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Over 4,000,000 people in the US are living with blood disorders such as leukemia, myeloma, bone marrow failures, and anemia. Since Hematopoietic Stem Cells (HSCs) self-renew and differentiate into all blood cell types, HSC transplantation is often the only therapy available to treat these disorders. However, a shortage of bone marrow donors and complications after allogeneic transplantation leads to a failure rate >65%. The possibility of generating patient-derived HSCs (iHSCs) from induced pluripotent stem cells (iPSCs) would make HSC transplantation therapies widely available to patients, circumventing the complications of allogenic transplantations. However, current protocols generate extremely low amounts of human iHSCs, and Hematopoietic Progenitor Cells (iHPCs), which lack self-renewal and long-term engraftment capabilities, overpower the system, making this product unsuitable for clinical applications or for the study of stem cell function. Although current protocols attempt to mimic the inductive cues occurring during HSC development in vivo, inflammatory signaling, a critical driver of HSC fate discovered by us and others, has not been incorporated yet into these in vitro protocols. This is mainly due to previously unknown temporal requirements and incomplete understanding of its function, which we have uncovered recently (Campbell et al., Nat Communications 2024, PMID: 39242546). Here, we propose to integrate our comprehensive knowledge on inflammatory signaling dynamics during HSC development into current protocols of human iHSC production to increase stem cell yield and functional properties, facilitating the incorporation of this technology to future clinical interventions and opening numerous avenues to resolve the biochemical and molecular properties of human HSCs. To accomplish our goal, we will manipulate inflammatory signaling in a temporally precise stage-specific manner that mimics the natural in vivo kinetics. First, we will enhance hemogenic endothelium (HE) specification (the precursors of HSCs) by early activation of inflammatory signaling. Second, HE will be expanded while avoiding their differentiation through mid-stage inflammatory signaling inactivation. Last, iHSC delamination will be synchronized by abrupt inflammatory signaling activation to perform one harvest time that will reduce differentiation and labor time. iHSC properties will also be tested functionally. Our results have the potential to positively disrupt the field by facilitating the eluded goal of efficient iHSC generation for disease modeling and autogenic transplantation.
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