  • the cells of the disclosed method are pluripotent stem cells (PSCs).
  • the PSCs of the present invention include any pluripotent cell, including for example, an embryonic stem cell (ESC), an embryonic germ cell (EGC), a blastocyst, an induced pluripotent stem cell (iPSC) or a somatic cell.
  • the naive pluripotent stem cell is selected from the group consisting of an ESC, an EGC, a blastocyst, an iPSC or a somatic cell.
  • the primed pluripotent stem cell is selected from the group consisting of an ESC, an EGC, a blastocyst, an iPSC or a somatic cell.
  • the PSCs of the present invention may be derived from an animal such as a mammal, preferably a human, but includes and is not limited to non-human primates, murines (i.e., mice and rats), canines, felines, equines, bovines, ovines, porcines, caprines, etc.
  • the PSC is a human PSC.
  • ESCs can be obtained by any method known in the art.
  • ESCs can be isolated from a blastocyst-stage or epiblast-stage embryo.
  • ESCs have been obtained from many species, including, but limited to, mouse (Mills et al. (2001) Trends Genet. 17:331- 339); rat (lannaccone et al. (1994) Dev. Biol. 163 :288-292); non-human primate (Thomson et al. (1995) Proc. Natl. Acad. Sci. USA 92:7844-7848); and human (Thomson et al., (1998) Science 282(5391): 1145-1147).
  • hESCs Human ESCs
  • IVVF in vitro fertilization
  • some methods involve removing a single cell from an embryo during the IVF process. Chung et al. (2008) Cell Stem Cell 2(2): 113-117. This method allows for the generation of a hESC line without concomitant destruction of the embryo. It will also be appreciated that many commercially available hESCs are available and can be used according to the embodiments of the disclosure.
  • Exemplary hESC cell lines include but are not limited to cell lines listed on the National Institutes of Health Embryonic Stem Cell Registry such as CHB-1-6, 8-12
  • the embryonic cell line for use in the disclosed methods is the H9 (WA09) human embryonic stem cell line.
  • EGCs Embryonic germ cells
  • germ cells gametes
  • EGCs are pluripotent in vitro; however EGCs have yet to be shown to be pluripotent in vivo.
  • EGCs are prepared from the primordial gem cells obtained from fetuses using techniques well- known in the art. Shamblott et al. (1998) Proc. Natl. Acad. Sci USA 95: 13726-13731.
  • iPSCs are cells derived from non-pluripotent cells such as somatic cells (e.g.
  • fibroblasts fibroblasts, peripheral blood mononucleocytes, and keratinocytes
  • multipotent stem and progenitor cells e.g., CD34+ hematopoietic progenitors, neural progenitor cells, and adipose- derived stem cells
  • exemplary reprogramming genes include but are not limited to OCT4, SOX2, KLF4, cMYC, NANOG, LIN28, REX1, Zfp296, D MT3B, AURKB, PRMT5, SETD7, and Glisl .
  • Techniques for the production of iPSCs are well-known in the art (see, e.g., Yu, J. et al.
  • the iPSC cell line for use in the disclosed methods is sHDF#l .
  • the stem cells are adult stem cells, for example, amniotic stem cells, neural stem cells, hepatic stem cells, hematopoietic stem cells, umbilical cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells, pancreatic stem cells, and placental stem cells.
  • adult stem cells for example, amniotic stem cells, neural stem cells, hepatic stem cells, hematopoietic stem cells, umbilical cord blood stem cells, epidermal stem cells, gastrointestinal stem cells, endothelial stem cells, muscle stem cells, mesenchymal stem cells, pancreatic stem cells, and placental stem cells.
  • the stems cells comprise a cell line. In some embodiments, the stem cells comprise a population of stem cells.
  • Nonlimiting examples of signaling pathway modulators, media supplements, media components, media additives, and/or growth factors include Activin A (03-0001 Stemgent), Advanced DMEM (12491023 Gibco), Alk5 inhibitor II (ALX-270-445-M005 ENZO life science), B27 (Minus Insulin) (A18956-01 Gibco), BMP4 (314-BP-050 R&D), CHIR99021 (4423 Tocris), Cyclopamine (SI 146 Selleckchem), DAPT (D5942 Sigma), Dexamethasone (D8893 Sigma), DMEM (11995065 Gibco), Dorsomorphin (1435934-00-1 Axon Medchem), E8 (A1517001 Gibco), FGF10 (100 - 26 Peprotech), FGF2 (HRP 0011-1 BioPioneer Inc.), IMDM (12440053 Gibco), ITS 100X (41400-045 Gibco), Lipid 100X (11905-031
  • Equivalents of each of the media supplements, component, additives, and/or growth factors listed herein include but are not limited to (i) recombinant versions of the media supplements, component, additives, and/or growth factors, (ii) truncations of the media supplements, component, additives, and/or growth factors, and (iii) other media supplements, component, additives, and/or growth factors that modulate the same signaling pathway as the media supplement, component, additive, and/or growth factor.
  • CDM-PVA medium contains DMEM-F12 (Gibco) and IMDM (Gibco) mixed 1 : 1, 1% Glutamax, 1% concentrated lipids (Gibco), 450 mM Monohioglycerol (Sigma), 1% Insulin-Transferrin-Selenium ITS supplement (Gibco), and lmg/mL Polyvinylalcohol (Sigma).
  • Medium can be stored at 4°C for up to 1 month. This media is used for PS and DE (Stage 1) differentiation.
  • Endoderm (DE, Stage lb) with 100 ng/mL ActivinA, 10 ⁇ LY294002, 20 ng/mL FGF2, 100 nM Dorsomorphin (Axon Medchem) in CDM-PVA.
  • Advanced DMEM (Gibco) medium is used for stages 2-4.
  • stage 2 posterior foregut
  • cells were cultured for 3 days in 10 ⁇ SB431542 (Selleckchem), 2 ⁇ Retinoic Acid (Sigma), 100 nM Dorsomorphin (Axon Medchem), and 50 ng/mL FGF10 (Peprotech) in Advanced DMEM (Gibco).
  • stage 3 Pancreatic Progenitor cells
  • cells were cultured for 3 days in 0.25 ⁇ Cyclopamine (Selleckchem), 2 ⁇ Retinoic Acid (Sigma), 100 nM Dorsomorphin (Axon Medchem), and 50 ng/mL FGF10 (Peprotech) in Advanced DMEM (Gibco).
  • stage 4 Pancreatic Endocrine Progenitors
  • cells were cultured for 3 days in 50 ng/mL FGF10 (Peprotech) in Advanced DMEM (Gibco) followed by 3 days in 1% B27 without insulin (Gibco) and 10 ⁇ DAPT (Sigma) in Advanced DMEM (Gibco).
  • DMEM (Gibco) is used for stage 5.
  • spheroids are induced from the Pancreatic Endocrine Progenitors obtained from stage 4 (see above) and matured for 6 days in 10 mM Nicotinamide (Sigma), 1 ⁇ Alk5 inhibitor II (Enzo Life Sciences), 10 mM dexamethasone (Sigma), and 1% B27 (without insulin) (Gibco) in DMEM (Gibco).
  • the efficiency of the differentiation process at each step is monitored through immunostaining, flow cytometry, gene expression analysis including gene expression arrays, quantitative PCR, and RNA sequencing, proteomics, or immunohistochemistry or assaying for the production of c-peptide upon glucose stimulation.
  • the relevant genes or surface markers for determining differentiation progress are noted at each stage below.
  • the differentiation protocol begins by adapting pluripotent cells, cell line, or population to media in vitronectin-coated plate(s) or plate(s) coated with an equivalent thereof for about 0.5 days, about 1 day, about 1.5 days, about 2 days, about 2.5 days, about 3 days, about 3.5 days, about 4 days, about 4.5 days, about 5 days, about 5.5 days, about 6 days, about 6.5 days, or about 7 days.
  • the pluripotent cells, cell line, or population is undifferentiated.
  • vitronectin-coated plates or an equivalent thereof due to the combinatorial role of extracellular proteins and specific signaling pathways that promote endoderm differentiation, while avoiding possible residual contaminants from fetal bovine serum used in previous works.
  • Equivalents of vitronectin include chimeric and recombinant vitronectin, as well as other matricellular proteins and other extracellular matrix (ECM) molecules and compositions that are capable of supporting growth and/or adhesion of cultured stem cells such as laminin, collagen, gelatin, fibronectin, entactin, nidogen-2, elastin, perlecan, and tropoelastin, and basement membrane matrices (e.g. Matrigel).
  • ECM extracellular matrix
  • ECMs with an arginine-glycine-aspartic acid ('RGD') motif serve as ligands for cellular integrins.
  • RGD ECMs include laminin, vitronectin, and fibronectin.
  • Laminin has demonstrated binding to integrin heterodimers including ⁇ , ⁇ 2 ⁇ 1, ⁇ 3 ⁇ 1, ⁇ , ⁇ 6 ⁇ 4, ⁇ 7 ⁇ 1, ⁇ 9 ⁇ 1, and ⁇ 3.
  • Vitronectin and fibronectin mediate cell adhesion through binding integrins such as ⁇ 3 ⁇ 1, ⁇ 5 ⁇ 1, ⁇ 8 ⁇ 1, ⁇ , ⁇ 3, ⁇ 5, and ⁇ .
  • ECMs ECMs, serums, and mixtures of various ECMs have also demonstrated ability to support cultured stem cells including Matrigel, an ECM mixture containing laminin, collagen type IV, heparin sulfate, proteoglycans, entactin, and nidogen.
  • the methods herein use vitronectin or equivalents that are devoid of animal products to avoid the presence of undefined factors that may increase experimental variability.
  • the concentration of vitronectin or the equivalent ranges from about 1 ⁇ g/mL to about 30 ⁇ g/mL, about 1 ⁇ g/mL to about 25 ⁇ g/mL, about 1 ⁇ g/mL to about 20 ⁇ g/mL, about 1 ⁇ g/mL to about 15 ⁇ g/mL, about 1 ⁇ g/mL to about 10 ⁇ g/mL, about 1 ⁇ g/mL to about 9 ⁇ g/mL, about 1 ⁇ g/mL to about 8 ⁇ g/mL, about 1 ⁇ g/mL to about 7 ⁇ g/mL, about 1 ⁇ g/mL to about 6 ⁇ g/mL, about 1 ⁇ g/mL to about 5 ⁇ g/mL, about 4 ⁇ g/mL to about 30 ⁇ g/mL, about 4 ⁇ g/mL to about 25 ⁇ g/mL, about 4 ⁇ g/mL to about 20 ⁇ g/mL
  • the concentration of vitronectin or an equivalent thereof is about 0.5 ⁇ g/mL, 1 ⁇ g/mL, about 2 ⁇ g/mL, about 5 ⁇ g/mL, about 10 ⁇ g/mL, or about 1 mg/mL.
  • the plate(s) are coated with vitronectin or an equivalent thereof at a coating concentration of about 0.01 ⁇ g/cm 2 , about 0.1 ⁇ g/cm 2 , about 0.2 ⁇ g/cm 2 , about 0.3 ⁇ g/cm 2 , about 0.4 ⁇ g/cm 2 , about 0.5 ⁇ g/cm 2 , about 0.6 ⁇ g/cm 2 , about 0.7 ⁇ g/cm 2 , about 0.8 ⁇ g/cm 2 , about 0.9 ⁇ g/cm 2 , about 1 ⁇ g/cm 2 , about 2 ⁇ g/cm 2 , about 3 ⁇ g/cm 2 , about 4 ⁇ g/cm 2 , about 5 ⁇ g/cm 2 , about 6 ⁇ g/cm 2 , about 7 ⁇ g/cm 2 , about 8 ⁇ g/cm 2 , about 9 ⁇ g/cm 2 , about 10 ⁇ g
  • the plate(s) are coated with vitronectin or an equivalent thereof at a coating concentration of about 0.5 ⁇ g/cm 2 .
  • the plate(s) are pre-coated with vitronectin or the equivalent thereof for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 8 hours, about 10 hours, about 12 hours, about 14 hours, about 16 hours, about 18 hours, about 20 hours, about 22 hours, about 24 hours, about 36 hours, about 48 hours, about 72 hours, or about 96 hours.
  • the plate(s) are coated overnight. Coating may occur at room temperature, 37°C in 5% C0 2 , at 4°C, at 2°C, or a combination thereof. In some
  • the coated plate(s) are pre-warmed to 37°C prior to use. Optimally,
  • Vitronectin-coated plates are not stored at 4°C for greater than 1 week.
  • plates are coated with vitronectin diluted in DPBS without Calcium and Magnesium (Gibco) 1 : 100 (final concentration: 5 ⁇ g/mL).
  • the final concentration of vitronectin on the plates may vary depending on the pluripotent stem cell line within a range of 0.1 to 1 ⁇ g/cm 2 of coating surface.
  • the diluted vitronectin is added to the well (1 mL per well of a 6 well plate or 0.5 mL per well of a 12 well plate), following the manufacturer's instructions.
  • the plate is left at room temperature in the tissue culture hood for 1 hour. Vitronectin coated plates can be tightly sealed with parafilm and stored at 4°C for up to 1 week. The supernatant was removed immediately before use.
  • the medium in Stage 0 is capable of maintaining the pluripotency of the stem cells while adapting them to the coated plates.
  • the medium is Essential 8TM ("E8") medium.
  • E8 medium is available, for example, from ThermoFisher Scientific (cat# A1517001).
  • E8 medium has been shown to maintain growth and pluripotency of hESCs, but it has been rarely used for maintenance and expansion of pluripotent cells for pancreatic differentiation.
  • E8 is a xeno-free and feeder-free medium specially formulated for human PSCs. It contains DMEM F-12, L-scorbic acid, selenium, transferrin, NaHCC , Insulin, FGF2, and TGFpi .
  • E8 medium lacks FGF2, and TGFp.
  • E8 has been used to scale up production of PSCs and shown to support PSC growth for >50 passages without any signs of karyotypic abnormalities, along with maintaining the ability of iPSCs to differentiate into all three germ line lineages.
  • the medium in Stage 0 is mTeSRlTM Medium available from, for example, Stem Cell Technologies (Cat# 05850/05857/05870/05875).
  • mTeSR media comprises DMEM F-12, L-scorbic acid, selenium, transferrin, NaHC0 3 , glutathione, L-glutamine, defined lipids, thiamine, trace elements B, trace elements C, ⁇ -mercaptoethanol, albumin, insulin, FGF2, TGFpi, pipecolic acid, LiCl, GAB A, and H 2 0.
  • the medium lacks FGF2, and TGFp.
  • the medium is mTeSR2TM Medium, available from, for example, Stem Cell Technologies (Cat#
  • mTeSRTMl contains a bovine albumin source that supports the long-term, feeder-free culture of human ES and iPS cells as well as the derivation of human iPS cells in feeder-free conditionsl5,16.
  • TeSRTM2 is an animal protein-free formulation that similarly supports the long-term, feeder-free culture of human ES and iPS cells.
  • the medium is PluriSTEMTM Human ES/iPSC Media, available, for example, from Millipore Sigma (cat#SCM130 or SCM132).
  • PluriSTEM is a complete small molecule based serum-free medium that enables feeder-free culture of human ES/iPS cells.
  • undifferentiated, pluripotent cells are maintained in vitronectin (5 ⁇ g/mL; Invitrogen) coated plates in E8 media (Gibco) until they reached approximately 80% confluency. At this point cells are split with 0.5 mM EDTA in a 1/6 ratio on vitronectin-coated plates forming homogeneous clumps. Passage PSCs using EDTA (50 mM solution) incubating cells for about 3 minutes at 37°C in humidified incubator. After EDTA incubation, EDTA suspension is removed and 1 mL of E8 media is added to each well of a 6 well plate.
  • Colonies are slightly scratched using a 1000 uL pipette tip and resuspended in 1 mL of media until formation of homogeneous clumps. Optimally, colonies are not resuspended greater than 3 times and are split in small clumps and then plated on vitronectin- coated plates in E8 culture medium in a 1 to 6 ratio.
  • the passaging method may affect the efficiency of differentiation. Timing and temperature of incubation in EDTA are important factors to obtain homogeneous size of the clumps, with an average cell number/clump between 100-200 cells. Some methods of passage such as trypsin, accutase or harsh treatments with EDTA may result in less optimal single-cell suspensions.
  • the day after splitting the cells are re-fed with fresh medium (E8). At this point, the differentiation process can be started on healthy cells less than about 50%, about 60%, about 70% , about 80%, or about 90% confluent after splitting. In some embodiments, cell cultures are no more than 70% confluent prior to starting the differentiation step.
  • PSCs can be plated at low density, medium density, or high density, as long as the population is homogeneously pluripotent.
  • the size of the colonies is homogeneous when cells are maintained in E8 media and in the early stages of DE differentiation. In some embodiments, the entire process of differentiation until Stage 4 (endocrine progenitors) may occur in the same wells, without replating.
  • the size of the colonies can influence the efficiency of endoderm differentiation, and in some embodiments the size of the colonies comprises between 200-1000 cells.
  • the density of PSCs in the culture ranges from about 1 cell/mL to about 10 8 cells/mL, 1 cell/mL to about 10 7 cells/mL, 1 cell/mL to about 10 6 cells/mL, 1 cell/mL to about 10 5 cells/mL, 1 cell/mL to about 10 4 cells/mL, 1 cell/mL to about 10 3 cells/mL, 1 cell/mL to about 10 2 cells/mL, 1 cell/mL to about 10 cells/mL, about
  • 10 4 cells/mL to about 108 cells/mL about 104 cells/mL to about 101 cells/mL, about 104 cells/mL to about 10 6 cells/mL, about 10 4 cells/mL to about 10 5 cells/mL, about 10 5 cells/mL to about 10 8 cells/mL, about 10 5 cells/mL to about 10 7 cells/mL, about 10 5 cells/mL to about 10 6 cells/mL, about 10 6 cells/mL to about 10 8 cells/mL, or about 10 6 cells/mL to about 10 7 cells/mL.
  • the concentration of cells is about 1 million cells/mL, about 2 million cells/mL, about 3 million cells/mL, about 4 million cells/mL, about 5 million cells/mL, about 6 million cells/mL, about 7 million cells/mL, about 8 million cells/mL, about 9 million cells/mL, about 10 million cells/mL, about 11 million cells/mL, about 12 million cells/mL, about 13 million cells/mL, about 14 million cells/mL, about 15 million cells/mL, about 16 million cells/mL, about 17 million cells/mL, about 18 million cells/mL, about 19 million cells/mL, about 20 million cells/mL, about 50 million cells/mL, or about 100 million cells/mL.
  • the concentration of cells is about 10 million per mL, about 20 million per mL, or about 30 million per mL.
  • the medium is changed every 6 hours, every 12 hours, daily, or every two days. In particular embodiments, the medium is changed daily.
  • Endoderm differentiation is a critical step in the differentiation process.
  • PSC lines that differentiate efficiently into endoderm systematically differentiate efficiently into pancreatic progenitors.
  • the stem cell is differentiated into an endoderm cell by modulation of all of the Activin, WNT, BMP, FGF, and AKT signaling pathways.
  • any one, two, three, or four of the Activin, WNT, BMP, FGF, or AKT signaling pathways are modulated.
  • endoderm differentiation comprises, consists of, or consists essentially of differentiation of PSCs into definitive endoderm cells expressing SOX17 and/or CXCR4.
  • PSCs from about 50%, about 60%, about 70%, about 80%, about 90%, or about 95% confluent cultures, maintained for no more than about 3, about 4, about 5, about 6, or about 7 days in E8 media in vitronectin or the equivalent thereof coated plates are first split onto fresh coated plates in the absence of serum, feeder cells and allowed to recover for about 12, about 18, about 24, about 30, about 36, about 40, about 48, or about 72 hours. The attachment rate and survival are considered when selecting the time for recovery.
  • the PSCs are cultured for about 2, about 3, or about 4 days to induce transition from mesendoderm to definitive endoderm.
  • PSC cultured in these culture conditions will differentiate first into primitive streak (PS) cells, as indicated by expression of Brachyury (T), EOMES, MIXL1 and the absence of expression of SOX2. Then, the cells will become positive for DE markers, including at least one of SOX17, CXCR4, FOXA2, and GATA4.
  • PS formation is induced by treating cells for about 12, about 18, about 24, about 30, or about 36 hours with ActivinA, CHIR99021, BMP4, LY294002, and FGF2, or equivalents of each thereof.
  • concentrations of each additive to the medium at Stage 1 islOO ng/mL ActivinA (Stemgent), 2 ⁇ CHIR99021 GSK-3 inhibitor (Tocris), 10 ng/mL BMP4 (R&D systems), 10 ⁇ LY294002 (Calbiochem) and 20 ng/mL FGF2 (BioPioneer).
  • cells are treated for about 12 hours, about 18 hours, about 24 hours, about 36 hours, or about 48 hours with ActivinA, LY294002, FGF2 and
  • the concentration of each additive is about 100 ng/mL ActivinA, about 10 ⁇ LY294002, about 20 ng/mL FGF2 and about 100 nM Dorsomorphin (Axon Medchem).
  • cells are grown in media comprising, consisting of, or consisting essentially of DMEM-F12 (Gibco) and IMDM (Gibco) mixed about 1 : 1, about 1% Glutamax, about 1% concentrated lipids (Gibco), about 450 mM Monohioglycerol (Sigma), about 1% Insulin-Transferrin-Selenium ITS supplement (Gibco), and aboutlmg/mL Polyvinylalcohol (Sigma) ("CDM/PVA media”).
  • media comprising, consisting of, or consisting essentially of DMEM-F12 (Gibco) and IMDM (Gibco) mixed about 1 : 1, about 1% Glutamax, about 1% concentrated lipids (Gibco), about 450 mM Monohioglycerol (Sigma), about 1% Insulin-Transferrin-Selenium ITS supplement (Gibco), and aboutlmg/mL Polyvinyl
  • the Activin signaling pathway is modulated by contacting the stem cell with ActA or an equivalent thereof for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 5 days, or alternatively from about 2 days to four days.
  • the amount of ActA or the equivalent thereof is about 100 ng/mL, or from about 1 ng/mL to about 1 ⁇ g/mL.
  • the concentration of ActivinA or an equivalent thereof in the tissue culture ranges from 0.001 to about 2 mg/mL, about 0.001 to about 1 mg/mL, about 0.01 mg/mL to about 10 mg/mL, about 0.01 mg/mL to about 5 mg/mL, about 0.01 mg/mL to about 2 mg/mL, about 0.01 mg/mL to about 1 mg/mL, about 1 mg/mL to about 25 mg/mL, about 1 mg/mL to about 20 mg/mL, about 1 mg/mL to about 15 mg/mL, about 1 mg/mL to about 10 mg/mL, about 1 mg/mL to about 9 mg/mL, about 1 mg/mL to about 8 mg/mL, about 1 mg/mL to about 7 mg/mL, about 1 mg/mL to about 6 mg/mL, about 1 mg/mL to about 5 mg/mL, about 4 mg/mL to about 50 mg/mL, about 4 mg/m/m
  • the concentration of ActivinA is about 100 ng/mL.
  • the WNT signaling pathway is modulated by contacting the stem cell with CHIR99021 or an equivalent thereof during about the first 24 hours of differentiation.
  • the amount of CHIR99021 or an equivalent thereof comprises about 2 ⁇ , or alternatively, from about 1 ⁇ to about 10 ⁇ , or alternatively from about 0.1 ⁇ to about 3 ⁇ .
  • the stem cell is contacted with CHIR99021 or an equivalent thereof for about the first 1 hour to about the first 48 hours of differentiation.
  • the concentration of CHIR99021 or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to about 7
  • the BMP signaling pathway is modulated by contacting the stem cell with BMP4 or an equivalent thereof for about the first 24 hours of culture followed by about 48 hours of contacting the stem cell with dorsomorphin or an equivalent thereof.
  • the amount of BMP4 or an equivalent thereof is about 10 ng/mL, or alternatively from about 1 ng/mL to 100 ng/mL.
  • the amount of dorsomorphin is about 100 nM, or alternatively from about 1 nM to 1 ⁇ .
  • the cell is contacted with BMP4 or an equivalent thereof from about 1 hour to about 48 hours, or from about 1 day to about 4 days.
  • the cell is contacted with dorsomorphin after the contact with BMP4, or alternatively, before contact with BMP4 or concurrently with contact with BMP4.
  • the contact with dorsomorphin may be from about 1 hour to about 48 hours, or alternatively from about 1 day to about 4 days.
  • the concentration of BMP4 or an equivalent thereof in the tissue culture ranges from 0.001 to about 2 ng/mL, about 0.001 to about 1 mg/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 2 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about
  • the AKT signaling pathway is modulated by contacting the stem cell with LY294002 for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 5 days, or alternatively from about 2 days to 4 days.
  • the amount of LY294002 comprises about 10 ⁇ , or alternatively, from about 1 ⁇ to about 10 ⁇ , or alternatively from about 0.1 ⁇ to about 1 ⁇ .
  • the concentration of LY294002 or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to about
  • the FGF signaling pathway is modulated by contacting the stem cell with an effective amount of FGF2 for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 4 days.
  • the amount of FGF2 is about 20 ng/mL, or from about 1 ng/mL to about 1 ⁇ g/mL.
  • the concentration of FGF2 or an equivalent thereof in the tissue culture ranges from 0.001 to about 2 ng/mL, about 0.001 to about 1 mg/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 2 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about
  • the concentration of Dorsomorphin or an equivalent thereof in the tissue culture ranges from about 1 nM to about 1000 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 250 nM, about 1 nM to about 200 nM, about 1 nM to about 150 nM, about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 30 nM to about 500 nM, about 30 nM to about 300 nM, about 30 nM to about 250 nM, about 30 nM to about 200 nM, about 30 nM to about 150 nM
  • the concentration of Dorsomorphin or an equivalent thereof is about 100 nM.
  • cells are fed with CDM/PVA + Activin (lOOng/ml) + FGF2 (20 ng/ml) + BMP4 (10 ng/ml) + LY294002 (10 mM) + CHIR99021 (2 u M).
  • the efficiency of the differentiation of the stem cell into an endoderm cell during stage 1 is carefully monitored by assaying expression of DE markers through gene expression analysis, immunostaining, flow cytometry, western blot, immunohistochemistry, or any other method known in the art to determine the fraction of cells expressing DE markers.
  • At least 60%, at least 70%, at least 80%, at least 90%), or at least 95%> of the cells express DE markers such as SOX17 (SRY-box 17, Entrez 64321; RefSeq M_022454), CXCR4 (C-X-C chemokine receptor type 4, Entrez 7852, RefSeq M_003467), FOXA2 (Hepatocyte nuclear factor 3-beta, Entrez 3170, RefSeq
  • GATA4 Transcription factor GATA-4, Entrez 2626, RefSeq
  • At least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the cells should express at least one of CXCR4 and SOX17.
  • the endoderm cell may be further differentiated to produce a posterior foregut (PF) cell by modulation of signaling pathways selected from the BMP, TGFP, or FGF signaling pathways.
  • this second stage of differentiation further comprises contacting the endoderm cell with retinoic acid or an equivalent thereof.
  • the endoderm cell is differentiated by modulation of all of the BMP, TGFp, and FGF signaling pathways, or alternatively, one or two of the BMP, TGFP, and FGF signaling pathways are modulated.
  • the endoderm cell is cultured during differentiation into an PF cell in a culture system comprising Advanced DMEM culture medium.
  • the TGFP signaling pathway is modulated by contacting the endoderm cell with SB431542 for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 4 days.
  • the amount of SB431542 comprises about 10 ⁇ , or alternatively, from about 1 ⁇ to about 10 ⁇ , or alternatively from about 0.1 ⁇ to about 1 ⁇ .
  • the concentration of SB431542 or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to about 7
  • the endoderm cell is contacted with retinoic acid for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 5 days, or alternatively from about 2 days to 4 days.
  • the cell is contacted with about 2 ⁇ retinoic acid, or alternatively from about 0.1 ⁇ to about 1 ⁇ , or alternatively from about 1 ⁇ to about 100 ⁇ retinoic acid.
  • the concentration of Retinoic Acid or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to
  • the concentration of Retinoic Acid or an equivalent thereof is about 2 ⁇ .
  • the FGF signaling pathway is modulated by contacting the endoderm cell with FGF10 for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 4 days.
  • the amount of FGF10 is about 50 ng/mL, or alternatively from about 1 ng/mL to about 100 ng/mL or from about 100 ng/mL to 1 ⁇ g/mL.
  • the concentration of FGF10 or an equivalent thereof in the tissue culture ranges from 0.001 to about 2 ng/mL, about 0.001 to about 1 mg/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 2 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about
  • the concentration of SB431542 or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to about 7
  • the concentration of SB431542 or an equivalent thereof is about 10 ⁇ .
  • the BMP signaling pathway is modulated by contacting the endoderm cell with dorsomorphin or an equivalent thereof for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 4 days.
  • the amount of dorsomorphin comprises about 100 nM, or alternatively, from about 1 nM to about 1 ⁇ , or alternatively from about 0.1 nM to about 10 ⁇ .
  • the concentration of Dorsomorphin or an equivalent thereof in the tissue culture ranges from about 1 nM to about 1000 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 250 nM, about 1 nM to about 200 nM, about 1 nM to about 150 nM, about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 30 nM to about 500 nM, about 30 nM to about 300 nM, about 30 nM to about 250 nM, about 30 nM to about 200 nM, about 30 nM to about 150 nM
  • cells are cultured for about 1, about 2, about 3, about 4, or about 5 days in Advanced DMEM supplemented with SB431542 (Selleckchem), Retinoic Acid (Sigma), Dorsomorphin (Axon Medchem), and FGF10 (Peprotech).
  • concentration of each supplement is about 10 ⁇ SB431542 (Selleckchem), 2 ⁇ Retinoic Acid (Sigma), 100 nM Dorsomorphin (Axon Medchem), and 50 ng/mL FGF10 (Peprotech) on days 4-6.
  • the efficiency of the differentiation of the endoderm cell into an posterior foregut during stage 2 is carefully monitored by assaying expression of PF markers through gene expression analysis, immunostaining, flow cytometry, western blot, immunohistochemistry, or any other method known in the art to determine the fraction of cells expressing PF markers.
  • At least 60%, at least 70%, at least 80%>, at least 90%, or at least 95% of the cells express at least one of HNFIB (HNF1 homeobox A, Entrez gene 6927, RefSeq M_000545), HNF4A (Hepatocyte nuclear factor 4 alpha, Entrez 3172, RefSeq M 000457) and significantly suppress expression of at least one of HHEX
  • HHEX Hematopoietically-expressed homeobox protein HHEX, Entrez 3087, RefSeq M 002729
  • HOXA3 Homeobox protein Hox-A3, Entrez 3200, RefSeq M 030661
  • CDX2 CDX2
  • the PF cell expresses HNFip and HNF4A but does not express OCT4, NANOG, HHEX, HOXA3, nor CDX2.
  • the PF cell may be further differentiated to produce a pancreatic progenitor cell by modulation of signaling pathways selected from BMP, Sonic hedgehog, or FGF signaling pathways. In some embodiments, all of the BMP, Sonic hedgehog, or FGF signaling pathways are modulated. Alternatively, one or two of the BMP, Sonic hedgehog, or FGF signaling pathways are modulated.
  • pancreatic specification consists of differentiation of posterior foregut cells into pancreatic progenitors through inhibition of Sonic Hedgehog signaling with Cyclopamine. In some embodiments, this third stage of differentiation further comprises contacting the PF cell with retinoic acid or an equivalent thereof.
  • the PF cell is cultured during differentiation into a pancreatic progenitor cell in a culture conditions comprising Advanced DMEM culture medium.
  • cells are cultured for about 1, about 2, about 3, about 4, or about 5 days in Advanced DMEM supplemented with Cyclopamine (Selleckchem), Retinoic Acid (Sigma), Dorsomorphin (Axon Medchem) and FGF10 (Peprotech).
  • the Sonic hedgehog signaling pathway is modulated by contacting the PF cell with cyclopamine or an equivalent thereof for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to four days.
  • the amount of cyclopamine comprises about 0.25 ⁇ , or alternatively, from about 0.1 ⁇ to about 1 ⁇ , or alternatively from about 0.01 ⁇ to about 10 ⁇ .
  • the concentration of Cyclopamine or an equivalent thereof in the tissue culture ranges from about 0.01 ⁇ to about 100 ⁇ , about 0.01 ⁇ to about 5 ⁇ , about 0.1 ⁇ to about 4 ⁇ , about 0.1 ⁇ to about 3 ⁇ , about 0.1 ⁇ to about 2 ⁇ , about 0.1 ⁇ to about 1 ⁇ , about 0.1 ⁇ to about 0.5 ⁇ , about 0.1 ⁇ to about 0.25 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8
  • the PF cell is contacted with retinoic acid for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 5 days, or alternatively from about 2 days to 4 days.
  • the PF cell is contacted with about 2 ⁇ retinoic acid, or alternatively from about 0.1 ⁇ to about 1 ⁇ , or alternatively from about 1 ⁇ to about 100 ⁇ retinoic acid.
  • the concentration of Retinoic Acid or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to
  • the concentration of Retinoic Acid or an equivalent thereof is about 2 ⁇ .
  • the BMP signaling pathway is modulated by contacting the PF cell with dorsomorphin or an equivalent thereof for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 4 days.
  • the amount of dorsomorphin comprises about 100 nM, or alternatively, from about 1 nM to about 1 ⁇ , or alternatively from about 0.1 nM to about 10 ⁇ .
  • the concentration of Dorsomorphin or an equivalent thereof in the tissue culture ranges from about 1 nM to about 1000 nM, about 1 nM to about 500 nM, about 1 nM to about 400 nM, about 1 nM to about 300 nM, about 1 nM to about 250 nM, about 1 nM to about 200 nM, about 1 nM to about 150 nM, about 1 nM to about 100 nM, about 1 nM to about 90 nM, about 1 nM to about 80 nM, about 1 nM to about 70 nM, about 1 nM to about 60 nM, about 1 nM to about 50 nM, about 1 nM to about 20 nM, about 30 nM to about 500 nM, about 30 nM to about 300 nM, about 30 nM to about 250 nM, about 30 nM to about 200 nM, about 30 nM to about 150 nM
  • the FGF signaling pathway is modulated by contacting the PF cell with FGF 10 or an equivalent thereof for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to four days.
  • the amount of FGF 10 is about 50 ng/mL, or alternatively from about 1 ng/mL to about 100 ng/mL or from about 100 ng/mL to 1 ⁇ g/mL.
  • the concentration of FGF 10 or an equivalent thereof in the tissue culture ranges from 0.001 to about 2 ng/mL, about 0.001 to about 1 mg/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 2 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about
  • the concentration of the supplements is 0.25 ⁇
  • Cyclopamine (Selleckchem), 2 ⁇ Retinoic Acid (Sigma), 100 nM Dorsomorphin (Axon Medchem) and 50 ng/mL FGF 10 (Peprotech).
  • the efficiency of the differentiation into a pancreatic progenitor (PP) cell during stage 3 is carefully monitored by assaying expression of PP markers through gene expression analysis, immunostaining, flow cytometry, western blot, immunohistochemistry, or any other method known in the art to determine the fraction of cells expressing PP markers.
  • at least 60%, at least 70%, at least 80%>, at least 90%, or at least 95% of the pancreatic progenitor cells express PDX1 (pancreatic and duodenal homeobox 1, Entrez 3651, Refseq M_000209).
  • stage 4 cells are differentiated into endocrine pancreas cells.
  • this step consists in driving differentiation of pancreatic progenitors into endocrine pancreatic buds through inhibition of Notch signaling by DAPT (g-secretase inhibitor) or an equivalent thereof in the presence of FGF10 or an equivalent thereof.
  • DAPT g-secretase inhibitor
  • the pancreatic progenitor cell may be further differentiated to produce an endocrine pancreas cell by modulation of the FGF signaling pathway and/or modulation of the Notch signaling pathway.
  • modulation of the Notch signaling pathway may occur before, after, or concurrently with modulation of the FGF signaling pathway.
  • the pancreatic progenitor cell may be further contacted with B-27 or an equivalent thereof.
  • the pancreatic progenitor cell is cultured during
  • the Notch signaling pathway is modulated by contacting the pancreatic progenitor cell with DAPT for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 4 days.
  • the pancreatic progenitor cell is contacted with about 10 ⁇ DAPT, or alternatively from about 0.1 ⁇ to about 1 ⁇ , or alternatively from about 1 ⁇ to about 100 ⁇ DAPT.
  • the concentration of DAPT or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to about 7
  • the pancreatic progenitor cell is contacted with about 1% B- 27 without insulin for 3 days or alternatively from about 1 day to about 3 days, or
  • the concentration of B27 or an equivalent thereof is about 0.1% to about 1%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 9%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 100%), about 5% to about 15%, or about 1% to about 20%.
  • the concentration of B27 or an equivalent thereof is about 1%, about 5%, about 10%, or about 20%.
  • the FGF signaling pathway is modulated by contacting the pancreatic progenitor cell with FGF 10 for about 3 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to four days.
  • the amount of FGF 10 is about 50 ng/mL, or alternatively from about 1 ng/mL to about 100 ng/mL or from about 100 ng/mL to 1 ⁇ g/mL.
  • the concentration of FGF 10 or an equivalent thereof in the tissue culture ranges from 0.001 to about 2 ng/mL, about 0.001 to about 1 mg/mL, about 0.01 ng/mL to about 10 ng/mL, about 0.01 ng/mL to about 5 ng/mL, about 0.01 ng/mL to about 2 ng/mL, about 0.01 ng/mL to about 1 ng/mL, about 1 ng/mL to about 25 ng/mL, about 1 ng/mL to about 20 ng/mL, about 1 ng/mL to about 15 ng/mL, about 1 ng/mL to about 10 ng/mL, about 1 ng/mL to about 9 ng/mL, about 1 ng/mL to about 8 ng/mL, about 1 ng/mL to about 7 ng/mL, about 1 ng/mL to about 6 ng/mL, about 1 ng/mL to about
  • Advanced DMEM supplemented with FGF10 (Peprotech).
  • cells are fed for about 3 days in Advanced DMEM supplemented with B27 without insulin (Gibco) and DAPT (Sigma).
  • the cells are fed for 3 days with 50 ng/mL FGF10 (Peprotech).
  • cells are fed for 3 days in Advanced DMEM
  • DAPT DAPT
  • the efficiency of the differentiation into a endocrine pancreas (EP) cell during stage 4 is carefully monitored by assaying expression of EP markers through gene expression analysis, immunostaining, flow cytometry, western blot,
  • the cells can be identified as proliferative and /or characterized by expression of KX6.1 (Homeobox protein Nkx-6.1, Entrez 4825,
  • cells further express at least one of EURO-D1 (Neurogenic differentiation 1, Entrez 4760, NM_002500) and NGN3 (neurogenin 3 Entrez 50674, NM_020999).
  • EURO-D1 Neuroogenic differentiation 1, Entrez 4760, NM_002500
  • NGN3 Neurogenin 3 Entrez 50674, NM_020999
  • emerging "bud-structures” can be visualized in 2D cultures. Those structures are highly positive for NKX6.1 expression. Proliferation can be determined, for example, by growth rate, detecting cells with increased DNA content from DNA synthesis, BrdU analyses, or any other method known in the art.
  • the endocrine pancreas cell may be further differentiated into an islet precursor cell by transitioning the cell to three-dimensional culture conditions and modulation of the TGFP signaling pathway.
  • the endocrine pancreas cell is further contacted with dexamethasone or an equivalent thereof and/or nicotinamide or an equivalent thereof.
  • the endocrine pancreas cell is further contacted with B27 or an equivalent thereof.
  • endocrine pancreas progenitor cells are differentiated into islet precursors.
  • the differentiation process can take from 6 to 12 days.
  • the endocrine pancreas cell is cultured during differentiation into an islet precursor cell in culture conditions comprising DMEM culture medium.
  • DMEM media is used in Stage 5 of differentiation is not Advanced DMEM.
  • Advanced DMEM contains insulin that can be taken up by cells during differentiation, causing artifacts and false positive results during insulin and/or c- peptide staining.
  • endocrine progenitors obtained from stage 4 are subjected to form spheroids and maturate for about 4, about 5, about 6, or about 7 days in the presence of Nicotinamide (Sigma), Alk5 inhibitor II (Enzo Life Sciences), and dexamethasone (Sigma) in B27 (without insulin) in DMEM media.
  • Nicotinamide Sigma
  • Alk5 inhibitor II Enzo Life Sciences
  • dexamethasone Sigma
  • the endocrine pancreas cell is contacted with nicotinamide for about 6 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 12 days.
  • the endocrine pancreas cell is contacted with about 10 mM nicotinimide, or alternatively from about 0.1 mM to about 1 mM, or alternatively from about 1 mM to about 100 mM
  • the concentration of Nicotinamide or an equivalent thereof in the tissue culture ranges from about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 2 mM, about 3 mM to about 50 mM, about 3 mM to about 30 mM, about 3 mM to about 25 mM, about 3 mM to about 20 mM, about 3 mM to about 15 mM
  • the endocrine pancreas cell is contacted with dexamethasone for about 6 days, or alternatively from about 1 day to about 3 days, or alternatively from about 0.5 days to 6 days, or alternatively from about 2 days to 12 days.
  • the endocrine pancreas cell is contacted with about 10 ⁇ dexamethasone, or alternatively from about 0.1 ⁇ to about 1 ⁇ , or alternatively from about 1 ⁇ to about 100 ⁇
  • the concentration of dexamethasone or an equivalent thereof in the tissue culture ranges from about 1 mM to about 100 mM, about 1 mM to about 50 mM, about 1 mM to about 40 mM, about 1 mM to about 30 mM, about 1 mM to about 25 mM, about 1 mM to about 20 mM, about 1 mM to about 15 mM, about 1 mM to about 10 mM, about 1 mM to about 9 mM, about 1 mM to about 8 mM, about 1 mM to about 7 mM, about 1 mM to about 6 mM, about 1 mM to about 5 mM, about 1 mM to about 2 mM, about 3 mM to about 50 mM, about 3 mM to about 30 mM, about 3 mM to about 25 mM, about 3 mM to about 20 mM, about 3 mM to about 15
  • the concentration of dexamethasone or an equivalent thereof is about 10 mM.
  • the endocrine pancreas cell is contacted with about 1% B-27 without insulin for about 6 days or alternatively from about 1 day to about 9 days, or alternatively from about 0.5 days to 3 days, or alternatively from about 2 days to 12 days.
  • the concentration of B27 or an equivalent thereof is about 0.1% to about 1%, about 1% to about 2%, about 1% to about 3%, about 1% to about 4%, about 1% to about 5%, about 1% to about 6%, about 1% to about 7%, about 1% to about 8%, about 1% to about 9%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 100%), about 5% to about 15%, or about 1% to about 20%.
  • the concentration of B27 or an equivalent thereof is about 1%, about 5%, about 10%, or about 20%.
  • the TGFP signaling pathway is modulated by contacting the endocrine pancreas cell with 1 ⁇ Alk5 inhibitor II for about 6 days or alternatively from about 1 day to about 9 days days, or alternatively from about 0.5 days to 3 days, or alternatively from about 2 days to 12 days.
  • the endocrine pancreas cell is contacted with about 1 ⁇ Alk5 inhibitor II, or alternatively from about 0.1 ⁇ to about 10 ⁇ , or alternatively from about 1 ⁇ to about 100 ⁇ Alk5 inhibitor II.
  • the concentration of Alk5 inhibitor II or an equivalent thereof in the tissue culture ranges from about 1 ⁇ to about 100 ⁇ , about 1 ⁇ to about 50 ⁇ , about 1 ⁇ to about 40 ⁇ , about 1 ⁇ to about 30 ⁇ , about 1 ⁇ to about 25 ⁇ , about 1 ⁇ to about 20 ⁇ , about 1 ⁇ to about 15 ⁇ , about 1 ⁇ to about 10 ⁇ , about 1 ⁇ to about 9 ⁇ , about 1 ⁇ to about 8 ⁇ , about 1 ⁇ to about 7 ⁇ , about 1 ⁇ to about 6 ⁇ , about 1 ⁇ to about 5 ⁇ , about 1 ⁇ to about 2 ⁇ , about 3 ⁇ to about 50 ⁇ , about 3 ⁇ to about 30 ⁇ , about 3 ⁇ to about 25 ⁇ , about 3 ⁇ to about 20 ⁇ , about about 3 ⁇ to about 15 ⁇ , about 3 ⁇ to about 10 ⁇ , about 3 ⁇ to about 9 ⁇ , about 3 ⁇ to about 8 ⁇ , about 3 ⁇ to about
  • Nicotinamide (Sigma), 1 ⁇ Alk5 inhibitor II (Enzo Life Sciences), and 10 mM
  • dexamethasone Sigma in 1% B27 (without insulin).
  • the efficiency of the differentiation into a islet precursor cell (IP) during stage 5 is carefully monitored by assaying expression of IP markers through gene expression analysis, immunostaining, flow cytometry, western blot, immunohistochemistry, or any other method known in the art to determine the fraction of cells expressing IP markers.
  • IP markers include gene expression analysis, immunostaining, flow cytometry, western blot, immunohistochemistry, or any other method known in the art to determine the fraction of cells expressing IP markers.
  • at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the IP cells express markers specific to insulin producing beta cells including but not limited to INS, PDX1, and NGN3.
  • secretion of C-peptide (a short 31- amino-acid polypeptide that connects insulin's A-chain to its B-chain in the proinsulin molecule) can be tested to validate maturation and functionality of insulin producing cells.
  • the efficiency of the differentiation process is monitored through immunostaining, flow cytometry, gene expression analysis, or assaying for the production of c-peptide upon glucose stimulation.
  • the stage 4 progenitor cells are trypsinized using 0.05 Trypsin-EDTA (Invitrogen) for 5 minutes at 37°C.
  • the trypsin is neutralized by adding 10%FBS in DMEM media and pelleting the cells by centrifugation at about 400 x g for about 5 minutes.
  • the cells are resuspended in stage 5 media and transferred to U bottom low attachment 96 well plates (Prime Surface), splitting the contents of one 12 well plate of pancreatic endocrine progenitors into 4 low attachment 96 well plates, with 125 uL of cell suspension per well.
  • Each well of the 96 well plate contained about 1000-2000 total cells.
  • the 96 well plates were centrifuged at about 800 x g for about 10 minutes.
  • stage 4 progenitors are trypsinized with 0.05 Trypsin-EDTA (Invitrogen) for 5 minutes at 37°C.
  • pelleted cells are resuspended in the media described above and transferred to low-attachment U-bottom 96 well plates in a 1 ⁇ 2 split ratio; 1000-2000 cells per spheroid equivalent.
  • Low-attachment plates are centrifuged for about 10 minutes at 2000 rpms. After one day in the incubator, spheroids are collected and transferred into low-attachment plates (Costar) and matured for an additional about 5, about 6, or about 7 days, with a media change every 1-2 days.
  • Stage 1 Differentiation In order to increase the number of SOX17 positive cells and decrease OCT4 positive cells at the end of DE differentiation, Applicants tested combination of CHIR99021 treatments, and two AKT inhibitors: LY294002 and PI- 103 in two surface coated plates (Gelatin FBS and Vitronectin). Undifferentiated clusters (U) were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, with or without 10 nM LY294002 and 60 nM PI- 103, and with or without 2 uM CHIR99021.
  • FIG. 1 A cells were maintained 2 more days with 100 ng/mL Activin A, 10 ng/mL BMP4 and 20 ng/mL FGF2 in CDM-PVA media as shown in FIG. 1 A.
  • FIG. IB cells were immunostained for OCT4 and SOX17 in DE cells in LY294002, PI-103 or control (non-AKT inh) cells.
  • FIG. 1C qPCR analysis of SOX17, OCT4, MIXL1 and GOOSECOID was performed in CHIR99021 treated or not treated cells during primitive streak (PS) commitment.
  • FIG. ID an experimental scheme was developed to test the importance of inhibiting BMP4 and AKT signaling pathways after PS formation.
  • Cells were treated with BMP4 or Noggin during days 2 and 3 of DE. Undifferentiated colonies were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, 10 nM LY294002 and 2 uM CHIR99021 to induce PS formation. After this treatment, cells were maintained 2 more days with 100 ng/mL Activin A, 20 ng/mL FGF2 and with 200 ng/mL of Noggin or with 10 ng/mL BMP4 in CDM-PVA media. qPCR analysis show expression of HHEX, FOXA2, HNF4A and SOX17 in gelatin or vitroenctin coated plates during DE commitment.
  • FIG. 2A shows the experimental scheme. Undifferentiated clusters were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, 10 nM LY294002 and 2 uM CHIR99021 to induce PS formation.
  • stage 2 cells were differentiated in the presence of 2 uM RA, 10 ng/mL FGF10, 50 ng/mL Noggin and with or without 10 uM SB431542 during 3 days.
  • qPCR analysis was performed to assay expression of HNFIB, OCT4, SOX2, HOXA3, HHEX and CDX2 in stage 2 cells treated or not treated with 10 uM SB431542.
  • FIG. 2C immunostaining of OCT4, HNFIB and HNF4A of stage 2 cells was performed after blockade of TGFbeta with 10 uM SB431542.
  • Stage 3 differentiation To test the importance of inhibiting the BMP4 signaling pathway after PS formation, Applicants treated cells with a small molecule dorsomorphin (DM) to substitute the actions of Noggin. Experimental scheme comparing Cho et al descriptions and our improved protocol is shown in FIG. 3 A. Undifferentiated clusters were treated during 24 hours with a cocktail of 100 ng/mL Activin A, 10 ng/mL BMP4, 20 ng/mL FGF2, 10 nM LY294002 and 2 uM CHIR99021 to induce PS formation.
  • DM dorsomorphin
  • cells were maintained 2 more days in CDM-PVA media with 100 ng/mL Activin A, 20 ng/mL FGF2, 10 nM LY294002 and 10 ng/mL BMP4 (control conditions) or with a gradient of 25-200 ng/mL of Noggin or 50-500 nM dorsomorphin.
  • Applicants noticed a toxic effect of 500 nM dorsomorphin conditions.
  • stages 2 and 3 cells were maintained with 50 ng/mL of Noggin for control conditions (BMP4 during Stagel) or with a gradient of 50-200 ng/mL of Noggin or 50-200 nM dorsomorphin for improved protocol.
  • FIG. 3B qPCR analysis of MIXL1 and NANOG in Stage 1 cells in a gradient of Noggin or dorsomorphin is shown in FIG. 3B.
  • Cyclopamine or SANTl is shown in FIG. 3D.
  • qPCR analysis shows expression of PDX1, HNF6, RFX6 and NGN3 in stage 3 cells treated combined actions of Cyclopamine or SANTl with Activin A.
  • FIGs. 5A-C The protocol for pancreatic differentiation is performed in 5 stages, with Stages 1-4 in 2D conditions, followed by formation of 3D spheroids for Stage 5.
  • FIGs. 5A-C The protocol for pancreatic differentiation is performed in 5 stages, with Stages 1-4 in 2D conditions, followed by formation of 3D spheroids for Stage 5.
  • Stage 5 spheroids demonstrate the morphology of the Stage 5 spheroids, as well as their expression of mRNA and protein markers of pancreatic beta cells.
  • Stage 5 spheroids were embedded in paraffin and sectioned in slides of 5 microns. Slides were stained with hematoxylin and eosin. Scale bar: 50 microns.
  • FIGs. 5D-E show that the combined 2D (Stages l-4)/3D (Stage5) approach results in higher expression of pancreatic endoderm markers compared to purely 2D (Stages 1-5) or purely 3D (Stages 1-5) approaches. This data demonstrates that the purely 3D approach results in aberrant expression of hepatic markers. Images of 2D and 3D St5 cells.
  • Glucose stimulatory insulin release (GSIR) of Stage 5 cells after transplantation is shown in FIG. 5G.
  • Hierarchical clustering, multigroup comparison is shown in FIG. 5H.
  • Principal component analysis of Stage 5 (St5), Fetal Pancreas (FP), and Adult Islets (AI) is shown in FIG. 51.
  • Qluocore, multigroup comparison q 0.01.
  • Gene clusters selected on heatmap of FIG. 5H is shown in FIG. 5J.
  • Example 2 Treatment of Diabetes in Humans using iPSCs
  • the disclosed methods are useful for treating pancreatic diseases, disorders, or conditions including but not limited to juvenile onset (Type I) diabetes mellitus, including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes).
  • Type I diabetes mellitus including pediatric insulin-dependent diabetes mellitus (IDDM), and adult onset diabetes mellitus (Type II diabetes).
  • IDDM pediatric insulin-dependent diabetes mellitus
  • Type II diabetes Type II diabetes.
  • CRISPR/Cas9 methods or other methods of introducing iPSC genes to the fibroblasts may be used.
  • colonies resembling human ES cells are selected and expanded. The expanded cells are tested for expression of expressed pluripotency markers such as alkaline phosphatase (AP) activity, OCT4, NANOG, and SOX2.
  • AP alkaline phosphatase
  • the cells are adapted to vitronectin coated plates and E8 medium as described above.
  • the cells are then subjected to the five stages of differentiation for 21 days ((i) endoderm differentiation, (ii) posterior foregut specification, (iii) pancreatic specification, (iv) endocrine pancreas specification, and (v) islet precursors) as described above.
  • the resulting islet precursor cells derived from the patient's iPSCs are harvested and tested for their ability to secrete C-peptide.
  • C-peptide release is measured by incubating the cells in Krebs-Ringer solution with Hepes and bicarbonate (KRBH). The cells are first incubated for one hour in KRBH buffer.
  • the cells are incubated in the presence of 2.5 mM D-glucose for 1 hour followed by 20 mM D-glucose for another hour.
  • the culture supernatant is then harvested and the C-peptide levels are measured by ELISA.
  • the karyotype of the cells is assayed using methods well known in the art to confirm that the cells have remained normal chromosome number throughout the expansion and differentiation process.
  • the isolated islet precursor cells are then combined with a suitable excipient such as saline and administered to the diabetic patient through infusion into the patient's liver or pancreas, intravenous, intraperitoneal, or intraarterial injection, or other method of administration known in the art.
  • a suitable excipient such as saline
  • the cells may also be administered by the creation and/or implantation of an artificial organ such that the transplanted cells can reside in an
  • Islet precursor cells may be delivered via one or more administrations. Optimally, a total of at least 800,000 islet equivalents are administered to a single patient. Administration of the cells may optionally be combined with
  • immunosuppressive therapies to prevent the patient's immune system from attacking the graft.
  • the efficiency of islet cell engraftment may be tested by measuring the patient's insulin levels, assaying the patient's blood glucose levels including fasting plasma glucose, random plasma glucose, and the oral glucose tolerance test, testing the patient's hemoglobin AlC, or any other method of measuring insulin response well known in the art.
  • the efficacy of treatment may be determined by detecting an improvement in the patient's blood glucose levels such as fasting plasma glucose, random plasma glucose, the oral glucose tolerance test, or the patient's hemoglobin AlC.
  • FgflO is essential for maintaining the proliferative capacity of epithelial progenitor cells during early pancreatic organogenesis. Development (Cambridge, England) 128, 5109-5117. doi: 10.1038/385257a0
  • Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. Journal of Clinical Investigation 92, 1459-1466. doi: 10.1172/JCI 116723

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Abstract

L'invention concerne des méthodes se rapportant à la différenciation de cellules souches en cellules pancréatiques in vitro. L'invention concerne également des cellules et des populations de cellules produites selon les méthodes décrites. L'invention concerne en outre des méthodes de traitement de maladies ou d'affections liées au pancréas à l'aide de cellules produites selon les méthodes décrites.
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WO2019051281A1 (fr) * 2017-09-07 2019-03-14 StemoniX Inc. Différenciation efficace de cellules souches humaines en endoderme définitif
CN113234664A (zh) * 2021-05-11 2021-08-10 澳门大学 一种胰腺祖细胞的制备方法及其应用
WO2024033299A1 (fr) * 2022-08-08 2024-02-15 Spiber Technologies Ab Dérivation in vitro d'îlots pancréatiques à partir de cellules souches pluripotentes humaines

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TOUBOUL, T ET AL.: "Stage-Specific Regulation of the WNT/Beta-Catenin Pathway Results in Improved Differentiation of hESCs to Functional Hepatocytes", J HEPATOL., vol. 64, no. 6, June 2016 (2016-06-01), pages 1315 - 1326, XP029538552 *
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Cited By (5)

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Publication number Priority date Publication date Assignee Title
WO2019051281A1 (fr) * 2017-09-07 2019-03-14 StemoniX Inc. Différenciation efficace de cellules souches humaines en endoderme définitif
CN113234664A (zh) * 2021-05-11 2021-08-10 澳门大学 一种胰腺祖细胞的制备方法及其应用
CN113234664B (zh) * 2021-05-11 2024-05-10 澳门大学 一种胰腺祖细胞的制备方法及其应用
WO2024033299A1 (fr) * 2022-08-08 2024-02-15 Spiber Technologies Ab Dérivation in vitro d'îlots pancréatiques à partir de cellules souches pluripotentes humaines
WO2024033300A1 (fr) * 2022-08-08 2024-02-15 Spiber Technologies Ab Formation d'îlots 3d à partir de cellules progénitrices endocrines

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