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

通过选择和使用骨髓间充质干细胞的高软骨形成亚群来改善软骨再生

• 批准号: MR/K015648/2
• 负责人: Anthony Hollander
• 金额: $ 42.33万
• 依托单位: University of Liverpool
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2014
• 资助国家: 英国
• 起止时间: 2014 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=MR%2FK015648%2F2
• 关键词: Improved; cartilage; regeneration; through; selection

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.

该赠款的目的是开发一种可靠且可预测的干细胞疗法来治疗软骨疾病。骨髓中的间充质干细胞（MSC）可用于生成软骨细胞（软骨细胞），以直接植入或用于在组织工程方案中使用，可用于在实验室中创建新的软骨。但是，当使用从一个患者到另一个患者的细胞时，软骨形成的结果有明显的变化。我们已经表明，当使用来自同一患者的MSC的不同克隆人群时，软骨形成也有明显的变化。通过选择这些克隆中最大的软骨基因和最小的软骨，我们能够对MSC或多或少能够形成软骨表达的所有基因进行比较。通过分析差异表达的基因，我们已经鉴定出可以用作最软体动物MSC的细胞表面标记。它在未分化的MSC克隆上的表达水平提高，与三维支架上的软骨分化和组织工程后的工程软骨的较高体积和质量有关。将MSC分离为表达标记的人和未在细胞表面表达标记的人的分离，可以比较每个亚群的软骨生成能力。通过这种方式，我们已经表明，表达标记的亚种群显然比不表达标记的亚种群更重要（即更好地形成软骨）。此外，对特定胶原蛋白的测量结果表明，增加软骨的形成与新软骨的钙化风险增加无关（这通常是使用骨髓干细胞制造软骨的固有问题）。这些新颖的观察结果为我们提供了开发一种用于生产MSC的方法的独特机会，这些方法可预测其形成软骨的能力可预测。此选定的细胞可以用于软骨组织工程程序中，或者可以将其直接植入无需体外组织形成而直接植入软骨病变中。现在，我们希望开发出强大的方法来隔离表达新标记的干细胞，以便我们可以开发一种细胞生产方法，该方法在良好的制造实践（GMP）设置中，如果要使用细胞来治疗患者，则需要为监管原因而进行。然后，我们希望通过关节软骨损伤的绵羊模型来测试这一人群。我们还希望研究表达标记物抑制免疫反应的细胞能力。骨髓间充质干细胞通常能够抑制免疫反应的某些方面，但我们尚不知道该特性是否保留在表达新标记物的细胞中。这些信息对于决定将来是否基于Patietns自己的细胞或捐赠细胞制定软骨修复策略可能至关重要。最后，我们希望确定标记是否是一种被动分子，恰好与软骨形成有关，还是在干细胞形成软骨的方式中起着机械作用。

项目成果

The Wnt5a Receptor, Receptor Tyrosine Kinase-Like Orphan Receptor 2, Is a Predictive Cell Surface Marker of Human Mesenchymal Stem Cells with an Enhanced Capacity for Chondrogenic Differentiation.

• DOI: 10.1002/stem.2691
• 发表时间: 2017-11
• 期刊: Stem cells (Dayton, Ohio)
• 作者: Dickinson SC;Sutton CA;Brady K;Salerno A;Katopodi T;Williams RL;West CC;Evseenko D;Wu L;Pang S;Ferro de Godoy R;Goodship AE;Péault B;Blom AW;Kafienah W;Hollander AP
• 通讯作者: Hollander AP

Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue.

• DOI: 10.1038/ncomms8405
• 发表时间: 2015-06-17
• 期刊: Nature communications
• 影响因子: 16.6
• 作者: Armstrong JPK;Shakur R;Horne JP;Dickinson SC;Armstrong CT;Lau K;Kadiwala J;Lowe R;Seddon A;Mann S;Anderson JLR;Perriman AW;Hollander AP
• 通讯作者: Hollander AP