EXPANSION OF HUMAN STEM CELLS WITH TRANSPLANTATION MODEL

描述（改编自申请人摘要）：总体目标 本课题旨在开发无菌条件下的试剂和培养条件， 将支持造血干细胞的离体扩增 细胞（HSC）用于短期和长期移植。 研究 本文将评估保留所需的培养条件， 具有体内短期和长期再增殖活性的HSC 利用新近建立的绵羊子宫内移植模型， 人类造血 本提案中的可行性数据显示， 在无血清培养基中培养的人骨髓CD 34+细胞 含有低水平的细胞因子的细胞保持了长期的重新增殖， 细胞 相反，在类似条件下培养的CD 34+细胞， 在血清存在下，仅表现出短期骨髓再生 活动 在第一阶段的工作中，调查人员建议延长 这些研究比较了没有血清的培养条件， 含血清培养条件。申请人建议分析 对于1）细胞周期，来自每种培养条件的细胞群 状态，2）活细胞数，3）GM-CFC，和4）群体动态 与髓样细胞分化相比， 使用免疫表型分析。 这些信息将是有价值的， 将维持所需的培养条件与 耗尽长期移植所需的细胞群。 第二阶段的建议将扩大这些研究，包括脐带缆 血液正常人骨髓和动员外周血干细胞 开发和优化培养试剂和条件 没有血清，这是体外扩增长期 移植细胞 第二阶段的工作将集中在细胞因子混合物， 浓度效应和暴露时间，以扩大长期 移植细胞 这些研究将可用于利用离体 在治疗疾病的临床方案中， 在设计HSC离体培养的临床方案中是非常宝贵的 进行移植

DESCRIPTION (Adapted from applicant's abstract): The overall goal of this project is to develop the reagents and culture conditions devoid of serum that will support the ex vivo expansion of hematopoietic stem cells (HSC) for short-term and long-term engraftment. The studies presented here will evaluate the culture conditions necessary to retain HSC capable of in vivo short-term and long-term repopulating activity using the recently development sheep in utero transplantation model for human hematopoiesis. Feasibility data presented in this proposal show that CD34+ cells from human bone marrow cultured in serum-free medium containing low levels of cytokines retained the long-term repopulating cells. In contrast, CD34+ cells cultured under similar conditions, but in the presence of serum, exhibited only short-term marrow repopulating activity. In the Phase I effort, the investigator proposes to extend these studies comparing the culture conditions devoid of serum with serum containing culture condition. The applicants propose to analyze the cell populations from each culture condition for 1) cell cycle status, 2) viable cell number, 3) GM-CFC, and 4) population dynamics of the more primitive HSC versus differentiation of the myeloid cells using immunophenotype analysis. Such information will be valuable in correlating the culture condition necessary to maintain and those that deplete the population of cells necessary for long-term engraftment. The Phase II proposal will be to expand these studies to include cord blood normal human bone marrow and mobilized peripheral blood stem cells and to develop and optimize the culture reagents and conditions devoid of serum, necessary for ex vivo expansion of the long- term engrafting cells. The Phase II effort will focus on cytokine mixtures, concentration effects and times of exposure to expand the long-term engrafting cells. Such studies will be available in utilizing ex vivo culture in clinical protocols for the treatment of disease, and will be invaluable in designing the clinical regimens for ex vivo culture of HSC for transplantation.