Differentiation of human pluripotent stem cells into cells expressing mature hepatocyte markers by 2D and bioreactor culture

A method for differentiation of human pluripotent stem cells into cells expressing mature hepatocyte markers comprising:

• i) culturing human pluripotent stem (hPS) cells in a 2D feeder-free culture system in a low serum culture medium comprising Activin A to produce cells differentiating towards definitive endoderm (DE);
• ii) seeding the cells of step i) in a bioreactor;
• iii) perfusing the bioreactor with one or more culture media selected from a medium comprising low serum, bFGF and Activin A;
• a medium comprising serum, bFGF and Activin A;
• or a medium comprising serum, aFGF, bFGF, BMP2 and BMP4 to induce differentiation of DE into cells expressing AFP at about day 17;
• and
• iv) perfusing the bioreactors containing the AFP expressing cells of step ii) with a culture medium comprising bFGF, HGF, Oncostatin M, and Dexamethasone or in a culture medium comprising HGF, Oncostatin M, Dexamethasone, and DMSO, wherein resulting cells express AFP, albumin and urea;
• wherein the perfused cells of step (ii) differentiate into cells that express the mature hepatic markers Albumin, CYP3A4, UGT2B7, OATP-2, ADH1A, UGTIA6, CYP2C9, CYP2C19, and CYP2D6.