Extracellular matrix control mesenchymal stem cell fate

描述(申请人提供)：骨髓间充质干细胞(MSCs)可复制以产生相同的子代干细胞，并可分化为许多不同类型的细胞，包括成骨细胞。然而，骨髓间充质干细胞在组织培养塑料上培养时会失去其独特的特性。这表明，在这样的培养系统中，缺乏有助于保留干细胞特性的骨髓微环境的关键特征--这种情况损害了对骨髓间充质干细胞的研究，并限制了它们的治疗潜力。在其他组织中，细胞外基质(ECM)通过调节干细胞对生长因子的暴露，在控制干细胞行为方面发挥着重要作用。细胞外基质中的一种蛋白多糖成分Biglycan在维持MSC的数量和功能方面起着重要作用。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上培养的MSCs保留了它们的干细胞特性，包括自我更新的能力，维持未分化状态，以及对外源性BMP-2促成骨细胞作用的反应性。这一发现可能是因为有证据表明，ECM隔离了基质细胞分泌的内源性BMP-2。上述观察结果构成了一个假设的基础，即由骨髓基质细胞产生的ECM至少部分地通过调节细胞对控制其复制和分化的因素的暴露来特异性地调控MSCs的自我更新和分化为特定谱系的能力。利用免疫染色和共聚焦显微镜鉴定小鼠骨髓基质细胞来源的ECM的主要胶原蛋白、黏附蛋白和蛋白多糖成分；并确定基质细胞来源的ECM在保留MSC特性方面是否独一无二。此外，还将测定基质细胞来源的ECM中促成骨因子的数量，包括IGF-I、TGF-β、PDGF、VEGF和BMP-2；并将研究双聚糖缺乏对该ECM中这些生长因子水平的影响。最后，在这种ECM上生长的MSCs是否保持其干细胞特性的问题将在移植到免疫缺陷小鼠后在体内进行测试。这些研究将为研究与年龄相关的骨丢失是否部分归因于骨髓细胞外基质变化引起的MSC异常行为奠定基础。

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.