Project Summary Mesenchymal stem/stromal cells (MSCs) are under intensive investigation as therapeutics for treating a diverse array of human diseases, and while some trials have demonstrated significant patient benefits, most have yielded suboptimal outcomes. Currently, most clinical trials employ similar manufacturing schemes to treat medically unrelated diseases, which reflects knowledge gaps in the critical quality attributes that confer potency and undervalues the impacts of donor selection and manufacturing on potency. We previously identified TWIST1 as a biomarker that quantifies inter-donor differences in stem/progenitor (growth, CFU-F activity, tri-lineage potential) and paracrine (pro-angiogenic, anti-inflammatory, immuno-modulatory) functions of MSCs, thereby linking these functions mechanistically. We then developed a Clinical Indications Prediction (CLIP) scale based on TWIST1 that predicts inter-donor differences in MSC potency for a range of activities to produce MSC products tailored to specific disease indications or patient populations. Most recently, we showed that TWIST1 levels correlate with donor stature, that MSCs from short statured donors exhibit poor growth and potent immuno- modulatory activity, and that cGMP manufacturing platforms are biased against these donor populations. Based on these findings, the central goals of this application are to further refine the CLIP scale and establish it as an industry wide platform to inform donor selection and manufacturing processes to produce MSC-based therapeutics of defined potency. To accomplish these goals, aim 1 will delineate epigenetic mechanisms that confer inter-donor differences in TWIST1 levels and correlate outcomes with donor stature to establish stature as a critical quality attribute that confers potency. MSCs from donors of varying stature will also be exposed to agents that license pro-angiogenic (FGF2), anti-inflammatory (IL1, TNF) and immuno-modulatory (IFNG) activities and their impacts on TWIST1 expression will be quantified to determine if the CLIP scale predicts their sensitivity to these agents. These data will establish the importance of donor selection on MSC potency, which to date has been largely overlooked. Aim 2 will subject MSCs from donors of variable stature to large-scale expansion using standard (academic) and proprietary (industry) cGMP manufacturing platforms and quantify impacts on potency pre- and post-manufacturing using well-established pre-clinical models. Methods (small molecules, gene editing) to modulate TWIST1 levels post-manufacturing to alter potency will also be explored. By revealing how biases in donor selection/manufacturing impact product potency these studies will highlight limitations of current manufacturing platforms that reduce MSC therapeutic efficacy. Lastly, aim 3 will determine if the CLIP scale predicts inter-donor differences in MSC-derived extracellular vesicle (EV) biogenesis, cargo content, and potency. Expanding ...