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

描述(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。它们沿多个肌肉骨骼谱系分化的能力使其成为一种有吸引力的自体细胞替代来源。然而，在实际使用之前，这些细胞必须首先从其他类型的细胞中分离出来，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不到1%，这就需要昂贵而耗时的单层扩增来获得足够的细胞数量，用于临床治疗。需要替代的浓缩策略，可以通过谱系潜力来识别大量细胞。在这个项目中，我们将研究基于机械和基因表达的方法来丰富成骨和成脂的谱系。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记物是否表明干细胞的再生能力。原子力显微镜将被用来量化单个MSCs在成骨和成脂分化之前、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，允许根据机械相似性将细胞分成组织特定的组。将使用已建立的成骨和成脂体外方案来比较分选细胞群的组织构建能力。(2)开发和实现活细胞分子信标分析，根据谱系特异性基因表达区分分化细胞和未分化细胞。信标，一旦结合到目标mRNA分子，将识别新分离的骨髓间充质干细胞的成骨和成脂潜力。高表达细胞将通过荧光激活的细胞分选得到丰富，并对其分化能力进行评估。(3)探讨机械和基因表达生物标记物在体外和体内对谱系特异性细胞的富集性。初步研究结果表明，分子信标可以识别一组广泛的能够进行谱系特异性分化的细胞，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。分选的细胞群体将首先在单层中评估合成的基质的数量和类型，最终在活体小鼠模型中进行评估。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组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某些方面，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.