Methods of engineering human induced pluripotent stem cells to produce liver tissue
AssigneeUniversity of Pittsburgh—Of the Commonwealth System of Higher Education
InventorsAlejandro Soto-Gutierrez, Tomoji Mashimo, Alexandra Sylvie Collin de l'Hortet, Eduardo Cervantes Alvarez, Jorge Guzman Lepe, Kan Handa, Kazuki Takeishi, Yang Wang, Branimir Popovic
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Abstract
Methods are disclosed herein for producing human hepatocytes from human induced pluripotent stem cells. Also provided are transgenic rats for the expansion of human hepatocytes, such as those produced using the methods disclosed herein.
Claims40
A method for producing human hepatocytes, comprising:
- a) culturing human induced pluripotent stem cells (iPSC) in a first medium comprising activin A, fibroblast growth factor (FGF)-2 and bone morphogenic protein (BMP)-4 for about 2 to about 3 days, such that mesendoderm cells are obtained;
- b) culturing the mesendoderm cells in a second medium comprising activin A, and in absence of FGF-2 and BMP-4, for about 2 to about 3 days, such that definitive endoderm cells are obtained;
- c) culturing the definitive endoderm cells in a third medium comprising dimethyl sulfoxide (DMSO), and hepatocyte growth factor (HGF), wherein the third medium is a low glucose medium, for about eight to about 14 days, such that hepatic progenitors are obtained;
- and
- d) culturing the hepatic progenitors in a fourth medium comprising HGF, urso deoxycholic acid, cholesterol, palmitic acid, oleic acid, and rifampicin, wherein the fourth medium is a low glucose medium such that
- human hepatocytes are obtained.
A method for producing human hepatocytes, comprising
- a) culturing human induced pluripotent stem cells (iPSC) in a first medium comprising activin A, fibroblast growth factor (FGF)-2 and bone morphogenic protein (BMP)-4 for about 2 to about 3 days, such that mesendoderm cells are obtained;
- b) culturing the mesendoderm cells in a second medium comprising activin A, and in absence of FGF-2 and BMP-4, for about 2 to about 3 days, such that definitive endoderm cells are obtained;
- c) culturing the definitive endoderm cells in a third medium comprising dimethyl sulfoxide (DMSO), and hepatocyte growth factor (HGF), wherein the third medium is a low glucose medium for about eight to about 14 days, such that hepatic progenitors are obtained;
- d) transplanting the hepatic progenitors into a liver of a transgenic Rag2−/− Il2rg−/− rat whose genome comprises a nucleic acid molecule encoding a fusion protein operably linked to a liver-specific promoter, wherein the fusion protein comprises FKBP12 and caspase 9, and the transgenic Rag2−/− Il2rg−/− rat is capable of expanding human hepatocytes in its liver;
- and
- e) harvesting human hepatocytes from the liver of the transgenic Rag2−/− Il2rg−/− rat.
A transgenic Rag2−/− Il2rg−/− rat whose genome comprises a nucleic acid molecule encoding a fusion protein operably linked to a liver-specific promoter, wherein the fusion protein comprises FKBP12 and caspase 9, and the rat is capable of expanding human hepatocytes in its liver.
Dependencies are inferred from the claim text.