Stem cells from a variety of sources hold enormous potential to revolutionize regenerative medicine and to understand disease, but identifying and manipulating the multitude of factors that control their fate remains a significant challenge. In the case of post-natal stem cells, the niche, defined as the in vivo microenvironment in which the cells reside, provides a significant influence on the regulation of fate decisions. Building on this concept, a particularly exciting area of research is the development of artificial stem cell niches. Current state-of-the-art artificial niches, which typically involve the use of instructive biomaterials as 2D and 3D culture substrates, have generated promising results. However, they lack a critical anatomic feature of many adult stem cell niches: proximity to the vasculature. Many adult stem cells reside near the vasculature in vivo, including neural stem cells, mesenchymal stem cells (MSCs) from bone marrow and adipose tissue, and hematopoietic stem cells. Given the conservation of this anatomic location, we hypothesize that recreating the perivascular niche ex vivo can regulate, enhance, and even restore the multilineage potential of adult stem cells. The objective of this R21 application is to explore this concept in more depth by creating a new perivascular interface as an innovative artificial niche for bone marrow-derived MSCs. Our approach builds logically on our published ability to generate robust capillary networks in vitro in 3D hydrogel-based cultures, and our data demonstrating that MSCs occupy perivascular locations in these in vitro capillary networks. Aim 1 will determine if MSC contact with a well-defined capillary network in vitro maintains their ability to express surface markers indicative of their multilineage potential. Aim 2 will assess if contact with capillary vessels enhances their multilineage differentiation potential when compared to culture-expanded MSCs. Finally, Aim 3 will determine if culturing aged MSCs within the artificial perivascular niche can restore their stem cell properties. Successful completion of these proposed aims would yield two important, perhaps paradigm- shifting, potential outcomes in the long run. First, by recreating key features of the perivascular environment ex vivo, the tools and approaches pioneered here could potentially help efforts to elucidate how stem cells in a variety of tissues are maintained and instructed within the body. Second, validating the innovative concept that capillary networks are instructive (beyond their ability to nourish tissues) will potentially enable broad expansion of this idea into a variety of clinical/translational efforts to regenerate tissues. PUBLIC HEALTH RELEVANCE: Many adult stem cells reside near blood vessels in the body. This project seeks to understand the significance and implications of that anatomic location, and to use this insight to develop a new method to control stem cell properties outside of the body. Understanding how blood vessels regulate stem cells, and using that knowledge to control their function provides the opportunity to impact bone regeneration strategies specifically, and any stem cell-based therapy more generally.