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

间充质干细胞(MSCs)因其具有向成骨、软骨和肌细胞分化的能力而被作为骨再生医学的治疗药物。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的异质性，其特征是供体之间的差异性以及供体内表型和功能特征的异质性。不幸的是，由于有效性和安全性的考虑，目前通过外源性生长因子预适应或基因操作来提高MSC骨修复能力的策略在FDA的批准潜力有限。在这里，我们提出了新的非侵入性方法，通过生物物理刺激和异种培养中的衰老清除来改善MSC的功能。该项目的目的是确定是否可以通过(I)非侵入性的生物力学信号和(Ii)调节不同来源的MSC群体中的细胞衰老来提高MSC的骨修复能力。我们提出了三个目标：(I)确定非侵入性机械信号是否促进骨髓间充质干细胞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，(Iii)使用单细胞RNA测序确定MSC的异质性，并将关键的异质性参数与体内新骨形成的结果相关联。为了实现这些目标，MSCs将被提供低幅度振动(LMV)，并在小鼠临界大小的颅骨缺损中评估体外成骨分化和体内骨形成。其次，异种培养的MSCs具有明显的衰老细胞和相关的分泌表型，将用降钙剂处理，然后在颅骨缺损模型中评估成骨分化能力、细胞因子和营养因子的分泌以及体内骨愈合。第三，我们将使用单细胞基因组学方法研究与MSC的增殖、分化和细胞表面表型等关键特征相关的基因表达差异。解开可能决定功能潜力的干细胞亚群对于选择合适的异基因细胞疗法捐赠者至关重要。创新地使用LMV和感觉神经降解剂作为移植前条件以获得均质和有效的MSC准备对于开发成功的基于细胞的颅骨组织修复再生疗法具有重要意义。

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.