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
Life of this patent
Estimating the term…
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.
Claims42
A method for producing human hepatocytes, comprising:
- a) culturing human induced pluripotent stem cells (iPSC) in a first medium comprising an effective amount of activin A, fibroblast growth factor (FGF)-2 and bone morphogenic protein (BMP)-4 for about 2 to about 3 days, to produce mesendoderm cells;
- b) culturing the mesendoderm cells in a second medium comprising an effective amount of activin A, and in absence of FGF-2 and BMP-4, for about 2 to about 3 days, to produce definitive endoderm cells;
- c) culturing the definitive endoderm cells in a third medium comprising an effective amount of dimethyl sulfoxide (DMSO), and hepatocyte growth factor (HGF), wherein the third medium is a low glucose medium, for about eight to about 14 days, to produce hepatic-specified cells;
- and
- d) culturing the hepatic-specified cells in a fourth medium comprising an effective amount of HGF, urso deoxycolic acid, cholesterol, palmitic acid, oleic acid, and rifampicin, wherein the fourth medium is a low glucose medium, and
- thereby producing the human hepatocytes.
A method for producing human hepatocytes, comprising
- a) culturing human induced pluripotent stem cells (iPSC) in a first medium comprising an effective amount of activin A, fibroblast growth factor (FGF)-2 and bone morphogenic protein (BMP)-4 for about 2 to about 3 days, to produce mesendoderm cells;
- b) culturing the mesendoderm cells in a second medium comprising an effective amount of activin A, and in the absence of FGF-2 and BMP-4, for about 2 to about 3 days, to produce definitive endoderm cells;
- c) culturing the definitive endoderm cells in a third medium comprising an effective amount of dimethyl sulfoxide (DMSO), and hepatocyte growth factor (HGF), wherein the third medium is a low glucose medium for about eight to about 14 days, to produce hepatic progenitor cells;
- d) transplanting the hepatic progenitor cells into a liver of an immunocompromised non-human transgenic animal;
- and
- e) harvesting human hepatocytes from the liver of the immunocompromised non-human transgenic animal.
A transgenic rat, wherein the transgenic rat is a Rag2−/−2rg−/− rat harboring a transgene comprising a liver specific promoter operably linked to a nucleic acid molecule encoding a fusion protein, wherein the fusion protein comprises FKBP12 and caspase 9, and wherein human hepatocytes can be expanded in a liver of the transgenic rat.
Dependencies are inferred from the claim text.