Protocols for making hepatocytes from embryonic stem cells
First Claim
1. A process for differentiating primate pluripotent stem (pPS) cells into mature hepatocytes in at least three discrete steps, comprising:
- a) culturing undifferentiated pPS cells under conditions which cause differentiation of the cells into cells having characteristics of fetal endoderm;
b) culturing the cells from a) under conditions which cause differentiation of fetal endoderm cells into cells having characteristics of hepatocyte progenitor cells;
c) culturing the cells from b) under conditions which cause differentiation of hepatocyte progenitor cells into cells having characteristics of mature hepatocytes;
thereby producing a cell population that has at least five of the following characteristics;
antibody-detectable expression of α
1-antitrypsin (AAT);
antibody-detectable expression of albumin;
absence of antibody-detectable expression of a-fetoprotein;
RT-PCR detectable expression of asialoglycoprotein receptor (ASGR);
evidence of glycogen storage;
evidence of cytochrome p450 enzyme activity;
evidence of glucose-6-phosphatase enzyme activity; and
the morphological features of hepatocytes.
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Abstract
This disclosure provides a newly developed strategy and particular options for differentiating pluripotent stem cells into cells of the hepatocyte lineage. Many of the protocols are based on a strategy in which the cells are first differentiated into early germ layer cells, then into hepatocyte precursors, and then into mature cells. The cells obtained have morphological features and phenotypic markers characteristic of human adult hepatocytes. They also show evidence of cytochrome p450 enzyme activity, validating their utility for commercial applications such as drug screening, or use in the manufacture of medicaments and medical devices for clinical therapy.
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Citations
20 Claims
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1. A process for differentiating primate pluripotent stem (pPS) cells into mature hepatocytes in at least three discrete steps, comprising:
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a) culturing undifferentiated pPS cells under conditions which cause differentiation of the cells into cells having characteristics of fetal endoderm; b) culturing the cells from a) under conditions which cause differentiation of fetal endoderm cells into cells having characteristics of hepatocyte progenitor cells; c) culturing the cells from b) under conditions which cause differentiation of hepatocyte progenitor cells into cells having characteristics of mature hepatocytes; thereby producing a cell population that has at least five of the following characteristics;
antibody-detectable expression of α
1-antitrypsin (AAT);antibody-detectable expression of albumin; absence of antibody-detectable expression of a-fetoprotein; RT-PCR detectable expression of asialoglycoprotein receptor (ASGR); evidence of glycogen storage; evidence of cytochrome p450 enzyme activity; evidence of glucose-6-phosphatase enzyme activity; and the morphological features of hepatocytes. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 13, 14)
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11. A process for differentiating human embryonic stem (hES) cells into hepatocyte lineage cells, comprising:
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a) culturing the undifferentiated pluripotent cells with DMSO, b) culturing the cells from a) with a histone deacetylase inhibitor in the absence of growth factors; and
thenc) culturing the cells from b) with a histone deacetylase inhibitor in combination with one or more growth factors thereby differentiating human embryonic stem (hES) cells into hepatocyte lineage cells. - View Dependent Claims (12)
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15. A process for differentiating human embryonic stem (hES) cells into hepatocyte lineage cells, comprising:
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a) culturing undifferentiated hES cells with DMSO; b) culturing the cells from a) with one or more growth factors in combination with Oncostatin M; and
thenc) culturing the cells from b) with hepatocyte growth factor (HGF) thereby differentiating human embryonic stem (hES) cells into heQatocyte lineage cells. - View Dependent Claims (16, 17)
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18. A process for differentiating human embryonic stem (hES) cells into hepatocyte lineage cells, comprising:
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a) culturing undifferentiated hES cells with FGF 8 or a bone morphogenic protein (BMP); b) culturing the cells from a) with Oncostatin M, and then c) culturing the cells from b) with HGF thereby differentiating human embryonic stem (hES) cells into hepatocyte lineage cells. - View Dependent Claims (19, 20)
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Specification