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

我们利用我们实验室建立的数据库系统，研究了人胎盘和脐带血是否可以成为再生医学的新细胞来源。首先，采用组织块法分离胎儿和母体组织中的成纤维细胞。取自胎儿的细胞与母体细胞无污染。由于脐血移植所需的资料，如婴儿的人类白细胞抗原配型、脐带血中的病毒和细菌污染、供者的家族史等，我们选择了胎儿部分的绒毛作为间充质细胞的来源。取人胎盘间充质干细胞(HPDMSCs)，用DMEM+10%胎牛血清培养。CD29、CD44、CD73、CD105、CD90、HLAI类细胞表达阳性，CD31、CD34、CD45、Tie-II…表达阴性More和HLAII类抗原。在成骨诱导条件下，hPDMSCs可向成骨细胞分化。在软骨分化方面，使用颗粒培养系统。诱导培养过程中，颗粒体积增大，光亮透明，石蜡切片甲苯胺蓝染色和II型胶原免疫组织化学染色均为阳性。细胞向脂肪细胞和神经样细胞分化的能力也得到证实。将诱导后的细胞移植到裸鼠和大鼠的皮下和骨软骨缺损处，观察其在体内的成软骨作用。我们尝试通过Lenti载体转导hTERT和Bmi-1来延长hPDMCs的寿命，永生化细胞保持了向成骨、成软骨、成脂细胞分化的潜能。当脐带血的体积大于60ml时，从脐带血中分离间充质细胞的成功率可达70%，且从分娩到细胞分离的时间小于5h。这些细胞表现出高度的成骨、成软骨分化，但成脂能力较低。在裸鼠和大鼠体内观察其成骨和成软骨情况。因此，我们的结果表明，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。较少

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