Culture And Characterization Of Mouse Hepatic Stem Cells

Since a long-term contraversy, doubts on the existance of hapetic stem cells (HSCs) now have been removed, but its biological characteristics remains to be well documented, partially due to methods of research on them, which have been focused on in vivo studies of murine livers. A defined cell culture based model is urged to be established and expected to throw light on the entity of HSCs and promote studies on liver development and concerned medical problems. To meet this demand, we tried to establish methods of in vitro cultivation on hepatic stem cells from mouse fetal livers and adult livers respectively. Firstly, to confirm the existance of HSCs, cells isolated from mouse fetal livers were transplanted intravenously into lethally irradiated sygnernic mice, 12 days later, a few nodules appeared in the obviously enlarged spleens of recipient mice, and the number of the nodules is correlated to that of transplanted cells. Morphology and immunohistochemistry analysis show that the splenic nodules are composed of various types of cells, and cells in some nodules express AFP, CD34 and CKI9, which confirmed the very existence of HSCs in the mouse fetal liver. Then, cells isolated from mouse fetal livers were cultured in vitro and in diffusion chambers in vivo respectively, cell colonies expressing AFP, CD34 and Albumin were obtained, and subculture was performed 4? generations. Besides, according to possible localization of HSCs in adult mouse liver, technique of portal vein filling digestion was developed to isolate cells from the adult mouse liver, an epithelial cell line was established expanding from several small hepatic cells. Results of cell transplantation show that it could expand in the recipient mice to form splenic nodules, whatsmore cells in most of the nodules express albumin and CKI9, which suggest this cell line has characteristics of stem cells, that is, self-renew capability and multipotential differentiation capability. Recently, experiments on murine and human bone marrow transplantation showed that bone marrow cells could give rise to oval cells, and then hepatocytes, which draws us to investigate the potential source of HSCs. After we transplanted fate Mx-cre~ bone marrow cells into female recipient mice, both PCR and immunohistochemistry analysis show that some hepatocytes and ductular cells are Mx-cre positive, white Mx-cre positive cells were also found in several other tissues, such as kidney, spleen and cutis. In a summary, development of methods and establishment of the cell line as well as related explorations in our experiments found basements for further research on HSCs.