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来治疗软骨疾病。骨髓间充质干细胞（MSCs）可用于生成软骨细胞（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，当使用不同患者的细胞时，软骨形成的结果有明显的差异。我们已经证明，当使用来自同一患者的不同克隆MSCs群体时，软骨形成也存在显着差异。通过选择这些克隆中最具软骨性和最不具软骨性的，我们已经能够对MSCs表达的所有基因进行比较，这些基因或多或少能够形成软骨。通过对差异表达基因的分析，我们已经确定了一个可以作为大多数软骨干细胞的细胞表面标记物。在未分化的间充质干细胞克隆中，其表达水平的提高与软骨分化和三维支架组织工程后工程化软骨的体积和质量的提高有关。将MSCs分离为在细胞表面表达标记物和不表达标记物的细胞，可以比较每个亚群的软骨形成能力。通过这种方式，我们已经表明，表达该标记的亚群明显比不表达该标记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的数据表明，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）。这些新颖的观察结果为我们提供了一个独特的机会来开发一种方法来生产MSCs，这种方法可以预测MSCs形成软骨的能力。这种选定的细胞群可以用于软骨组织工程程序，或者它们可以直接植入软骨病变，而不需要体外组织形成。我们现在希望开发强大的方法来分离表达新标记的干细胞，这样我们就可以开发一种符合良好生产规范（GMP）的细胞生产方法，如果细胞用于治疗患者，这将是监管原因所要求的。然后，我们希望在绵羊关节软骨损伤模型中测试这一群体。我们还希望研究表达该标记物的细胞抑制免疫反应的能力。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。这一信息对于决定将来是否发展基于患者自身细胞或捐赠细胞的软骨修复策略至关重要。最后，我们希望确定该标记物是与软骨形成有关的被动分子，还是在干细胞形成软骨的过程中起机制作用。

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.