骨髓源性肝干细胞的筛选、扩增和分化

Objective To explore the feasibility of direct separation, selective proliferation and differentiation of BDLSC from hone marrow cells with culture systems containing cholestatic serum in vitro.Methods Whole bone marrow cells of rats cultured in routine medium were replaced with con-ditioning selection media containing eholestatic sera of different concentrations after they attached to the plates.The optimal concentration of cholestatic serum was determined according to the outcome of the selected cultures.Then the selected BDLSC were induced to proliferating culture system and dif-ferentiating culture system.Each passage of the proliferated stem cells was subjected to flow cytome-try for detection of stem cell markers.The morphology and phenotypie markers of BDLSC were char-acterized using immunohistochemistry, RT-PCR and electron microscopy.The metabolic functions of differentiated cells were also determined by glycogen staining and urea assay.Results The 50 ml/L eholestatie serum was the optimal concentration for the selection of BDLSC at which BDLSC could sur-vive while the other populations of the bone marrow cells could not.The purified BDLSC could prolif-erate for 6 passages and maintained stable markers in our proliferating system.When the culture sys-tem changed to differentiating system, hepatocyte-like colony-forming units (HCFU) were formed.HCFU expressed markers of embryonic hepatoeytes (AFP, albumin and cytokeratin 8/18), biliary cells (cytokeratin 19), hepatocyte functional proteins (transthyretin and eytoehrome P450-2b1), and hepatocyte nuclear factors (HNF-1α and HNF-3β).They also had glycogen storage and urea synthesis functions, two of the critical features of hepatocytes.Conclusion BDLSC can be selected directly from whole bone marrow cells and pure BDLSC can also proliferate for 6 passages.The differentiated cells have hepatocyte-like phenotype and function.BDLSC will he a new way to provide a readily avail-able alternate source of cells for clinical hepatocyte therapy.

Selection, proliferation and differentiation of bone marrow derived liver stem cells