Biology/Transplantation of Multipotent Marrow Stem Cells

描述（由申请人提供）：本研究的长期目标是在开发新型细胞疗法中利用骨髓干细胞的多谱系分化能力。该应用建立在全骨髓（BMT）的临床移植导致供体细胞植入成骨细胞瘤（一种遗传性骨疾病）患者的骨中的证明的基础上。随后移植分离的基因标记的间充质干细胞也导致供体细胞在骨中的植入，但与BMT相比水平低得多，这表明骨髓中成骨活性的主要来源并不存在于骨髓干细胞的塑料粘附群体中。因此，在小鼠BMT模型中移植逆转录病毒转导的供体细胞，以鉴定负责BMT对骨生成的意外作用的假定干细胞。除血液外，塑料非粘附性骨髓细胞产生了骨中20-50%的成骨细胞。逆转录病毒整合位点研究的克隆分析显示，一个单一的非粘附性骨髓细胞产生了血细胞和骨细胞，表明存在多能骨髓造血和成骨分化能力。目的1旨在定义这种细胞的表型和发育潜力，使用GFP表达转基因小鼠作为候选骨髓细胞亚群的供体，其特性将在小鼠移植模型中进行测试和比较。最强候选者的多谱系分化能力将在单细胞移植后通过严格的克隆分析（逆转录病毒整合位点的比较）来验证。相关的实验将确定在干细胞的承诺，以分化成骨细胞途径中的辅助细胞的要求。在目的2中，目标是在恒河猴模型中检验我们的工作假设，研究基因标记的CD 34+和CD 34-（SP）非粘附骨髓细胞克隆再生造血和成骨区室的能力。这一目标的结果将为基于骨髓干细胞的治疗人类骨疾病或损伤的可行性提供有价值的估计。最后，在目标3中，将努力鉴定可能刺激骨髓干细胞向骨细胞谱系分化的生长因子，从而解决组织（成体）干细胞低水平植入的一般问题。总的来说，这个为期5年的项目产生的数据将有助于澄清干细胞生物学中的几个悬而未决的问题，以及具有明显多谱系潜力的骨髓干细胞的治疗相关性。

DESCRIPTION (provided by applicant): The long-range goal of this research is to exploit the multilineage differentiation capacity of bone marrow stem cells in the development of novel cellular therapies. This application builds on the demonstration that clinical transplantation of whole bone marrow (BMT) results in donor cells engraftment in the bones of patients with osteogenesis imperfecta, a genetic bone disorder. Subsequent transplantation of isolated gene-marked mesenchymal stem cells also led to donor cell engraftment in bone but at a much lower level that seen with BMT, suggesting that the major source of osteogenic activity in bone marrow is not represented in the plastic-adherent population of marrow stem cells. Thus, a murine BMT model was transplanted with retroviraly-transduced donor cells in an effort to identify the putative stem cell responsible for the unexpected effect of BMT on osteogenesis. In addition to blood, the plastic-nonadherent marrow cells gave rise to 20-50% of the osteoblasts identified in bone. Clonal analysis by retroviral integration site studies revealed that a single nonadherent marrow cell had given rise to both blood and bone cells, indicating the existence of a multipotent marrow with both hematopoietic and osteopoietic differentiation capacity. Aim 1 seeks to define the phenotype and developmental potential of this cell, using GFP-expressing transgenic mice as donors of candidate marrow cell subsets whose properties will be tested and compared in a murine transplantation model. The multilineage differentiation capacity of the strongest candidates will be verified by rigorous clonal analysis (comparison of retroviral integration sites) after single-cell transplantation. Related experiments will determine the requirement for accessory cells in the commitment of stem cells to differentiate in the osteocytic pathway. In Aim 2, the goal is to test our working hypothesis in a rhesus macaque monkey model, investigating the capacity of gene-marked CD34+ and CD34- (SP) nonadherent marrow cells to clonally regenerate the hematopoietic and osteopoietic compartments. Results of this aim will provide valuable estimates of the feasibility of marrow stem cell-based therapy for human bone diseases or injury. Finally, in Aim 3, efforts will be made to identify growth factors that might stimulate the differentiation of marrow stem cells toward the osteocytic lineage, thus addressing the general issue of low-level engraftment by tissue (adult) stem cells. Collectively, the data generated by this 5-year project will help to clarify several outstanding questions in stem cell biology and the therapeutic relevance of marrow stem cells with apparent multilineage potential.