Direct hepatic differentiation of mouse embryonic stem cells induced by valproic acid and cytokines.
OBJECTIVE
To develop a protocol for direct hepatic lineage differentiation from early developmental progenitors to a population of mature hepatocytes.


METHODS
Hepatic progenitor cells and then mature hepatocytes from mouse embryonic stem (ES) cells were obtained in a sequential manner, induced by valproic acid (VPA) and cytokines (hepatocyte growth factor, epidermal growth factor and insulin). Morphological changes of the differentiated cells were examined by phase-contrast microscopy and electron microscopy. Reverse transcription polymerase chain reaction and immunocytochemical analyses were used to evaluate the gene expression profiles of the VPA-induced hepatic progenitors and the hepatic progenitor-derived hepatocytes. Glycogen storage, cytochrome P450 activity, transplantation assay, differentiation of bile duct-like structures and tumorigenic analyses were performed for the functional identification of the differentiated cells. Furthermore, FACS and electron microscopy were used for the analyses of cell cycle profile and apoptosis in VPA-induced hepatic differentiated cells.


RESULTS
Based on the combination of VPA and cytokines, mouse ES cells differentiated into a uniform and homogeneous cell population of hepatic progenitor cells and then matured into functional hepatocytes. The progenitor population shared several characteristics with ES cells and hepatic stem/progenitor cells, and represented a novel progenitor cell between ES and hepatic oval cells in embryonic development. The differentiated hepatocytes from progenitor cells shared typical characteristics with mature hepatocytes, including the patterns of gene expression, immunological markers, in vitro hepatocyte functions and in vivo capacity to restore acute-damaged liver function. In addition, the differentiation of hepatic progenitor cells from ES cells was accompanied by significant cell cycle arrest and selective survival of differentiating cells towards hepatic lineages.


CONCLUSIONS
Hepatic cells of different developmental stages from early progenitors to matured hepatocytes can be acquired in the appropriate order based on sequential induction with VPA and cytokines.
Dong
Xue-Jun
XJ
Molecular Medicine Center of Shaoxing People's Hospital, The First Affiliate Hospital of Shaoxing University, Shaoxing 312000, Zhejiang Province, China.
Zhang
Guo-Rong
GR
Zhou
Qing-Jun
QJ
Pan
Ruo-Lang
RL
Chen
Ye
Y
Xiang
Li-Xin
LX
Shao
Jian-Zhong
JZ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D002453:Cell Cycle / Q000187:drug effects; D002454:Cell Differentiation / Q000187:drug effects*; D019070:Cell Lineage; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006003:Glycogen / Q000378:metabolism; D017228:Hepatocyte Growth Factor / Q000494:pharmacology*; D022781:Hepatocytes / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D007328:Insulin / Q000494:pharmacology*; D051379:Mice; D023421:Models, Animal; D014635:Valproic Acid / Q000494:pharmacology*
D004791:Enzyme Inhibitors; D007328:Insulin; D014635:Valproic Acid; D017228:Hepatocyte Growth Factor; D006003:Glycogen; D003577:Cytochrome P-450 Enzyme System