High-yield, lineage-specific enrichment of living mesenchymal stem cells

描述（由申请人提供）：间充质干细胞（MSCs）为基于细胞的再生治疗提供了令人兴奋的可能性。它们沿多种肌肉骨骼谱系分化的能力使它们作为自体细胞的替代来源具有吸引力。然而，在实际使用之前，这些细胞必须首先与其他细胞类型分离，事实证明，使用基于抗体的分选方法很难做到这一点。典型的细胞产量通常低于1%，这就需要昂贵且耗时的单层扩增来获得临床治疗所需的足够细胞数量。需要替代富集策略，可以通过谱系潜力识别大量细胞。在这个项目中，我们将研究基于机械和基因表达的方法来富集成骨和脂肪谱系。为了实现该项目的总体目标，我们将：(1)确定活的单细胞机械生物标志物是否表明干细胞的再生能力。原子力显微镜将用于量化单个MSCs在成骨和脂肪分化之前，期间和之后的弹性和粘弹性特性。这些数据将提供分类参数，允许基于机械相似性将细胞分离成组织特异性组。分类细胞群的组织构建能力将使用已建立的体外成骨和脂肪生成方案进行比较。(2)开发并实施活细胞分子信标试验，根据谱系特异性基因表达区分分化细胞和非分化细胞。信标在与目标mRNA分子结合时发出荧光，将识别从抽脂液中新分离的MSCs的成骨和成脂潜能。高表达细胞将通过荧光激活细胞分选富集，并评估其分化能力。(3)在体外和体内研究机械和基因表达生物标志物联合富集谱系特异性细胞的方法。初步研究结果表明，分子信标可以识别一系列具有谱系特异性分化能力的细胞，而机械生物标志物可以预测分化反应的稳健性。这两种技术的结合有望丰富高度再生的组织特异性细胞。分选的细胞群将被评估合成基质的数量和类型，首先，在单层中，最终，在体内小鼠模型中。除了发现机械性能如何与分化和谱系潜力相关外，这项工作还将产生一套广泛适用于肌肉骨骼研究的分子信标。我们期望实验结果能够阐明干细胞群体中细胞异质性的各个方面，同时也促进基于细胞的临床治疗的实际实施。这项工作将通过布朗大学和罗德岛医院的合作进行。

DESCRIPTION (provided by applicant): Mesenchymal stem cells (MSCs) pose exciting possibilities for cell-based regenerative therapies. Their capability to differentiate along multipe musculoskeletal lineages makes them attractive as an alternative source of autologous cells. However, these cells must first be separated from other cell types before practical use, which has proven difficult using antibody-based sorting approaches. Typical cell yields are often less than 1%, which necessitates expensive and time-consuming monolayer expansion to obtain sufficient cell numbers for clinical therapies. Alternative enrichment strategies are needed that can identify large numbers of cells by lineage potential. In this project, we will investigate mechanical- and gene expression-based approaches for osteogenic and adipogenic lineage enrichment. To accomplish the overall goals of the project we will: (1) Determine whether live, single-cell mechanical biomarkers indicate the regenerative capacity of stem cells. Atomic force microscopy will be used to quantify the elastic and viscoelastic properties of individual MSCs before, during, and after osteogenic and adipogenic differentiation. These data will provide sorting parameters that allow for separation of cells into tissue-specific groups based on mechanical similarity. The tissue building capacity of sorted cell populations will be compared using established in vitro protocols for osteogenesis and adipogenesis. (2) Develop and implement a live-cell molecular beacon assay that distinguishes differentiating cells from non-differentiating cells based on lineage- specific gene expressions. Beacons, which fluoresce upon binding to target mRNA molecules, will identify osteogenic and adipogenic potential in freshly isolated MSCs from lipoaspirate. Highly expressing cells will be enriched via fluorescence-activated cell sorting and evaluated for their differentiation capabilities. (3) Investigate the combination of mechanical and gene expression biomarkers for enrichment of lineage-specific cells in vitro and in vivo. Preliminary findings suggest that molecular beacons can identify a broad set of cells capable of lineage-specific differentiation while mechanical biomarkers can predict the robustness of the differentiation response. The combination of these two techniques is expected to enrich for highly regenerative, tissue-specific cells. Sorted cell populations will be evaluated for the amount and type of matrix synthesized, first, in monolayer and, ultimately, in an in vivo mouse model. In addition to discovering how mechanical properties are associated with differentiation and lineage potential, this work will generate a set of molecular beacons broadly applicable to musculoskeletal investigations. We expect experimental findings to elucidate aspects of cellular heterogeneity in stem cell populations while also facilitating the practical implementation of cell-based clinical therapies. This work wil be conducted through collaborations between Brown University and Rhode Island Hospital.