Gfi1 dynamics define EHT onset and functionally distinct HSC stemness, and a subset of adult bone marrow HSCs retained expression and exhibited superior repopulating and self-renewal capacity.
Abstract
Gfi1 regulates endothelial-to-hematopoietic transition (EHT) and hematopoietic stem cell (HSC) maintenance; however, its expression dynamics remain unclear. We generated a non-disruptive Gfi1-T2A-mScarlet reporter to track Gfi1 expression. At E10.5, reporter selectively marked EHT in the dorsal aorta, umbilical artery, and vitelline artery. At E9.5, mScarlet-positive endothelial cells were present in the umbilical and vitelline arteries but were scarce in the dorsal aorta, indicating vascular-bed-specific differences in hemogenic activation. Gfi1 was broadly expressed in fetal liver HSCs, while higher expression levels identified HSCs with enhanced multilineage reconstitution and preferential T-cell output. Although Gfi1 expression declined during fetal-to-adult maturation, a subset of adult bone marrow HSCs retained expression and exhibited superior repopulating and self-renewal capacity. Bulk and single-cell transcriptomic analyses linked this subset to dormant and fetal-associated programs, including imprinted genes. Reduced chromatin accessibility at a conserved Gfi1 +26.5kb putative enhancer correlated with developmental Gfi1 downregulation. Thus, Gfi1 dynamics define EHT onset and functionally distinct HSC stemness.
Transient developmental signaling perturbations reshape hematopoietic trajectories produce sustained effects on hematopoietic and immune function into adulthood, including diminished inflammatory gene expression, reduced clonal complexity, and impaired regenerative capacity.
Marta Mastrogiovanni, Anastasia Nizhnik, Joaquín Cantón-Sandoval et al.· bioRxiv· 0 citations
Using mice carrying a mutation in the CBFβ2 isoform, it is demonstrated that CBFβ2 haploinsufficiency rewires transcriptional regulatory circuits in adult bone marrow progenitors, rendering them permissive for an early life-restricted Tγδ17 cells.
Michela Frascoli, Alyssa Berthelette, Joonsoo Kang et al.· Journal of Immunology· 0 citations
Hematopoietic stem and progenitor cells (HSPCs) arise from hemogenic endothelial cells (HECs) via the endothelial-to-hematopoietic transition (EHT). As a signature gene of hematopoietic stem cell-primed HECs, Mycn is highly expressed in these cells alongside its paralog, Myc. However, their roles and underlying mechanisms in EHT remain unclear. Here, we demonstrate that endothelial-specific deletion of Mycn, but not Myc, impairs HSPC formation in mouse embryos. Single-cell transcriptomics and functional assays reveal that Mycn deficiency specifically attenuates the HEC-to-HSPC transition but not thereafter. We also establish a mosaic analysis strategy to distinguish Mycn deletion states in mutant embryos, enabling precise characterization. Unlike control HECs that downregulate adhesion signatures during their specification, Mycn-deficient HECs aberrantly upregulate adhesion pathways. Inhibiting focal adhesion kinase, a critical modulator of cell adhesion, rescues HSPC production in Mycn-deficient explant cultures. These findings uncover a regulatory mechanism whereby Mycn promotes HSPC generation from endothelial cells by suppressing adhesion signaling.
Chi Wang, Zhe Chen, Mei-Lin Jiang et al.· Cell Reports· 0 citations
During development, transplantation, and homeostasis, hematopoietic stem cells (HSCs) migrate to niches to (re)constitute and maintain the hematopoietic system. A solid mechanistic understanding of their movement is essential but remains poorly examined. Here, we used long-term single-cell imaging and tracking to quantify how motility changes as immature HSCs differentiate into multipotent progenitors (MPPs), establishing motility as an early indicator of differentiation. HSCs and early MPPs exhibit rapid, non-directional movement, while more differentiated MPPs move more slowly and directed. Interestingly, ex vivo expanded HSCs retain HSC characteristics with more non-directional movement, suggesting it as a defining HSC trait. Moreover, we show that movement phenotypes can identify cells with higher HSC potential in long-term cultures. HSCs feature a dedicated cytoskeletal architecture characterized by elevated expression of microtubule- and actin-regulating proteins, and by 3D imaging we show that this coincides with reduced cytoskeletal dynamics. FSCN1, a cytoskeletal regulator prevalent in immature cells, controls HSC locomotion by modulating cell shape, and influences cell cycle kinetics and HSC attributes. Finally, THPO, but not SCF or CXCL12, triggers HSC motility through PI3K/AKT signaling.
Germán Camargo Ortega, Timosh Rueff, Melinda Kondorosy et al.· Communications Biology· 0 citations
Ex vivo expansion of human hematopoietic stem cells (HSCs) holds promise for overcoming their limited availability, a major barrier to broader clinical application. Although recent advances in culture systems can increase HSC numbers, these conditions frequently impair self-renewal and induce myeloid bias, and the underlying molecular mechanisms remain poorly understood. Here, we performed single-cell multiome sequencing (scMultiome-seq) on human umbilical cord blood-derived CD34⁺ hematopoietic stem and progenitor cells to co-profile transcriptional and epigenetic adaptations within the same cells during ex vivo culture. Our analyses revealed reduced transcriptional and epigenetic HSC signatures, accompanied by markedly increased activity of myeloid-associated transcription factor motifs, providing molecular insight into the functional decline and myeloid bias of cultured HSCs. We further observed substantial functional heterogeneity among phenotypically defined HSCs following culture. To address these limitations, we established a niche-mimetic culture system that integrates intrinsic and extrinsic bone marrow regulatory cues, including pharmacologic inhibition of the m6A reader YTHDF2 using the small molecule Y13-27, a three-dimensional microenvironment, and N-cadherin-mediated adhesion. This condition (3D-NcadP-Y) robustly preserved long-term repopulating capacity. When combined with the self-renewal agonist UM729, the resulting platform (3D-NcadP-Y-UM) uniquely enabled the expansion of serially transplantable long-term HSCs with balanced multilineage potential. scMultiome-seq and cellular analyses demonstrated that this condition preserves transcriptional and epigenetic long-term HSC signatures, maintains multilineage-associated transcription factor motifs, and limits excessive cell-cycle activation. Together, these findings elucidate molecular mechanisms underlying culture-induced HSC dysfunction and establish a niche-mimetic strategy for expanding functional human long-term HSCs while preserving key features of stemness.
Xinjian Mao, Ning Zhang, Xi C. He et al.· Blood· 0 citations
Hematopoietic stem and progenitor cells (HSPCs) arise from a specialized subset of arterial endothelial cells, the hemogenic endothelium (HE), during embryonic development and sustain blood production throughout life. Notch signalling is a key regulator of this process: its ligand Jag1 promotes HSPC formation, whereas Dll4 promotes arterial identity. However, how the activities of these ligands are temporally coordinated during HSPC emergence remains unresolved. Here we demonstrate that Dll4 is required prior to circulation onset, acting by dampening MAPK signalling to drive the transition from pre-HE to HE fate and enabling HE differentiation towards HSPCs. Subsequently, after circulation starts, Jag1a acts to maintain gene expression in HE and support transition to HSPC fate. Jag1a activity depends on blood flow-induced shear stress and rescues HSPC loss caused by impaired flow. Thus, rather than playing opposing roles, Dll4 and Jag1a act sequentially and coordinately to drive the endothelial-to-hematopoietic transition and promote HSPC emergence.
Dashuai Wu, B. Edginton-White, Dorothee Bornhorst et al.· bioRxiv· 0 citations
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