DEVELOPMENT OF REAGENTS FOR STEM CELL DIFFERENTIATION

这项提案的总体目标是开发化学定义的 支持造血干细胞增殖的试剂和 差异化。这样的试剂在血液学方面会很有价值。 研究，特别是骨髓移植、基因治疗和 净化。在第一阶段的工作中，我们已经开发出了 由化学定义的成分组成，不含任何衍生的蛋白质 来自人类来源，强烈支持CD34+细胞增殖和 差异化。我们在第二阶段的工作中建议优化这些 骨髓、脐带体外培养试剂及培养条件 外周血和动员的CD34+细胞。第二阶段的工作 将侧重于细胞因子混合物、浓度效应和培养时间 与细胞总数、CFU-GM形成、细胞周期 动力学和免疫表型分析。短期和长期 培养细胞的重建能力将使用 大动物宫内绵羊模型用于人类造血。这类研究 在开发化学定义的试剂方面将是无价的， 最重要的是去除从人类血液中提取的蛋白质 易患各种疾病的成分，因此FDA将其召回。 这些试剂将促进临床方案的开发 用细胞疗法治疗疾病。 建议的商业应用： 支持干细胞扩增的已定义试剂将对 基因治疗的研究者和临床医生，骨髓 移植和净化。

The overall goal of this proposal is to develop chemically defined reagents that support hematopoietic stem cell proliferation and differentiation. Such reagents would be valuable in hematological research, especially for bone marrow transplantation, gene therapy and purging. During the Phase I effort, we have developed reagents that are composed of chemically defined components, free of any proteins derived from human sources which strongly support CD34+ cell proliferation and differentiation. We propose in the Phase II effort to optimize these reagents and culture conditions for ex vivo culture of bone marrow, cord blood and mobilized peripheral blood CD34+ cells. The Phase II effort will focus on cytokine mixtures, concentration effects and culture time frames with respect to total cell number, CFU-GM formation, cell cycle kinetics, and immunophenotype analysis. The short-term and long-term reconstituting ability of the cultured cells will be assessed using the large animal in utero sheep model for human hematopoiesis. Such studies will be invaluable in developing reagents that are chemically defined, and most importantly for removal of proteins derived from human blood components that are subject to various diseases and thus FDA recall. These reagents will foster the development of clinical protocols for the treatment of disease using cellular therapies. PROPOSED COMMERCIAL APPLICATION: Defined reagents that support stem cell expansion will be valuable to the researcher and clinician for gene therapy, bone marrow transplantation and purging.