RiPS细胞用于脊髓损伤髓鞘修复的研究

One important pathophysiology and dysfunction of spinal cord injury (SCI) is the demyelination caused by the apoptosis of oligodendrocytes (OLs). Oligodendrocyte precursor cell (OPCs) transplantation for the remyelinating is an efficacious way to treat SCI. IPS cells induced by mRNA transfection (RiPS cells) provide a safe cell source for OPCs as no change in the genome. In the present study, we plan to build a bacterial artificial chromosome: BAC-OG-Neo-OE which can exist in human cells for a long time as a episome and can specifically express GFP in OLIG2-positive cells, and it will not recombine with the host genome, so it is a safe BAC for transgenic. This BAC will be tranfected into RiPS cells we have already induced, and then establish a protocol for the differentiation of RiPS cells to OPCs, and in the appropriate time of differentiation filter the GFP-positive OPCs by cell sorting utilizing the specific expression of OLIG2 in OPCs. The high-purity OPCs obtained by cell sorting are then transplanted into SCI rat model to study their survival, migration, differentiation, and remyelination in the injury area. This study will help to open up a new way to enrich high-purity interest cells from the directed differentiation of RiPS cells by this safe transgenic technology, and it will accumulate some important experimental evidences for the SCI treatment using RiPS cells.

脊髓损伤(SCI)后一项重要病理特征是少突胶质细胞凋亡后引起的脱髓鞘，少突胶质前体细胞(OPCs)移植修复髓鞘将是治疗SCI的重要途径之一。mRNA转染诱导的iPS细胞(RiPS细胞)因无基因组改变而成为OPCs安全的细胞来源。在本研究中，我们拟构建在人细胞中以附加体形式长期存在且能在OLIG2+细胞中特异表达GFP的载体:BAC-OG-Neo-OE，该BAC不会重组到人基因组而具有很好的安全性。将该BAC导入我们已经成功诱导的人RiPS细胞，建立RiPS细胞向OPCs分化的实验流程，在分化过程中利用OLIG2在OPCs特异表达的特点流式筛选GFP+的OPCs。将所获高纯度OPCs移植到SCI大鼠模型，研究其在损伤区域的存活、迁移、分化和参与髓鞘修复的情况。本研究将为利用安全的转基因技术从RiPS细胞定向分化中获得高纯度目的细胞开辟新的思路，为RiPS细胞用于SCI治疗积累重要的实验依据。

采用DNA-free的方法获得iPS细胞是其应用于再生治疗的重要前提之一。在本项目中，我们采用四种多潜能基因的mRNA和蛋白质组合使用的方法获得了RiPS细胞，体内和体内分化均证明了其分化为三个胚层组织细胞的潜能。接着我们构建了在人细胞中以附加体形式长期存在且能在OLIG2+细胞中特异表达GFP的载体:BAC-OG-Neo-OE，该BAC不会重组到人基因组而具有很好的安全性。将该BAC导入我们已经成功诱导的RiPS细胞，获得了安全的转基因细胞克隆。我们针对胚胎干细胞和iPS细胞向OPC定向诱导分化效率低且时程太长的难题利用转基因RiPS细胞建立了高效的OPCs分化策略，在分化的三个阶段均找到了加速分化的策略。RA和SHH联合使用加速了RiPS细胞分化为神经上皮细胞（NE）的速度。EGF和bFGF协同作用加快了NE分化为pre-OPC的时间，并且提高了分化效率。特别关键的是，星形胶质细胞条件培养基将pre-OPC发育为OPC的时间由传统的60天缩短到24天左右。所以，我们的分化策略只需要45天左右的时间就可以获得高纯度的GFP+的OPC，远远优于传统的将近100天的分化策略。将所获高纯度OPCs移植到大鼠脊髓损伤（SCI）模型，发现其在损伤区域可以存活、分化并参与髓鞘修复。本研究为利用安全的iPS细胞用于SCI治疗积累了重要的实验依据。