Daley, George Q.Li, Christopher2026-06-0920262026-06-052026Li, Christopher. 2026. Programming Hematopoietic Stem Cell Fate from Human Pluripotent Stem Cells: Mechanistic and Functional Dissection of Endothelial-to-Hematopoietic Transition. Doctoral Dissertation, Harvard University Graduate School of Arts and Sciences.32702169https://dash.harvard.edu/handle/1/42740404The derivation of hematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) is a longstanding cardinal objective—indeed, even a “holy grail”—in the hematology and developmental biology fields, with manifold applications to clinical and therapeutic translation, particularly for hematologic malignancies and blood disorders. Despite significant advances in dissecting the mechanisms governing HSC emergence in development, existing differentiation platforms—including transcription factor-mediated reprogramming and embryoid body-based systems—have failed to reproducibly yield robust, bona fide hPSC-derived HSCs that are endowed with the potential for long-term multilineage engraftment of primary and secondary recipients and defined with rigorous clonal analysis. This limitation reflects an incomplete understanding of the developmental processes and signaling cues governing HSC specification during embryonic hematopoiesis, as well as the inability of hPSC differentiation platforms to faithfully recapitulate these processes in vitro. One of the crucial and conserved developmental processes that gives rise to HSCs in vivo is the “endothelial-to-hematopoietic transition” (EHT), a complex and carefully orchestrated process by which adherent hemogenic endothelial cells lining the ventral wall of the dorsal aorta adopt hematopoietic characteristics and round morphology, budding off into circulation as non-adherent HSCs that subsequently colonize the fetal live and bone marrow. However, the precise role—and sufficiency—of EHT in enabling the generation of functional HSCs from hPSCs in vitro remains unclear. In this dissertation, I construct an integrated experimental framework to characterize, perturb, and optimize the attempted engineering of HSC fate from hPSCs. First, to enable systemic identification of putative regulators of hematopoietic specification in an unbiased and high-throughput manner, I leverage pooled CRISPR interference (CRISPRi) screening and a library of sgRNAs targeting every known chromatin modulator and epigenetic factor in the human genome—and apply this powerful CRISPRi screen during hematopoietic differentiation. Using upregulation of HSC-associated markers including PROM1 as a phenotypic readout, we identify epigenetic, cell cycle, and ubiquitin pathway regulators—including KDM1A, HDAC5, CUL2, and CDK1—as putative modulators of hematopoietic stem cell identity, establishing a functional platform for interrogating regulators of HSC specification in human iPSC differentiation. Second, I integrate candidate-driven strategies to define and attempt to optimize the engineering of HSC specification from hPSCs. We develop an HLF reporter and ultrasensitive AkaLuciferase reporter to enable in vitro and in vivo monitoring of hematopoietic stem and progenitor cell emergence and migration, particularly in murine xenotransplantation. We also employ machine learning to uncover HSC identity metaprograms as a surrogate for long-term mouse transplantation and functional engraftment. Modulation of inflammatory signaling demonstrates that while interferon-γ induces HSC-associated programs such as MHC Class II expression, sustained inflammation impairs the ability of cells to engraft murine recipients, whereas glucocorticoid modulation yields modest improvements in multilineage engraftment. Finally, I optimize a 2D directed differentiation platform that gives rise to HLF+ HOXA5-9+ multipotent hematopoietic progenitors by eliciting the induction of EHT through a defined cytokine combination and find that the de novo induction of EHT enhances hematopoietic specification, generating previously inaccessible populations of developmentally distinct hematopoietic cells including a floating fraction of progenitors with increased multilineage potential as measured by colony forming units (CFUs) and increased efficiency of hematopoietic commitment into erythroid, myeloid, and lymphoid lineages, as well as a separate adherent population of cells that exhibit enrichment of HSC signature genes and enhanced potential for in vivo engraftment (albeit myeloid-restricted). Taken together, this work establishes a scalable platform for dissecting complex regulators of HSC emergence and provides a set of tools to optimize ongoing attempts at engineering functional human HSCs.application/pdfenBiologyThesis or Dissertation2026-06-090000-0002-7000-4184