Phenotypic & Functional Analysis of Human Schwann Cells for Potency Assay Dvlpmt

描述(申请人提供)：雪旺细胞(SC)是细胞移植治疗的一种细胞类型，旨在恢复受损的中枢神经系统和三叉神经节的功能。在动物模型中的研究一直表明，干细胞可以缩小脊髓损伤后形成的空洞的大小；促进损伤部位的轴突再生和髓鞘再生轴突，从而促进功能恢复。2012年7月，FDA批准了一项I期临床试验，以测试自体成人神经来源的人干细胞移植在脊髓损伤患者中的安全性。在细胞治疗中使用原代干细胞的可行性依赖于细胞培养方法的可用性，从而能够从周围神经活检组织中获得大量具有移植质量的纯化干细胞。对人类干细胞产品的特性和功能的准确评估对于确定细胞的潜在治疗价值至关重要。事实上，移植前对细胞功能的评估是临床测试后期产品释放的法规要求。尽管有大量文献记载这份报告 对啮齿动物干细胞的研究，对人类干细胞的研究很少。这至少在一定程度上是由于培养的人类干细胞在最初为啮齿动物细胞设计的标准功能分析中表现不佳。该项目致力于克服体外试验中的技术障碍，以评估人类干细胞的生物学活性。由于干细胞的特性和功能密不可分，我们建议通过开发简单的分析方法来评估干细胞分化为髓鞘细胞的潜力，方法是在给予cAMP之前和之后鉴定分化标记物，cAMP是一种指导分化的信号(目标1)。由于轴突的包膜和髓鞘可能是干细胞在体外和体内功能的两个最关键的决定因素，我们还建议通过对影响干细胞和共培养系统神经元组件的现有方案进行合理的修改，来发展人干细胞和神经元的髓鞘共培养(目标2)。识别体外扩增的干细胞与其分化为髓鞘形成细胞的潜能相关的最重要的细胞特征将使我们能够 开发和实施可靠的检测方法，评估细胞的治疗价值。如果成功，允许在体外量化髓鞘形成的共培养系统可以作为干细胞效力的直接测量。

DESCRIPTION (provided by applicant): The Schwann cell (SC) is a cell type of choice for cell transplantation therapies that aim to restore function in the injured CNS and PNS. Work in animal models has consistently shown that SCs reduce the size of the cavities that form after spinal cord injury; promote axon regeneration across the lesion site and myelinate regenerating axons, thereby improving functional recovery. In July 2012, the FDA approved a Phase I clinical trial to test the safety of autologous adult nerve-derived human SC transplantation in patients suffering from spinal cord injury. The feasibility to use primary SCs in cell therapy relies on th availability of cell culture methods rendering high numbers of transplantation quality purified SCs from peripheral nerve biopsies. An accurate assessment of the identity and function of the human SC product is essential to determine the potential therapeutic value of the cells. Indeed, the assessment of cell function prior to transplantation is a regulatory requirement for product release in later phases of clinical testing. Even though an extensive literature exists that report studies on rodent SCs, studies on human SCs are scarce. This is at least in part due to the poor performance of cultured human SCs in standard functional assays that were originally designed for rodent cells. This project seeks to overcome technical barriers in the development of in vitro assays to evaluate the biological activity of human SCs. Because SC identity and function are inextricably intertwined, we propose to begin addressing this challenge by developing simple analytical methods to evaluate the differentiating potential of SCs to become myelinating cells by the identification of differentiation markers before and after administration of cAMP, an instructing signal for differentiation (Aim 1). Because ensheathment and myelination of axons are perhaps the two most critical determinants for SC function in vitro and in vivo, we also propose to develop myelinating co-cultures of human SCs and neurons by implementing rational modifications to current protocols affecting both the SC and the neuronal components of the co-culture system (Aim 2). Identifying the most important cellular characteristics of in vitro expanded SCs relevant to their differentiating potential into myelin-forming cells will allow us to develop and implement reliable assays that assess the therapeutic value of the cells. If successful, co-cultures systems that allow for a quantification of myelin formation in vitro can serve as a direct measure of SC potency.