Improved cartilage regeneration through the selection and use of highly chondrogenic subpopulations of bone marrow mesenchymal stem cells

这项资助的目的是开发一种可靠和可预测的干细胞疗法，用于治疗软骨疾病。来自骨髓的间充质干细胞（MSC）可用于产生软骨细胞（软骨细胞），用于直接植入或用于组织工程方案，可用于在实验室中创建新的软骨。然而，当使用来自一个患者的细胞到另一个患者时，软骨形成的结果存在显著差异。我们已经表明，当使用来自同一患者的不同克隆MSC群体时，软骨形成也存在显著变化。通过选择这些克隆中最具软骨形成性和最不具软骨形成性的克隆，我们已经能够对或多或少能够形成软骨的MSC表达的所有基因进行比较。通过对差异表达基因的分析，我们已经确定了一个可以用作最软骨形成MSC的细胞表面标志物的基因。其在未分化的MSC克隆上表达水平的增加与在三维支架上软骨分化和组织工程化后工程化软骨的更高体积和质量相关。将MSC分离成在其细胞表面上表达标志物的MSC和不表达标志物的MSC允许比较每个亚群的软骨形成能力。以这种方式，我们已经表明，表达标记物的亚群明显比不表达标记物的亚群更能形成软骨（即更能形成软骨）。此外，对特定胶原的测量提供了数据，表明软骨形成的增加与新软骨钙化的风险增加无关（这通常是使用骨髓干细胞制造软骨时的固有问题）。这些新的观察结果给了我们一个独特的机会，以开发一种方法来生产的MSC，是可预测的，在他们的能力提高，形成软骨。这种选定的细胞群体可以用于软骨组织工程程序或他们可以直接植入到软骨病变，而不需要在体外组织形成。我们现在希望开发用于分离表达新标志物的干细胞的稳健方法，使得我们可以开发在良好制造规范（GMP）设置中的细胞生产方法，如果细胞用于治疗患者，则出于监管原因将需要所述GMP设置。然后，我们希望在关节软骨损伤的绵羊模型中测试该群体。我们还希望研究表达标记物的细胞抑制免疫应答的能力。骨髓间充质干细胞通常能够抑制免疫反应的某些方面，但我们还不知道这种特性是否保留在表达新标记的细胞中。这些信息可能是决定未来是否开发基于患者自身细胞或捐赠细胞的软骨修复策略的关键。最后，我们希望确定标记物是否是一种被动分子，碰巧与软骨形成有关，或者它是否在干细胞形成软骨的方式中起着机械作用。

The objective of this grant is to develop a reliable and predictable stem cell therapy for the treatment of diseases of cartilage. Mesenchymal stem cells (MSCs) from bone marrow can be used to generate chondrocytes (cartilage cells) for direct implantation or for use in tissue engineering protocols that can be used to create new cartilage in the laboratory. However there is a marked variation in the outcome of cartilage formation when using cells from one patient to another. We have shown that there is also marked variation in cartilage formation when using different clonal populations of MSCs from the same patient. By selecting the most chondrogenic and least chondrogenic of these clones we have been able to undertake a comparison of all the genes expressed by MSCs that are more or less able to form cartilage. Through analysis of differentially expressed genes we have identified one that can be used as a cell surface marker of the most chondrogenic MSCs. Its increased level of expression on undifferentiated MSC clones is associated with a higher volume and quality of engineered cartilage after chondrogenic differentiation and tissue engineering on three-dimensional scaffolds. Separation of MSCs into those expressing the marker and those not expressing the marker on their cell surface allows comparison of the chondrogenic capacity of each subpopulation. In this way we have shown that the subpopulation expressing the marker is clearly more chondrogenic (ie better able to form cartilage) than the subpopulation that does not express the marker. Furthermore, measurement of specific collagens has provided data suggesting that the increased cartilage formation is not associated with an increased risk of calcification of the new cartilage (this is usually an inherent problem when using bone marrow stem cells to make cartilage). These novel observations give us the unique opportunity to develop a methodology for the production of MSCs that are predictable in their improved capacity to form cartilage.This selected population of cells could be used in cartilage tissue engineering procedures or they could be implanted directly into cartilage lesions without the need for in vitro tissue formation. We now wish to develop robust methods for isolation of stem cells expressing the new marker so that we can develop a cell production method that in a Good Manufacturing Practice (GMP) setting that will be required for regulatory reasons if the cells are to be used to treat patients. We then wish to test this population in a sheep model of articular cartilage damage. We also wish to investigate the capacity of cells expressing the marker to suppress immune responses. Bone marrow mesenchymal stem cells are normally able to suppress some aspects of the immune response but we do not yet know if this property is retained in those cells expressing the new marker. This information may be critical in deciding whether in future to develop strategies for cartilage repair based on the patietns own cells or donated cells. Finally we wish to establish if the marker is a passive molecule that happens to be related to cartilage formation or if it plays a mechanistic role in the way stem cells form cartilage.