Hepatocyte production by forward programming
The invention generally features methods for providing hepatocytes from a variety of cell sources, particularly pluripotent stem cells, therapeutic compositions featuring such cells, and methods of using them for the treatment of subjects.
1. A method of producing hepatocytes by forward programming of stem cells, comprising transfecting the stem cells with at least one exogenous expression cassette comprising at least the hepatocyte programming factor genes FOXA2, HHEX, HNF1A and GATA4, thereby producing hepatocytes by forward programming of the stem cells.
2. The method of claim 1 , wherein the stem cells are mesenchymal stem cells, hematopoietic stem cells, or induced pluripotent stem cells.
3. The method of claim 1 , wherein the number of the hepatocyte programming factor genes are five or six.
4. The method of claim 3 , wherein the stem cells are further transfected with T-box transcription factor (TBX3).
5. The method of claim 1 , wherein the stem cells or progeny cells thereof further comprise a reporter expression cassette comprising a hepatocyte-specific transcriptional regulatory element operably linked to a reporter gene.
6. The method of claim 5 , wherein the hepatocyte-specific transcriptional regulatory element is a promoter of albumin, α-1-antitrypsin (AAT), cytochrome p450 3A4 (CYP3A4), apolipoprotein A-I, or APOE.
7. The method of claim 1 , wherein the hepatocytes comprise the following hepatocyte characteristics:
(i) expression of one or more hepatocyte markers including glucose-6-phosphatase, albumin, α-1-antitrypsin (AAT), cytokeratin 8 (CK8), cytokeratin 18 (CK18), asialoglycoprotein receptor (ASGR), alcohol dehydrogenase 1, arginase type I, cytochrome p450 3A4 (CYP3A4), liver-specific organic anion transporter (LST-1), or a combination thereof;
(ii) activity of glucose-6-phosphatase, CYP3A4, bile production or secretion, urea production, or xenobiotic detoxification;
(iii) hepatocyte morphological features; and
(iv) in vivo liver engraftment in an immunodeficient subject.
8. The method of claim 1 , further comprising selecting or enriching for hepatocytes.
9. The method of claim 1 , wherein the stem cells or progeny cells thereof are cultured in a medium comprising one or more growth factors including Oncostain M (OSM).
10. The method of claim 9 , wherein the medium further comprises hepatocyte growth factor (HGF).
11. The method of claim 1 , wherein the stem cells and progeny cells thereof are cultured in a medium essentially free of fibroblast growth factor (FGF).
12. The method of claim 1 , wherein the stem cells and progeny cells thereof are cultured in a medium essentially free of epidermal growth factor (EGF).
13. The method of claim 1 , wherein the stem cells and progeny cells thereof are cultured in a medium essentially free of nicotinamide.
14. The method of claim 1 , comprising obtaining the hepatocytes less than or about 10 days after culturing in said conditions.
15. The method of claim 14 , comprising obtaining the hepatocytes less than or about 5 days after culturing in said conditions.
16. A method of assessing a compound for a pharmacological or toxicological effect on a hepatocyte, comprising:
a) contacting the hepatocyte provided by the method in accordance with claim 1 with the compound; and
b) assaying a pharmacological or toxicological effect of the compound on the hepatocyte.