Isolation, culture, induction and differentiation of fetal bone marrow-derived postembryonic pluripotent stem cells / 中国组织工程研究

ABSTRACT

BACKGROUND: At present, studies show that a kind of stem cell community which in many kinds of organizations can differentiate into tissue cells of different embryonic layers; but those are different from embryonic stem cells, embryonic stem cell will lose the part differentiation potential gradually during the development of pregnancy, and will present some special phenotypes or the molecular markers, as CD105 and so on, will cell it postembryonic pluripotent stem cells.OBJECTIVE: To study the isolation of postembryonic pluripotent stem cells from fetal bone marrow, proliferative culture in vitro, induction and differentiation; transplantation to the liver of SCID mice with hepatic failure, and detect therapy effects.DESIGN, TIME AND SETTING: Cell observation and animal randomization experiment which was completed in the Ministry of Health of Cell Engineering Technology Research Center, Tianjin Third Central Hospital from March 2003 to March 2005.MATERIALS: The postembryonic pluripotent stem cells were extracted from thighbone and shinbone of 22-week old fetuses under sterile circumstance. Adult female SCID mice ware regarded as the recipients. CD105 immunornagnetic beads were provided by Miltenyi Biotec, Germany; mouse-anti-human albumin by Sigma, USA; basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) by PEPROTECH, UK.METHODS: Postembryonic pluripotant stem cells obtained from fetal bone marrow were isolated using density gradient centrifugation and micromagnetic beads technique. The hepatocyte-like cells were induced and differentiated with culture media containing HGF (30 ng/mL) and bFGF (20 ng/mL). Twenty-four SCID mice were randomly divided into experimental group and control group with 12 mice in each group. Hepatic injury models were established with intraperitoneal injection of D-galactosamine On the next day, about 106 CD105(+) cells were perfused into liver in situ in the experimental group, and about 106 CD105(-) cells or isovolumic culture medium were perfused in the control group.MAIN OUTCOME MEASURES: Two, seven days, one and three months after the transplantation of cells, human albumin expression in the liver tissue was detected by immunohistochemistry.RESULTS: The immunocytochemical assay of the cells after micromagnetic beads selection showed that the CD105 expression was slightly positive; the doubling time of the cells in the logarithmic growth period was around 30 hours; after being expanded for 10 population doublings, the cells entered decline period. The cells were transplanted into SCID mice's liver; 3 months later, the human serum albumin in the mouse liver was assessed by using monoclonal antibody of mouse-anti-human serum albumin, dotted or small focal expression of the protein could be detected. However, any expression was not observed in the control group.CONCLUSION: The bone marrow-derived pluripotent stem cells are able to transform to hepatocytas in the hepatic microenvironment.