Myeloid-restricted Progenitors Research Articles

Multiple cell populations generate macrophage progenitors in the early yolk sac.

Yolk sac (YS) CSF1 receptor positive (CSF1R+) cells are thought to be the progenitors for tissue-resident macrophages present in various tissues. The YS progenitors for tissue-resident macrophages are referred to as erythroid-myeloid progenitors (EMPs). However, diverse types of hematopoietic progenitors are present in the early YS, thus it is not precisely known which type of hematopoietic cell gives rise to the CSF1R+ lineage. In this study, an analysis was conducted to determine when CSF1R+ progenitors appeared in the early YS. It showed that CSF1R+ cells appeared in the YS as early as embryonic day 9 (E9) and that the earliest hematopoietic progenitors that differentiate into CSF1R+ cells were found in E8. Since these progenitors possessed the capability to generate primitive erythroid cells, it was likely that primitive erythroid lineages shared progenitors with the CSF1R+ lineage. Mutual antagonism appears to work between PU.1 and GATA1 when CSF1R+ cells appear in the early YS. One day later (E9), multiple progenitors, including myeloid-restricted progenitors and multipotent progenitors, in the YS could immediately generate CSF1R+ cells. These results suggest that EMPs are not an exclusive source for the CSF1R+ lineage; rather, multiple hematopoietic cell populations give rise to CSF1R+ lineage in the early YS.

EPCR Signaling Controls the Activity of Hematopoietic Stem Cells Independent of Coagulation Regulation

BackgroundAll mature blood cells are derived from multipotent hematopoietic stem cells (HSCs) which are activated to meet the demand of the host during inflammation and injury. The endothelial cell protein C receptor (EPCR) is a marker for primitivity and quiescence of HSCs but the relative contributions of EPCR signaling versus anticoagulant functions in HSC maintenance are incompletely defined.AimsWe aimed to dissect functions of EPCR by studying anticoagulant and signaling function in HSC of EPCR C/S mice carrying a single intracellular point mutation abolishing normal trafficking of EPCR through endo-lysosomal compartments. We assessed the contributions of EPCR signaling to stem cell maintenance by analyzing HSC mobilization and leukemia progression.MethodsWe studied the frequency and cell cycle activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) by multicolor flow cytometry. Furthermore, we analyzed changes in hematopoiesis in steady state, after granulocyte colony stimulating factor (G-CSF)-induced mobilization, in the context of aging and in the context of leukemia, using the MLL-AF9-induced acute myeloid leukemia (AML) model.ResultsHSCs, lungs and isolated lung-derived smooth muscle cells of EPCR C/S mice showed protein expression levels and anticoagulant function indistinguishable from wildtype (WT). We found an increase of circulating HSCs in the peripheral blood of EPCR C/S mice compared to control under steady state conditions. Isolated HSC displayed diminished polarization of CDC42 and VLA-4 (α 4β 1 integrin) affinity to VCAM-1 in EPCR C/S versus strain-matched EPCR wt mice, indicating that EPCR signaling directly controls HSC retention via integrin affinity to the BM niche. In addition, we noticed a higher cell cycle activity in myeloid-restricted progenitors of EPCR C/S mice compared to control. G-CSF treatment led to increased mobilization of both BM neutrophils and HSCs into the peripheral blood of EPCR C/S mice compared to EPCR wt mice. A myeloid bias was also seen in serially transplanted aged mice, resulting in increased frequencies of myeloid-biased progenitors in the BM of EPCR C/S mice compared to control mice, accompanied by an increase of circulating neutrophils in the blood. Consistent with higher cell cycle activity of myeloid progenitors and an overall increase of myeloid-biased output in EPCR C/S mice, induction of AML by retroviral transduction of EPCR C/S BM cells with MLL-AF9-expressing retrovirus resulted in an increase of cell cycle activity of Lin - MLL-AF9 + leukemic BM blasts and a higher leukemic load in the peripheral blood of mice transplanted with MLL-AF9 + EPCR C/S BM compared to control. As a result, MLL-AF9 + EPCR C/S leukemia showed a more aggressive disease with shortened survival times compared to control. In contrast, chemotherapy of MLL-AF9 + EPCR C/S leukemia reduced leukemic load in the peripheral blood and decelerated disease progression. These data demonstrate that increased leukemia cell cycle activity conferred chemosensitivity.ConclusionWith a site-specific EPCR mutant knock-in mouse, we here demonstrate that EPCR signaling and anticoagulant function can be separated. We provide direct evidence that EPCR signaling plays a crucial role in maintaining HSC retention via VLA-4 affinity to VCAM-1, controls cell cycle activity and myeloid output in normal, stress-induced, and malignant hematopoiesis with implications for therapeutic approaches in acute myeloid leukemia. DisclosuresRuf: MeruVasimmune: Other: Ownership Interest; ARCA bioscience: Consultancy, Patents & Royalties; ICONIC Therapeutics: Consultancy.

Acute and chronic waves of myeloid cell maturation and myelopoiesis with high fat diet

Abstract Diet-induced obesity is associated with an infiltration of monocytes into the adipose tissue in male mouse models. These monocytes differentiate into inflammatory macrophages that drive insulin resistance. This increase in circulating monocytes originates in the bone marrow (BM) where hematopoietic stem cells (HSC) and myeloid-restricted progenitors (GMPs and PreGMs) expand. HSCs isolated from BM of male mice on high-fat diet (HFD) for 16 weeks show myeloid lineage skewing that lasts through successive bone marrow transplants (BMT). This suggests that HFD induces changes to the HSC niche - a multicellular structure that produces regulatory signals that are critical for maintaining the HSC pool. While BM adipocytes expand with HFD exposure, the niche cells promoting myelopoiesis with HFD are unknown. We chose to investigate two cell populations that have been implicated in driving myelopoiesis in other conditions; the BM endothelial cell (BMEC), a critical HSC niche cell, and neutrophils. We hypothesize that HFD induces HSC expansion and reprogramming through neutrophil-BMEC communication. Using 60% HFD in C57Bl/6J mice we evaluated circulating and BM neutrophils and Ly6C monocytes; HSCs and progenitors, and BMECs during short- and long-term HFD exposure. We found that as early as 24 hours after mice went on HFD there is a significant spike in circulating mature neutrophils that return to normal levels by 72 hours only to expand again by 16 weeks HFD. Ly6Chi monocytes rose only after 1 month of HFD exposure. BMECs do not appear to be altered in number with HFD in short- or long-term assays. Further qualitative analysis of BMECs, MAT, and neutrophils are necessary to determine which cells are critical for regulating the HSC niche in HFD.

Sequentially inducible mouse models reveal that Npm1 mutation causes malignant transformation of Dnmt3a-mutant clonal hematopoiesis

Clonal hematopoiesis (CH) is a common aging-associated condition with increased risk of hematologic malignancy. Knowledge of the mechanisms driving evolution from CH to overt malignancy has been hampered by a lack of in vivo models that orthogonally activate mutant alleles. Here, we develop independently regulatable mutations in DNA methyltransferase 3A (Dnmt3a) and nucleophosmin 1 (Npm1), observed in human CH and AML, respectively. We find Dnmt3a mutation expands hematopoietic stem and multipotent progenitor cells (HSC/MPPs), modeling CH. Induction of mutant Npm1 after development of Dnmt3a-mutant CH causes progression to myeloproliferative disorder (MPD), and more aggressive MPD is observed with longer latency between mutations. MPDs uniformly progress to acute myeloid leukemia (AML) following transplant, accompanied by a decrease in HSC/MPPs and an increase in myeloid-restricted progenitors, the latter of which propagate AML in tertiary recipient mice. At a molecular level, progression of CH to MPD is accompanied by selection for mutations activating Ras/Raf/MAPK signaling. Progression to AML is characterized by additional oncogenic signaling mutations (Ptpn11, Pik3r1, Flt3) and/or mutations in epigenetic regulators (Hdac1, Idh1, Arid1a). Together, our study demonstrates that Npm1 mutation drives evolution of Dnmt3a-mutant CH to AML and rate of disease progression is accelerated with longer latency of CH.

MLL-Menin Inhibition Reverses Pre-Leukemic Progenitor Self-Renewal Induced By NPM1 Mutations and Prevents AML Development

Nucleophosmin (NPM1) mutant acute myeloid leukemia (AML) is the most common type of cytogenetically normal AML. The mutant NPM1 gene (NPM1c) co-occurs with other mutations such as DNMT3AR882 and is associated with distinctive HOX gene expression. While DNMT3A mutations are detected in the most primitive stem cell compartment, NPM1 mutations are found in committed progenitors and differentiated myeloid cells but are absent from the stem cell compartment. This suggests that NPM1c may induce self-renewal in myeloid progenitors as a critical step in the development of AML.To address this hypothesis, we used a conditional NPM1c mutant knock-in mouse model (NPM1cflox-cA/+). We activated NPM1c in hematopoietic cells by treatment of NPM1cflox-cA/+, Mx1-Cre mice with pIpC. NPM1c expression induced a stem cell gene expression program including HoxA, HoxB and Meis1 in granulocyte-macrophage progenitors (GMPs) and multipotent progenitors (MPPs) in just 4 weeks. Functionally, progenitors expressing NPM1c showed enhanced self-renewal in serial replating assays (GMP, 4 rounds; MPPs, >8 rounds), in comparison to their wildtype counterparts that could not be replated. Progenitors isolated from DNMT3aR878H/+ (homolog to human R882H) mutant knock-in mice showed no enhanced Hox expression or self-renewal properties while NPM1cflox-cA/+/DNMT3aR878H/+ double mutant cells performed similarly to NPM1c single mutants. To determine if NPM1c directly induced stem cell gene expression in progenitor cells, Cre-negative NPM1cflox-cA/+ GMPs and MPPs were transduced with retroviral Cre. This led to activation of HoxA/HoxB/Meis1 expression and enhanced replating capacity, thus demonstrating activation of stem cell like programs and activity in progenitor cells.We further evaluated the self-renewal potential of myeloid progenitors expressing NPM1c in vivo by transplantation. Wildtype GMPs engrafted at low levels 4 weeks post-transplant (~1% blood chimerism) and were undetectable at later time points. In contrast, NPM1c positive GMPs engrafted at higher percentages (average ~10%) and about half of the recipients showed >1% engraftment for over 12 weeks. These pre-leukemic, NPM1c-expressing GMPs engrafted in secondary recipients, indicating that they had been transformed to long-term myeloid restricted progenitors (LT-GMPs). We observed AML in primary (~8-month post-transplant) as well as secondary (~3-month post- transplant) LT-GMP engrafting mice. These results demonstrate that NPM1c has the ability to initiate self-renewal in committed myeloid progenitor cells as a step in the progression to AML.We have previously shown that inhibition of the interaction between the histone methyltransferase MLL1 and Menin (MEN1) reverses leukemogenic gene expression in NPM1c AML. We assessed whether MLL-MEN1 inhibition could inhibit NPM1c induced progenitor self-renewal and prevent AML development. We used a recently developed selective and orally bioavailable Menin-MLL inhibitor (VTP-50469) to target MLL1/MEN1 in pre-AML and AML in vivo. To test whether NPM1cflox-cA/+ and NPM1cflox-cA/+/DNMT3aR878H/+ double mutant LT-GMPs could be eradicated before overt signs of AML were observed, we treated secondary LT-GMP mice with VTP-50469 for 6 weeks with no evidence of toxicity. Remarkably, LT-GMP engraftment was rapidly reduced to <1% in the blood. Mutant cells remained undetectable for at least 2 months post-treatment while untreated mice succumbed AML. Gene expression analysis of VTP-50469 treated LT-GMPs in vivo showed a rapid loss of stem cell gene expression such as Meis1 (-2.6 fold) and Mecom/Evi-1 (-3 fold) after 5 days of treatment. VTP-50469 was also effective against NPM1c mouse AMLs as well as human PDX models as shown by dramatic reduction of leukemic engraftment and increased survival in inhibitor treated mice. In summary, NPM1 mutations induce stem cell gene expression and self-renewal in myeloid progenitors during progression to AML. Targeted inhibition of MLL1/MEN1 reverses this stem cell signature and self-renewal in myeloid progenitors and effectively reverses aberrant self-renewal in pre-leukemic cells before the development of frank AML. These findings highlight the importance of progenitor self-renewal during progression to AML and also suggests a strategy for intervention/prevention in patients with DNMT3A mutations and clonal hematopoiesis that acquire NPM1c mutations. DisclosuresMcGeehan:Syndax Pharmaceuticals Inc.: Employment. Levine:Roche: Consultancy, Research Funding; Janssen: Consultancy, Honoraria; Prelude: Research Funding; Gilead: Honoraria; Imago: Equity Ownership; Epizyme: Patents & Royalties; Isoplexis: Equity Ownership; Novartis: Consultancy; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Loxo: Consultancy, Equity Ownership; C4 Therapeutics: Equity Ownership; Celgene: Consultancy, Research Funding. Vassiliou:Celgene: Research Funding; KYMAB: Consultancy, Equity Ownership.

Lysine Methyltransferase Kmt5a Restricts Myeloid-Biased Output of Lymphoid-Primed Multipotent Progenitors

Distinct, lineage-biased subsets of multipotent progenitor cells (MPP) dynamically respond to the demands of the hematopoietic system to replenish mature hematopoietic cells as needed. It currently remains unclear as to whether distinct epigenetic mechanisms regulate lineage-specific expansion and differentiation from MPPs. Focusing on lymphoid-primed multipotent progenitor cells (LMPP/MPP4), we performed a lentiviral shRNA screen of 15 epigenetic factors, selected based on differential expression between myeloid-restricted and lymphoid-restricted progenitors. Following a 48 hour infection with lentiviral shRNA constructs or a non-targeting control, the lineage potential of lymphoid-primed multipotent progenitors was interrogated by myeloid and lymphoid colony forming unit (CFU) assays. From this screen, knockdown of the lysine methyltransferase Kmt5a most dramatically altered lineage output from lymphoid-primed multipotent progenitors through an expansion of myeloid lineage colonies without altering lymphoid colony production. To confirm target specificity, two independent shRNA hairpins targeting distinct locations of the Kmt5a transcript demonstrated that knockdown of Kmt5a (97.1% and 99.5% versus non-targeting control shRNA) increased macrophage colony production by 1.94 and 1.95 fold, respectively (P < 0.01 and P < 0.05, n = 3). Preliminary single cell culture experiments support that the enhanced myeloid lineage output from lymphoid-primed multipotent progenitors occurs at the single-cell level through increased cloning efficiency of myeloid-biased cells. Our results suggest that Kmt5a functions to restrict myeloid lineage output from lymphoid-primed multipotent progenitors. Mechanistically, KMT5A is responsible for monomethylation of histone H4K20 and the methylation of non-histone proteins (ex. p53K376). Our ongoing work aims to distinguish between these histone and non-histone targets to determine the precise mechanisms restricting myeloid lineage output from lymphoid-primed multipotent progenitors. This work has direct implications for a better understanding of the molecular drivers of transient myeloid lineage reprogramming of lymphoid-primed multipotent progenitors during hematopoietic regeneration, age associated myeloid lineage skewing of hematopoiesis, and myeloid malignancies. DisclosuresNo relevant conflicts of interest to declare.

Angiogenin Promotes Hematopoietic Regeneration by Dichotomously Regulating Quiescence of Stem and Progenitor Cells

Regulation of stem and progenitor cell populations iscritical in the development, maintenance, and regeneration of tissues. Here, we define a novel mechanism by which a niche-secreted RNase, angiogenin (ANG), distinctively alters the functional characteristics of primitive hematopoietic stem/progenitor cells(HSPCs) compared with lineage-committed myeloid-restricted progenitor (MyePro) cells. Specifically, ANG reduces the proliferative capacity of HSPC while simultaneously increasing proliferationofMyePro cells. Mechanistically, ANG induces cell-type-specific RNA-processing events: tRNA-derived stress-induced small RNA (tiRNA) generation in HSPCs and rRNA induction in MyePro cells, leading to respective reduction and increase in protein synthesis. Recombinant ANG protein improves survival ofirradiated animals and enhances hematopoietic regeneration of mouse and human HSPCs in transplantation. Thus, ANG plays a non-cell-autonomous role in regulation of hematopoiesis by simultaneously preserving HSPC stemness and promoting MyePro proliferation. These cell-type-specific functions of ANG suggest considerable therapeutic potential.

GATA2 regulates dendritic cell differentiation

AbstractDendritic cells (DCs) are critical immune response regulators; however, the mechanism of DC differentiation is not fully understood. Heterozygous germ line GATA2 mutations induce GATA2-deficiency syndrome, characterized by monocytopenia, a predisposition to myelodysplasia/acute myeloid leukemia, and a profoundly reduced DC population, which is associated with increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. To define the role of GATA2 in DC differentiation and function, we studied Gata2 conditional knockout and haploinsufficient mice. Gata2 conditional deficiency significantly reduced the DC count, whereas Gata2 haploinsufficiency did not affect this population. GATA2 was required for the in vitro generation of DCs from Lin−Sca-1+Kit+ cells, common myeloid-restricted progenitors, and common dendritic cell precursors, but not common lymphoid-restricted progenitors or granulocyte-macrophage progenitors, suggesting that GATA2 functions in the myeloid pathway of DC differentiation. Moreover, expression profiling demonstrated reduced expression of myeloid-related genes, including mafb, and increased expression of T-lymphocyte–related genes, including Gata3 and Tcf7, in Gata2-deficient DC progenitors. In addition, GATA2 was found to bind an enhancer element 190-kb downstream region of Gata3, and a reporter assay exhibited significantly reduced luciferase activity after adding this enhancer region to the Gata3 promoter, which was recovered by GATA sequence deletion within Gata3 +190. These results suggest that GATA2 plays an important role in cell-fate specification toward the myeloid vs T-lymphocyte lineage by regulating lineage-specific transcription factors in DC progenitors, thereby contributing to DC differentiation.

Promotion of Expansion and Differentiation of Hematopoietic Stem Cells by Interleukin-27 into Myeloid Progenitors to Control Infection in Emergency Myelopoiesis.

Emergency myelopoiesis is inflammation-induced hematopoiesis to replenish myeloid cells in the periphery, which is critical to control the infection with pathogens. Previously, pro-inflammatory cytokines such as interferon (IFN)-α and IFN-γ were demonstrated to play a critical role in the expansion of hematopoietic stem cells (HSCs) and myeloid progenitors, leading to production of mature myeloid cells, although their inhibitory effects on hematopoiesis were also reported. Therefore, the molecular mechanism of emergency myelopoiesis during infection remains incompletely understood. Here, we clarify that one of the interleukin (IL)-6/IL-12 family cytokines, IL-27, plays an important role in the emergency myelopoiesis. Among various types of hematopoietic cells in bone marrow, IL-27 predominantly and continuously promoted the expansion of only Lineage−Sca-1+c-Kit+ (LSK) cells, especially long-term repopulating HSCs and myeloid-restricted progenitor cells with long-term repopulating activity, and the differentiation into myeloid progenitors in synergy with stem cell factor. These progenitors expressed myeloid transcription factors such as Spi1, Gfi1, and Cebpa/b through activation of signal transducer and activator of transcription 1 and 3, and had enhanced potential to differentiate into migratory dendritic cells (DCs), neutrophils, and mast cells, and less so into macrophages, and basophils, but not into plasmacytoid DCs, conventional DCs, T cells, and B cells. Among various cytokines, IL-27 in synergy with the stem cell factor had the strongest ability to augment the expansion of LSK cells and their differentiation into myeloid progenitors retaining the LSK phenotype over a long period of time. The experiments using mice deficient for one of IL-27 receptor subunits, WSX-1, and IFN-γ revealed that the blood stage of malaria infection enhanced IL-27 expression through IFN-γ production, and the IL-27 then promoted the expansion of LSK cells, differentiating and mobilizing them into spleen, resulting in enhanced production of neutrophils to control the infection. Thus, IL-27 is one of the limited unique cytokines directly acting on HSCs to promote differentiation into myeloid progenitors during emergency myelopoiesis.

GATA-2 Regulates Dendritic Cell Differentiation

(Background) Dendritic cells (DCs) are critical regulators of the immune response, but their differentiation mechanism remains unclear. Heterozygous germline GATA-2 mutations in humans cause MonoMAC syndrome, characterized by monocytopenia and predisposition to myelodysplasia/acute myeloid leukemia. In this syndrome, DC count decreases profoundly, with an increased susceptibility to viral infections, impaired phagocytosis, and decreased cytokine production. In the present study, we analyzed the role of GATA-2 in DC differentiation and the underlying molecular mechanisms.(Method)Gata2 haploinsufficient mice (Gata2+/−: Tsai et al. Nature 1994) and tamoxifen-inducible Gata2-knockout mice (Gata2flox/flox/ER-Cre: Charles et al. Molecular Endocrinology 2006) were used. To generate conditional Gata2 knockouts in vivo, Gata2flox/flox/ER-Cre mice were intraperitoneally injected with 1-μg tamoxifen on days 1-3 and 8-10 and evaluated on days 20-22. Isolation of splenic DCs and bone marrow (BM) precursors, including LSK (Lin- Sca1+ Kit+ cell), CMP (common myeloid-restricted progenitor), GMP (granulocyte-macrophage progenitor), CLP (common lymphoid-restricted progenitor), and CDP (common dendritic cell precursor), were separated with both MACS (Miltenyi Biotech) and BD FACSAria II (BD Biosciences). For the in vitro analysis of Gata2-knockout, BM cells were cultured with CD45.1+ BM feeder cells from SJL mice (The Jackson Laboratory) with FLT3L (200 ng/mL) and 4-hydroxytamoxifen (Sigma). For transcription profiling, SurePrint G3 mouse GE microarray (Agilent) was used, and the data was subsequently analyzed with ImmGen database (http://www.immgen.org). Promoter assay was conducted with Dual Luciferase Reporter Assay system (Promega). Quantitative chromatin immunoprecipitation (ChIP) analysis was performed using CMP fraction and erythroid-myeloid-lymphoid (EML) hematopoietic precursor cell line (ATCC) with antibodies to GATA-2 (sc-9008, Santa Cruz Biotechnology).(Results) Quantitative RT-PCR analysis showed abundant Gata2 expression in LSK and CMP fractions, with detectable expression in GMP, CLP, and CDP fractions and in vitro differentiated DCs. Although the DC count did not change in Gata2 haploinsufficient mice, it significantly and profoundly decreased in Gata2 conditional knockout mice. To examine the role of GATA-2 during DC differentiation, we knocked out Gata2 during in vitro DC differentiation, starting from LSK, CMP, GMP, CLP, and CDP fractions obtained from Gata2flox/flox/ER-Cre mice. Gata2 knockout significantly decreased CD11c+ DC counts from LSK, CMP, and CDP fractions, while those from CLP and GMP were unaffected, implying the importance of GATA-2 during DC differentiation in the pathway from LSK to CDP via CMP, not via CLP nor GMP.To elucidate the underlying molecular mechanisms, we performed expression profiling with control and Gata2 -knockout DC progenitors from CMP of Gata2flox/flox/ER-Cre mice. Gata2 knockout caused >5-fold upregulation and downregulation of 67 and 63 genes, respectively. Although genes critical for the DC differentiation, e.g., Spi1, Ikzf1, and Gfi1, were not detected among the GATA-2-regulated gene ensemble, we found significant enrichment of myeloid-related and T lymphocyte-related genes among the downregulated and upregulated gene ensembles, respectively. We focused on Gata3 upregulation (7.33-fold) as a potential key mechanism contributing to Gata2 knockout-related impaired DC differentiation. Quantitative ChIP analysis with both CMP fraction and EML cell line demonstrated obvious GATA-2 chromatin occupancy at the consensus GATA-binding motif within Gata3+190 kb, which was conserved with human. Furthermore, addition of Gata3 +190 kb region to the Gata3 promoter (~0.5 kb) significantly decreased luciferase activity, which was significantly recovered by the deletion of GATA sequence within Gata3 +190 kb, in EML cells.(Conclusion) GATA-2 seems to play an important role for cell fate specification toward myeloid versus T lymphocytes, and thus contributing to the DC differentiation. Our data offer a better understanding of the pathophysiology of MonoMAC syndrome. DisclosuresFujiwara:Chugai Pharmaceuticals. Co., Ltd.: Research Funding. Fukuhara:Gilead Sciences: Research Funding. Ishizawa:GSK: Research Funding; Takeda: Research Funding; Celgin: Speakers Bureau; Kyowa Kirin: Research Funding; Celgin: Research Funding; Janssen: Research Funding; Takeda: Speakers Bureau; Kyowa Kirin: Speakers Bureau; Pfizer: Speakers Bureau.

Brief Report: Single-Cell Analysis Reveals Cell Division-Independent Emergence of Megakaryocytes From Phenotypic Hematopoietic Stem Cells.

Despite increasingly stringent methods to isolate hematopoietic stem cells (HSCs), considerable heterogeneity remains in terms of their long-term self-renewal and differentiation potential. Recently, the existence of long-lived, self-renewing, myeloid-restricted progenitors in the phenotypically defined HSC compartment has been revealed, but these cells remain poorly characterized. Here, we used an in vitro single-cell analysis approach to track the fate of 330 long-term HSCs (LT-HSC; Lin- cKit+ Sca-1+ CD150+ CD48- CD34-) cultured for 5 days under serum-free basal conditions. Our analysis revealed a highly heterogeneous behavior with approximately 15% of all phenotypic LT-HSCs giving rise to megakaryocytes (Mk). Surprisingly, in 65% of these cases, Mk development occurred in the absence of cell division. This observation suggests that myeloid-restricted progenitors may not derive directly from LT-HSCs but instead could share an identical cell surface marker repertoire.

Label retention selects for hematopoietic stem cells in aged bone marrow and distinguishes HSCs from myeloid restricted progenitors

Loss of immune cell function, increased myeloid cell output, and increased incidence of myeloproliferative neoplasms all characterize aged hematopoiesis. Studies have shown that many of these characteristics are due to the aging hematopoietic stem cell (HSC) compartment. The HSC compartment consists of discreet subsets of HSCs that differ in their differentiation potential, and HSCs with myeloid-biased output accumulate in aged individuals contributing to increased myeloid cell production. Additionally, phenotypic HSCs are known to accumulate in the aged but demonstrate functional defects in homing and repopulation after transplantation.

Clonal Analysis Unveils Self-Renewing Lineage-Restricted Progenitors Generated Directly from Hematopoietic Stem Cells

Consensus holds that hematopoietic stem cells (HSCs) give rise to multipotent progenitors (MPPs) of reduced self-renewal potential and that MPPs eventually produce lineage-committed progenitor cells in a stepwise manner. Using a single-cell transplantation system and marker mice, we unexpectedly found myeloid-restricted progenitors with long-term repopulating activity (MyRPs), which are lineage-committed to megakaryocytes, megakaryocyte-erythroid cells, or common myeloid cells (MkRPs, MERPs, or CMRPs, respectively) in the phenotypically defined HSC compartment together with HSCs. Paired daughter cell assays combined with transplantation revealed that HSCs can give rise to HSCs via symmetric division or directly differentiate into MyRPs via asymmetric division (yielding HSC-MkRP or HSC-CMRP pairs). These myeloid bypass pathways could be essential for fast responses to ablation stress. Our results show that loss of self-renewal and stepwise progression through specific differentiation stages are not essential for lineage commitment of HSCs and suggest a revised model of hematopoietic differentiation.

Extramedullary Myelopoiesis in Malaria Depends on Mobilization of Myeloid-Restricted Progenitors by IFN-γ Induced Chemokines

Resolution of a variety of acute bacterial and parasitic infections critically relies on the stimulation of myelopoiesis leading in cases to extramedullary hematopoiesis. Here, we report the isolation of the earliest myeloid-restricted progenitors in acute infection with the rodent malaria parasite, Plasmodium chabaudi. The rapid disappearance of these infection-induced myeloid progenitors from the bone marrow (BM) equated with contraction of the functional myeloid potential in that organ. The loss of BM myelopoiesis was not affected by the complete genetic inactivation of toll-like receptor signaling. De-activation of IFN-γ signaling completely abrogated the contraction of BM myeloid progenitors. Radiation chimeras of Ifngr1-null and control BM revealed that IFN-γ signaling in an irradiation-resistant stromal compartment was crucial for the loss of early myeloid progenitors. Systemic IFN-γ triggered the secretion of C-C motif ligand chemokines CCL2 and CCL7 leading to the egress of early, myeloid-committed progenitors from the bone marrow mediated by their common receptor CCR2. The mobilization of myeloid progenitors initiated extramedullary myelopoiesis in the spleen in a CCR2-dependent manner resulting in augmented myelopoiesis during acute malaria. Consistent with the lack of splenic myelopoiesis in the absence of CCR2 we observed a significant persistence of parasitemia in malaria infected CCR2-deficient hosts. Our findings reveal how the activated immune system mobilizes early myeloid progenitors out of the BM thereby transiently establishing myelopoiesis in the spleen in order to contain and resolve the infection locally.

Hematopoietic Stem Cell Subtypes Expand Differentially during Development and Display Distinct Lymphopoietic Programs

Adult hematopoietic stem cells (HSCs) with serially transplantable activity comprise two subtypes. One shows a balanced output of mature lymphoid and myeloid cells; the other appears selectively lymphoid deficient. We now show that both of these HSC subtypes are present in the fetal liver (at a 1:10 ratio) with the rarer, lymphoid-deficient HSCs immediately gaining an increased representation in the fetal bone marrow, suggesting that the marrow niche plays a key role in regulating their ensuing preferential amplification. Clonal analysis of HSC expansion posttransplant showed that both subtypes display an extensive but variable self-renewal activity with occasional interconversion. Clonal analysis of their differentiation programs demonstrated functional and molecular as well as quantitative HSC subtype-specific differences in the lymphoid progenitors they generate but an indistinguishable production of multipotent and myeloid-restricted progenitors. These findings establish a level of heterogeneity in HSC differentiation and expansion control that may have relevance to stem cell populations in other hierarchically organized tissues.

The earliest intrathymic precursors of CD8α+ thymic dendritic cells correspond to myeloid‐type double‐negative 1c cells

The dendritic cells (DCs) present in lymphoid and non-lymphoid organs are generated from progenitors with myeloid-restricted potential. However, in the thymus a major subset of DCs expressing CD8α and langerin (CD207) appears to stand apart from all other DCs in that it is thought to derive from progenitors with lymphoid potential. Using mice expressing a fluorescent reporter and a diphtheria toxin receptor under the control of the cd207 gene, we demonstrated that CD207(+) CD8α(+) thymic DCs do not share a common origin with T cells but originate from intrathymic precursors that express markers that are normally present on all (CD11c(+) and MHCII molecules) or on some (CD207, CD135, CD8α, CX3CR1) DC subsets. Those intrathymic myeloid-type precursors correspond to CD44(+) CD25(-) double-negative 1c (DN1c) cells and are continuously renewed from bone marrow-derived canonical DC precursors. In conclusion, our results demonstrate that the earliest intrathymic precursors of CD8α(+) thymic DCs correspond to myeloid-type DN1c cells and support the view that under physiological conditions myeloid-restricted progenitors generate the whole constellation of DCs present in the body including the thymus.

The myeloid 7/4-antigen defines recently generated inflammatory macrophages and is synonymous with Ly-6B

This study aimed to identify the inflammation-associated 7/4-antigen, which is highly expressed on neutrophils, inflammatory monocytes, some activated macrophages, as well as on bone marrow myeloid-restricted progenitors. The high expression on inflammatory cells is suggestive of a role in inflammation and makes the 7/4-antigen a potential target for the manipulation of inflammatory cells. Consistent with this, the 7/4-antibody mediates specific depletion of 7/4-expressing neutrophils and monocytes. We have identified the 7/4-antigen as a 25- to 30-kDa GPI-anchored glycoprotein synonymous with the Ly-6B.2 alloantigen. We characterized the expression of Ly-6B during the inflammatory reaction induced by zymosan. During the later stages of an experimental, acute, self-resolving inflammatory response, we found that Ly-6B is differentially expressed on macrophages. Ly-6B-expressing macrophages also express more MHCII, CIITA, CCR2, Ly-6C, and CD62L than the Ly-6B-negative macrophages, which in turn, express more of the resident tissue macrophage marker SIGN-R1 and higher CD11b and F4/80. Ly-6B-expressing macrophages incorporate more BrdU than their Ly-6B-negative contemporaries when fed during the resolution phase of the acute inflammatory response. Thus, Ly-6B expression on mature macrophages defines a subset of recently generated inflammatory macrophages that retain monocytic markers and is hence a surrogate marker of macrophage turnover in inflammatory lesions. The definition of the 7/4:Ly-6B antigen will allow further characterization and specific modulation of Ly-6B-expressing cells in vivo.

DNA Methyltransferase 1 Is Essential for and Uniquely Regulates Hematopoietic Stem and Progenitor Cells.

Abstract 392DNA methylation is essential for development and plays crucial roles in a variety of biological processes. The DNA methyltransferase Dnmt1 serves to maintain parental cell methylation patterns on daughter DNA strands in mitotic cells, however, the precise role of Dnmt1 in regulation of quiescent adult stem cells is not known. To examine the role of Dnmt1 in adult hematopoietic stem cells (HSCs), we crossed Dnmt1fl/fl mice with Mx1-Cre transgenic mice, and by injection of poly(I)-poly(C) we selectively deleted Dnmt1 in the hematopoietic system (Dnmt1Δ/Δ). In Dnmt1Δ/Δ mice, peripheral blood counts and mature multilineage composition of the bone marrow was found to be normal. Interestingly, specific defects were observed in Dnmt1Δ/Δ HSC self-renewal as assessed by long-term and secondary competitive transplantation, in retention of Dnmt1Δ/Δ HSCs within the bone marrow niche, and in the ability of Dnmt1Δ/Δ HSCs to give rise to multilineage hematopoiesis. Loss of Dnmt1 also had unique impact on myeloid progenitor cells (including common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythrocyte progenitors), regulating their cycling and transcriptional lineage fidelity. To determine the molecular mechanisms underlying these defects, we performed global gene expression microarray analysis and bisulfite sequencing of select loci (IAP, Car1, and Gata1) in purified populations of control and Dnmt1Δ/Δ long-term HSCs, short-term HSCs/multipotent progenitor cells, and myeloid restricted progenitor cells. Through this approach, we demonstrate that loss of Dnmt1 has cell type-specific molecular consequences. For example, demethylation of the Car1 and Gata1 loci in Dnmt1Δ/Δ long-term HSCs is not sufficient to activate gene transcription, whereas demethylation of these genes in Dnmt1Δ/Δ short-term HSCs is associated with activation of transcription. In Dnmt1Δ/Δ myeloid restricted progenitor cells, we observed increases in DNA methylation at specific gene loci such as Car1, indicating that methylation can be established by other methyltransferases in the absence of Dnmt1. Our global gene expression microarray analysis clearly demonstrates that Dnmt1 regulates expression of distinct gene families in these closely related, primitive hematopoietic populations. We were unable to attribute specific functional defects in Dnmt1Δ/Δ hematopoietic stem and progenitor cells to alterations in expression of previously characterized genes, supporting the existence of novel, uncharacterized regulators of HSC and progenitor cell function to be explored from candidates in our data set. We conclude that maintenance methylation induced by Dnmt1 appears to be especially important for HSC and progenitor cell state transitions, such as the stepwise differentiation of long-term HSCs to multipotent progenitors, multipotent progenitors to myeloid restricted progenitors, stem cell mobilization, and regulating cell cycle entry. These findings establish a unique and critical role for Dnmt1 in the primitive hematopoietic compartment. Furthermore, our evidence suggests that epigenetic regulation, at least with respect to DNA methylation, of adult stem cells is distinct from embryonic stem cells and other somatic cell types. Disclosures:No relevant conflicts of interest to declare.

Cell-intrinsic and Vector-related Properties Cooperate to Determine the Incidence and Consequences of Insertional Mutagenesis

In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient gamma-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of approximately 10(-4). Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10(-6)). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore, our study offers perspectives for refinement of animal experiments in the assessment of vector-related genotoxicity.

SCL Expression at Critical Points in Human Hematopoietic Lineage Commitment

The stem cell leukemia (SCL or tal-1) gene was initially identified as a translocation partner in a leukemia that possessed both lymphoid and myeloid differentiation potential. Mice that lacked SCL expression showed a complete block in hematopoiesis; thus, SCL was associated with hematopoietic stem cell (HSC) function. More recent studies show a role for SCL in murine erythroid differentiation. However, the expression pattern and the role of SCL during early stages of human hematopoietic differentiation are less clear. In this study we chart the pattern of human SCL expression from HSCs, through developmentally sequential populations of lymphoid and myeloid progenitors to mature cells of the hematopoietic lineages. Using recently defined surface immunophenotypes, we fluorescence-activated cell-sorted (FACS) highly purified populations of primary human hematopoietic progenitors for reverse transcription-polymerase chain reaction (RT-PCR) analysis of SCL expression. Our data show that SCL mRNA is easily detectable in all hematopoietic populations with erythroid potential, including HSCs, multipotential progenitors, common myeloid progenitors, megakaryocyte/erythrocyte progenitors, and nucleated erythroid lineage cells. SCL mRNA expression was present but rapidly downregulated in the common lymphoid progenitor and granulocyte/monocyte progenitor populations that lack erythroid potential. SCL expression was undetectable in immature cells of nonerythroid lineages, including pro-B cells, early thymic progenitors, and myeloid precursors expressing the M-CSF receptor. SCL expression was also absent from all mature cells of the nonerythroid lineages. Although low levels of SCL were detected in lymphoid- and myeloid-restricted progenitors, our studies show that abundant SCL expression is normally tightly linked with erythroid differentiation potential.