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.