In Vitro Differentiation Of Embryonic Stem Cells Into Hepatocytes And Characterization Of A Novel Progenitor Cell

Previous reports have shown that the most widely used approach of in vitro differentiating embryonic stem cells into hepatocytes is by forming embryoid bodies, followed by coculture with cytokins and cellular matrix to induce the development and maturation process of hepatocytes. However, because at least some of the cells of EBs are not terminally differentiated, this material is not useful for transplantation. Moreover, EB differentiation is a long time process. Therefore, more attention has been laid on the direct in vitro differentiation from ES cells to hepatocytes. Here we established a methodology for the direct differentiation of functional hepatocytes from an adherent monoculture of ES cells. We also discussed the character and function of a sort of pluripotent progenitor cells that we found in the process of differentiation.The first part of the experiment is the induction and characterization of a sort of oval progenitor cells. When treated with 2.5mM reagent A, the morphology of ES cells changed significantly. By the following culture in medium without regent A, a sort of oval progenitor cells appeared and proliferated rapidly. RT-PCR analysis showed that this kind of progenitor cells expressed AFP, Nestin and CK19 mRNA. Immunocytochemistry analysis also revealed that the progenitor cells are AFP, CK19, Nestin positive. Therefore, we primarily defined the progenitor cells as AFP~+/Nestin/CK19~+ progenitor cells.The second part is the characterization and identification of the AFP~+/Nestin~+/CK19~+ progenitor cells, including their differentiation abilities into hepatocytes, bile canaliculi like cells and neuron like cells. For the differentiation of AFP~+/Nestin~+/CK19~+progenitor cells into hepatocytes, we cultured the progenitor cells with two kinds of cell culture medium with different cytokines. One culture medium contained low concentration of HGF, Dex and ITS, the other one supplemented with higher concentration of HGF, bFGF, OSM and Dex. After 15 days culture, we detected liver-specific gene expression, such as TAT, HNF4 and ALB mRNA expression in the cells treated with low concentration of cytokines. Immunocytochemistry analysis showed that these cells display AFP and low ALB expression. PAS reaction indicated these cells synthesize glycogen in vitro. However, the cells treated with higher concentration of cytokines showed a strong expression of ALB in vitro, which suggested that these cells display the characteristics of mature hepatocytes. When cultured the AFP~+/Nestin~+/CK19~+ progenitor cells on Matrigel in William E culture medium supplemented with 100ng/ml HGF for 7 days, we observed bile canaliculi like structure in vitro. Our experiment also showed that the AFP~+/Nestin~+/CK19~+progenitor cells have the potential to differentiate into neuron like cells when cultured with culture medium containing RA. In conclusion, we isolated and identified a sort of progenitor cells expressing AFP, Nestin and CK19 during the in vitro differentiation process of embryonic stem cells. These progenitor cells have multi-potential to differentiate into mature hepatocytes, bile canaliculi like cells and neuron like cells when cultured in appropriate conditions and special cellular matrix.