[Biological characteristics and induced differentiation ability of in vitro expanded umbilical cord blood mesenchymal stem cells].

OBJECTIVE: Mesenchymal stem cells (MSCs) from bone marrow are capable of differentiating into cells of different tissue lineages such as bone, cartilage, and adipose tissue and are the best candidates for tissue engineering. It is well accepted that umbilical cord blood (UCB) is a source for hematopoietic stem cells. However, controversy exists as to whether UCB contains MSCs and can serve as a source of MSCs. Therefore, the aim of this study was to explore the biological characteristics and inducing differentiation ability of in vitro expanded UCB MSCs.

METHODS: UCB was collected on normal full term delivery of infants with informed consent (n = 35) obtained from the mothers. Mononuclear cells (MNCs) were isolated from UCB by gravity centrifugation and cultured with DMEM including 10% fetal bovine serum. The morphology was observed under microscope per day. Cytochemical staining was carried out and flow cytometry was used to examine the surface antigen phenotype. Fifth passage cells were transferred into a different medium and osteogenic differentiation, adipogenic differentiation, and neurogenic differentiation were assessed.

RESULTS: MSCs could be isolated and cultured from MNCs of a few UCB sources. These cells displayed fibroblast-like morphology. They withstood over 20 passages without significant structural changes. These MSCs were negative for alkaline phosphatase (ALP) staining and positive for alpha-naphthol butyric acid esterase (NBE) staining. Expression of CD(29), CD(44)and CD(105), especially the human MSCs-specific markers SH-2 and SH-3 were observed, but CD(3), CD(14), CD(19), CD(34) and CD(45) could not be found, indicating that these cells were not of hematopoietic origin. Exposure of these MSCs to serum-free osteogenic condition, they could differentiate into bone cells and form mineralized matrix as evidenced by Alizarin red staining 2 weeks later. When these UCB-derived MSCs were cultured in adipogenic medium, morphologic changes in cells as well as the formation of neutral lipid vacuoles were noticeable as early as 1 week after induction and visualized by staining with oil-red O. Surprisingly, these MSCs were also able to differentiate into neuroglial-like cells. Morphology of these induced cells resembled that of neurons. Immunocytochemistry showed that they expressed Nestin and neuron-specific enolase (NSE), but not glial fibrillary acidic protein (GFAP).

CONCLUSION: UCB does contain MSCs. These MSCs, which are multipotent, could be isolated and cultured from a few UCB sources. UCB might serve as an alternative source of MSCs to bone marrow.