Hematopoietic potential arises in mammalian embryos before adult-repopulating hematopoietic stem cells (HSCs). At embryonic day 9.5 (E9.5), we show the first murine definitive erythro-myeloid ...progenitors (EMPs) have an immunophenotype distinct from primitive hematopoietic progenitors, maturing megakaryocytes and macrophages, and rare B cell potential. EMPs emerge in the yolk sac with erythroid and broad myeloid, but not lymphoid, potential. EMPs migrate to the fetal liver and rapidly differentiate, including production of circulating neutrophils by E11.5. Although the surface markers, transcription factors, and lineage potential associated with EMPs overlap with those found in adult definitive hematopoiesis, they are present in unique combinations or proportions that result in a specialized definitive embryonic progenitor. Furthermore, we find that embryonic stem cell (ESC)-derived hematopoiesis recapitulates early yolk sac hematopoiesis, including primitive, EMP, and rare B cell potential. EMPs do not have long-term potential when transplanted in immunocompromised adults, but they can provide transient adult-like RBC reconstitution.
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•Yolk sac definitive erythro-myeloid progenitors (EMPs) have a unique immunophenotype•EMPs are distinct from primitive hematopoiesis, non-HSC B cells, and immature HSCs•ESC maturation recapitulates yolk sac primitive, EMP, and B cell hematopoiesis•EMPs colonize the fetal liver and differentiate rapidly, including neutrophil production
Yolk-sac-derived definitive erythro-myeloid progenitors (EMPs) can be prospectively identified and distinguished from HSC-independent primitive and B cell hematopoietic emergence. Embryonic stem cell maturation produces yolk-sac-like HSC-independent hematopoietic potential. EMPs colonize and differentiate in the early fetal liver, including granulopoiesis. In adults, EMPs can provide transient adult-like RBC reconstitution.
Adult-repopulating hematopoietic stem cells (HSCs) emerge in low numbers in the midgestation mouse embryo from a subset of arterial endothelium, through an endothelial-to-hematopoietic transition. ...HSC-producing arterial hemogenic endothelium relies on the establishment of embryonic blood flow and arterial identity, and requires β-catenin signaling. Specified prior to and during the formation of these initial HSCs are thousands of yolk sac-derived erythro-myeloid progenitors (EMPs). EMPs ensure embryonic survival prior to the establishment of a permanent hematopoietic system, and provide subsets of long-lived tissue macrophages. While an endothelial origin for these HSC-independent definitive progenitors is also accepted, the spatial location and temporal output of yolk sac hemogenic endothelium over developmental time remain undefined. We performed a spatiotemporal analysis of EMP emergence, and document the morphological steps of the endothelial-to-hematopoietic transition. Emergence of rounded EMPs from polygonal clusters of Kit(+) cells initiates prior to the establishment of arborized arterial and venous vasculature in the yolk sac. Interestingly, Kit(+) polygonal clusters are detected in both arterial and venous vessels after remodeling. To determine whether there are similar mechanisms regulating the specification of EMPs with other angiogenic signals regulating adult-repopulating HSCs, we investigated the role of embryonic blood flow and Wnt/β-catenin signaling during EMP emergence. In embryos lacking a functional circulation, rounded Kit(+) EMPs still fully emerge from unremodeled yolk sac vasculature. In contrast, canonical Wnt signaling appears to be a common mechanism regulating hematopoietic emergence from hemogenic endothelium. These data illustrate the heterogeneity in hematopoietic output and spatiotemporal regulation of primary embryonic hemogenic endothelium.
Natural killer (NK) cells are a critical component of the innate immune system. However, their ontogenic origin has remained unclear. Here, we report that NK cell potential first arises from ...Hoxaneg/low Kit+CD41+CD16/32+ hematopoietic-stem-cell (HSC)-independent erythro-myeloid progenitors (EMPs) present in the murine yolk sac. EMP-derived NK cells and primary fetal NK cells, unlike their adult counterparts, exhibit robust degranulation in response to stimulation. Parallel studies using human pluripotent stem cells (hPSCs) revealed that HOXAneg/low CD34+ progenitors give rise to NK cells that, similar to murine EMP-derived NK cells, harbor a potent cytotoxic degranulation bias. In contrast, hPSC-derived HOXA+ CD34+ progenitors, as well as human cord blood CD34+ cells, give rise to NK cells that exhibit an attenuated degranulation response but robustly produce inflammatory cytokines. Collectively, our studies identify an extra-embryonic origin of potently cytotoxic NK cells, suggesting that ontogenic origin is a relevant factor in designing hPSC-derived adoptive immunotherapies.
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•NK cell potential arises from erythro-myeloid progenitors (EMPs) in the yolk sac•EMP-derived NK cells, similar to fetal NK cells, have a potent degranulation response•hPSC differentiation yields 2 distinct CD34+ populations, each with NK cell potential•hPSC-derived EMP-like NK cells are more potently cytotoxic than adult CD16+ NK cells
NK cell potential is thought to arise from lymphoid progenitors; however, in parallel studies of murine embryos and human pluripotent stem cells, Dege et al. demonstrate that NK cells with a potent cytotoxic degranulation response arise from erythro-myeloid progenitors.
Sorting of endocytic ligands and receptors is critical for diverse cellular processes. The physiological significance of endosomal sorting proteins in vertebrates, however, remains largely unknown. ...Here we report that sorting nexin 3 (Snx3) facilitates the recycling of transferrin receptor (Tfrc) and thus is required for the proper delivery of iron to erythroid progenitors. Snx3 is highly expressed in vertebrate hematopoietic tissues. Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates due to impaired transferrin (Tf)-mediated iron uptake and its accumulation in early endosomes. This impaired iron assimilation can be complemented with non-Tf iron chelates. We show that Snx3 and Vps35, a component of the retromer, interact with Tfrc to sort it to the recycling endosomes. Our findings uncover a role of Snx3 in regulating Tfrc recycling, iron homeostasis, and erythropoiesis. Thus, the identification of Snx3 provides a genetic tool for exploring erythropoiesis and disorders of iron metabolism.
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► Snx3 is highly expressed in vertebrate hematopoietic tissues ► Silencing of Snx3 results in anemia and hemoglobin defects in vertebrates ► Snx3 and Vps35 physically interact with Tfrc ► Snx3 is required for endosomal recycling of Tf-Tfrc complex
Megakaryocyte (MK) development in the bone marrow progresses spatially from the endosteal niche, which promotes MK progenitor proliferation, to the sinusoidal vascular niche, the site of terminal ...maturation and thrombopoiesis. The chemokine stromal cell-derived factor-1 (SDF-1), signaling through CXCR4, is implicated in the maturational chemotaxis of MKs toward sinusoidal vessels. Here, we demonstrate that both IV administration of SDF-1 and stabilization of endogenous SDF-1 acutely increase MK-vasculature association and thrombopoiesis with no change in MK number. In the setting of radiation injury, we find dynamic fluctuations in marrow SDF-1 distribution that spatially and temporally correlate with variations in MK niche occupancy. Stabilization of altered SDF-1 gradients directly affects MK location. Importantly, these SDF-1-mediated changes have functional consequences for platelet production, as the movement of MKs away from the vasculature decreases circulating platelets, while MK association with the vasculature increases circulating platelets. Finally, we demonstrate that manipulation of SDF-1 gradients can improve radiation-induced thrombocytopenia in a manner additive with earlier TPO treatment. Taken together, our data support the concept that SDF-1 regulates the spatial distribution of MKs in the marrow and consequently circulating platelet numbers. This knowledge of the microenvironmental regulation of the MK lineage could lead to improved therapeutic strategies for thrombocytopenia.
•SDF-1 acutely affects megakaryocyte spatial distribution in the bone marrow at steady state and in the setting of radiation injury.•SDF-1-directed localization of megakaryocytes into the vascular niche increases platelet output.
Red blood cells (RBCs), responsible for oxygen delivery and carbon dioxide exchange, are essential for our well-being. Alternative RBC sources are needed to meet the increased demand for RBC ...transfusions projected to occur as our population ages. We previously have discovered that erythroblasts derived from the early mouse embryo can self-renew extensively ex vivo for many months. To better understand the mechanisms regulating extensive erythroid self-renewal, global gene expression data sets from self-renewing and differentiating erythroblasts were analyzed and revealed the differential expression of Bmi-1. Bmi-1 overexpression conferred extensive self-renewal capacity upon adult bone-marrow-derived self-renewing erythroblasts, which normally have limited proliferative potential. Importantly, Bmi-1 transduction did not interfere with the ability of extensively self-renewing erythroblasts (ESREs) to terminally mature either in vitro or in vivo. Bmi-1-induced ESREs can serve to generate in vitro models of erythroid-intrinsic disorders and ultimately may serve as a source of cultured RBCs for transfusion therapy.
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•Bmi-1 promotes the extensive self-renewal of adult murine erythroblasts•Transfusion of iESRE leads to fully mature RBCs that circulate for 6–7 weeks in vivo
Palis and colleagues determined that Bmi-1 is preferentially expressed in self-renewing erythroblasts and that Bmi-1 promotes the extensive self-renewal of erythroblasts derived from adult murine bone marrow, which normally have limited self-renewal capacity. Importantly, Bmi-1-induced self-renewing erythroblasts maintain their capacity to terminally mature into enucleated red cells both in vitro and in vivo.
Megakaryocyte endoreplication, maturation and platelet production have all been reported to be affected by developmental stage (Elagib, Exp Hematol 2018). Additionally, we have recently found that ...P-selectin expression and function are altered in fetal and neonatal murine platelets (Stolla, Blood Adv 2019), suggesting that both megakaryopoiesis and platelet function are developmentally regulated. The developmental regulation of megakaryopoiesis can have important implications regarding platelet production in premature and full-term neonates, platelet regeneration following cord blood transplantation, and the in vitro generation of platelets from iPS cells (Elagib, Exp Hematol 2018). To further explore differences between fetal and adult megakaryocytes, we analyzed primary murine megakaryocytes in the fetal liver and adult bone marrow, and found that fetal megakaryocytes have lower average ploidy as well as increased cell size and initial accumulation of surface GP1bbeta expression at 2N and 4N stages. These latter findings indicate that cytoplasmic maturation begins at lower ploidy stages in fetal versus adult murine megakaryocytes. Consistent with findings in cultured human cord blood versus adult blood-derived progenitors (Liu, Blood 2015), we found lower ploidy and earlier cytoplasmic maturation in cultures of fetal versus adult megakaryocytes. These findings suggest the existence of a cell intrinsic developmental program of megakaryocyte maturation. Global gene expression studies of cultured megakaryocytes derived from fetal liver versus adult bone marrow progenitors revealed differential expression of Gata1, Cyclin D1, Cyclin D2, and Igf2bp3, supporting their proposed roles in fetal versus adult megakaryopoiesis (Klusmann, Genes Dev 2010; Liu, Blood 2011; Elagib, J Clin Invest 2017). Igf1R transcripts were increased in fetal megakaryocytes consistent with their sensitivity to IGF signaling (Klusmann, Genes Dev 2010). In addition, we found increased expression of Lin28b, a negative regulator of Let7 miRNAs, as well as multiple downstream targets of Let7 miRNAs in fetal liver-derived megakaryocytes. Induction of Lin28b expression in adult mice (Zhu, Cell 2011) led to increased expression of Gata1, Cyclin D1, Igf2bp3 and IGF1R in bone marrow-derived megakaryocytes. Interestingly, adult megakaryocyte average ploidy was decreased and the sensitivity to IGF1R inhibition was increased by Lin28b induction, suggesting that the Lin28b axis regulates key aspects of fetal versus adult megakaryopoiesis.
The largest category of upregulated genes in adult versus fetal megakaryocyte transcriptomes was immune-related genes, including PPBP, a neutrophil chemotactic cytokine, and beta2M, which we have recently shown to mediate platelet-monocyte interactions (Hilt, JCI Insight 2019). Consistent with the transcriptomic data, neonatal platelets express less MHCI on their surface (an indirect indication of less platelet beta2M protein expression) and release less PPBP post-activation when compared to adult platelets. Activated neonatal platelets induce less neutrophil migration and activation when compared to activated adult platelets. In addition, the releasates from activated neonatal versus adult platelets differentially altered the balance of proinflammatory versus proreparative signaling in monocytes. Finally, the induction of Lin28b expression in adult mice resulted in platelets that express less MHCI, release less PPBP, and induce less neutrophil chemotaxis. Taken together, our studies support the hypothesis that megakaryocyte maturation and resulting platelet-immune cell interactions are developmentally regulated, in part, through a Lin28b-mediated program.
Palis:Rubius Therapeutics: Consultancy.
Hematopoietic development during mammalian embryogenesis is comprised of a restricted primitive program of primitive erythroid, megakaryocytic, and macrophage lineages, and a definitive program of ...definitive erythroid, myeloid and lymphoid potential emerging from hematopoietic stem cell (HSC)-independent and dependent processes. Interestingly, progenitors of natural killer (NK) cells, but not B- or T-cells, have been found in the early human yolk sac, suggesting that NK cells may arise from HSC-independent sources.
NK cells recognize and kill virally infected cells and tumor cells, making them a highly desirable cell-type for adoptive immunotherapy. To bypass donor-related issues, human pluripotent stem cell (hPSC)-derived NK cells offer the possibility of uniform activity in a renewable “off-the-shelf” cell product. As the differentiation of hPSCs recapitulates early developmental processes, we sought to characterize the developmental origin of hPSC-derived NK cells. Studies in mice indicate that NK cells in the adult are derived from hematopoietic stem cells (HSCs) that commit to a lymphoid differentiation pathway. However, while NK cells, like HSCs, have been found in the fetal liver, the developmental origin of the fetal NK cell lineage remains poorly understood.
We have developed a stage-specific hPSC differentiation method that separates WNT-independent (WNTi) hematopoietic progenitors that harbor “primitive” hematopoietic potential from WNT-dependent (WNTd) erythro-myeloid-(T-)lymphoid “definitive” hematopoietic progenitors. Using this system, we find that CD34+ cells from both populations harbor NK cell potential. NK cells from hPSC WNTi progenitors (WNTi-NK cells) mature rapidly, are significantly more granular, and express very high levels of CD16 in comparison to their hPSC WNTd counterparts (WNTd-NK cells) and cord blood-derived NK (cbNK) cells. Further, WNTi CD34+ progenitors always gave rise to a granulocyte population alongside NK cells, suggesting they may be derived from a myeloid progenitor. Both WNTi-NK and WNTd-NK cells robustly respond to tumor targets, antibody-dependent cell-mediated cytotoxicity (ADCC), and PMA/ionomycin stimulation in comparison to cbNK cells. In all cases, WNTi-NK cells exhibited a strong bias for cytolytic degranulation over cytokine production, while WNTd-NK cells were biased for IFNg secretion. Similarly, WNTi-NK cells exhibit superior ADCC-mediated cell killing of Raji cells.
We then turned to the well-characterized murine embryo to determine whether HSC-independent NK cell progenitors are developmentally conserved. Assessing NK cell potential via explant culture, we found that as early as E7.5, yolk sac explants give rise to NK cells, as well as primitive and definitive erythroid progenitors. Further, we find that murine E9.5 yolk sac kit+CD41+CD16/32+ erythro-myeloid progenitors (EMP) give rise to NK cells ex vivo. Similar to hPSC WNTi-NK cells, EMP-derived NK cells were larger and more granular, and emerged alongside a granulocyte population in explant culture. Thus, the murine yolk sac harbors unique NK cell potential, from a committed myeloid progenitor, prior to HSC emergence.
Collectively, these studies suggest that ontological origin is an unexpectedly important consideration in the design of hPSC-derived NK cell-based therapeutics, and raise new questions regarding the potential of early hematopoietic progenitors in the mammalian embryo.
Fehniger:Celgene: Research Funding; Cyto-Sen Therapeutics: Consultancy; Altor BioScience: Research Funding; Affimed: Research Funding; NIH/NCI: Other: R01 CA205239, P50CA171963. Palis:Rubies Therapeutics: Consultancy.
Myb-null murine embryos lack definitive erythropoiesis but can produce primitive erythroid cells, allowing their survival to embryonic day 15 (E15) (Mucenski et al., Cell, 1991). Myb expression has ...been used to detect emerging HSC and is required for HSC maintenance (Lieu et al., PNAS 2009; North et al., Cell 2009). These data have led to the model that myb function is required for definitive hematopoiesis. Interestingly, macrophages and megakaryocytes are still detected in myb-null embryos and, as both of these lineages are also components of primitive hematopoiesis, it is proposed that these cells are not definitive in origin. Myb-independent macrophages infiltrate fetal tissues and have been implicated as a self-renewing source for several adult tissue-resident macrophage populations (Schulz et al., Nature 2012; Gomez Perdiguero et al., Glia 2013; Hoeffel et al., Immunity 2015). We tested the hypothesis that definitive hematopoiesis is entirely myb-dependent by examining two distinct sources of definitive erythroid/myeloid potential: 1) HSCs that emerge from hemogenic endothelium, including the AGM region at E10.5 in mice and 2) HSC-independent definitive EMP that emerge after primitive hematopoiesis from yolk sac hemogenic endothelium beginning at E8.25 (Frame et al., Stem Cells 2016). By E9.5, EMP can be prospectively isolated based on immunophenotype and contain all the erythroid/myeloid progenitor activity present at this time (McGrath et al., Cell Reports 2015). Surprisingly, we found normal numbers of immunophenotypic EMP in E9.5 Myb-null yolk sacs, and immunohistochemical analysis confirmed their emergence from Runx1+ hemogenic endothelium. At E10.5, reduced numbers of Myb-null EMP were found not only in the yolk sac, but also in the bloodstream and the liver. This decrease correlated with fewer hemogenic clusters in the yolk sacs of Myb-null embryos, as well as alterations in their cell-cycle status. The presence of significant numbers of immunophenotypic EMP suggests they could serve as an alternate source of Myb-null macrophages. Clonal analysis of sorted EMP confirmed that Myb function is necessary for erythroid and granulocyte progenitors, but Myb-null EMP retain normal plating efficiencies for macrophage progenitors. Indeed, Myb-null EMP generate only macrophages in liquid culture, lacking not only erythroid and granulocyte cells, but also Ly6C+ monocytes. Consistent with these results, RNA sequencing analysis of Myb-null versus wildtype EMP demonstrated decreased expression of genes in pathways associated with cellular growth, as well as erythroid and granulocyte fates. We further determined that Myb is not required for the emergence of immunophenotypic pre-HSC in the AGM region of E10.5 embryos. In addition, there were normal numbers of clusters in the aorta of E10.5 myb-null embryos. We were also able to detect lineage-, Kit+ (LK) cells in E14.5 livers as previously reported (Sumner et al., Oncogene 2000). While LK numbers were reduced, the Sca1+ (LSK) subset was present in normal numbers. Like EMP, sorted Myb-null E10.5 pre-HSC, as well as E14.5 liver LK and LSK, lacked erythroid or granulocyte CFC activity, but retained normal CFC-M plating efficiencies. In addition, Ly6C+ monocytes were not observed in liquid cultures of sorted Myb-null E10.5 pre-HSC, which produced only macrophages in vitro, or in Myb-null E14.5 livers. Together these data indicate that Myb is not required for hematopoietic emergence of definitive EMP or HSC, but does facilitate the expansion of these definitive stem/progenitor cells and is required for erythroid and granulocyte differentiation. Additionally, EMP and HSC contain Myb-independent macrophage potential, which does not appear to differentiate from a monocyte intermediate.
Palis:Rubius Therapeutics: Consultancy.
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