Production of virus-receptive pluripotent stem cell (PSC)-derived hepatocytes
The present disclosure provides methods for maturing hepatocytes comprising culturing with cyclic adenosine monophosphate and a Janus kinase inhibitor. There is also provided a method for screening inhibitors of hepatitis B virus infection and/or replication.
1. An in vitro method of producing virus-receptive pluripotent stem cell-derived hepatocytes comprising:
(a) obtaining pluripotent stem cell (PSC)-derived hepatocytes; and
(b) culturing the PSC-derived hepatocytes in media comprising cyclic adenosine monophosphate (cAMP) and a Janus kinase inhibitor (JAKi), thereby producing virus receptive hepatocytes.
2. The method of claim 1 , wherein the cAMP and JAKi are cultured sequentially in different media.
3. The method of claim 2 , wherein the cAMP is administered in a first medium and the JAKi is administered in a second medium.
4. The method of claim 1 , wherein the pluripotent stem cell is human.
5. The method of claim 1 , wherein the pluripotent stem cell is an embryonic stem cell.
6. The method of claim 1 , wherein the pluripotent stem cell is an induced pluripotent stem cell.
7. The method of claim 1 , wherein the JAKi is 2-(1,1-Dimethylethyl)-9-fluoro-3,6-dihydro-7H-benz[h]-imidaz[4,5-f]isoquinolin-7-one (JAK Inhibitor 1), 1,2,3,4,5,6-Hexabromocyclohexane, or (E)-4,4′-(1,2-Diethyl-1,2-ethenediyl)bis[2-[(diethylamino)methyl]-phenol (9CI), 4,4′-(3E)-Hex-3-ene-3,4-diylbis{2-[(diethylamino)methyl]phenol} (NSC33994).
8. The method of claim 1 , wherein the JAKi is present at a concentration of about 0.1 μM to about 5 μM.
9. The method of claim 8 , wherein the JAKi is present at a concentration of about 1 μM.
10. The method of claim 1 , wherein the cAMP is present at a concentration of about 0.1 mM to about 3 mM.
11. The method of claim 10 , wherein the cAMP is present at a concentration of about 1 mM.
12. The method of claim 1 , wherein the virus receptive hepatocytes have an increased expression of at least one hepatocyte maturation or viral infectivity gene relative to expression in the PSC-derived hepatocytes.
13. The method of claim 12 , wherein the hepatocyte maturation or viral infectivity gene is selected from the group consisting of UGT1A1, PPARGC1A, TAT, PCK1, NR13, SLC10A1, GSTA2, GLYAT and MT1M.
14. The method of claim 1 , wherein cAMP is present in the medium from about 2 days to about 6 days.
15. The method of claim 1 , wherein the JAKi is present in the medium from about 1 day to about 3 days.
16. The method of claim 1 , wherein the media is serum-free or defined media.
17. The method of claim 1 , wherein the virus receptive hepatocytes have an enhanced ability to support a hepatitis B virus (HBV) infection and/or a hepatitis C virus (HCV) infection relative to the PSC-derived hepatocytes.
18. The method of claim 1 , further comprising infecting the virus receptive hepatocytes with hepatitis B virus.
19. The method of claim 18 , wherein the virus receptive hepatocytes have an increase in secretion of at least one HBV surface antigen.
20. The method of claim 19 , wherein the at least one HBV surface antigen is HBsAG or HBeAG.
21. The method of claim 20 , wherein the increase in surface antigen secretion is at least 2 fold relative to PSC-derived hepatocytes infected by HBV.
22. The method of claim 20 , wherein the increase in surface antigen secretion is at least 5 fold relative to PSC-derived hepatocytes infected by HBV.
23. The method of claim 20 , wherein the increase in surface antigen secretion is at least 10 fold relative to PSC-derived hepatocytes infected by HBV.
24. The method of claim 18 , wherein the virus receptive hepatocytes produce hepatitis B virus capable of infecting other virus receptive hepatocytes.