To supporting growth and functional differentiation of adult stem cells into hepatocytes in a well-controlled manner, we performed differentiation of human bone marrow mesenchymal stem cells (hBMSCs) to hepatocytes-like cells on a constructed 3-dimensional (3D) nanofibrous biocompatible scaffold.
Methods
After characterization of the hBMSCs isolated from human bone marrow, the performance of the cells seeded and their proliferation on the scaffold was evaluated by scanning electron microscopy (SEM) and 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Different approaches such as immunocytochemistry, reverse transcriptase polymerase chain reaction (RT-PCR), and biochemical assays were used to estimate the ability of hBMSC-derived cells to express hepatocyte-specific markers.
Results
Scanning electron micrographs and MTT analysis revealed the cells were able to expand and remained biologically and metabolically active for 21 days. Immunocytochemical analysis of albumin and α-fetoprotein showing the accumulation of these markers in differentiated cells was confirmed by RT-PCR. Additional markers such as cytochrome P450 3A4, cytokeratin-18, and cytokeratin-19 detected by RT-PCR showed progressive expression during 3 weeks of differentiation on 3D scaffold. The hepatocyte-like cells displayed several characteristics of metabolic functions as judged by production of albumin, urea, transferrin, serum glutamic pyruvic transaminase (SGPT), and serum oxaloacetate aminotransferase (SGOT). Levels of above-mentioned markers, except SGOT in differentiated cells on scaffold, were found to be significantly greater than in the 2D culture system (p<0.05).
Conclusion
Overall data suggest that the engineered nanofibrous scaffold is a conductive matrix for functional hBMSC-derived hepatocyte-like cells and is promising for maintenance of hepatocytes suitable for implantation.
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