Abstract
The prevalence of skeletal ailments is a growing concern for our aging society. Nearly 80 million Americans over the age of 50 are affected by osteoporosis, osteoarthritis, or low bone density. Effective long-term treatments for bone loss or diseases such as osteoporosis are lacking. Osteoporosis is caused by imbalanced bone resorption during bone remodeling altering the microarchitecture of the tissue and increasing the risk for fracture. Fragility fractures caused by osteoporosis often require surgical intervention, hospitalization, and rehabilitation. Skeletal integrity is closely tied to the proper regulation of bone-forming skeletal stem cells (SSCs) which have only been discovered within the past decade due to advancements in cell sorting and lineage tracing. SSCs have been characterized as multipotent stem cells that give rise to bone, cartilage, and hematopoiesis supportive stroma. More recently, stem cell aging has been implicated in skeletal health decline. Aged SSCs display dysfunctional lineage output that favors the production of stroma over osteochondrogenic lineages. Aged pro-inflammatory stroma in bone promotes bone resorption thereby accelerating bone loss. The Ambrosi lab has found that SSC aging coincides with the upregulation of the gene WISP2/CCN5. Here, the goal was to assess whether the increased expression of WISP2 in SSCs mediates the aging phenotype and if it can be targeted to improve bone-stem cell function and skeletal tissue during aging. We hypothesize that excess production of WISP2 drives alterations in clonal activity and differentiation potential which in turn leads to detrimental changes in the bone marrow microenvironment. We employed in vitro, in vivo, and in situ approaches to investigate the effects of WISP2 overexpression in SSCs. In vitro osteogenesis was evaluated with Alizarin Red S (ARS) staining to determine the effect of WISP2 overexpression on osteogenic differentiation and mineralization. Our results suggest that WISP2 overexpression impairs osteogenesis as a result of decreased mineralization. To spatially map WISP2 expression in the long bones of mice, we employed two in situ approaches including immunohistochemical (IHC) confocal fluorescence microscopy and RNAscope. Our IHC results showed slightly stronger WISP2 detection in bone marrow, periosteum, and growth plate of aged (24-months) compared to young (2-months) femurs. However, due to potential nonspecific antibody binding, we employed an additional in situ method, RNAscope, to target WISP2 mRNA. Our RNAscope results detected strong and specific WISP2 mRNA expression in the growth plate, bone marrow, periosteum, and articular cartilage of aged femurs. To assess if WISP2 overexpression impairs ossicle formation and bone volume in vivo we used an adeno-associated viral (AAV) vector to overexpress WISP2 in primary mouse SSCs and then performed renal capsule transplantation and serial bone marrow transplantation experiments. Based on Movat’s Pentachrome staining of explanted kidney grafts, our results indicate WISP2 overexpression impairs ossicle formation as substantial reduction in mineralized bone was found in ossicles derived from WISP2 overexpressing SSCs compared to controls. To assess bone volume and resorption we performed Hematoxylin and Eosin staining and Tartrate-Resistant Acid Phosphatase staining on samples from bone marrow transplant recipients. Our histological analysis revealed a significant decrease in bone volume and increase in osteoclast activity suggesting WISP2 overexpression promotes bone resorption contributing to the loss bone volume. Through the application of viral transduction in both human and mouse SSCs, we have demonstrated that WISP2 might be a translationally relevant therapeutic target that is implicated in SSC aging that could be leveraged to improve SSC function and skeletal parameters during aging.