Long-term in vitro culture and hepatocytic differentiation of human umbilical cord-derived mesenchymal stem cells / 中国组织工程研究

ABSTRACT

OBJECTIVE: To investigate biological characteristics of human umbilical cord-derived mesenchymal stem cells, and to explore the possibility of hepatocyte-like cells differentiation.METHODS: The umbilical cord was provided by healthy term birth woman in Tianjin Third Central Hospital. Mesenchymal stem cells were isolated from human umbilical cord by enzyme digestion method. Cells were passaged at 80%-90% confluent. The ninth passage of cells at a density of 5×10~(10)/L were seeded in 12-well culture plate and incubated with DMEM containing hepatocyte growth factor, fibroblast growth factor-4 and oncostatin for 28 days. Cell growth activity was detected by MTT method; cell cycle was detected by flow cytometry; surface immunological marker in MSC was detected by immunocytochemical stain and flow cytometry; specific surface phenotype of hepatocyte was detected by immunocytochemical staining. Function characteristic of hepatocyte was determined by staining for glycogen.RESULTS: MSCs were isolated from human umbilical cord and presented with fibroblastic morphology. 80% of cells were at G_0/G_1 phase with good growth activity and stably passaged over 20 times. These cells were positive for CD29, CD105, and Vimentin, but negative for CD34 and CD31. MSCs were induced to hepatocyte-1 ike cells that were positive for alpha fetoprotein, CK18, CK19 at 1 week and albumin at 3 weeks. At 4 weeks, induced cells were positive for glycogen staining.CONCLUSION: MSCs isolated from human umbilical cord can be cultured in a long periods time in vitro and are able to differentiate into functional hepatocyte-like cells.