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不同，以及用于判断分化程度的方法的不同，对于从移植的祖细胞(干细胞)群体中成功地进行多组织分化仍然存在争议。越来越多地认识到，当使用组织特异性供体表达的标记基因时，可能无法验证基于标准组织学标准的植入或普遍存在的遗传标记的表达。根据我们在不同移植方案中使用成骨细胞限制性GFP报告转基因小鼠的经验，我们同意这一观点。在全骨髓移植或全身注射骨髓基质细胞时，我们没有发现成骨细胞植入的证据。然而，当基质细胞被注入髓内间隙时，成骨细胞有很强的分化和整合到宿主骨中。使用这种类型的移植方案，四种不同颜色的GFP转基因小鼠，以及一种快速、敏感和特异的组织学方法来评估成骨细胞植入，我们将把骨祖细胞群体的位置映射到假想的起源于血管母细胞的谱系图上。这种成体祖细胞具有造血细胞、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来植入并支持新骨形成一段临床意义上的时间。将在五个临床相关的组织来源(骨髓、骨组织、肌肉、脂肪和新生儿脐带)中产生这种类型的细胞的证据，并测试体内成骨细胞分化对新骨形成的最佳条件。鉴定和丰富负责最佳成骨的细胞的方法将通过基于表面抗原或早期谱系发育中活跃的启动子-GFP转基因表达的FACS分选来进行。对富集群的微阵列研究可能会为进一步富集祖细胞提供其他策略。这笔赠款将对来自5个不同组织的细胞进行骨移植的成骨细胞潜能进行逐一比较。它将评估浓缩方案的有效性，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化图联系起来，这对骨病的细胞和基因治疗应用是有用的。

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.