الفهرس 
Stem cell basicsOnly 14 pages are availabe for public view
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Abstract
Objective: Liver cell transplantation and cellular-based therapies evolved as viable clinical alternatives to whole organ transplantation required in some liver diseases. Differentiation of stem cells into hepatocyte like cells would provide a renewable source of exogenous hepatocytes for drug toxicity testing and cell-based therapeutics. Many protocols of in vitro differentiation of stem cells into hepatocyte like cells had been employed to check the ability of differentiation as well as the functional efficacy of differentiated cells. But still the exploration of an easy and affordable one is needed to facilitate MSC differentiation into hepatocytes.
In this study, we employ three different protocols of differentiation of bone marrow mesenchymal stem cells (MSCs) into hepatocyte-like cells with a view to developing easier and affordable protocol using liver extract verses expensive growth factors cocktails.
Materials and Methods: MSCs were isolated from rat’s bone marrow using the plastic adherence technique. Cells were then subcultured till passage 3 for their purification and proliferation. Colony forming unit fibroblast (CFU-F) assay and immunological characterization was done through the flowcytometric analysis of CD29, CD90 and CD45 surface markers. Flasks then were divided into four groups, first; control group cultured in proliferative media. Second group; cells cultured in hepatocytic differentiation media 1 (HDM) 1 containing FGF-4. Third group; cells cultured in HDM2 containing FGF-4 and HGF and finally fourth group; cultured in HDM3 containing liver extract instead of the previously mentioned growth factors. Cells were tested for their differentiation morphologically and functionally through assessing Urea secretion, glycogen storage and gene expression of albumin by RT-PCR.
Results: Flowcytometric analysis showed that after passage 3, the cells do not express hematopoietic marker CD 45 but express the two mesenchymal markers CD29 and CD90. Cells cultured in HDM2 and HDM3showed morphological changes of hepatocyte like cells in day 12 and day 15 respectively, these changes didn’t appear neither in control group nor in cells cultured in HDM1. Urea production and secretion by hepatocytes like cells were detected at various time points throughout differentiation. In the HDM 2 and 3 groups cells produced urea 3 d later, and in a time-dependent manner. On d 24, the amount of urea produced by differentiated bone marrow cells reached the maxium amount and was detected with a concentration of 23.06±11.9 mmol/L on HDM 2, and 20.89±10.44 mmol/L on HDM 3. Cells in the control and HDM 1 groups did not secret any urea. Intracellular glycogen accumulation was analyzed by staining the cells with the PAS reagent. On d 24 accumulation of glycogen was detected in the HDM 2 and 3, while no accumulation of glycogen was found in the other groups. Albumin mRNA was detected by RT-PCR, the results showed that the cells cultivated in HDM 2 and 3 expressed albumin, while in control group and HDM 1 did not.
Conclusion: The usage of liver extract in the differentiation of MSCs into hepatocyte like cells proved to be almost as effective as the usual used cocktail of HGF and FGF, yet more affordable and easier approach, thus useful in providing a renewable source of exogenous hepatocytes for drug toxicity testing and cell-based therapeutics.