Biology/Transplantation of Multipotent Marrow Stem Cells

描述(申请人提供)：这项研究的长期目标是在新的细胞疗法的开发中开发骨髓干细胞的多系分化能力。这一应用建立在临床全骨髓移植(BMT)导致供体细胞植入成骨不全患者的骨骼中的证明之上，成骨不全是一种遗传性骨骼疾病。随后，分离的标记基因的间充质干细胞移植也导致了供体细胞在骨中的植入，但水平比骨髓移植低得多，这表明骨髓中成骨活性的主要来源并不是骨髓干细胞的塑料贴壁群体。因此，将逆转录病毒转导的供体细胞移植到小鼠BMT模型中，试图确定BMT对成骨产生意外影响的可能干细胞。除血液外，在骨中发现的成骨细胞中，有20%-50%来自塑料非黏附的骨髓细胞。逆转录病毒整合位点的克隆分析表明，单个非贴壁骨髓细胞同时分化为血细胞和骨细胞，表明存在同时具有造血细胞和成骨分化能力的多潜能骨髓。目的1利用表达GFP的转基因小鼠作为候选骨髓细胞亚群的供体，在小鼠移植模型中对其特性进行测试和比较，以确定该细胞的表型和发育潜力。最强候选者的多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。相关实验将确定干细胞在骨细胞途径中分化所需的辅助细胞。在目标2中，目标是在恒河猴模型中验证我们的工作假设，调查基因标记的CD34+和CD34-(SP)非贴壁骨髓细胞克隆再生造血室和成骨室的能力。这一目标的结果将为以骨髓干细胞为基础的治疗人类骨骼疾病或损伤的可行性提供有价值的估计。最后，在目标3中，将努力确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(成人)干细胞低水平植入的一般问题。总而言之，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.