The differentiation of hepatocyte-like cells from monkey embryonic stem cells.
Embryonic stem cells (ESC) hold great potential for the treatment of liver diseases. Here, we report the differentiation of rhesus macaque ESC along a hepatocyte lineage. The undifferentiated monkey ESC line, ORMES-6, was cultured in an optimal culture condition in an effort to differentiate them into hepatocyte-like cells in vitro. The functional efficacy of the differentiated hepatic cells was evaluated using RT-PCR for the expression of hepatocyte specific genes, and Western blot analysis and immunocytochemistry for hepatic proteins such as alpha-fetoprotein (AFP), albumin and alpha1-antitrypsin (alpha1-AT). Functional assays were performed using the periodic acid schiff (PAS) reaction and ELISA. The final yield of ESC-derived hepatocyte-like cells was measured by flow cytometry for cells that were transduced with a liver-specific lentivirus vector containing the alpha1-AT promoter driving the expression of green fluorescence protein (GFP). The treatment of monkey ESC with an optimal culture condition yielded hepatocyte-like cells that expressed albumin, alpha1-AT, AFP, hepatocyte nuclear factor 3beta, glucose-6-phophatase, and cytochrome P450 genes and proteins as determined by RT-PCR and Western blot analysis. Immunofluorescent staining showed the cells positive for albumin, AFP, and alpha1-AT. PAS staining demonstrated that the differentiated cells showed hepatocyte functional activity. Albumin could be detected in the medium after 20 days of differentiation. Flow cytometry data showed that 6.5 +/- 1.0% of the total differentiated cells were positive for GFP. These results suggest that by using a specific, empirically determined, culture condition, we were able to direct monkey ESC toward a hepatocyte lineage.
Ma
Xiaocui
X
Department of Internal Medicine, Transplant Research Program, University of California, Davis Medical Center, Sacramento, California 95817, USA.
Duan
Yuyou
Y
Jung
Christine J
CJ
Wu
Jian
J
VandeVoort
Catherine A
CA
Zern
Mark A
MA
D000418:Albumins / Q000378:metabolism; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation / Q000502:physiology*; D002460:Cell Line; D003470:Culture Media / Q000494:pharmacology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000378:metabolism; D022781:Hepatocytes / Q000166:cytology / Q000502:physiology*; D006801:Humans; D008253:Macaca mulatta; D051379:Mice; D000515:alpha 1-Antitrypsin / Q000378:metabolism; D000509:alpha-Fetoproteins / Q000378:metabolism
D000418:Albumins; D003470:Culture Media; D000515:alpha 1-Antitrypsin; D000509:alpha-Fetoproteins; D049452:Green Fluorescent Proteins
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