Cell and Molecular Biology Core

核心B的目的是为分子/细胞分析、细胞和载体库以及三个项目和核心C下开展的研究所需的无动物产品试剂的生成提供关键支持。核心B的具体服务目标是：目标1：提供iPS和其他细胞系的细胞分析。Core B将负责提供iPS和分化细胞系中表达抗原的免疫组化染色的专业知识、协助和协调，以及核型分析。目的2：为iPS等细胞系的分子生物学研究提供基础。核心B将提供细胞系的分子分析，以验证其作为IPS、原代或分化细胞系的状态。这将包括基因表达谱，启动子甲基化和microRNA分析。核心将负责确认突变和校正细胞系的B-珠蛋白基因型和微卫星指纹图谱。一个目标是定义分子表达谱或特征，以监测和验证IPS体外分化为能够在体内重建骨髓的造血前体细胞。目的3：提供细胞和载体库，并评估细胞用于最终的治疗开发。预计生产的材料可能会转移到诊所，我们将建立一个集中的位置和数据库，以记录和维护关键细胞系、载体、试剂和其他辅助材料。这将与核心A一起完成。目的4：工程化人细胞系以替代用于将IPS细胞分化成造血细胞系的小鼠0 P9基质细胞。迄今为止使用的最有效的方法之一是采用IPS和hES细胞与小鼠基质0 P9细胞系的共培养。由于暴露于动物源性产品可能会引入对患者具有潜在不良安全后果的病原体，因此开发人基质细胞系以替代0 P9将是创建用于产生基因校正的HSC的无动物产品工艺的一个组成部分。目的5：将原代细胞系常规重编程为IPS细胞系，用于项目2和3的早期研究。体细胞将重编程为IPS细胞，用于基因连接（项目2），HCS分化（项目3）研究最初将使用携带4种重编程基因（OCT 4、SOX 2、KLF 4和cMYC）的逆转录病毒载体进行。随着新的重编程技术在项目1中的开发，它们将被应用于IPS细胞的生成。

The purpose of Core B is to provide critical support for molecular/cellular analyses, ceil and vector banking, and generation of animal-product-free reagents fbr research needs performed under the three Projects and Core C. The specific service aims of Core B are: Aim 1: To provide cellular analysis of iPS and other cell lines. Core B will be responsible for providing expertise, assistance and coordination of immunohistochemical staining for expression antigens in iPS and differentiated cell lines, and for analysis of karyotype. Aim 2: To provide molecular analysis of iPS and other cell lines. Core B will provide molecular analyses of cell lines to validate their status as IPS, primary, or differentiated cell lines. This will include gene expression profiles, promoter methylation, and microRNA analyses. The core will be responsible for confirming B-globin genotype for mutant and corrected cell lines and microsatellite fingerprint profiles. One goal is to define a molecular expression profile or signature to monitor and validate in vitro differentiation of IPS to hematopoietic precursor cells competent to repopulate bone marrow in vivo. Aim 3: To provide cell and vector banking and evaluate cells for eventual therapeutic development. In anticipation that materials produced may be transitioned to the clinic, we will establish a centralized location and database to document and maintain key ceil lines, vectors, reagents, and other ancillary materials. This will be done in conjunction with Core A. Aim 4: To engineer a human cell line to replace mouse 0P9 stromal cells used for differentiation of IPS cells into hematopoietic cell lines. One of the most effective methods used to date has employed co-culture of IPS and hES cells with the mouse stromal 0P9 cell line. Since exposure to animal derived products might introduce pathogens with potential adverse safety consequences for patients, development of a human stromal cell line to replace 0P9 will be one component of creating animal-product free processes for generating gene-corrected HSCs. Aim 5: To perform routine reprogramming of primary cell lines into IPS ceil lines for use in early stage research for projects 2 and 3. Somatic cells will be reprogrammed to IPS cells for the gene connection (Project 2) and HCS differentiation (Project 3) studies will initially be performed using retroviral vectors carrying 4 reprogramming genes (0CT4, S0X2, KLF4, and cMYC). As new reprogramming technologies are developed in Project 1 they will be applied for the generation of IPS cells.