In Vitro Hepatic Differentiation Of Mesenchymal Stem Cells From Human Fetal Bone Marrow

Hepatocyte transplantation is an effective treatment of liver failure and metabolic liver diseases, but its clinical application is restricted by the limited source of hepatocytes. With the rapid progression in the research of stem cells these years, it may open up new avenues in the treatment of liver diseases. Stem cells are ideal sources for tissue engineering and cell therapy because of their ability to self-renew and differentiate into multiple tissues. Mesenchymal stem cells (MSC), which are originated from embryonic mesoderm, have high plasticity and are considered as multipotent stem cells. Recent studies have demonstrated that MSC from bone marrow have the potential to transdifferentiate into hepatocyte-like cells. Because of MSC's advantages, such as their easy acquisition, abundance, easy separation and culture, rapid growth, no immunological rejection and easily to be transfected with exogenous genes, they may be ideal cells for cell therapy and gene therapy of liver diseases.Because the ability of MSC from fetal bone marrow to proliferate and differentiate is more powerful than that from adult bone marrow, we obtainedFMSC from ten fetuses with their gestational ages between 16 and 24 weeks and investigated their in vitro culture and biological properties. Cultured with HGF and FGF-4, FMSC were induced into a hepatic lineage and their morphology, surface markers, functions and biochemical properties were studied. We attempted to explore the condition under which FMSC can differentiate into hepatocyte-like cells and expected to provide a new source for hepatocyte transplantation to establish a foundation for the clinical applications of the cell therapy of liver diseases. Our study was as follows:1 The in vitro culture of mesenchymal stem cells from fetal bone marrow and their biological properties.FMSC were obtained from fetal bone marrow through density gradient centrifugation and selected by cell attachment. The morphology and ultrastructure of FMSC were observed. The cell cycle and surface markers were analyzed by flow cytometry, and the cell growth was investigated with the use of MTT.2 The hepatic differentiation of mesenchymal stem cells from human fetal bone marrow.FMSC, which were cultured on Matrigel with 20ng/ml HGF, 20ng/ml FGF-4, lOng/ml EGF, DMEM-LG and 2% FBS to simulate a microenvironment for hepatocyte differentiation, were induced into a hepatic lineage. The creep plates of induction culture cells and the culture supernatant were collected at various time points. The hepatic specific cell surface markers were detected by immunohistochemical and indirect immunofluorescence and the glycogen storage was analyzed by PAS staining. The ALB and urea production were tested with the undifferentiated FMSCs as negative control. Results: 1 FMSC were fibroblast-like cells and homogeneous, which were arrangedin a parallel or swirl pattern. The nucleocytoplasmic ratio was high, karyomorphism was irregular, cellular organelle such as mitochondrion and reticulum was few, and the nucleolus was large. FMSC proliferated quickly. The growth curve showed that the lag phase was about 24h and the average doubling time was 36h after subcultivation. It took 5 days for FMSCs to get confluence after each passage. The growth of FMSC was getting down after 15 passages.2 The results of flow cytometry showed that FMSC were positive for CD29 and CD44, but were negative for CD14, CD34 and CD45. 95% of FMSC were in G0/G1 phase, while only a small fraction of FMSC were in S + G2 +M phase.3 The shape of FMSC cultured on Matrigel with HGF and FGF-4 changed from spindle to polygon.4 In the beginning of the hepatic induction, immunofluorescence staining showed that AFP, ALB was positive, but CK18 was negative. After 12 days, AFP was expressed at a low level and FMSC were positive for CK18 and albumin. The immunohistochemical test showed similar results.5 Following the treatment of cytokines, the ALB and urea in supernatant increased in a time-dependent manner. Undifferentiated FMSC didn't produce ALB or urea.6 The glycogen storage increased in a time-dependent manner too, while undifferentiated FMSC were negative for PAS staining.Conclusion:1 It is easy and reliable to isolate FMSC through density gradient centrifugation and selected by cell attachment. Pure FMSC can be easily obtained. The FMSC proliferate stably and quickly and they may be an ideal source for cell therpy.2 When cultured on Matrigel with 20ng/ml HGF and 20ng/ml FGF-4, FMSC differentiate into hepatocyte-like cells. The differentiated cells were...