ABSTRACT Replicating the cascade of signals responsible for controlling stem cell behavior remains a critical challenge for biology and medicine. Hematopoiesis is the process where the body’s blood and immune cells are generated from a small number of hematopoietic stem cells (HSCs). HSC quiescence, self-renewal, and differentiation take place in, and are regulated by, unique regions of the bone marrow termed niches. HSCs are also the functional unit of therapeutic bone marrow transplants following myeloablative therapies. A major goal of the hematology community is to selectively expand HSCs without sacrificing a subpopulation of quiescent, long-term repopulating HSCs required for life-long hematopoiesis. Perivascular niches (PVNs) within the bone marrow are increasingly believed to present a constellation of matrix, biomolecular, and metabolic signals to support HSC expansion and quiescence, however their rarity and complexity can complicate direct in vivo examination. The long-term goal of this Stimulating Hematology Investigation – New Endeavors (SHINE) project is to advance a tissue engineering platform to achieve HSC expansion without exhaustion. In the previous funding period (R01DK099528), we established a tissue engineering ecosystem to examine the coordinated impact of niche- inspired biophysical signals and marrow-derived niche cells on HSC fate. We showed the kinetics of HSC-niche cell crosstalk can be manipulated via biomaterial design to dramatically alter HSC fate decisions. And we developed machine learning tools to identify secretome signals generated by niche-associated MSCs that enhance retention of quiescent HSCs. We build on these findings to investigate the coordinated effect of multicellular crosstalk, cell-mediated extracellular matrix remodeling, and hypoxic stress within the perivascular niche using biomimetic models of marrow sinusoidal vs. arteriolar vascular niches. The overall objective of this project is to define patterns of multicellular signaling and remodeling within an engineered PVN biomaterial in order to identify synthetic niches that promote HSC expansion without exhaustion. To address this goal we will first construct and thoroughly characterize an engineered perivascular niche (Aim 1). We will subsequently resolve patterns of niche remodeling and HSC-PVN crosstalk in response to hypoxia (Aim 2). And we will establish a microdroplet-based artificial marrow niche to encapsulate single murine HSCs in nanoliter-volume hydrogel droplets (Aim 3). Throughout, we will benchmark patterns of in vitro HSC expansion via the gold standard in vivo competitive repopulation assay. This proposed research is unified in our focus to use the well- characterized murine hematopoietic system to develop engineered niche technologies for HSC expansion. Consistent with score-driving criteria of the SHINE program, we will generate innovative tissue engineering infrastructure to define dynamic processes of remodeling and intercellul...