PROJECT SUMMARY/ABSTRACT Every year, >1 million patients undergo bone repair procedures in the United States. Autologous bone grafting remains the preferred treatment for bone defects, but this practice is limited by bone availability and donor site morbidity. Alternatively, the development of therapies that exploit the osteogenic potential of bone marrow- derived mesenchymal stem cells (bm-MSCs) continues to be a priority in regenerative medicine. However, efforts remain largely empirical due to a poor understanding of the mechanisms regulating bm-MSC osteogenic activity in vivo. Our overarching goal is to elucidate the mechanisms regulating ossification and develop therapeutic strategies for bone regeneration using autologous bm-MSCs. Previously, we showed that preserving human bm-MSCs' osteogenic potential depends on sustaining proximity to endothelial cells (ECs). More recently, we have found that the type of ECs drastically affects bm-MSC fate in vivo. Specifically, vascular networks lined by human trabecular bone arteriole ECs (tba-ECs) could spontaneously induce osteogenic differentiation of bm- MSCs. In contrast, non-bone ECs could not. Our Preliminary Data suggest that the expression of KITLG drives this unique osteoinductive potential. Indeed, silencing KITLG in tba-ECs completely abrogated osteogenesis upon implantation in vivo, whereas overexpressing KITLG in non-bone ECs conferred robust osteoinductive properties. Our data also suggest that KITLG expression in tba-ECs is regulated by type I interferon (IFN) signaling, a previously unknown link. Our central hypothesis is that a constitutive IFN-KITLG mechanism drives the distinct osteoinductive properties of human tba-ECs. We also postulate that educating induced pluripotent stem cells (iPSCs) could offer a plentiful source of surrogate tba-ECs, eliminating the need for harvesting autologous bone. To test these hypotheses, we propose three specific aims. In Aim-1, we will dissect the mechanism by which human tba-ECs mediates osteogenesis via KITLG expression. We will determine which KITLG isoform (soluble vs. membrane-bound) is indispensable and dissect the role of recruited c-Kit+ hematopoietic progenitor cells (c-Kit+ HPCs) in osteogenesis. In Aim-2, we will determine the molecular mechanism that regulates KITLG expression in human tba-ECs. We will use a CRISPR/Cas9 loss‐of‐function approach to silence components of the type I IFN pathway and unravel the interactions between IFN signaling mediators and the enhancer-promoter region of the KITLG gene. In Aim-3, we will pursue strategies to educate human iPSC-derived ECs to acquire osteoinductive function, including transient activation of KITLG and IFN signaling. In summary, these studies will define the cellular and molecular mechanisms by which human tba- ECs regulate the osteogenic differentiation of bm-MSCs and, in turn, ossification. This fundamental knowledge will form the foundation for strategies to promote bone repair and reg...