Differentiation Of Hematopoietic Stem/progenitor Cells Research Articles

Sheng Xue Ning as a Novel Agent that Promotes SCF-Driven Hematopoietic Stem/Progenitor Cell Proliferation to Promote Erythropoiesis.

Stimulating erythropoiesis is essential in the treatment of various types of anemia. Sheng Xue Ning (SXN) is commonly used in China as an iron supplement to treat iron deficiency anemia, renal anemia, and anemia in pregnancy. This research reports a novel effect of SXN in enhancing the proliferation of hematopoietic stem/progenitor cell (HSPC) to promote erythropoiesis in the bone marrow, which is distinct from conventional iron supplements that primarily aid in the maturation of red blood cells. Employing a model of hematopoietic dysfunction induced by X-ray exposure, we evaluated the efficacy of SXN in restoring hematopoietic function. SXN significantly promoted the recovery of peripheral erythroid cells and enhanced the proliferation and differentiation of Lin-/c-KIT+/Sca-1+ HSPC in mice exposed to X-ray irradiation. Our results showed that SXN elevated the expression of stem cell factor (SCF) and activated the SCF/c-KIT/PI3K/AKT signaling pathway, facilitating the proliferation and differentiation of HSPC. In vitro, SXN markedly enhanced the proliferation of bone marrow nucleated cell (BMNC) and the colony-forming capacity of BFU-E, CFU-E, and CFU-GM, while also elevating the expression of proteins involved in the SCF/c-KIT/PI3K/AKT pathway in BMNC. Additionally, SXN enhanced the proliferation and differentiation of mesenchymal stem cell (MSC) and increased SCF secretion. In conclusion, SXN demonstrates the capacity to enhance erythropoiesis by upregulating SCF expression, thereby promoting HSPC proliferation and differentiation via the SCF/c-KIT/PI3K/AKT pathway. SXN may offer a new strategy for improving the activity of HSPC and promoting erythropoiesis in the treatment of hematopoiesis disorders.

Liraglutide prevents hyperglycemia-induced senescence and proinflammatory monocyte differentiation in CD34+ hematopoietic stem cells

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Italian Ministry of Health- Ricerca Finalizzata Background Diabetes mellitus (DM) is known to promote aberrant activation of immune cells that contribute to the onset and progression of chronic inflammation and atherosclerosis in DM patients (1). This phenomenon, termed 'trained immunity', appears to be initiated in hematopoietic stem/progenitor cells (HSPCs). In recent years, there has been great interest in investigating the pleiotropic effects of glucagon-like peptide-1 receptor agonists (GLP-1RAs), including anti-inflammatory and immunological properties. We have recently demonstrated that HSPCs express the GLP-1R whose stimulation with the agonist liraglutide (LIRA) prevents oxidative stress and functional impairment induced by hyperglycemic conditions (HG) (2). On this basis, we hypothesized that LIRA treatment can also avert HG-induced senescence and proinflammatory myeloid differentiation of HSPCs. Purpose To evaluate senescence and inflammatory myeloid differentiation of CD34+ HSPCs after HG exposure in the presence or not of GLP1R agonist. Methods CD34+ HSPCs derived from cord blood of healthy donors were expanded in normal-glucose (NG; with 30 mM mannitol for osmotic control) or high-glucose (HG; 30 mM) and treated or not with LIRA (100nM) in serum-free medium plus cytokines (FLT3, SCF, IL3 and IL6) for up to 20 days. The expression of p21, p27, IL6, TNFα, and NFkB-p65 genes was assessed by qPCR. ROS production and CD34+ HSPC-derived monocyte subsets were evaluated by flow cytometry whereas cytokine secretion after LPS stimulation was measured by ELISA assay. Results LIRA treatment showed to significantly prevent the impairment of CD34+ HSPC proliferation as well as the upregulation of senescence-associated secretory phenotype (SASP) genes such as p21, p27, NFkB-p65, IL6, and TNFα in HG-CD34+ HSPCs when compared to HG cell alone (one-way ANOVA; p≤0.05). Interestingly, these pharmacological effects were associated with a significant reduction in HG-induced ROS production. Myeloid differentiation in vitro of HG-CD34+ HSPCs generated monocytes composed mainly of an inflammatory and highly reactive intermediate (CD14++CD16+) subpopulation characterized by higher LPS-induced release of IL6 and TNFα. LIRA treatment significantly prevented both the accumulation of intermediate (CD14++CD16+) monocyte subpopulation from differentiating HG-CD34+ HSPCs and their inflammatory phenotype (one-way ANOVA; p≤0.05). Conclusions These results demonstrate that HG exposure alters the differentiation program of CD34+ HSPCs towards more inflammatory immune cell components and suggest that GLP1R agonists are endowed with immunomodulatory properties that could be further developed and exploited for the treatment of immune system dysregulation in diabetes.

MicroRNA target homeobox messenger RNA in HIV induced hematopoietic inhibition.

Cytopenias are a common occurrence due to abnormal hematopoiesis persistent in patients suffering from and advancing with HIV/AIDS. In order to develop efficacious therapies against cytopenias, it is necessary to understand the mechanisms by which HIV infection affects the differentiation of hematopoietic stem-progenitor cells (HSPCs), causing hematopoietic inhibition, that leads to hematological disorders. Currently, only the antiretrovirals that are being used to treat HIV infection and indirectly lower the levels of virus replication also co-attenuate cytopenias. The evidence available suggests that this indirect efficacy may not prevail for the lifetime of the infected patients, and the acquired immunodeficiency can overtake the beneficial consequences of decreased virus replication. As cited in this article, we and our colleagues are the first to make a foray into the involvement of microRNAs and their use as potential interventional treatments for the cytopenias that occur with HIV/AIDS. Herein, we progressed further in the direction of the mechanisms of the involvement of homeobox gene regulation to cause cytopenias. We had previously shown that HIV-1 inhibits multi-lineage hematopoiesis of the CD34+ cells using SCID-hu Thy/Liv animals in vivo. Furthermore, we demonstrated that the virus-induced hematopoietic inhibition occurred despite the CD34+ cells being resistant to HIV-1 infection. We set out to search for the specific host factors secreted by CD4+ T-cells that likely participate in the inhibition of hematopoiesis of the HIV infection-resistant CD34+ cells. More recently, we reported the identification of virus-infected CD4+ thymocyte-secreted miRNA-15a and miRNA-24 and that their differential expression following HIV infection causes the indirect inhibition of hematopoiesis. We then hypothesized that the observed miRNA differential expression in the virus-infected T-cells causes the abnormal regulation of homeobox (HOX) gene-encoded transcriptomes in the CD34+ cells, affecting specific MAPK signaling and CD34+ cell fate, thereby disrupting normal hematopoiesis. We present that in HIV infection, miRNA-mediated post-transcriptional dysregulation of HOXB3 mRNA inhibits multi-lineage hematopoiesis, which translates into hematological disorders in virus-infected patients with HIV/AIDS. These observations portend specific microRNA candidates for potential efficacy against the virus-induced cytopenias that are otherwise not treatable by the existing HAART/ART regimens, which are primarily designed and applicable for the attenuation of virus replication.

Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation invivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the invivo effects of MSC transplantation.

Unraveling Pathophysiology and Hematopoiesis of Vexas Syndrome By Multi-Omics Analyses and Targeted Gene Editing

Background and Rationale VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome is a newly described, treatment-refractory, severe, highly prevalent disease (~1:4,000 males aged >50 years) caused by somatic mutations in the UBA1 (ubiquitin-activating enzyme 1) gene of hematopoietic stem/progenitor cells (HSPCs). VEXAS is hallmarked by vacuoles in myeloid/erythroid precursors and presents with systemic inflammation. About 50% patients develop hematologic malignancies, including myelodysplasia, contributing to the poor prognosis. The pathophysiology and impact of VEXAS syndrome on human hematopoiesis remain elusive. Moreover, the lack of suitable disease models hampers investigation of disease development and preclinical drug testing. To fill these gaps, here we: i) integrated multiparametric flow cytometry and multi-omics analyses on VEXAS patients to achieve comprehensive molecular and phenotypic characterization of hematopoiesis; and ii) successfully developed in vitro and in vivo models of VEXAS syndrome by cutting-edge targeted base editing strategies. Methods Multiparametric immunophenotypic analyses and single-cell transcriptomics were performed on peripheral blood and bone marrow (BM) cells from six VEXAS patients (p.Met41>Thr; p.Met41>Val; p.Met41>Leu; c.118-1 G>C) recruited by our Unit. Circulating monocytes were analyzed by whole RNA-sequencing (RNA-seq) and metabolomics. UBA1 mutations were introduced by gene editing technologies in myeloid cell lines, primary T cells and healthy human HSPCs. Engineered HSPCs were used to reconstitute the hematopoietic compartment of immunodeficient mice to generate a humanized VEXAS model. Variant allele frequency of UBA1 mutant cells was quantified by targeted sequencing in isolated hematopoietic lineages and HSPCs. Results i) Multiparametric immunophenotypic analyses of BM cells showed unbalanced composition of the HSPC compartment in VEXAS patients compared to age-matched healthy individuals, with lower abundance of stem cells, multipotent and lymphoid progenitors and expanded myeloid progenitors. This was paralleled by a significant increase of circulating myeloid-biased HSPCs and immature myeloid cells. Transcriptomic analyses of patient-derived peripheral monocytes showed sustained VEXAS-associated inflammatory signatures and heightened glycolysis, which were confirmed by metabolomic profiling. Single-cell transcriptomics on the BM hematopoietic compartment of VEXAS patients harboring different UBA1 mutations displayed genotype-dependent gene expression patterns. Single-cell RNA-seq of peripheral blood mononuclear cells from VEXAS patients is ongoing. ii) In vitro and in vivo models of VEXAS, generated by UBA1 gene editing, faithfully recapitulated patient hematopoiesis and pathophysiology . Editing efficiency approached 90% in wild-type myeloid cell lines, primary human T cells and HSPCs. While UBA1-mutant T cells were counterselected over time in culture, myeloid ones were enriched. Colony forming unit assays of UBA1-edited HSPCs revealed an exclusive myeloid output in vitro and a significant loss of the erythroid HSPC differentiation potential compared to mock-edited controls. Transplantation of edited HSPCs in immunodeficient mice resulted in a 100-fold reduction in circulating B cells, while NK and myeloid compartments were preserved. Human BM HSPCs were 5-fold lower than in control mice, largely myeloid-biased, and displayed abnormal vacuolar morphology. Concordantly, while myeloid cells and HSPCs were mainly (>80%) UBA1 mutated, the lymphoid compartment showed predominance of wild-type alleles. Strikingly, the distribution of UBA1-mutant cells across lineages in humanized mice mirrored the composition of our VEXAS patient cohort, where UBA1-mutant cells accounted for 90% of total myeloid cells and HSPCs and for less than 5% of lymphoid lineages. Conclusions Our findingsprovide evidence thatmutations in UBA1 drive accumulation of myeloid-biased HSPCs and immature myeloid precursors. Mutant lymphoid cells are negatively selected and their peripheral myeloid counterpart displays inflammatory activation. Gene editing-based in vitro and in vivo models phenocopy most hematopoietic features of the disease and hold promise to enable preclinical testing and validation of novel therapeutics to treat VEXAS syndrome.

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells

BackgroundIn vitro production of hematopoietic stem/progenitor cells (HSPCs) from human-induced pluripotent stem cells (hiPSCs) provides opportunities for fundamental research, disease modeling, and large-scale production of HLA-matched HSPCs for therapeutic applications. However, a comprehensive understanding of the signaling mechanisms that regulate human hematopoiesis is needed to develop a more effective procedure for deriving HSPCs from hiPSCs.MethodsIn this study, we investigate the role of YAP during the hematopoietic differentiation of hiPSCs to HSPCs and erythrocytes using the isogenic YAP-overexpressing (YAP-S5A) and YAP-depleting (YAP-KD) hiPSCs to eliminate the effects of a genetic background variation.ResultsAlthough YAP is dispensable for maintaining the self-renewal and pluripotency of these hiPSCs, it affects the early cell-fate determination and hematopoietic differentiation of hiPSCs. Depleting YAP enhances the derivation efficiency of HSPCs from hiPSCs by inducing the mesodermal lineage commitment, promoting hematopoietic differentiation, and preventing the differentiation toward endothelial lineage. On the contrary, the overexpression of YAP reduced HSPCs yield by inducing the endodermal lineage commitment, suppressing hematopoietic differentiation, and promoting the differentiation toward endothelial lineage.ConclusionsExpression of YAP is crucial for the differentiation of hiPSC-derived HSPCs toward mature erythrocytes. We believe that by manipulating YAP activity using small molecules, the efficiency of the large-scale in vitro production system for generating hematopoietic stem/progenitor cells for future therapeutic use could be improved.

Metabolomic characteristics in human CD34+ hematopoietic stem/progenitor cells exposed to polystyrene nanoplastics.

Nanoplastics is a major environmental concern and may cause potential harm to organisms. Previous studies have found that exposure to nanoplastics inhibited hematopoietic function, however, the effect of polystyrene nanoplastics (PSNPs) on the human CD34+ hematopoietic stem/progenitor cells (HSPCs) and its underlying mechanism remains unknown. In this study, the toxic effects were evaluated and the metabolites changes were systematically analyzed using the metabolomics study in combination with multivariate statistical analysis in HSPCs with PSNPs treatment. The results show that PSNPs could be uptake by cells, significantly decrease cell viability and cause cell membrane damage manifested as increased LDH release in cellular supernatant. Besides, the colony formation assay shows that PSNPs exposure can inhibit the proliferation and differentiation of HSPCs. Meanwhile, we found that PSNPs disturbed the metabolic activity, including amino acids, SCFAs, organic acids, fatty acids and carbohydrates, and mainly affect citrate cycle (TCA cycle) metabolism pathway. Those findings are helpful in evaluating the toxicity mechanisms and providing guidance in the selection of potential metabolism-related biomarkers of hematopoietic damage caused by nanoplastics exposure.

Unveiling the Biological Role of Peripheral Blood Human Circulating Hematopoietic Stem and Progenitor Cells

Although most hematopoietic stem/progenitor cells (HSPC) reside in the bone marrow (BM), few circulating HSPC (cHSPC) can be also found in the peripheral blood (PB) at steady state. The biological role and relationship with BM-resident counterpart in humans remain still not fully elucidated. In the present study, we phenotypically characterized cHSPC composition by applying multi-parametric flow cytometry on 110 PB and, as control, 48 BM samples of healthy donors (HD) of different age groups. These analyses were combined with cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) and ad hoc designed in vitro and in vivo assays to investigate the transcriptional and functional properties of steady-state cHSPC subpopulations with respect to BM counterpart. Moreover, to study circulating vs. resident HSPC differentiation potential in vivo in humans, we exploited integration site (IS) clonal tracking of cHSPC, BM HSPC and mature PB lineages isolated from patients treated with autologous HSPC-gene therapy (GT) at late follow-ups post-treatment (>2 years), once steady-state hematopoiesis is established. We observed that cHSPC show a progressive reduction in number during aging and a different composition than BM counterpart, with Multi Lymphoid Progenitors (MLP) displaying the highest PB circulation propensity. cHSPC were endowed with BM homing capability and multilineage differentiation potential both in vitro and in vivo, showing an early skewing toward erythroid differentiation after transplantation. Moreover, we detected a reduced long-term human cell engraftment in cHSPC- than BM HSPC-transplanted mice. This latter finding might be explained by the low primitive Hematopoietic Stem Cell (HSC) content and the transcriptional pre-activated state observed in steady-state PB HSC, as assessed in our single-cell RNA-sequencing analyses. Indeed, applying CITE-seq on PB- and BM-derived HSPC, we identified a unique transcriptional profile of both primitive and lineage-committed cHSPC subpopulations, characterized by lower replicative, metabolic and transcriptional activity, but increased differentiation-, adhesion- and immune response-priming than BM counterpart. Furthermore, we observed that PB phenotypic primitive HSC and Multipotent Progenitors (MPP) and Committed Myeloid Progenitors (CMP) were transcriptionally and functionally skewed toward erythroid lineages as compared to BM-resident counterpart. Finally, since upon gene correction each HSPC and its progeny became univocally marked by distinct IS, we exploited IS analyses from 5 patients treated with HSPC-GT at >2 years after GT to investigate the steady-state hematopoietic output of PB and BM HSPC in vivo in humans. In line with the enrichment of lymphoid transcriptional signature observed in trafficking HSPC, we found a higher IS sharing between PB mature lymphoid compartment and steady-state PB-derived HSPC. Moreover, a substantial fraction of trafficking HSPC displayed an enriched expression of the gene signature associated with thymus seeding progenitor type 1 (TSP1), a group of low-cycling immature lymphoid progenitors with thymus-emigrant properties. Although at lower level with respect to PB cells, we detected a TSP1 signature also in BM dataset, thus suggesting that few thymus-emigrant lymphoid progenitors originate in the BM and preferentially egress into the PB in order to seed lymphoid organs. Altogether, our findings indicate PB trafficking HSPC as a peculiar steady-state reservoir of low-cycling, pre-activated hematopoietic progenitors, which are endowed with BM homing and repopulation potential. The enrichment of phenotypic and functional erythroid-committed progenitors in cHSPC suggest a supportive role of these cells in sustaining extramedullary erythropoiesis. Furthermore, our results of IS analyses combined with the higher expression of TSP1 signature in PB vs. BM HSPC indicate the key function of steady-state trafficking HSPC in the seeding of the thymus, with the aim of locally differentiating into lymphoid progeny. Overall, our work represents one of the most comprehensive studies on cHSPC, unveiling fundamental insights on their biological role in humans.

Frizzled-6 promotes hematopoietic stem/progenitor cell mobilization and survival during LPS-induced emergency myelopoiesis

SummaryEmergency hematopoiesis involves the activation of bone marrow hematopoietic stem/progenitor cells (HSPCs) in response to systemic inflammation by a combination of cell-autonomous and stroma-dependent signals and leads to their release from bone marrow and migration to periphery. We have previously shown that FZD6 plays a pivotal role in regulating HSPC expansion and long-term maintenance. Now we sought to better understand the underlying mechanisms. Using lipopolysaccharide (LPS)-induced emergency granulopoiesis as a model, we show that failed expansion was intrinsic to FZD6-deficient HSPCs but also required a FZD6-deficient environment. FZD6-deficient HSPCs became more strongly activated, but their mobilization to peripheral blood was impaired and they were more susceptible to inflammatory cell death, leading to enhanced release of pro-inflammatory cytokines in the marrow. These studies indicate that FZD6 has a protective effect in the bone marrow to prevent an overactive inflammatory response and further suggest that mobilization improves HSPC survival during bone marrow inflammation.

MiR-486-5p and miR-22-3p Enable Megakaryocytic Differentiation of Hematopoietic Stem and Progenitor Cells without Thrombopoietin.

Megakaryocytes release submicron size microparticles (MkMPs) in circulation. We have shown that MkMPs target CD34+ hematopoietic stem/progenitor cells (HSPCs) to induce megakaryocytic differentiation, and that small RNAs in MkMPs play an important role in the development of this phenotype. Here, using single-molecule real-time (SMRT) RNA sequencing (RNAseq), we identify the synergetic effect of two microRNAs (miRs), miR-486-5p and miR-22-3p (highly enriched in MkMPs), in driving the Mk differentiation of HSPCs in the absence of thrombopoietin (TPO). Separately, our data suggest that the MkMP-induced Mk differentiation of HSPCs is enabled through JNK and PI3K/Akt/mTOR signaling. The interaction between the two signaling pathways is likely mediated by a direct target of miR-486-5p and a negative regulator of PI3K/Akt signaling, the phosphatase and tensin homologue (PTEN) protein. Our data provide a possible mechanistic explanation of the biological effect of MkMPs in inducing megakaryocytic differentiation of HSPCs, a phenotype of potential physiological significance in stress megakaryopoiesis.

3089 – WNT AND NOTCH SIGNALING REGULATION IN PRE-HEMOGENIC ENDOTHELIUM CONTROL HEMATOPOIETIC STEM AND PROGENITOR CELLS HETEROGENEITY.

Definitive hematopoietic stem/progenitor cells (HSPCs) are produced from a specialized population of endothelial cells in the embryonic aorta called hemogenic endothelial cells. This process, known as the endothelial-to-hematopoietic transition (EHT), is conserved across vertebrates, and is followed by HSPC differentiation in all blood lineages. Single-cell methods have uncovered substantial heterogeneity in lineage priming within nascent HSPCs from the aortic endothelium, however it is currently unknown how this diverse differentiation capacity is conferred. Such understanding could have important ramifications for bone marrow transplantation where heterogeneity in HSPC behavior is observed. Here we found that microRNA (miR) loss of function zebrafish mutant, miR-128 (miR-128-/-), has increased EHT, resulting in supernumerary lymphoid and erythroid primed HSPCs and relative mature cells. Correlatively, miR-128-deficient endothelium derived from human pluripotent stem cells show similar defective EHT, supporting miR-128’s functional conservation in human. Transcriptomic analysis of endothelial cells from wild type and miR-128-/- embryos revealed putative miR-128 target genes, cskn1a and jag1b, involved in the inhibition of the two master EHT signaling pathways, Wnt and Notch respectively. Additionally, responsive transgenic lines and single cell RNA sequencing revealed that Wnt and Notch signaling activity are decreased specifically in pre-hemogenic endothelium of miR-128-/-. Chemical inhibition of Wnt or de-repression of cskn1a promoted HSPCs with erythroid lineage commitment, while lymphoid cells number increased after de-repression of jag1b or blockage of Notch activity at the onset of EHT. This study sheds light on how the HSPC heterogeneity in lineage priming is programmed prior to endothelial transdifferentiation by miR-128-co regulation of Wnt and Notch. Definitive hematopoietic stem/progenitor cells (HSPCs) are produced from a specialized population of endothelial cells in the embryonic aorta called hemogenic endothelial cells. This process, known as the endothelial-to-hematopoietic transition (EHT), is conserved across vertebrates, and is followed by HSPC differentiation in all blood lineages. Single-cell methods have uncovered substantial heterogeneity in lineage priming within nascent HSPCs from the aortic endothelium, however it is currently unknown how this diverse differentiation capacity is conferred. Such understanding could have important ramifications for bone marrow transplantation where heterogeneity in HSPC behavior is observed. Here we found that microRNA (miR) loss of function zebrafish mutant, miR-128 (miR-128-/-), has increased EHT, resulting in supernumerary lymphoid and erythroid primed HSPCs and relative mature cells. Correlatively, miR-128-deficient endothelium derived from human pluripotent stem cells show similar defective EHT, supporting miR-128’s functional conservation in human. Transcriptomic analysis of endothelial cells from wild type and miR-128-/- embryos revealed putative miR-128 target genes, cskn1a and jag1b, involved in the inhibition of the two master EHT signaling pathways, Wnt and Notch respectively. Additionally, responsive transgenic lines and single cell RNA sequencing revealed that Wnt and Notch signaling activity are decreased specifically in pre-hemogenic endothelium of miR-128-/-. Chemical inhibition of Wnt or de-repression of cskn1a promoted HSPCs with erythroid lineage commitment, while lymphoid cells number increased after de-repression of jag1b or blockage of Notch activity at the onset of EHT. This study sheds light on how the HSPC heterogeneity in lineage priming is programmed prior to endothelial transdifferentiation by miR-128-co regulation of Wnt and Notch.

Novel Lentiviral Vectors for Gene Therapy of Sickle Cell Disease Combining Gene Addition and Gene Silencing Strategies

Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the sickle β S-globin chain. The sickle Hb (HbS, a 2β S2) polymerizes, leading to the formation of sickle-shaped red blood cells that cause vaso-occlusions and organ damage. Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LV) expressing an anti-sickling β-globin transgene (βAS LV) is a promising curative treatment; however, it is partially effective in SCD patients, who still present elevated HbS levels.Here, we aim to improve LVs to boost therapeutic β-like globin levels without increasing the mutagenic vector load in HSPCs. We developed 2 novel LVs expressing βAS together with an artificial microRNA (amiR) targeting either the fetal Hb (HbF) repressor BCL11A (βAS/amiRBCL11A) or the β S-globin (βAS/amiRHBB). By downregulating BCL11A, amiRBCL11A re-activates the expression of the endogenous anti-sickling fetal γ-globin, which, together with βAS, should improve the clinical course of SCD; β S-globin downregulation should favor βAS incorporation in Hb tetramers, increase therapeutic Hb levels and ameliorate the SCD phenotype.First, we developed βAS/amiRBCL11A LV by inserting the amiR in multiple position of the βAS intron 2 under the control of HBB promoter/enhancers to limit BCL11A downregulation to the erythroid lineage and reduce potential amiR-related cellular toxicity and off-target effects. We showed that amiR insertion site did not affect LV titer nor βAS expression in a human erythroid cell line (HUDEP2). BCL11A downregulation in HUDEP2 led to γ-globin gene de-repression and a high proportion of HbF + cells (RTqPCR, HPLC, flow cytometry). Importantly, the total amount of therapeutic β-like globins was substantially higher in βAS/amiRBCL11A LV- than in βAS LV-transduced cells, with no impairment in cell viability or erythroid differentiation.In parallel, we designed 17 amiRs targeting HBB and generated the corresponding βAS/amiRHBB LVs. We tested these LVs in HUDEP2 and selected 2 amiRs efficiently downregulating β-globin at mRNA and protein levels (RT-qPCR and Western Blot). Of note, we modified the βAS transgene by inserting silent mutations that prevent its recognition by the amiR (βASm).Finally, we tested βAS/amiRBCL11A and βAS/amiRHBB LVs in HSPCs from SCD patients. HSPC-derived erythroid cells transduced with βAS/amiRBCL11A LV showed increased HbF levels, although HbS levels remained high. To further reduce β S-globin levels, we targeted the β S-globin mRNA using the βAS/amiRHBB LV. Efficient HSPC transduction by βASm/amiRHBB LV led to a substantial decrease of β S-globin transcripts in HSPC-derived erythroid cells compared to the βAS LV-transduced cells (RTqPCR) at a VCN/cell of 2. Notably, the amiR specifically down-regulated β S-globin, without affecting βAS expression. In βASm/amiRHBB- vs βAS LV-transduced cells, HPLC analysis showed that β S-globin downregulation led to a significant decrease of HbS, which represented 58% and 71% of the total Hb, respectively). This was associated with a significant increase of the therapeutic Hb in βASm/amiRHBB LV- vs βAS LV-transduced erythroid cells (38% and 27% of the total Hb, respectively). Importantly, we observed a substantial reduction of the proportion of HbS-positive cells in βASm/amiRHBB- vs βAS LV-transduced samples (from 96% to 70%; Figure 1A). The increased incorporation of βAS in Hb tetramers and the decrease in β S-globin led to a better correction of the sickling phenotype in mature RBCs derived from HSPCs transduced with βASm/amiRHBB LV- compared to βAS LV (55% and 84% of sickling cells, respectively; Figure 1B). A clonal assay of hematopoietic progenitors showed no impairment in HSPC viability and differentiation towards the erythroid and myeloid lineages upon transduction with bifunctional LVs. βASm/amiRHBB LV showed a standard lentiviral integration profile. Finally, we performed RNAseq to further evaluate the safety of our therapeutic strategy.In conclusion, we created a LV able to concomitantly silence the β S-globin and express βAS, achieving clinically relevant levels of therapeutic Hb and efficient correction of the sickling phenotype. Therefore, the combination of gene addition and gene silencing strategies can improve the efficacy of current therapeutic approaches, representing a novel treatment for SCD. [Display omitted] DisclosuresCavazzana: Smart Immune: Other: co-founder.

The neurotransmitter receptor Gabbr1 regulates proliferation and function of hematopoietic stem and progenitor cells

AbstractHematopoietic and nervous systems are linked via innervation of bone marrow (BM) niche cells. Hematopoietic stem/progenitor cells (HSPCs) express neurotransmitter receptors, such as the γ-aminobutyric acid (GABA) type B receptor subunit 1 (GABBR1), suggesting that HSPCs could be directly regulated by neurotransmitters like GABA that directly bind to GABBR1. We performed imaging mass spectrometry and found that the endogenous GABA molecule is regionally localized and concentrated near the endosteum of the BM niche. To better understand the role of GABBR1 in regulating HSPCs, we generated a constitutive Gabbr1-knockout mouse model. Analysis revealed that HSPC numbers were significantly reduced in the BM compared with wild-type littermates. Moreover, Gabbr1-null hematopoietic stem cells had diminished capacity to reconstitute irradiated recipients in a competitive transplantation model. Gabbr1-null HSPCs were less proliferative under steady-state conditions and upon stress. Colony-forming unit assays demonstrated that almost all Gabbr1-null HSPCs were in a slow or noncycling state. In vitro differentiation of Gabbr1-null HSPCs in cocultures produced fewer overall cell numbers with significant defects in differentiation and expansion of the B-cell lineage. To determine whether a GABBR1 agonist could stimulate human umbilical cord blood (UCB) HSPCs, we performed brief ex vivo treatment prior to transplant into immunodeficient mice, with significant increases in long-term engraftment of HSPCs compared with GABBR1 antagonist or vehicle treatments. Our results indicate a direct role for GABBR1 in HSPC proliferation, and identify a potential target to improve HSPC engraftment in clinical transplantation.

TLR7/8 agonist treatment induces an increase in bone marrow resident dendritic cells and hematopoietic progenitor expansion and mobilization

There is accumulating evidence suggesting that toll-like receptor (TLR) signals play an important role in the regulation of hematopoietic stem/progenitor cells (HSPCs). TLR7/8 stimulation induces the myeloid differentiation of normal HSPCs and acute myeloid leukemia cells. However, the in vivo effect of TLR7/8 agonists on hematopoiesis is largely unknown. Here, we show that, similar to TLR4 and TLR2, treatment with the TLR7/8 agonist R848 induces an expansion of phenotypic hematopoietic stem cells (HSCs) with reduced repopulating potential and HSPC mobilization. In contrast to chronic TLR4 stimulation, treatment with R848 for 5 days did not induce a significant increase in myeloid-biased HSCs. Treatment with R848 results in a significant increase in classic dendritic cells (DCs) in the bone marrow, but a decrease in common dendritic cell progenitors and pre-DCs. Phenotypic analysis of DCs revealed that R848 treatment is associated with altered expression of certain chemokines, activation markers, and migratory receptors. Together, these data indicate that systemic administration of a TLR7/8 agonist has unique effects on hematopoiesis, including the expansion of DCs in the bone marrow, that might have clinical relevance to augment responses to certain immunotherapies, such as cancer vaccines and immune checkpoint blockade.

The Hematopoietic Stem/Progenitor Cell Response to Hemorrhage, Injury, and Sepsis: A Review of Pathophysiology.

Hematopoietic stem/progenitor cells (HSPC) have both unique and common responses following hemorrhage, injury, and sepsis. HSPCs from different lineages have a distinctive response to these "stress" signals. Inflammation, via the production of inflammatory factors, including cytokines, hormones, and interferons, has been demonstrated to impact the differentiation and function of HSPCs. In response to injury, hemorrhagic shock, and sepsis, cellular phenotypic changes and altered function occur, demonstrating the rapid response and potential adaptability of bone marrow hematopoietic cells. In this review, we summarize the pathophysiology of emergency myelopoiesis and the role of myeloid-derived suppressor cells, impaired erythropoiesis, as well as the mobilization of HSPCs from the bone marrow. Finally, we discuss potential therapeutic options to optimize HSPC function after severe trauma or infection.

Rapid and Reproducible Differentiation of Hematopoietic and T Cell Progenitors From Pluripotent Stem Cells.

Cell therapy using T cells has revolutionized medical care in recent years but limitations are associated with the difficulty of genome editing of the cells, the production of a sufficient number of cells and standardization of the product. Human pluripotent stem cells (hPSCs) can self-renew and differentiate into T cells to provide a standardized homogenous product of defined origin in indefinite quantity, therefore they are of great potential to alleviate limitations of therapeutic T cell production. The differentiation of hPSCs takes place in two steps: first the induction of hematopoietic stem/progenitor cells (HSPCs), then the induction of lymphopoiesis by Notch signaling. However, the differentiation of T cells from hPSCs can be difficult and lack reproducibility. One parameter that needs to be better assessed is the potential of DLL1 vs. DLL4 ligands of the Notch pathway to induce T cells. In addition, culture of hPSCs is labor-intensive and not compatible with GMP production, especially when they are cultured on feeder cells. Thus, the definition of a robust GMP-compatible differentiation protocol from hPSCs cultured in feeder-free conditions would increase the accessibility to off-the-shelf hematopoietic and T cell progenitors derived from hPSCs. In this article, we describe an efficient, rapid and reproducible protocol for the generation of hematopoietic and T cell progenitors in two steps: (1) generation of HSPCs from embryoid bodies (EB) in serum free medium and GMP-compatible feeder-free systems, (2) directed differentiation of hPSC-derived HSPCs into T-cell progenitors in the presence of bone marrow stromal cells expressing Notch-ligands OP9-DLL1 vs. OP9-DLL4.

A 3D construct based on mesenchymal stromal cells, collagen microspheres and plasma clot supports the survival, proliferation and differentiation of hematopoietic cells in vivo.

The hematopoietic niche is a specialized microenvironment that supports the survival, proliferation and differentiation of hematopoietic stem progenitor cells (HSPCs). Three-dimensional (3D) models mimicking hematopoiesis might allow in vitro and in vivo studies of the hematopoietic (HP) process. Here, we investigate the capacity of a 3D construct based on non-adherent murine bone marrow mononuclear cells (NA-BMMNCs), mesenchymal stromal cells (MSCs) and collagen microspheres (CMs), all embedded into plasma clot (PC) to support in vitro and in vivo hematopoiesis. Confocal analysis of the 3D hematopoietic construct (3D-HPC), cultured for 24h, showed MSC lining the CM and the NA-BMMNCs closely associated with MSC. In vivo hematopoiesis was examined in 3D-HPC subcutaneously implanted in mice and harvested at different intervals. Hematopoiesis in the 3D-HPC was evaluated by histology, cell morphology, flow cytometry, confocal microscopy and hematopoietic colony formation assay. 3D-HPC implants were integrated and vascularized in the host tissue, after 3months of implantation. Histological studies showed the presence of hematopoietic tissue with the presence of mature blood cells. Cells from 3D-HPC showed viability greater than 90%, expressed HSPCs markers, and formed hematopoietic colonies, in vitro. Confocal microscopy studies showed that MSCs adhered to the CM and NA-BMMNCs were scattered across the 3D-HPC area and in close association with MSC. In conclusion, the 3D-HPC mimics a hematopoietic niche supporting the survival, proliferation and differentiation of HSPCs, in vivo. 3D-HPC may allow evaluation of regulatory mechanisms involved in hematopoiesis.

Autism-Associated Chromatin Remodeler CHD8 Governs the Survival and Differentiation of Hematopoietic Stem/Progenitor Cells

Hematopoiesis is a strictly regulated process which depends on regulated proliferation, differentiation, and survival of hematopoietic stem/progenitor cells (HSPCs). Multiple signaling pathways, transcription factors and epigenetic machineries are involved in this important process. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in stem/progenitor cell regulation. Among them, the CHD8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of autism spectrum disorders. Whether CHD8 plays a role in hematopoietic cells remains unknown. In this study, to define the role of CHD8 in hematopoiesis and HSPC regulation, we carried out conditional deletion of CHD8 in the hematopoietic lineages using an interferon inducible Mx1-Cre; Chd8F/Fmouse model. As early as one week after polyI:C injection, the CHD8 knockout mice experienced drastic pancytopenia and bone marrow failure with a reduction of bone marrow (BM) cellularity to ~1/3 of that of WT controls. In the HSPC compartment, loss of CHD8 resulted in a depletion of Lin-c-Kit+ (LK) and Lin-Sca1+c-Kit+ (LSK) progenitors and a severe reduction of the GMP, MEP, MPP and CD150+CD48-LSK HSC populations. Accompanying the loss of HSPCs were dramatically increased apoptosis rate and cell cycle arrest in Chd8-/- cells. An examination of HSPC function by transplantation found that the BM cells from Chd8-/- mice could not engraft in the recipient mice that died ~20 days post-transplantation. The chimerism of Chd8-/- BM cells also reduced drastically in a competitive BM transplantation assay when induction of Chd8 deletion occurred after Mx1-Cre; Chd8F/Fcells were pre-engrafted at 1:1 ratio with WT BM cells. These results indicate that CHD8 is essential for hematopoiesis and HSPC survival and proliferation. To elucidate the underlying mechanism of CHD8 function in HSPCs, we performed complementary biochemical, genomic and genetic analyses of the WT (Chd8F/F) and Chd8-/-HSPC cells. Firstly, we found blood progenitor cells dominantly express the short form of CHD8 containing residues 1 to 751 which retains the N-terminal P53 binding domain, rather than the long form of the CHD8 protein. Secondly, we revealed by RNA-seq and subsequent RT-PCR analyses that the P53 signaling pathway signatures including P53, P21, Bax and Noxa are aberrantly activated in Chd8-/-HSPCs. At the protein level, P53 and P21 are significantly elevated in Chd8-/- LK cells. Thirdly, ATAC-seq analysis of the LSK cells revealed increased global chromatin accessibilities after CHD8 deletion, including the promoter regions of P21 and Noxa genes. By a "Cut & Run" assay using H3K4me3 antibody in Chd8F/Fand Chd8-/- LSK cells we have also seen significantly enhanced epigenetic H3K4me3 mark on the promoter region of P21 and Noxa. An anti-CHD8 "Cut & Run" further detected direct binding sites of CHD8 on P53 and P21, but not Noxa or Bax gene. Fourthly, we observed a direct binding interaction by co-immunoprecipitation between P53 and CHD8 proteins. Finally, genetic deletion of one allele of P53 gene in Chd8-/- HSPCs (P53+/-;Chd8-/-) was able to completely suppress the Bax/Noxa expression and apoptosis phenotype and rescue most of the HSC and early progenitor defects but not the later stage of differentiation defects and BM cellularity loss. Interestingly, deletion of both alleles of P53 gene (P53-/-;Chd8-/-) was able to further remove the differentiation block between LSK and LK cells observed in the P53+/-;Chd8-/-background which is associated with a P21 mediated G0 cell cycle phase arrest. These combined results suggest a mechanism of action that CHD8 works through a direct complex with P53 to regulate P21 gene expression which mediates cell cycle control of HSPC differentiation and an indirect CHD8 mediated P53 expression and subsequent P53 regulated Noxa/Bax expression which regulates HSPC survival. Thus, we identify the autism-associated CHD8 as an essential chromatin suppressor in regulating hematopoiesis through restricting the expression of P53 and chromatin accessibility of P53 downstream targets, Bax/Noxa and P21, to enable selective control of HSPC survival and differentiation, respectively. Disclosures No relevant conflicts of interest to declare.

GATA2-Dependent Developmental and Regenerative Networks

Coding and regulatory human GATA2 mutations that deregulate protein expression and/or function cause immunodeficiency that often progresses to MDS/AML (McReynolds et al., 2018). In the mouse, decreased GATA2 expression impairs hematopoietic stem/progenitor cell (HSPC) genesis and function (de Pater et al., 2013; Gao et al., 2013; Tsai et al., 1994). While prior studies demonstrated Gata2 +9.5 and -77 enhancers are essential for HSC emergence (+9.5) and/or progenitor cell fate (+9.5 and -77) (Johnson et al., 2012; Johnson et al., 2015; Mehta et al., 2017) and hematopoietic regeneration (+9.5) (Soukup et al., 2019), the mechanisms mediating these processes are not completely established. The -77 enhancer is required for fetal liver progenitors to undergo erythroid, megakaryocytic, granulocytic and monocytic differentiation. By contrast, progenitors with a -77 homozygous deletion (-77-/-) exhibit a predominant monocytic cell fate and generate macrophages ex vivo (Johnson et al., 2015). Using multiomic and single-cell strategies, we asked how this enhancer orchestrates a balance between fate-promoting and -suppressing circuitry in cell populations and single cells. Quantitative proteomics was conducted to discover the -77-regulated protein ensemble conferring multiple fates in a myeloid progenitor population [Common Myeloid Progenitor (CMP) and Granulocyte-Monocyte Progenitor (GMP)] from E14.5 fetal liver of -77+/+ and -77-/- mouse embryos. -77-/- progenitors exhibited decreased levels of GATA2 (4.7-fold) and proteins generated from GATA2 target genes (GATA1: 51-fold; HDC: 52-fold). The 202 proteins upregulated in -77-/- progenitors highlighted immune and inflammatory mechanisms, while the 232 downregulated proteins were linked to erythroid, megakaryocyte and granulocyte biology, indicative of loss of these fate potentials. Innate immune machinery was upregulated in -77-/- vs. -77+/+ progenitors, including interferon (IFN) signaling pathway components such as the IFN-inducible transcription factor and critical monocytic differentiation determinant Interferon Regulatory Factor 8 (IRF8; 2.7 fold higher) (Kurotaki et al., 2013) and diverse pathogen sensors. Expressing GATA2 at physiological levels in -77-/- progenitors normalized the aberrant transcriptome. Since -77 deletion downregulated Gata2 and upregulated Irf8, we tested whether this opposing expression pattern occurs in distinct and/or identical cells in the population using single cell transcriptomics. -77 deletion decreased Gata2 expression, which was anti-correlative with Irf8, and detailed single cell analyses indicated that -77 loss downregulates GATA2, corrupting the transcriptome/proteome, Irf8 expression increases, and IRF8 enables or drives the predominant monocytic differentiation. To determine how GATA2-dependent mechanisms governing progenitor fate relate to those guiding HSPC expansion and differentiation during regeneration, we utilized our +9.5 human disease Ets motif mutation that abrogates myeloablation-dependent GATA2 induction and hematopoietic regeneration in bone marrow (Soukup et al., 2019). While population RNA-seq with Lin-Sca1+c-Kit+ (LSK) cells revealed little to no perturbations in the steady-state, 5-FU treatment of mutants led to altered expression of only 14% of the transcripts regulated in regenerating wild type cells (423/2974). Gene Ontology analysis of differentially expressed genes indicated that genes dysregulated by the Ets motif mutation included those linked to cell cycle regulation and cellular proliferation. As LSKs consist of LT-HSCs, ST-HSCs, and MPPs, we used single-cell transcriptomics to elucidate defective regenerative circuits in individual cells. Analysis of 17000-20000 wild type and mutant LSK cells revealed GATA2-dependent mechanisms distinct from those mediating progenitor cell fate control in the fetal liver. These studies have revealed context-dependent GATA2 mechanisms governing developmental and regenerative hematopoiesis, which will enable the development of strategies to detect, diagnose, and treat GATA2-linked blood diseases. Disclosures No relevant conflicts of interest to declare.

A GPI-Anchored Protein, CD109, Protects Hematopoietic Progenitor Cells from Erythroid Differentiation Induced By TGF-β

[Background] Glycosylphosphatidylinositol-anchored proteins (GPI-APs) on hematopoietic stem progenitor cells (HSPCs) may have some roles in the negative regulation of the HSPC commitment induced by inflammatory cytokines given the fact that progenies of GPI(-) HSPC are often detected in patients with immune-mediated bone marrow (BM) failure. CD109, one of the GPI-APs expressed by keratinocytes and HSPCs in humans, serves as a TGF-β co-receptor and is reported to inhibit TGF-β signaling in keratinocytes; however, the role of CD109 on HSPCs remains unknown. We previously demonstrated that TGF-β induced erythroid differentiation of TF-1 cells, a myeloid leukemia cell line that expresses CD109, in a dose-dependent manner and that knockout of the CD109 gene resulted in erythroid differentiation of TF-1 cells cultured in fetal bovine serum-containing medium, suggesting an inhibitory role of CD109 in the erythroid differentiation of HSPCs induced by low levels of TGF-β (Blood, 2018. 132 (Suppl.1) :3874). However, as most CD109 KO TF-1 cells changed into erythroid cells, they were unsuitable for investigating the role of CD109 in the erythroid differentiation induced by TGF-β. To overcome this issue, we prepared TF-1 cells and cord blood (CB) HSPCs in which the CD109 expression was transiently downregulated, and attempted to further clarify the role of CD109. [Methods] TF-1 cells and CD34+ cells isolated from CB mononuclear cells were treated with siRNA that was complementary to CD109 mRNA. CD109 knockdown cells were cultured for 4 days in serum-free medium supplemented with stem cell factor, thrombopoietin, and erythropoietin with or without TGF-β. In separate experiments, TF-1 cells were treated with phosphatidylinositol-specific phospholipase C (PIPL-C) treatment for 1 hour and were incubated in the presence or absence of TGF-β. CD109 KO TF-1 cells were incubated in serum-free medium (StemPro-34 SFM) for 14 days and their phenotype was determined using flow cytometry (FCM). The erythroid differentiation of the cells was assessed by testing the expression of glycophorin A (GPA) and iron staining. [Results] The down-regulation of CD109 in TF-1 cells by the siRNA treatment increased GPA expression in response to 12 ng/ml of TGF-β from 1.77% to 35.6%. The transient depletion of GPI-APs by PIPL-C also augmented the GPA expression induced by TGF-β from 1.27% to 6.77%. In both BM of healthy individuals and CB, CD109 was more abundantly expressed in Lin-CD34+CD38-CD90+CD45RA- hematopoietic stem cells (HSCs) than in Lin-CD34+CD38-CD90-CD45RA- multipotent progenitors (MPPs) and Lin-CD34+CD38+ HSPCs (Fig. 1). The treatment of CB cells with siRNA reduced the CD109 expression in Lin-CD34+CD38+ cells from 55.9% to 23.1%. TGF-β induced the expression of GPA in Lin-CD34+CD38+CD123-CD45RA- megakaryocyte-erythrocyte progenitor cells (MEPs) of CD109 knockdown cells to a greater degree than the control counterpart (Fig. 2). During 14-day serum-free culture, GPA-positive CD109 KO TF-1 cells died, and similarly to WT TF-1 cells, most surviving CD109 KO TF-1 cells were GPA-negative. TGF-β treatment induced erythroid differentiation in CD109 KO TF-1 cells to a greater degree than in WT TF-1 cells. [Conclusions] CD109 plays a key role in the inhibition of TF-1 erythroid differentiation in response to TGF-β. CD109 may suppress TGF-β signaling, and the lack of CD109 may make PIGA-mutated HSPCs more sensitive to TGF-β, thus leading to the preferential commitment of the mutant erythroid progenitor cells to mature red blood cells in immune-mediated BM failure. Disclosures Yamazaki: Novartis Pharma K.K.: Honoraria; Sanofi K.K.: Honoraria; Nippon Shinyaku Co., Ltd.: Honoraria. Nakao:Novartis Pharma K.K: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Company Limited: Honoraria; Celgene: Honoraria; Ono Pharmaceutical: Honoraria; Chugai Pharmaceutical Co.,Ltd: Honoraria; Kyowa Kirin: Honoraria; Alaxion Pharmaceuticals: Honoraria; Ohtsuka Pharmaceutical: Honoraria; Daiichi-Sankyo Company, Limited: Honoraria; Janssen Pharmaceutical K.K.: Honoraria; SynBio Pharmaceuticals: Consultancy.