DEVELOPMENT OF REAGENTS FOR STEM CELL DIFFERENTIATION

该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， 分化 这些试剂在血液学方面是有价值的。 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，我们已经开发了试剂， 由化学成分确定的成分组成，不含任何蛋白质衍生物 来自人源，其强烈支持CD 34+细胞增殖， 分化 我们建议在第二阶段努力优化这些 用于骨髓、脐带和骨髓基质细胞的离体培养的试剂和培养条件 血液和动员的外周血CD 34+细胞。第二阶段的努力 将重点关注细胞因子混合物、浓度效应和培养时间 总细胞数、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 人造血大动物子宫内绵羊模型。 此类研究 在开发化学定义的试剂方面将是非常宝贵的， 并且最重要的是用于去除来自人血液的蛋白质 这些成分容易引起各种疾病，因此FDA会召回。 这些试剂将促进临床方案的发展， 使用细胞疗法治疗疾病。 拟定商业应用： 支持干细胞扩增的确定试剂将是有价值的， 研究人员和临床医生基因治疗，骨髓 移植和清除。

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.