ABSTRACT: Self-renewal and differentiation are inherent properties of hematopoietic stem cells that are necessary to support hematopoiesis. However, the underlying mechanisms, especially in human, remain unclear. Here, using the cynomolgus macaque as a surrogate model, we develop a new gating strategy to isolate with high purity transplantable cynomolgus HSCs and generated a single-cell transcriptomic map of cynomolgus HSCs and progenitor cells, that covers the gestational period not analyzed in human. We show that hematopoietic cells from the late-1st to early-3rd trimester fetal liver and late-2nd trimester and thereafter bone marrow has repopulating potential, closely mimicking humans. Unexpectedly however, we found unlike in human, cynomolgus HSCs express CD38 but not CD33, indicating that these cellular counterparts are molecularly distinct. Our transcriptomic analysis reveals the presence of a direct differentiation pathway from HSCs to megakaryocyte lineages, lineage-primed multipotent progenitors and also identified putative HSC surface markers. Taken together, our comprehensive dataset highlights not only the utility of cynomolgus monkeys as model systems to study hematopoiesis but also their potential for translational applications.
Project description:Increasing evidence links metabolic activity and cell growth to decline in hematopoietic stem cell (HSC) function during aging. The Lin28b/Hmga2 pathway controls tissue development and in the hematopoietic system the postnatal downregulation of this pathway causes a decrease in self renewal of adult HSCs compared to fetal HSCs. Igf2bp2 is an RNA binding protein and a mediator of the Lin28b/Hmga2 pathway, which regulates metabolism and growth signaling by influencing RNA stability and translation of its target genes. It is currently unknown whether Lin28/Hmga2/Igf2bp2 signaling impacts on aging-associated impairments in HSC function and hematopoiesis. Here, we analyzed homozygous Igf2bp2 germline knockout mice and wildtype control animals to address this question. The study shows that Igf2bp2 deletion rescues aging phenotypes of the hematopoietic system, such as the expansion of HSC numbers in bone marrow and the biased increase of myeloid cells in peripheral blood. This rescue of hematopoietic aging coincides with reduced mitochondrial metabolism and glycolysis in Igf2bp2-/- HSCs compared to Igf2bp2+/+ HSCs. Conversely, Igf2bp2 overexpression activates protein synthesis pathways in HSCs and leads to a rapid loss of self renewal by enhancing myeloid skewed differentiation in an mTOR/PI3K-dependent manner. Together, these results show that Igf2bp2 regulates energy metabolism and growth signaling in HSCs and that the activity of this pathways influences self renewal, differentiation, and aging of HSCs.
Project description:Human hematopoietic stem cell (HSC) ontogeny is poorly defined due to the inability to identify HSCs as they emerge and mature in different hematopoietic sites. We created a single-cell transcriptome map of human hematopoietic tissues from 1st trimester to birth and found that HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the AGM (aorta-gonad-mesonephros) region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. Comparison of HSCs from different maturation stages revealed the establishment of HSC transcription factor machinery upon HSC emergence, whereas their surface phenotype evolved throughout development. HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens, reflecting nascent HSC identity, and acquisition of HSC maturity markers PROM1/CD133 and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ hemogenic endothelial (HE) cells, which arose from IL33+ALDH1A1+ arterial endothelial subset, termed pre-HE. Spatial transcriptomics and immunofluorescence visualized this process in ventrally located intra-aortic hematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of AGM-like definitive HSPCs from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.
Project description:Somatic mutations of ASXL1 are frequently detected in age-related clonal hematopoiesis (CH). However, how ASXL1 mutations drive CH remains elusive. Using knockin (KI) mice expressing a C-terminally truncated form of ASXL1-mutant (ASXL1-MT), we examined the influence of ASXL1-MT on physiological aging in hematopoietic stem cells (HSCs). HSCs expressing ASXL1-MT display competitive disadvantage after transplantation. Nevertheless, in genetic mosaic mouse model, they acquire clonal advantage during aging, recapitulating CH in humans. Mechanistically, ASXL1-MT cooperates with BAP1 to deubiquitinate and activate AKT. Overactive Akt/mTOR signaling induced by ASXL1-MT results in aberrant proliferation and dysfunction of HSCs associated with age-related accumulation of DNA damage. Treatment with an mTOR inhibitor rapamycin ameliorates aberrant expansion of the HSC compartment as well as dysregulated hematopoiesis in aged ASXL1-MT KI mice. Our findings suggest that ASXL1-MT provokes dysfunction of HSCs, whereas it confers clonal advantage on HSCs over time, leading to the development of CH.
Project description:Under stress hematopoiesis, previous studies have suggested the migration of hematopoietic stem cells (HSCs) from bone marrow (BM) to extramedullary sites such as spleen. However, there is little direct evidence of HSC migration from BM to spleen. Here, we induced myeloablation via 5-fluorouracil (5-FU) and showed the direct evidence of HSC migration from BM to spleen during hematopoietic regeneration via a photoconvertible fluorophore. We found that during hematopoietic regeneration, there were mechanistic differences for HSC migration during early (day 3 to 8) and late phase (day 11 to 14). During steady-state, HSCs preferentially migrated to BM rather than spleen, but during the early phase HSC migration to spleen predominated. Indeed, in the early phase, HSC mobilization from BM was induced in G-CSF-dependent manners, while HSCs in the late phase gained significantly enhanced cell-autonomous motility, which was independent of chemotaxis. Collectively, HSC migration was dynamically changed during hematopoietic regeneration.
Project description:A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age
Project description:Recent years have seen exploration into the hematopoietic stem cell (HSC) niche in adult bone marrow (BM). Due to their relatively rare occurrence, research on splenic HSCs and their role in hematopoiesis under steady-state conditions has been limited. However, studies have been taken up to understand extramedullary hematopoiesis (EMH) in spleen under stress and pathological conditions. Moreover, activation of EMH also has been linked to mobilized BM derived HSCs, and contribution from the splenic HSCs has not been clear. Our studies have identified a myofibroblastic niche in spleen that supports HSCs under homeostatic conditions. To assess the maintenance and regulation of HSCs in spleen tissue by niche cells, we performed label-free mass spectrometry (MS) based quantitative proteomic analysis. The splenic HSC niche cells including capsular and trabecular myofibroblasts (or CTMs/TBs), Lin-CD45- stromal cell population (or STs) and HSPCs (Lin-CD45+c-kit+Sca-1+) were isolated by fluorescence-activated cell sorting (FACS). The sorted cells were then lysed in lysis buffer, sonicated, thermally denatured, reduced, alkylated and tryptic digests were processed, desalted and analyzed on nano-LC coupled with high resolution mass spectrometer. Using Proteome Discoverer program and computational analysis performed on the global protein expression we examined the protein-protein interactions, cell-cell interactions and molecular pathways enriched in each cell population. This study demonstrates the molecular components HSC niche and presents a model to uncover novel hematopoietic regulators crucial for improving the function of adult and pluripotent stem cell derived HSCs.
Project description:Uterine Natural Killer (uNK) cells regulate essential developmental processes at the maternal-fetal interface during early pregnancy. Uterine NK cell functions are tightly regulated during early placentation to stimulate trophoblast invasion and remodel uterine spiral arteries. Access to human 1st and 2nd trimester uterine tissues allowed us to investigate the transcriptional changes in uNK cells during the early stages of early human pregnancy. Microarray analysis identified 97 upregulated genes in 2nd compared to 1st trimester purified uNK cells of which the majority (61%) clustered as interferon-stimulated-genes (ISG), with ISG15 and ISG20 being upregulated profoundly. Type I interferons (IFNα/β), but not type II interferon (IFNɣ) increased expression of the identified interferon target genes ISG15 and ISG20 in uNK cells in vitro. Moreover, the cytokine-like protein ISG15 stimulated in vitro trophoblast invasion. Second trimester uNK cells promoted trophoblast invasion in vitro, whereas both 1st and 2nd trimester uNK cells stimulated endothelial tube formation. IFNα but not IFNβ stimulation of 1st trimester uNK cells enhanced their capacity to promote trophoblast invasion. In conclusion, the uNK cell interferon transcriptome is upregulated during the 2nd trimester allowing uNK cells to promote trophoblast invasion. Type I interferon signaling regulates uNK cell-induced trophoblast invasion via induction of effector molecules like ISG15. First trimester uNK cells can be induced to act like second trimester uNK cells by IFNα with respect to promotion of trophoblast invasion. Key words: Gene expression profiling ■ uterine natural killer cells ■ extravillous trophoblast invasion ■ interferon alpha ■ ISG15
Project description:Gene expression analyses of Tif1b, Hp1a or Hp1g knockdown hematopoietic progenitors. Growth of hematopoietic stem cells (HSCs) are significantly impaired upon knockdown of Hp1a and Hp1g. Results provide insight into the role of these factors in hematopoiesis. HSCs (CD34- c-Kit+ Sca1+ Lineage- ) were transduced with lentivirus expressing shRNA against Tif1b, Hp1a or Hp1g, and cultured for 14 days. Then, GFP+ c-Kit+ Lineage- hematopoietic progenitors were sorted and subjected into microarray analysis
Project description:The hierarchical model of hematopoiesis posits that self-renewing, multipotent hematopoietic stem cells (HSCs) give rise to all blood cell lineages. While this model accounts for hematopoiesis in transplant settings, its applicability to steady-state hematopoiesis remains to be clarified. Here we used inducible clonal DNA barcoding of endogenous adult HSCs to trace their contribution to major hematopoietic cell lineages in unmanipulated animals. Whereas the majority of barcodes were unique to a single lineage, we also observed frequent barcode sharing between multiple lineages, specifically between lymphocytes and myeloid cells. These results suggest a spectrum of multipotent and unipotent HSCs that collectively drive continuous hematopoiesis in the adult, and highlight a close relationship of myeloid and lymphoid development.
Project description:Blood flow promotes hematopoiesis, Definitive hematopoietic stem cells can not be assessed in circulation negative Ncx-/- mice. This study looks at the impact of lack of blood flow on the earliest blood cell precursors, the pro-HSCs. E9.5 was chosen as this is the time point pro-HSCs are normally generated. Cells from the hemogenic endothelium, the endothelium and pro-HSCs were sequenced.