Identification of novel regulators of HSC specification and maturation

Identification of novel regulators of HSC specification and maturation Project Abstract Hematopoietic stem cells (HSCs) are defined by self-renewal and multilineage differentiation capacities. HSCs sustain lifelong hematopoiesis and are exploited for hematopoietic transplantation to replace damaged or diseased blood systems in patients with hematologic disorders. Despite the widespread usage, transplantation remains unattainable for many due to the paucity of appropriate immunologically matched donors. De novo production of HSCs from patient-specific induced pluripotent stem cells (iPSCs) has been proposed as a means to circumvent this problem, but it remains an extremely inefficient process. Current approaches result in the abundant creation of hematopoietic progenitors that possess limited differentiation abilities and lack sufficient transplantation capacity. During development, there is simultaneous de novo production of these limited-capacity progenitors alongside HSCs. One of the major limitations in the field is the inability to distinguish these cells at the time of formation, which makes it difficult to define the factors distinctly critical for HSC generation. Through single-cell RNA-sequencing of nascent hematopoietic stem and progenitor cells from developing zebrafish, we identified genes that discriminate HSCs from progenitors at the time of their formation. We also developed new in situ lineage-tracing and regenerative assays in zebrafish larvae to define functional differences between the differentiation kinetics and self-renewal of these cell subsets. Here, we plan to exploit the genetic malleability of zebrafish coupled with our novel assays to discover new regulators uniquely required for HSC formation. The SHINE-II mechanism (PAS-18-730) is dedicated to supporting new research directions in their early stages. Consistent with this aim, this project takes our laboratory in a new direction away from our focus on RNA processing in hematopoiesis and into the realm of understanding de novo production of bona fide HSCs. Our experience in zebrafish genetics and developmental biology along with our establishment of new assays to functionally distinguish nascent HSCs from HSC-independent progenitors make us well-positioned to accomplish our goal of identifying novel factors that uniquely regulate HSC specification and functional maturation. Identifying this program will provide fundamental knowledge on the acquisition of self-renewal and multipotency attributes and could provide guidance to improve ex vivo HSC production from mammalian iPSCs.