Adult Stem Cells: Osteoblast Differentiation and Engraftment

描述（由申请人提供）：由于组织来源和细胞制备的差异以及用于判断分化水平的方法的差异，关于移植祖细胞（干细胞）群体的多组织分化的成功仍然存在争议。越来越多的人认识到，当利用组织特异性供体表达的标记基因时，基于标准组织学标准或普遍存在的遗传标记表达的移植可能无法验证。基于我们在各种移植方案中使用成骨细胞限制性GFP报告基因转基因小鼠的经验，我们同意这一观点。我们没有发现在全骨髓移植或骨髓基质细胞全身注射时细胞植入成骨细胞的证据。然而，当骨髓间质细胞被注入骨髓腔时，成骨细胞分化和并入宿主骨。使用这种类型的移植方案，四种可区分的GFP转基因小鼠颜色，以及快速、敏感和特异性的组织学方法来评估成骨细胞的植入，我们将绘制成骨细胞群体的位置到一个假设的起源于成血管细胞的谱系图。这种成体祖细胞具有造血、内皮细胞和成骨细胞分化的特性，并被认为具有足够的自我更新潜力，可以在临床显著的一段时间内植入并支持新骨形成。这类细胞在五个临床相关组织来源（骨髓、骨组织、肌肉、脂肪和新生儿脐带）中的证据将被生成，并在体内测试最适合新骨形成的成骨细胞分化。基于表面抗原或在早期谱系发育过程中活跃的启动子- GFP转基因基因表达的FACS分选，将寻求鉴定和富集负责最佳成骨的细胞的方法。富集群体的微阵列研究可能提示进一步富集最佳祖细胞的其他策略。这项资助将提供5种不同组织细胞骨移植的骨祖细胞潜能的头部对比。它将评估富集方案的有效性，并制定一种策略，将祖细胞的位置与一个假设但可测试的谱系进展图联系起来，从真正的胚胎干细胞到具有足够成体干细胞特性的细胞，以用于骨疾病的细胞和基因治疗应用。

DESCRIPTION (provided by applicant): Controversy still persists as to success of multi-tissue differentiation from a transplanted progenitor (stem) cell population either due to differences in tissue source and cell preparation and to methods used to judge the level of differentiation. Increasingly it is appreciated that engraftment based on standard histological criteria or expression of a ubiquitous genetic marker may not be validated when a tissue specific donor expressed marker gene is utilized. We concur with this opinion based on our experience using osteoblast restricted GFP reporter transgenic mice in a variety of transplantation protocols. We can find no evidence for osteoblast engraftment by cells in a total bone marrow transplant or when marrow stromal cells are injected systemically. However there is robust osteoblast differentiation and incorporation into host bone when the stromal cells are administered in the intramedullary space. Using this type of transplantation protocol, four distinguishable colors of GFP transgenic mice and a rapid, sensitive and specific histological method for assessing osteoblast engraftment, we will map the position of an osteoprogenitor cell population to a hypothesized lineage map that originates from a hemangioblast. This adult progenitor cell has the properties of hematopoietic, endothelial and osteoblast differentiation and is hypothesized to have sufficient self-renewal potential to engraft and support new bone formation for a clinically significant period of time. Evidence for this type of cell in five clinically relevant tissue sources (bone marrow, bone tissue, muscle, fat and newborn umbilical cord) will be generated and tested for in vivo osteoblast differentiation optimal for new bone formation. Methods to identify and enrich for the cells responsible for optimal osteogenesis will be pursued by FACS sorting based on surface antigens or expression of promoter - GFP transgenes active during early lineage development. Microarray studies of the enriched population may suggest other strategies to further enrich for the optimal progenitor cells. This grant will provide a head to head comparison of the osteoprogenitor potential for bone transplantation of cells from 5 different tissues. It will assess the effectiveness of enrichment protocols and develop a strategy to relate the position of the progenitor cells to a hypothesized but testable map of lineage progression from a true embryonic stem cell to a cell with sufficient adult stem cell properties to be useful in cell and gene therapy applications for diseases of bone.