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 from harvesting the bone. Alternatively, the development of therapies that exploit the osteogenic potential of bone marrow-derived mesenchymal stem cells (bmMSCs) continues to be a priority in osteoregenerative medicine. However, efforts remain largely empirical due to poor understanding of the mechanisms regulating bmMSC engraftment and osteogenic activity in vivo. Our long-term goal is to develop a regenerative therapy that is based on bioengineering an osteoinductive niche for human bmMSCs. We have found that the in vivo preservation of human bmMSC osteogenic potential depends on sustaining proximity to endothelial cells (ECs) and on the timely engraftment of bmMSCs as perivascular cells (Lin et al., PNAS 2014). We have also found that vascular networks bioengineered using human iliac crest trabecular bone ECs (bECs) could spontaneously induce osteogenic differentiation of bmMSCs at ectopic sites. In contrast, ECs from other human tissues could not. In addition, we have identified five candidate genes (BMP2, BMP7, NOG, KITLG, MSX1) differentially upregulated in bECs. Our overarching hypothesis is that bioengineered microvessels lined with bECs serve as stable niches for bmMSCs and autonomously drive osteogenesis via regulation of specific osteoinductive genes. Moreover, we postulate that induced pluripotent stem cells (iPSCs) could offer a plentiful source of surrogate bECs, eliminating the need for harvesting autologous trabecular bone. To test these hypotheses and to elucidate the precise osteoinductive factors whereby human trabecular bECs uniquely regulate osteogenesis, we propose three Specific Aims. In Aim-1, we will bioengineer vascular networks with human bECs and bmMSCs and determine the capacity to regenerate critical-sized orthotopic bone defects. In Aim-2, we will determine the factors responsible for the unique in vivo osteoinductive potential of human trabecular bECs. We will knockout each candidate bEC gene and will determine the effect on in vivo osteogenesis. To this end, we will use a luciferase-reporter driven by the human osterix promoter to measure bmMSC osteogenic activity via bioluminescence. In Aim-3, we will determine conditions to generate surrogate bECs from iPSCs. We will examine whether iPSC-derived ECs (iECs) acquire osteoinductive properties upon transplantation into bone sites and are in turn able to autonomously regulate the osteogenic activity of bmMSCs in vivo. We will use our murine calvarial bone defect model to determine the extent of in vivo osteogenic education of iECs b...