Methods and compositions for culturing endoderm progenitor cells in suspension
First Claim
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1. A method of producing human self-renewing endoderm progenitor cells comprising:
- a) culturing undifferentiated human induced pluripotent stem cells (iPS cells) in a defined, serum-free, feeder cell and feeder cell conditioned media free, medium comprising Activin A or Nodal; and
b) culturing the iPS cells of step a) in defined, serum-free, feeder-cell free medium, the media comprising Activin A, BMP4, VEGF and FGF-2 to produce definitive endoderm (DE) cells, said cells characterized as express both CXCR4 and CD117; and
c) washing the DE cells of step b); and
d) culturing the washed DE cells of step a) with BMP4, VEGF, FGF2, and EGF in defined, serum-free, feeder-cell free medium to selectively promote the growth of self-renewing endoderm progenitor cells (EPC cells), wherein the EPC cells are characterized as express CD34 and HNF4α
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Abstract
Provided herein are methods for the in vitro differentiation of induced pluripotent stem cells, which have been expanded and/or maintained under defined conditions, into endodermal precursor cells (EPCs) that are capable of producing mono-hormonal beta cells.
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Citations
32 Claims
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1. A method of producing human self-renewing endoderm progenitor cells comprising:
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a) culturing undifferentiated human induced pluripotent stem cells (iPS cells) in a defined, serum-free, feeder cell and feeder cell conditioned media free, medium comprising Activin A or Nodal; and b) culturing the iPS cells of step a) in defined, serum-free, feeder-cell free medium, the media comprising Activin A, BMP4, VEGF and FGF-2 to produce definitive endoderm (DE) cells, said cells characterized as express both CXCR4 and CD117; and c) washing the DE cells of step b); and d) culturing the washed DE cells of step a) with BMP4, VEGF, FGF2, and EGF in defined, serum-free, feeder-cell free medium to selectively promote the growth of self-renewing endoderm progenitor cells (EPC cells), wherein the EPC cells are characterized as express CD34 and HNF4α
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21. A method of producing human self-renewing endoderm progenitor cell aggregates comprising:
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a) culturing human pluripotent stem cells in defined, feeder-free, serum-free medium comprising Activin A or Nodal, and BMP4 and one or both of VEGF and FGF-2, to effect the induction of endoderm cells; and b) culturing the endoderm cells in a culture with BMP4, VEGF, FGF2, and EGF or homologs thereof in serum-free medium to effect the formation of self-renewing endoderm progenitor cell (EPC) aggregates. - View Dependent Claims (22, 23, 28, 29, 31)
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24. A method of producing human self-renewing endoderm progenitor cells comprising:
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a) culturing human induced pluripotent stem (iPS) cells in a defined, feeder-free, serum-free medium comprising Activin A, BMP4 and one or both of VEGF and FGF-2; and b) culturing the resultant cells from step a) in a culture with Activin A, BMP4, VEGF, FGF2, and EGF in defined, feeder-free, serum-free medium to selectively promote the growth of self-renewing endoderm progenitor cells (EPCs). - View Dependent Claims (25, 32)
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Specification