Study On Isolation And Basic Characterization Of Wharton’s Jelly Mesenchymal Stem Cells From Human Umbilical Cord

Stem cells, existing in single or cluster, possess self-renewal capacity and differentiation potential. Mesenchymal stem cells (MSCs) derive from mesoderm and located in differentiated or specialized various tissue. Under appropriate conditions, MSCs can be committed differentiation into mesoderm, ectoderm and endoderm lineages, including adipocytes, osteoblasts, chondrocytes, endothelium-like cells, muscle cells, hepatocyte like cells, insulin-producing stem cells, neuron-like cells and glial-like cells, which can be widely involved in the process of injury repair of tissue and organ. Thus, MSCs in recent years become a research hotspot. Previous studies demonstrated that Wharton’s jelly (WJ) of umbilical cord contains a plenty of MSCs that maintain high proliferate potential as well as differentiation. However, the standard culture system and the "gold standard marker" are still wanted to be investigated. In this study, we focused on the developing the procedure of rapid, simple and cost-effective cultured amplification system of WJ-MSCs from human umbilical cord, and explored its basic biological characterization. This work contributes to the technical platform and theoretical foundation for research and clinical application, and provides seed cells as a source of regenerative medicine.Objective:To explore the explant culture of WJ-MSCs and basic biological characteristics by observing the morphology and growth characteristics, detecting surface marker as well as differential potency.Methods:The umbilical cord organization of healthy full-term caesarean section was collected under sterile condition. The vessels and the umbilical cord membrane were removed. The WJ was fully cleaned by PBS and cut into the size of approximately0,5～1.0mm3, then plant them in culture flasks using L-DMEM with100ml/L fetal bovine serum. The WJ-MSCs were subcultured with0.125g/L Trypsin. Morphology and growth characteristics were observed under the inverted phase contrast microscope. The growth curve of the P3WJ-MSCs was drawn by cell counting. Vimentin was tested by immunocytochemistry. The cell phenotype of WJ-MSCs was detected by flow cytometry. WJ-MSCs were cultured in induction medium containing IBMX and forskolin in6.5h. The differentiated cells were confirmed by the expression of GFAP, NF, nestin and NSE with immunocytochemical staining.Results:The Wharton’s jelly organization kept still in3days. Under the inverted phase contrast microscope, some cells swam out from the tissue block for6days or so, which belong to fibroblast-like cell appearing long spindle shaped, short rod lie or flat shaped fibroblast-like cells. Then about10days, cells scattered in clone. WJ-MSCs after subculture presented in a uniform spindle morphology shape, showing parallel growth or vortex growth. WJ-MSCs possess strong proliferation ability after freeze-thawed. P3WJ-MSCs for2days were in latent phase, followed by logarithmical proliferation from3days, reached the growth platform at6days. Vimentin was strongly expressed positive. The expression rate of CD44、CD90、 CD105、CD34and CD45was100%,100%,99.7%,0.5%and0.1%, respectively. The morphology of WJ-MSCs in committed induction medium changed to round and with lipid vacuoles accumulated in the cytoplasm gradually, which could be stained by oil-red O. After committed induction with IBMX and forskolin in6.5hours, the differentiated cells forming the refractile appearance and neurite-like projections were positive with GFAP and NF, but Nestin and NSE were negative.Conclusions:WJ-MSCs can be effectively obtained from Wharton’s jelly with the planting culture, which displayed a fibroblast-like morphology, and possessed MSCs surface markers and multiple differentiation capability. It is an ideal seed cells as a source of cell transplantation as well as tissue engineer.