MM： 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 mou...MM： 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 hepa- tocytes, 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 dif- ferentiated 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 to-wards hepatic lineages. CONCLUSION： 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.展开更多
基金Supported by A grant from Medicine and Health Key Project of Zhejiang Province, Science and Technology Foundation of Ministry of Health of the People’s Republic of China, No. WKJ2007-2-037Shaoxing Key Project for Science and Technology, No. 2007A23008, 2005141
文摘MM： 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 hepa- tocytes, 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 dif- ferentiated 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 to-wards hepatic lineages. CONCLUSION： 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.
