Reversing Skeletal Aging by Restoring Functional Skeletal Stem Cell Diversity

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PROJECT SUMMARY Advancing age and obesity are tightly linked to the increasing global incidence of skeletal diseases. Osteoporosis in particular poses a major public health threat for over 54 million Americans as it is interrelated with high fracture rates. Osteoporosis-related hip fractures are invariably associated with significant morbidity and strikingly, a 58% mortality rate in the elderly within the first year of injury. This problem is compounded by a lack of efficient preventive and medical therapies for age-related bone disease free of major side effects. Recent studies have revealed adult stem cell populations within bone that could be potentially targeted as a regenerative source to maintain and restore skeletal health. However, breakthroughs in stem cell based-regenerative strategies have been hampered by the inability to isolate bona fide stem cell populations which could serve as starting points for targeted approaches. Our group has helped delineate highly purified skeletal stem cell (SSC) lineages crucial for maintaining normal bone homeostasis and regeneration following injury. My latest results suggest that aging shifts lineage determination of stem cells thereby contributing to a decline of regenerative capacity and providing a rationale that skeletal aging is caused by SSC dysfunction. My preliminary data further provides evidence for the existence of multiple SSC subtypes (SSC diversity) present in limb long bones. Transcriptomic analysis at the single-cell level shows that SSCs undergo aging-induced molecular and compositional changes coinciding with functional heterogeneity. I have identified Wnt1 Inducible Signaling Pathway Protein 2 (Wisp2) that is specifically upregulated in the aged SSC lineage. Wisp2 significantly impairs bone formation when applied to SSC in vitro or fractures of young mice in vivo. Thus, Aim1 of this proposal is to determine the role of age-related changes in SSC diversity including the relative proportion of SSC subtypes and their functional heterogeneity to skeletal integrity using a variety of methods for spatio-temporal single-cell analysis. In Aim2 I will examine the mechanism of stem cell-based skeletal aging through Wisp2, which I hypothesize, may regulate SSC diversity and heterogeneity. Proposed experiments will also address its potential connection to new concepts of stem cell aging such as adverse clonal skeletogenesis. Importantly, I will determine the unexplored identity of the Wisp2 receptor in SSCs by highly sensitive proximity-dependent labeling to identify targetable pathways involved in SSC-mediated skeletal aging. The guidance and research environment provided by the expert mentors from Stanford and the NIH is cutting-edge and highly relevant to the purpose of this proposal allowing implementation of the latest transcriptomic and proteomic methods, including SmartSeq2 single-cell RNA- sequencing, RNAScope, and TurboID, to interrogate the proposed aims. These studies will establish a new paradigm for understanding skeletal disease from the perspective of SSC diversity and should facilitate the development of new preventive, diagnostic, and therapeutic approaches to tackle skeletal disorders.