Noninvasive preconditioning of mesenchymal stem cells to improve potency for bone repair

间充质干细胞（MSC）由于其分化为成骨、成软骨和成肌谱系的能力而被用作骨再生医学中的疗法。然而，治疗结果差异很大。这种不一致的基础尚不清楚，但可以归因于MSC异质性，其特征在于供体间变异性和供体内表型和功能特征的异质性。不幸的是，目前的策略，以提高骨髓间充质干细胞的潜力，通过预处理与外源性生长因子或遗传操作骨修复有限的潜力，FDA批准，由于疗效和安全性问题。在这里，我们提出了新的非侵入性的方法来改善MSC的功能，利用生物物理刺激和衰老清除异源文化。该项目的目的是确定MSC用于骨修复的效力是否可以通过（i）非侵入性生物力学信号和（ii）调节异质MSC群体中的细胞衰老来提高。我们提出了三个目标：（i）确定非侵入性机械信号是否促进MSCs的成骨分化及其骨修复能力。(ii)确定MSC衰老对骨修复能力的影响，以及（iii）使用单细胞RNA测序定义MSC异质性，并将关键异质性参数与体内新骨形成结果相关联。为了实现这些目标，MSC将被递送低幅度振动（LMV），并在小鼠中的临界大小的颅骨缺损内评估体外成骨分化和体内骨形成。其次，将用衰老清除剂处理以显著存在衰老细胞和相关分泌表型为特征的MSC的异源培养物，然后评估成骨分化能力、细胞因子和营养因子分泌以及颅骨缺损模型中的体内骨愈合。第三，我们将使用多个供体MSC的单细胞基因组学方法研究与MSC关键特征（如增殖、分化和细胞表面表型）相关的基因表达差异。解开干细胞亚群，可能决定功能的潜力将是至关重要的选择合适的供体异基因细胞治疗。所提出的LMV和senolytics作为移植前预处理以实现均质和有效的MSC制剂的创新用途在开发用于颅组织修复的成功的基于细胞的再生疗法中具有重要意义。

Mesenchymal stem cells (MSCs) are trialed as therapies in bone regenerative medicine owing to their ability to differentiate into osteogenic, chondrogenic and myogenic lineages. However, therapeutic outcomes vary greatly. The basis for this inconsistency is unclear but can be attributed to MSC heterogeneity characterized by donor-to-donor variability and intra-donor heterogeneity in phenotypic and functional characteristics. Unfortunately, current strategies to improve MSC potency for bone repair via preconditioning with exogenous growth factors or genetic manipulation have limited potential for FDA approval owing to efficacy and safety concerns. Here, we propose novel noninvasive approaches to improve MSC functionality using biophysical stimulation and senescence clearance in heterogenous cultures. The objective of this project is to determine whether MSC potency for bone repair can be improved by (i) non-invasive, biomechanical signals and (ii) modulating cellular senescence in heterogeneous MSC populations. We propose three aims: (i) determine if non-invasive mechanical signaling promotes osteoblastic differentiation of MSCs and their bone-repair potency. (ii) determine the impact of MSC senescence on bone-repair potency and, (iii) define MSC heterogeneity using single-cell RNA sequencing and correlate key heterogeneity parameters with in vivo outcomes of new bone formation. To execute these aims, MSCs will be delivered low magnitude vibrations (LMV) and assessed for in vitro osteogenic differentiation and in vivo bone formation within critical-sized calvarial defect in mice. Secondly, heterogenous cultures of MSCs characterized by significant presence of senescent cells and associated secretory phenotype will be treated with senolytics followed by assessment for osteogenic differentiation capacity, cytokine and trophic factor secretion, and in vivo bone healing in a calvarial defect model. Thirdly, we will investigate gene expression differences related to key MSC characteristics such as proliferation, differentiation, and cell surface phenotype using single cell genomics approach on multiple donor MSCs. Unraveling stem cell subpopulations that may determine functional potency will be critical for the selection of suitable donors for allogeneic cell therapies. The proposed innovative use of LMV and senolytics as pre-transplantation conditioning to achieve homogeneous and potent MSC preparations holds significance in developing successful cell-based regenerative therapies for cranial tissue repair.