Abstract Long-term hematopoietic stem cells (LT-HSCs) extensively self-renew to maintain the pool of HSCs for a lifelong blood production and differentiate to generate all mature blood cells. Self-renewing LT-HSCs regenerate the adult blood systems after transplantation, which is a curative therapy for many diseases, including immunodeficiencies and leukemias. In addition, gene therapies with engineered HSCs are adopted as curative therapies for an increasing number of diseases, such as sickle cell disease, hemoglobinopathies, autoimmunity, and inheritable genetic disorders. Enhancing the efficiency of HSC ex vivo expansion and preserving HSC reconstitution capacity after ex vivo expansion has broad therapeutic implications. However, in vitro culture of LT-HSC has been limited by a loss of functional stem cells over time. This functional decline is at least partially due to the dysregulation of proteostasis in cultured HSCs. There is growing evidence that dysregulated protein homeostasis is associated with HSC exhaustion in ex vivo culture. Endoplasmic reticulum associated degradation (ERAD) is a critical component of protein homeostasis, to ensure protein quality control by degrading inappropriately folded or assembled proteins in ER. ERAD complexes recognize misfolded secretory and membrane proteins in ER and dislocate them to cytosol for proteasomal degradation. Our recent studies show that ERAD genes are enriched in quiescent and metabolically inactive HSCs as compared to proliferative and activated HSCs. Furthermore, ERAD deficiency via knockout of Sel1L, an important component of the Sel1L/Hrd1 ERAD complex, leads to hyperproliferation of HSCs that results in loss of HSC reconstituting capacity and depletion of HSCs. Importantly, we observed decreased Se1L expression and accumulation of the ERAD substrate after HSC culture, suggesting ERAD activity is decreased in cultured HSC. ERAD deficiency via Sel1L knockout leads to a decrease of the number of HSCs after in vitro culturing and enhancing ERAD via overexpression of Sel1L increased HSC expansion after ex vivo culture, supporting the hypothesis that ERAD promotes HSC ex vivo expansion. To identify small molecular compounds that activate ERAD activity, we have designed and optimized high throughput cell-based screening system using an ERAD activity reporter and preliminary screening of the FDA repurposing library resulted in 4 chemicals that increase ERAD activity. All 4 compounds are HSP90 inhibitors, some of which have been previously shown to enhance HSC ex vivo expansion, providing evidence that enhancing ERAD may promote HSC ex vivo expansion. Here we propose studies to identify modulators of ERAD activity via a novel small molecular compound library and test the hypothesis that loss of ERAD activity is associated with the loss of functional HSC in ex vivo culture systems and that restoring ERAD function will improve HSC maintenance in culture and function following transplantation. Ou...