| Objective: Mesenchymal stem cells (MSCs) are population of cells which exist in many tissues and organs and have capability of expansion, self-renewal and differentiation into multiple cell lineages. Among the research field of MSCs, bone marrow derived MSCs (BM-MSCs) are most intensively investigated. A series of reports revealed that BM-MSCs could differentiate into neural cells in vivo and in vitro, and improve functional recovery after transplantation into animal models of neurological diseases. However, it has also indicated that MSCs are rare in bone marrow (1/104～1/105 mononuclear cells). As a result, they are difficult to isolate, purify and expand this population. Moreover, there are significant drops in cell number of BM-MSC and proliferative capacity with age. Taken together, it is of vital importance to find an alternative cell source for replacement of defective neural tissue resulting from neurological diseases. In this study, we have made an attempt to isolate the MSCs from umbilical cord, and established a series of methods of isolation, long-term culture and neural differentiation. In addition, we have also analyzed the immunophenotype of UC-MSCs to offer a novel cell source for cell replacement of neurological diseases. Methods: The full-term umbilical cords from healthy children were washed with PBS and umbilical cord arteries and vein were stripped off. Then remaining parts of umbilical cord were diced into small fragments of 12mm in diameter and plated in growth medium consisting of low glucose-Dulbecco's Modified Essential Media (LG-DMEM), 20% FBS, 25mM L-glutamine (L-Glu), 100 units/ml penicillin, and 100 μg/ml streptomycin. 35 days after primary culture, great number of cells came out of fragments and became adherent to the flasks. When fibroblast-like cells reached confluence around two weeks, they were detached with 0.20%trypsin /0.02%EDTA solution and were passaged. After trypsization, the adherent cells were analyzed as follows: ⑴Flow cytometry analysis by FACScalibur flow cytometry with mouse monoclonal phycoerythrin (PE) or fluorescein isothiocyanate (FITC) labeled antibodies, which includes CD13-PE, CD14-FITC, CD29-PE, CD31-FITC, CD34-PE, CD38-PE, CD44-FITC, CD45-PE, CD90-FITC, CD105-FITC, CD133-PE, CD166-PE and HLA-DR-PE. Mouse FITC or PE conjugated IgG1 were used as controls; ⑵Assay of adipogenic and osteogenic differentiation were performed after plating the cells into 6-well plates at a density of 3,000 cells/cm2. For adipogenic differentiation, 1 μmol/L dexamethasone, 5 μg/mL insulin, 0.5 mmol/Lisobutylmethylxanthine, and 60 μmol/L indomethacin were added to the growth medium. For osteogenic differentiation, 0.1 μmol/L dexamethasone, 0.05 mmol/L ascorbic acid-2-phosphate and 10 mmol/L β-glycerophosphate were added to the growth medium. Both osteogenic and adipogenic media were changed twice a week for two weeks. After two weeks of induction, cells were fixed with 4% paraformaldehyde and stained with Oil red O and Alizarin red (Sigma) to view neutral lipid vacuoles in adipocytes and calcium deposition in osteocytes, respectively; ⑶In analysis of neural differentiation potential of UC-MSCs, we used two protocols, namely, neurotrophic factors(bFGF,BDNF,I-T-S ) and combination of bFGF and antioxidants. Phase-contrast microscope was used to record morphology changes, immuno-fluorescence was applied to view phenotype changes and RT-PCR was used to reflect the levels of mRNA expression of neural markers. Results: Three to five days after primary culture, adherent cells with a homogenous fibroblastic morphology came out of fragments and circled fragments, forming a radiating appearance. Two weeks after plating, when the cells reached 80%～90% confluence, they were detached with 0.20% trypsin /0.02% EDTA solution and were passaged at a ratio of 1: 2 to 1: 3. Several hours after plating, cells became adherent and formed a compact, whirling-like appearance in low power field. After 20 passages, thses cells maintainedtheir homogenous fibroblastic appearance and growth char...
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