Mechanisms Underlying the Specification of Definitive Hematopoiesis

Sammanfattning: Hematopoietic stem cells (HSCs) maintain blood through self-renewal and differentiation. Although HSC transplantation is the only cure for various blood disorders, generating and maintaining HSCs in vitro remains challenging, partly due to a limited understanding of the cellular and molecular mechanisms underlying human HSC ontogeny. In embryos, definitive HSCs arise from hemogenic endothelium via an endothelial-to-hematopoietic transition (EHT) in the aorta-gonad-mesonephros (AGM) region and placenta. In humans, limited access to embryos hinders the study of this process. Exploring new methods to mimic hematopoietic development in vitro may shed light on the regulators and mechanisms of human HSC specification in vivo.In my thesis, I outlined a protocol for generating hemogenic-like cells with hematopoietic potential from human dermal fibroblasts (HDFs) through direct cell reprogramming. HDFs were transduced with lentiviruses encoding GATA2, GFI1B, and FOS transcription factors (TFs). These three TFs activate hemogenic and hematopoietic transcriptional programs in HDFs, recapitulating EHT and leading to the generation of hematopoietic progeny capable of short-term engraftment in mice. Notably, I showed that the three TFs induce the expression of the HSC marker CD9 at early stages of reprogramming. Thus, human hemogenic reprogramming offers a tractable platform for identifying new markers and regulators of human HSC development. I then combined hemogenic reprogramming with CRISPR/Cas9 knockout screening to identify regulators. I transduced HDFs with lentivirus encoding Cas9 and a single guide RNA library targeting over 100 genes related to HSC function. In parallel, I optimized the delivery of the three TFs in a single polycistronic vector at a defined stoichiometry, where high levels of GATA2 and GFI1B induced reprogramming efficiently. After Cas9-edited cells underwent hemogenic reprogramming, my colleagues and I isolated both successfully and unsuccessfully reprogrammed cells based on the expression of CD49f and CD9 for next-generation sequencing. Surprisingly, we identified two markers of hemogenic endothelium and HSCs, CD34 and CD44, as barriers to hemogenic reprogramming, while STAG2 was uncovered as a facilitator of the process. These results suggest that commitment to human hemogenic and hematopoietic identity may benefit from time-wise inhibition of CD34 and CD44 signaling.Finally, I set out to uncover a less appreciated role of TFs in vivo using definitive hematopoiesis as a model. Several TFs remain bound to chromatin during mitosis and mark specific genomic sites – a mechanism termed “mitotic bookmarking”. Mitotic retention and bookmarking have been associated with the maintenance of pluripotency, cell reprogramming, and the preservation of somatic lineages in vitro, but the relevance for lineage commitment in vivo remains to be addressed. Here, I assessed the mitotic retention of hemogenic reprogramming TFs using fluorescent fusion proteins and subcellular protein quantification. Live-cell imaging and western blotting showed that GATA2 remains bound to chromatin in mitosis via C-terminal zinc finger-mediated DNA binding, as opposed to GFI1B and FOS. Moreover, GATA2 bookmarks a subset of its interphase sites with a higher density of GATA2 motifs, which include key regulators of hematopoietic fate. To uncover the role of GATA2 at mitotic exit in vivo, we generated a mouse model with the mitosis-degradation domain of cyclin B1 inserted upstream the Gata2 gene. Remarkably, homozygous mice died during development, partially phenocopying Gata2 null mice, which die at the onset of definitive hematopoiesis. Interestingly, removing GATA2 at mitosis-to-G1 transition impacts AGM and placental hematopoiesis but not yolk sac hematopoiesis. Altogether, these findings implicate GATA2 as a mitotic bookmarker critical for definitive hematopoiesis and underscore a dependency on bookmarkers for in vivo lineage commitment.Overall, my thesis provides new insights on the molecular mechanisms underlying the specification of definitive hematopoiesis. In the future, harnessing these mechanisms may enable the faithful generation of patient-tailored HSCs to meet clinical demands.