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

我们研究了人类胎盘和脐带血是否可以成为再生医学的新细胞来源，利用我们实验室建立的银行系统。首先，通过外植体法分离来自胎儿和母体部分的成纤维细胞样细胞。胎儿部分来源的细胞未被母体细胞污染。由于脐血库中记录了婴儿HLA分型、脐血中病毒和细菌污染、供者家族史等脐血移植所需的信息，因此本研究选择胎儿绒毛组织作为间充质细胞来源。将来自绒毛膜绒毛的细胞（人胎盘来源的间充质干细胞：hPDMSC）用DMEM +10%FBS培养。这些细胞的表型与来自骨髓和脂肪组织的细胞相似，CD29、CD44、CD73、CD105、CD90、HLA-I类阳性表达，而CD31、CD34、CD45、TIE-II阴性表达 ...更多信息 和HLA-II类抗原。hPDMSC在成骨诱导条件下可向成骨细胞分化。对于软骨形成分化，使用沉淀培养系统。在诱导培养过程中，细胞团大小逐渐增大，有光泽，透明，石蜡切片甲苯胺蓝染色和II型胶原免疫组化染色阳性。在细胞中也证实了向脂肪细胞和神经样细胞的分化能力。将诱导后的hPDMCs移植到裸鼠和大鼠膝关节骨软骨缺损处，观察hPDMCs的成软骨能力。我们尝试用慢病毒载体介导hTERT和Bmi-1基因转染hPDMCs，以延长hPDMCs的寿命，使hPDMCs保持向成骨、成软骨、成软骨细胞的分化潜能，在此基础上，建立了从脐血中分离间充质细胞的方法。当CB体积大于60ml且从分娩到分离间充质细胞的时间小于5h时，从CB分离间充质细胞的成功率可达70%。这些细胞表现出高的成骨、成软骨分化，但低的脂肪形成。采用裸鼠和大鼠体内成骨和软骨形成实验观察其成骨和软骨形成情况。因此，我们的研究结果表明，人胎盘和脐带血可以作为再生医学的同种异体间充质干细胞来源。少

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