Extracellular matrix control mesenchymal stem cell fate

描述（由申请人提供）：骨髓间充质干细胞（MSC）复制产生相同的子干细胞，并可分化为许多不同的细胞类型，包括成骨细胞。然而，当在组织培养塑料上培养时，MSC失去了它们独特的特性。这表明，在这种培养系统中，骨髓微环境的一个关键特征，即促进干细胞特性的保留是缺失的-这种情况损害了MSC的研究，并限制了其治疗潜力。在其他组织中，细胞外基质（ECM）通过控制干细胞暴露于生长因子而在控制干细胞行为中起重要作用。还已知ECM的蛋白聚糖组分双糖链蛋白聚糖（biglycan）在MSC数量和功能的维持中起重要作用。初步研究表明，在由小鼠骨髓来源的基质细胞制成的无细胞ECM上培养MSC保留了它们的干细胞特性，包括自我更新的能力、未分化状态的维持以及对外源性BMP-2的促成骨细胞作用的响应性。这一发现可以解释的证据表明，ECM隔离内源性BMP-2分泌的基质细胞。上述观察结果形成了以下假设的基础，即由骨髓基质细胞产生的ECM特异性地控制MSC自我更新和分化成特定谱系的能力，至少部分地通过调节细胞暴露于控制其复制和分化的因子。实验提出，以确定主要的胶原蛋白，粘附蛋白和蛋白多糖成分的小鼠骨髓基质细胞衍生的ECM使用免疫染色和共聚焦显微镜，并确定是否基质细胞衍生的ECM是独特的，其能力，以保持MSC的属性。此外，将测定基质细胞衍生的ECM中促成骨细胞生成因子（包括IGF-I、TGF-β、PDGF、VEGF和BMP-2）的量;并将研究双糖链蛋白聚糖缺乏对该ECM中这些生长因子水平的影响。最后，在移植到免疫缺陷小鼠体内后，将在体内测试在该ECM上生长的MSC是否保留其干细胞特性的问题。这些研究将为研究年龄相关的骨丢失是否部分是由于骨髓ECM改变引起的MSC行为异常奠定基础。

DESCRIPTION (provided by applicant): Mesenchymal stem cells (MSCs) of the bone marrow replicate to produce identical daughter stem cells and can differentiate into many different cell types inculding osteoblasts. However, MSCs lose their unique properties when cultured on tissue culture plastic. This indicates that a critical feature of the marrow microenvironment that facilitates retention of stem cell properties is missing in such culture systems - a situation that impairs the study of MSCs and limits their therapeutic potential. In other tissues, the extracellular matrix (ECM) plays an important role in controlling stem cell behavior by governing their exposure to growth factors. It is also known that a proteoglycan component of the ECM, biglycan, plays an important role in the maintenance of MSC number and function. Preliminary studies indicated that culture of MSCs on a cell-free ECM made by murine marrow-derived stromal cells preserved their stem cell properties, including the capacity to self-renew, the maintenance of an undifferentiated state, and responsiveness to the pro-osteoblastogenic effect of exogenous BMP-2. This finding may be explained by evidence that the ECM sequestered endogenous BMP-2 secreted by the stromal cells. The above observations form the basis of the hypothesis that the ECM made by marrow stromal cells specifically governs the ability of MSCs to both self-renew and differentiate into a particular lineage, at least in part, by regulating exposure of cells to factors that control their replication and differentiation. Experiments are proposed to identify the principal collagen, adhesion protein and proteoglycan components of murine marrow stromal cell-derived ECM using immunostaining and confocal microscopy; and to determine whether the stromal cell-derived ECM is unique in its ability to preserve MSC properties. In addition, the amount of pro-osteoblastogenic factors, including IGF-I, TGF-beta, PDGF, VEGF, and BMP-2, in the stromal cell-derived ECM will be determined; and the impact of biglycan deficiency on the level of these growth factors in this ECM will be investigated. Finally, the question of whether MSCs grown on this ECM retain their stem cell properties will be tested in vivo following transplantation into immunodeficient mice. These studies will set the stage for investigating whether age- related bone loss is due in part to aberrant MSC behavior caused by alteration in the ECM of the bone marrow.