Generation of hepatocytes from pluripotent stem cells
Methods are provided for producing differentiated cells from stem cells, including producing hepatocytes. Compositions thereof are also provided, as are methods of treating a liver disorder.
1. A method of producing differentiated cells with hepatocyte-like function comprising culturing pluripotent stem cells in a cell culture medium comprising:
L-cysteinglutathione disulfide,
γ-Glu-Cys,
DL-kynurenine,
D-penicillamine disulfide,
phenacetin,
phytosphingosine HCl,
pyridoxal HCl,
and optionally, an additional antibiotic;
wherein the differentiated cells are characterized by:
(a) decreased Octamer-binding transcription factor 4 (OCT4) expression relative to undifferentiated stem cells;
(b) increased vimentin expression relative to the undifferentiated stem cells;
(c) increased synthesis of albumin relative to the undifferentiated stem cells;
(d) increased synthesis of urea relative to the undifferentiated stem cells;
(e) epithelial morphology relative to the undifferentiated stem cells;
(f) increased metabolism of ethoxyresorufin into resorufin relative to the undifferentiated stem cells; and
(g) increased cytochrome P450 activity relative to the undifferentiated stem cells.
2. The method of claim 1 , wherein the additional antibiotic is tetracaine HCl.
3. The method of claim 1 , wherein the cell culture medium does not contain the additional antibiotic.
4. The method of claim 1 , wherein culturing the pluripotent stem cells in the cell culture medium is for a period of from 1 hour to 2 weeks.
5. The method of claim 1 , further comprising wherein culturing the pluripotent stem cells in the cell culture medium is for a period of from 24 hours to 2 weeks.
6. The method of claim 1 , further comprising wherein culturing the pluripotent stem cells in the cell culture medium is for a period of 2 weeks.
7. The method of claim 1 , wherein the differentiated cells are produced from human embryonic stem cells (hESC).
8. The method of claim 1 , wherein the differentiated cells are prepared from induced pluripotent stem cells (iPSC).
9. The method of claim 1 , wherein culturing the pluripotent stem cells is in DMEM/F12 medium.
10. The method of claim 9 , wherein the DMEM/F12 medium further comprises 2% Knock-out Serum Replacer (KSR), 1% B27 supplement, 2 mM L-glutamine, 0.1 mM MEM Non-Essential Amino Acids (NEAA), and 1% penicillin-streptomycin.
11. The method of claim 1 , wherein the cell culture medium substantially omits conditioned medium from fetal hepatocytes (FH-CM).
12. The method of claim 1 , wherein the concentration of each of L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine, D-penicillamine disulfide, phenacetin, phytosphingosine HCl, and pyridoxal HCl, is from 1 μM to 10 μM.
13. The method of claim 12 , wherein the cell culture medium comprises tetracaine HCL in a concentration of from 1 μM to 10 μM.
14. The method of claim 1 , wherein the concentration of each of L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine, D-penicillamine disulfide, phenacetin, phytosphingosine HCl, and pyridoxal HCl is 1 μM.
15. The method of claim 1 , wherein the concentration of each of L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine, D-penicillamine disulfide, phenacetin, phytosphingosine HCl, and pyridoxal HCl is 5 μM.
16. The method of claim 1 , wherein the concentration of each of L-cysteinglutathione disulfide, γ-Glu-Cys, DL-kynurenine, D-penicillamine disulfide, phenacetin, phytosphingosine HCl, and pyridoxal HCl is 10 μM.
17. The method of claim 14 , wherein the cell culture medium comprises tetracaine HCL in a concentration of 1 μM.
18. The method of claim 15 , wherein the cell culture medium comprises tetracaine HCL in a concentration of 5 μM.
19. The method of claim 16 , wherein the cell culture medium comprises tetracaine HCL in a concentration of 10 μM.