Adult Stem Cells: Osteoblast Differentiation and Engraftment

描述（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，关于从移植的祖（干）细胞群成功分化多组织仍然存在争议。越来越多地认识到，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的表达的植入可能无法验证。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。我们在全骨髓移植中或全身注射骨髓基质细胞时，没有发现成骨细胞植入的证据。然而，当在髓内空间中施用基质细胞时，存在稳健的成骨细胞分化和并入宿主骨中。使用这种类型的移植协议，四个可区分的颜色的GFP转基因小鼠和一个快速，灵敏和特异性的组织学方法评估成骨细胞植入，我们将映射的位置骨祖细胞群体的假设谱系地图，起源于成血管细胞。这种成体祖细胞具有造血、内皮和成骨细胞分化的特性，并且被假设具有足够的自我更新潜力以在临床上显著的时间段内移植和支持新骨形成。将在五种临床相关组织来源（骨髓、骨组织、肌肉、脂肪和新生儿脐带）中生成这种类型细胞的证据，并测试其体内成骨细胞分化，以最佳地形成新骨。鉴定和富集负责最佳成骨的细胞的方法将通过基于表面抗原或在早期谱系发育期间有活性的启动子- GFP转基因的表达的FACS分选来进行。富集群体的微阵列研究可能建议进一步富集最佳祖细胞的其他策略。这项资助将提供一个头对头的比较骨祖细胞的潜力骨移植的细胞从5个不同的组织。它将评估富集方案的有效性，并开发一种策略，将祖细胞的位置与从真正的胚胎干细胞到具有足够成体干细胞特性的细胞的谱系进展的假设但可测试的地图相关联，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.