Placental and umbilical cord blood derived mesenchymal cell for regenerative medicine and construction of a banking system for mesenchymal cell.

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We examined the possibility whether human placenta and cord blood can be a new cell source for regenerative medicine utilizing the bank system that has been established in our laboratory. First, fibroblast-like cells from fetal and maternal part were isolated by an explant method. The cells derived from fetal part were without contamination with maternal cells. Since the information required for cord blood transplantation, such as HLA typing of infant, viral and bacterial contamination in cord blood units, family history of the donor etc, were recorded in the bank, we chose chorionic villi from the fetal part as the source of mesenchymal cells in this study. The cells derived from chorionic villi (human placenta derived mesenchymal stem cells: hPDMSCs) were cultured with DMEM+10%FBS. The phenotypes of those cells were similar to those derived from bone marrow and adipose tissue; positive expression of CD29, CD44, CD73, CD105, CD90, HLA-class I, but negative for CD31, CD34, CD45,TIE-II … More and HLA-class II antigen. The hPDMSCs could differentiate to osteoblast in the osteogenic induction condition. For chondrogenic differentiation, a pellet culture system was used. The size of the pellet increased with glistening and transparency during the induction culture, and toluidine blue staining and type-II collagen immuno-histochemical staining were positive in paraffin section of the pellets. Differentiation ability to adipocyte and neural-like cell were also confirmed in the cells. The chondrogenesis of hPDMCs was also detected in vivo by transplantation of induced cells in a scaffold at subcutaneous and osteochondral defected knee of nude mice and rat.We attempted to prolong the life span of hPDMCs by transduction with hTERT and Bmi-1 using lenti-vector, the immortalized cells kept the differentiation potential into osteogenic, chondrogenic, adipogenic cells.Then we established the method for isolation of mesenchymal cell from cord blood (CB). The success rate of isolation of mesenchymal cell from CB increased up to 70% when the volume of CB was more than 60ml and the time between deliveries and cell isolation was shorter than 5 hours. Those cells showed high osteogenic, chondrogenic differentiation, but low for adipogenesis. The osteogenesis and chondrogensis were also observed in vivo using nude mice and rat. Thus our results indicated that human placenta and umbilical cord blood can be allogeneic mesenchymal stem cell sources for regenerative medicine. Less