Potential Of Hematopoietic Stem Cells Research Articles

Endothelial MAPK Activation Disrupts Hematopoiesis By Depleting Plasma SCGF

Adult hematopoietic stem cells (HSCs) are defined by their ability to undergo self-renewal and maintain the capacity to generate all of the mature hematopoietic cell types within the blood and immune system. Maintenance of the HSC is dependent upon its cell-intrinsic properties, as well as the extrinsic cues from the bone marrow (BM) microenvironment. Within the BM microenvironment there exists a diverse array of cellular hubs that comprise the HSC niche, including vascular endothelial cells (ECs), perivascular stromal cells, osteoblasts, sympathetic nerves, and cells part of the hematopoietic hierarchy such as macrophages, granulocytes, and megakaryocytes. Our group has demonstrated that BMECs are indispensable in supporting HSC self-renewal and differentiation into lineage-committed progeny in order to maintain balanced hematopoiesis. We have built upon this finding and have recently demonstrated that suppression of vascular inflammation via EC-specific inhibition of the canonical NF-kB signaling pathway resulted in significant enhancement of steady state and regenerative hematopoiesis, in part by interfering with MAPK signaling. Furthermore, we have shown in an in vitro setting that overexpression of MAPK in ECs results in differentiation of HSCs via the upregulation of pro-differentiation paracrine factors. Based on this data, we hypothesized that MAPK overexpression within ECs promotes severe defects in the hematopoietic system and we set out to develop strategies to overcome these deficiencies and enhance hematopoietic function.To formally test if endothelial MAPK signaling impacts the ability of the BM endothelial niche to support functional hematopoiesis, we generated a mouse model in which the MAPK signaling pathway was selectively activated in ECs (CDH5-MAPK mice) and found that CDH5-MAPK mice manifested a profound decline in their HSC and progenitor potential resulting from loss of vascular integrity and increased inflammation in BMECs. Additionally, we found that CDH5-MAPK mice resulted in an increase in inflammation and a decrease in overall frequency in other critical BM niche cells, including Lepr+, Nestin+, and osteoblast cells. Suppression of the canonical NF-kB signaling pathway in ECs of CDH5-MAPK mice led to a complete restoration of their hematopoietic deficiencies and restored the proper function, inflammatory profile, and frequency in all BM niche cells. We analyzed expression of known pro-HSC factors, such as KitL and Cxcl12, and found no differences within BMECs or other BM niche cells. However, proteomic analysis of CDH5-MAPK mice revealed the deficiency of a novel, plasma-borne pro-hematopoietic factor, Stem Cell Growth Factor-a (SCGF), which was restored following EC-specific inhibition of NF-kB signaling. SCGF was recently described as a potent osteogenic factor than can rejuvenate bone homeostasis in aged mice. Analysis of bone health in CDH5-MAPK mice revealed a significant defect in bone homeostasis, suggesting a critical need in maintaining the proper cellular cross-talk between BMECs and other BM niche constituents. Infusion of recombinant SCGF into CDH5-MAPK mice led to a complete recovery of their functional HSC and progenitor defects, restoration of their vascular integrity and bone health, and suppression of inflammation within the microenvironment. Furthermore, we demonstrated that infusion of SCGF has potent therapeutic properties that enhance hematopoietic recovery and increase the functionality of the HSC following myelosuppressive insult in both control and CDH5-MAPK mice. Utilizing our genetic models, we were able to establish a role for SCGF in supporting the instructive capacity of the endothelial niche, and their interaction with other critical components of the HSC niche, to facilitate the maintenance and expansion of HSCs and improve their functional output following hematopoietic insults. We anticipate that future studies directed towards a thorough characterization of the core endothelial signaling networks will offer novel therapeutic approaches to accelerate the regeneration of the HSC pool by promoting vascular regeneration and enhancing the pro-HSC function of BMECs. These therapies could be utilized following myeloablation as well as to expand transplantable, repopulating HSCs ex vivo, thereby ameliorating life-threatening pancytopenia associated with chemo-irradiation regimens. DisclosuresNo relevant conflicts of interest to declare.

Vascular E-Selectin Acts As a Gatekeeper Inducing Commitment and Loss of Self-Renewal in HSC Transmigrating through the Marrow Vasculature

Hematopoietic stem cells (HSC) reside in specific peri-vascular niches in the bone marrow (BM). We have shown interactions with the inflammatory vascular adhesion molecule E-(endothelial)-selectin awakens HSC (Nat Med 2012). We now report that BM vascular cell-surface E-selectin expression is strongly upregulated during HSC mobilization regimens raising the question of a role for endothelial E-selectin in HSC transplant outcome.G-CSF was administered to cohorts of E gene-deleted or wildtype C57BL/6 mice together with E-selectin antagonist Uproleselan (Upro, GMI-1271). We found absence (Sele-/- mice) or therapeutic blockade (Upro) of E-selectin alone in steady-state hosts did not alter levels of circulating peripheral blood (PB) HSC. In contrast absence or therapeutic blockade of E-selectin strongly synergized with mobilizing regimens such as G-CSF or cyclophosphamide+G-CSF by boosting long-term engraftment and reconstitution potential of mobilized blood. The effect was pronounced boosting reconstitution potential over G-CSF-alone-mobilized blood 24-fold (<p=0.0001; analyzed by Poisson Statistics following serial-dilution long-term competitive-reconstitution [LT-CR] transplantation assays of 4 PB dilutions). Despite the increased reconstitution potential no change in mobilized phenotypic HSC nor in colony forming cell numbers were observed. Together these results suggest the significant 24-fold boost in peripheral blood reconstitution potential induced by Upro + G-CSF is due to increases in HSC potency alone.Two hypotheses may explain how therapeutic E-selectin-blockade promotes potency of mobilized HSC;•Dampening of inflammatory activation at the BM niche induced by G-CSF that drive HSC exhaustion,•Directly preventing adhesion-mediated HSC commitment during trans-endothelial transmigration from BM into blood.To investigate inflammatory mediator profiles in the BM, cohorts of mice were administered saline control or G-CSF ± Upro (125ug/kg G-CSF, 40mg/kg Upro BiD for 3 days) then femoral BM fluids flushed for cytokine profiling (LegendPlex). As anticipated, G-CSF administration significantly increased concentration of classic pro-inflammatory cytokines (TNF-α, IL-1β, IL-23, IFN-β) in BM associated with HSC activation and loss of quiescence in the BM. No similar boost in inflammatory mediators was observed in BM from mobilized mice co-administered Upro. Similarly pronounced metabolic changes could be observed in BM HSC following in vivo G-CSF administration (such as a significant doubling in HSC Mitochondrial Membrane potential [MOMP] suggesting increased energy demands with HSC activation) was similarly reversed in vivo by Upro co-administration. Together these results suggest blockade of E-selectin on activated vasculature significantly dampens BM HSC activation following G-CSF administration - potentially shielding HSC self-renewal potential.Next we investigated whether the transient interactions with endothelial E-selectin during trans-endothelial transmigration from BM into blood also directly affects HSC potency. BM HSPC harvested from G-CSF plus Upro-treated mice were loaded in transwells pre-coated with recombinant adhesion molecules (P-selectin, E-selectin and CD14-Fc as control) and HSC induced to transmigrate through coated pores towards CXCL12 gradient. After 3hr cell numbers were enumerated and exactly 100 transmigrated HSC from each well transplanted/recipient in a LT-CR transplant assay. Analysis of % CD45.2+ donor HSC reconstitution confirmed a pronounced 10-fold drop in reconstitution potential of HSC transmigrated through E-selectin compared to P-selectin or control coated transwells (p=0.0027) confirming that transient adhesive interactions with E-selectin, such as would occur on activated vasculature during HSC transmigration from BM into the blood, strongly impact subsequent HSC reconstitution upon transplantation.In summary, transient interactions between intravasating HSC with E-selectin on BM vasculature inadvertently compromises reconstitution potential of up to 96% of conventionally harvested HSC, indicating an unexpected disadvantage with current HSC harvesting procedures. These studies also point the way forward to a simple remedy, administration of E-selectin antagonist (Upro) together with G-CSF during HSC mobilization, to improve HSC transplant outcomes. DisclosuresWinkler:GlycoMimetics: Patents & Royalties. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Marlton:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Perkins:Novartis Oncology: Honoraria. Levesque:GlycoMimetics: Equity Ownership, Patents & Royalties.

Pathogenesis Study of Down Syndrome Prone to Leukemia Using iPSCs from Trisomy 21 Patients

AimsDown syndrome (DS) is the most frequent single cause of human birth defects and intellectual disability (ID). Its etiology has been known for over 50 years, DS is caused by trisomy of chromosome 21 (Ts21). In addition, there are more evidences in clinical show that patients of DS are also have high incidence of leukemia, but its underlying mechanisms have yet to be discovered. Elucidation of these mechanisms has been hindered by the difficulties in isolating and expanding enough hematopoietic stem cells (HSCs) from the patients. Circular RNAs (CircRNAs) are a novel type of endogenous noncoding RNAs that reported to play important roles in biological and pathological processes. Our present study aims to investigate the pathogenesis of Down Syndrome prone to leukemia using iPSCs from DS patients and the modulation mechansim of key target gene and circRNAs.MethodsTo overcome the limitation of clinical sample resources, we developed Ts21-induced pluripotent stem cells (iPSCs) from mononuclear cells using Episomal vectors which highly express Oct4,Sox2 and Klf4. Under certain conditions, these Ts-21 specific iPSCs could be further induced into any cell types, including HSCs. Next, we induced the patient-specific iPSCs differentiate into HSCs under serum free and feeder-free condition in vitro. By using flow cytometry, qRT-PCR and CFU assays, we have made well comparisons between HSCs derived from TS-21 iPSCs and control iPSCs, and expolre the pathological feature and mechanisms. By performing circRNA-seq analysis, we identified key circRNA and its host gene regulatory networks in DS with hematopoietic disorder. On the basis of that, we can further search for the potential drug targets that effective against leukemia of DS patients.ResultsWe show here by gene sequencing, karyotyping and immunohistochemistry staining, the initial characterization of the patient-specific induced pluripotent stem are confirmed to be fully reprogrammed and they still carry the original mutation. These cells can be used for screening approaches and can be modified for loss- and gain-of-function strategies to identify pathways and test candidate mechanisms. We have observed that myeloid differentiation potential of HSCs derived from DS patients was significantly higher than normal patinets. DS-iPSCs exhibited a 2-4 fold increase in a population of CD43+/GPA+ hematopoietic cells, accompanied by increased multilineage colony-forming potential in CFU assays.During this process, has_circ_0082802 was significantly up-regulated. After knockdown of has_circ_0082802 using shRNAs, the erythroid differentiation level as well as cell proliferation were decreased. The potential modulation mechansim of them will be further explored.ConclusionWe established the cellular models of DS patients and derived differentiation into different cell types in vitro, which has the similar function with patient HSCs. On the basis of that, we found has_circ_0082802 may play important role in erythroid differentiation. We can further search for the potential drug targets thateffective against leukemia of DS patients. This approach would provide a powerful cell resource for clinical research and a useful model for the study of the mechanisms of DS-AML DisclosuresNo relevant conflicts of interest to declare.

Single Cell Transcriptomics Maps the Embryonic Emergence of HSC and Identifies Intercellular Interactions Regulating HSC Genesis

Hematopoietic stem cells (HSC) are generated during a transient window of embryonic development from endothelial-like hemogenic precursors within specific arterial vessels such as the aorta of the AGM (aorta-gonad-mesonephros region). During HSC emergence, hemogenic precursors must acquire and maintain HSC-defining properties such as the ability to self-renew, home, and provide multilineage hematopoiesis, properties which distinguish rare HSC from a multitude of other hematopoietic progenitors arising simultaneously in the developing embryo. However, the precise niche-derived signals necessary and sufficient to support the acquisition and maintenance of these properties remains poorly defined. Toward identification of these signals, we generated a platform consisting of endothelial cells from the embryonic AGM (AGM-EC) which supports the specification and self-renewal of engrafting HSC from clonal embryo-derived hemogenic precursors in vitro. Using this platform to assay functional HSC potential at the single cell level, we determined a phenotype (VE-Caderin+CD61+EPCRhigh) that encompasses the population of hemogenic precursors during their asynchronous transition to HSC between E9.5 and E11.5 in murine embryonic development. To elucidate the transcriptional changes associated with the emergence of HSC from hemogenic precursors, we analyzed the global transcriptional profiles of FACS-purified VE-Caderin+CD61+EPCRhigh cells at various stages of embryonic development by single cell RNA-sequencing and reconstructed their developmental trajectory in “pseudotime” based on incremental changes in their transcriptional profiles. Complementary analysis of AGM-EC by bulk and single cell RNA-sequencing revealed a unique transcriptional profile of niche endothelial cells supporting HSC development enriched for immune/inflammatory signals. Combining the transcriptional profiles of emerging HSC with niche AGM-EC, we have identified candidate ligand-receptor pairs regulating intercellular interactions during HSC specification and self-renewal and have begun to validate the functional importance of these interactions in supporting HSC generation from hemogenic precursors in vitro. We expect these studies will enhance our understanding of the unique signal pathways necessary for the development of functional HSC, a critical step toward engineering HSC in vitro for clinical applications in disease modeling, drug discovery, and gene modification to identify novel therapies for hematologic and immunologic disorders. DisclosuresRafii:Angiocrine Bioscience: Equity Ownership.

Chemotherapy-induced niche perturbs hematopoietic reconstitution in B-cell acute lymphoblastic leukemia

BackgroundConsiderable efforts have been devoted toward the uncovering of the molecular mechanisms underlying the maintenance of hematopoietic stem cells (HSCs) by the normal bone marrow (BM) niche. Previously, we demonstrated that a chemotherapy-induced niche, which is mainly composed of mesenchymal stem cells (MSCs), protects the residual B-cell acute lymphoblastic leukemia (B-ALL) cells from the insult of chemotherapeutic drugs. However, the roles of chemotherapy-induced niche on HSCs functions in B-ALL remain unclear.MethodsWe established an oncogenic N-MYC-driven B-ALL mouse model, which were subsequently treated with common chemotherapy drug cytarabine (Ara-C) and daunorubicin (DNR). After treatment, the structures of the BM niche were imaged by immunofluorescence staining. Then, the self-renewal and differentiation capability of the MSCs in the BM after Ara-C and DNR treatment were studied by ex vivo culture and gene expression analysis with RNA-seq and qRT-PCR. The effects of chemotherapy-induced niche on the hematopoietic reconstitution of HSCs were determined with series transplantation assay. Furthermore, the cell cycle, ROS level, mitochondrial membrane potential and cell apoptosis of HSCs were detected by flow cytometry.ResultsThe MSCs, which is the main component of chemotherapy-induced BM niche, have decreased self-renewal capability and are prone to differentiate into adipocytes and chondrocytes. The results of gene expression analysis with RNA-seq showed that the MSCs have reduced levels of cytokines, including SCF, CXCL12, ANGPT1, VCAM1, and IL7. Furthermore, the chemotherapy-induced niche perturbed the hematopoietic reconstitution of HSCs in our N-MYC-driven B-ALL mouse model by promoting HSCs to enter cell cycle and increasing intracellular ROS levels and mitochondrial membrane potential of HSCs, which lead to the cell apoptosis of HSCs.ConclusionsChemotherapy-induced BM niche perturbs the hematopoietic reconstitution of HSCs by increasing intracellular ROS level and inducing cell apoptosis.

Motives for joining an unrelated HSC donor registry: description, categorization, and association with donor availability.

A challenge for registries of unrelated hematopoietic stem cell (HSC) donors is to recruit and retain sufficient donors for patients needing transplants. Many registered HSC donors opt-out when called as a potential match for a patient. Anecdotal evidence suggests that motives for joining a registry may be linked to the donation decision. The primary goals of this investigation were to describe the range of motives for joining a registry and to examine donor availability by motive type. A diverse sample of 357 potential HSC donors from the Anthony Nolan registry in the UK was asked about their motives for joining and their decision to continue or opt-out after matching a patient was recorded. Motives for joining (N = 557) were first coded and categorized into 17 specific motive types and then arranged along a spectrum from internal to external. Internal values-based motives were most prevalent and availability was highest among potential donors expressing these motives (92%) and lowest among those expressing external motives (pressure, incentives; 0%). Although further research is needed to confirm these findings, they suggest that registries should assess donors' motives for joining at recruitment in order to conduct follow-up to ensure commitment among those potential members expressing external motives.

3015 - Hierarchically Related Lineage-Restricted Fates of Multipotent Hematopoietic Stem Cells

Self-renewing bone marrow hematopoietic stem cells (HSCs) secure blood cell replenishment in homeostasis and upon stress stimuli. Single cell transplantations uncovered considerable heterogeneity among HSCs (Müller-Sieburg et al. Blood 2002; Dykstra et al. Cell Stem Cell 2007; Challen et al. Cell Stem Cell 2010), also supported by findings in unperturbed hematopoiesis (Busch et al. Nature 2015; Pei et al. Nature 2017; Rodriguez-Fraticelli et al. Nature 2018). Expression of the megakaryocytic (Mk) von Willebrand factor gene (Vwf) enriches for platelet-biased HSCs (Sanjuan-Pla et al. Nature 2013). Some studies also suggested direct generation of unipotent self-renewing Mk progenitors within the phenotypic HSC compartment, implicating uncoupling between self-renewal and multipotency (Yamamoto et al. Cell 2013; Haas et al. Cell Stem Cell 2015). In this study (Carrelha et al. Nature 2018), sensitive tracking of progenitors and mature cells of the Mk/platelet (P), erythroid (E), myeloid (M), B and T cell lineages upon single HSC transplantation revealed a stable hierarchical framework for lineage-restricted fates of HSCs. The Vwf- HSC compartment includes long-term (LT) and short-term (ST) multilineage HSCs, with lymphoid-biased output resulting from ST-HSC exhaustion. In addition to multilineage HSCs, the Vwf+ compartment includes distinct subsets adopting PEMB-restricted, PEM-restricted, PE-restricted and P-restricted fates. Sustained self-renewal potential and unfulfilled multipotency of these fate-restricted HSCs is revealed through serial transplantation and in vitro assays, respectively. The only unilineage-fate subset of serially-transplantable HSCs replenish exclusively the Mk lineage branch of the hematopoietic tree, and no LT-HSC subset was found contributing exclusively to another single lineage. Genetic lineage tracing with Vwf-Cre inducible mouse model also supports an important role for platelet-biased HSCs in unperturbed hematopoiesis. Our findings uncover a limited repertoire of LT-HSC subsets, defined by hierarchically organised propensities for replenishment of a restricted set of blood lineages, prior to loss of self-renewal and multipotency.

Lineage tracing of murine adult hematopoietic stem cells reveals active contribution to steady-state hematopoiesis

AbstractCharacterization of hematopoietic stem cells (HSCs) has advanced largely owing to transplantation assays, in which the developmental potential of HSCs is assessed generally in nonhomeostatic conditions. These studies established that adult HSCs extensively contribute to multilineage hematopoietic regeneration upon transplantation. On the contrary, recent studies performing lineage tracing of HSCs under homeostatic conditions have shown that adult HSCs may contribute far less to steady-state hematopoiesis than would be anticipated based on transplantation assays. Here, we used 2 independent HSC-lineage–tracing models to examine the contribution of adult HSCs to steady-state hematopoiesis. We show that adult HSCs contribute robustly to steady-state hematopoiesis, exhibiting faster efflux toward the myeloid lineages compared with lymphoid lineages. Platelets were robustly labeled by HSCs, reaching the same level of labeling as HSCs by 1 year of chase. Our results support the view that adult HSCs contribute to the continuous influx of blood cells during steady-state hematopoiesis.

HDAd5/35++ Adenovirus Vector Expressing Anti-CRISPR Peptides Decreases CRISPR/Cas9 Toxicity in Human Hematopoietic Stem Cells

We generated helper-dependent HDAd5/35++ adenovirus vectors expressing CRISPR/Cas9 for potential hematopoietic stem cells (HSCs) gene therapy of β-thalassemia and sickle cell disease through re-activation of fetal γ-globin expression (HDAd-globin-CRISPR). The process of CRISPR/Cas9 gene transfer using these vectors was not associated with death of human CD34+ cells and did not affect their in vitro expansion and erythroid differentiation. However, functional assays for primitive HSCs, e.g., multi-lineage progenitor colony formation and engraftment in irradiated NOD/Shi-scid/interleukin-2 receptor γ (IL-2Rγ) null (NSG) mice, revealed toxicity of HDAd-globin-CRISPR vectors related to the prolonged expression and activity of CRISPR/Cas9. To control the duration of CRISPR/Cas9 activity, we generated an HDAd5/35++ vector that expressed two anti-CRISPR (Acr) peptides (AcrII4 and AcrII2) capable of binding to the CRISPR/Cas9 complex (HDAd-Acr). CD34+ cells that were sequentially infected with HDAd-CRISPR and HDAd-Acr engrafted at a significantly higher rate. Target site disruption frequencies in engrafted human cells were similar to those in pre-transplantation CD34+ cells, indicating that genome-edited primitive HSCs survived. In vitro differentiated HSCs isolated from transplanted mice demonstrated increased γ-globin expression as a result of genome editing. Our data indicate that the HDAd-Acr vector can be used as a tool to reduce HSC cytotoxicity of the CRISPR/Cas9 complex.

Thymic B cell development is controlled by the B potential of progenitors via both hematopoietic-intrinsic and thymic microenvironment-intrinsic regulatory mechanisms.

BackgroundHematopoietic stem cells (HSCs) derived from birth through adult possess differing differentiation potential for T or B cell fate in the thymus; neonatal bone marrow (BM) cells also have a higher potential for B cell production in BM compared to adult HSCs. We hypothesized that this hematopoietic-intrinsic B potential might also regulate B cell development in the thymus during ontogeny.MethodsFoxn1lacZ mutant mice are a model in which down regulation of a thymic epithelial cell (TEC) specific transcription factor beginning one week postnatal causes a dramatic reduction of thymocytes production. In this study, we found that while T cells were decreased, the frequency of thymic B cells was greatly increased in these mutants in the perinatal period. We used this model to characterize the mechanisms in the thymus controlling B cell development.ResultsFoxn1lacZ mutants, T cell committed intrathymic progenitors (DN1a,b) were progressively reduced beginning one week after birth, while thymic B cells peaked at 3–4 weeks with pre-B-II progenitor phenotype, and originated in the thymus. Heterochronic chimeras showed that the capacity for thymic B cell production was due to a combination of higher B potential of neonatal HSCs, combined with a thymic microenvironment deficiency including reduction of DL4 and increase of IL-7 that promoted B cell fate.ConclusionOur findings indicate that the capacity and time course for thymic B-cell production are primarily controlled by the hematopoietic-intrinsic potential for B cells themselves during ontogeny, but that signals from TECs microenvironment also influence the frequency and differentiation potential of B cell development in the thymus.

Inflammation-Associated Cytokines IGFBP1 and RANTES Impair the Megakaryocytic Potential of HSCs in PT Patients after Allo-HSCT

Prolonged isolated thrombocytopenia (PT) is a severe complication in patients after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Whether the megakaryoctic potential of hematopoietic stem cells (HSCs) in bone marrow is intact and what factors drive the pathological process of PT remain elusive. A retrospective study in patients (n = 285) receiving HSCT revealed that the occurrence of PT was approximately 8% and the number of platelets and megakaryocytes in PT patients is much lower compared with control subjects. To test whether the deficiency of thrombopoiesis was caused by the activities of HSCs, the megakaryocytic differentiation potential of HSCs before or after transplantation was assessed. Interestingly, a substantial decrease of megakaryocytic differentiation was observed 2 weeks after transplantation of HSCs in all of the allo-HSCT recipients. However, 4 weeks after transplantation, the ability of HSCs to generate CD41+CD42b+ megakaryocytes in successful platelet engraftment patients recovered to the same level as those of HSCs before implantation. In contrast, HSCs derived from PT patients throughout the postimplantation period exhibited poor survival and failed to differentiate properly. A protein array analysis demonstrated that multiple inflammation-associated cytokines were elevated in allo-HSCT recipients with PT. Among them, insulin-like growth factor–binding protein 1 and regulated on activation, normal T cell expressed and secreted were found to significantly suppress the proliferation and megakaryocytic differentiation of HSCs in vitro. Our results suggested that the occurrence of PT may be attributed, at least partially, to the damage to HSC function caused by inflammation-associated cytokines after HSCT. These findings shed light on the mechanism underlying HSC megakaryocytic differentiation in PT patients and may provide potential new strategies for treating PT patients after HSCT.

Junctional Adhesion Molecule 2 Intensifies T Lymphopoiesis of Hematopoietic Stem Cells By Facilitating Notch/Delta Signaling

Phenotypically described hematopoietic stem cells (HSCs) represent a functionally heterogeneous pool of primitive cells with conceivable potential to replenish and maintain the whole hematopoietic system. The diverse lineage potential of HSCs is supposed to play a significant role in the response to different kinds of hematopoietic stress. Since subcategorization of HSCs biased towards specific lineage(s) highly relies on the retrospective information, e.g. transplantation assay, exploring additional markers will allow us to understand further molecular mechanisms of HSC regulation such as activation and lineage choice but also the degree of correlation between them.Here, we show that the cell surface protein Junctional adhesion molecule 2 (Jam2) serves as an amplifier of the Notch/Delta signal thereby representing the higher T cell potential of HSCs. Flow cytometry analyses revealed that a subset of CD150+CD48-KSL cells in mouse bone marrow (BM) were positive for Jam2 (Jam2+HSC, 36.6 ±13.0%), while other Jam family member, Jam1 (F11r), was expressed on all HSCs and Jam3 was not detected. Transplantation assay using 30 Jam2+ or Jam2-HSCs revealed that Jam2+HSCs reconstituted lethally irradiated mice more efficiently than Jam2-HSCs (77.5 ±15.9 and 51.7 ±29.3% in peripheral blood, respectively). Lineage analyses revealed that Jam2+HSCs have a greater potential in lymphoid cell reconstitution, particularly T cells, whereas the chimerism in myeloid cells was not significantly different from Jam2-HSCs. This tendency of higher contribution to the T cell development was even more pronounced in the secondary transplantation experiments, where the contribution of Jam2+HSCs in T cells was close to 100%. Of note, most of Jam2+HSCs were in a dormant state, suggesting that the T cell potential of Jam2+HSCs is independent of the cell cycle progression.Jam2 has been reported to mediate the Notch signaling through an interaction with Jam1 (Kobayashi et al., Nature, 2014). In addition, Jam2+HSCs express Notch1 at a higher level than Jam2-HSCs (23.6 ±6.7 and 9.05 ±5.8%, respectively). We therefore analyzed the functional role of Jam2 in the Notch/Delta-oriented T cell production using a competitive feeder-free T cell culture system. At a low concentration of DLL1, that is insufficient to promote T cell production by itself, Jam2+HSCs effectively produced T cell lineages only in the presence of recombinant Jam1 protein, but not Jam2 or Jam3. In contrast, Jam2+HSCs did not require Jam1 protein with a higher concentration of DLL1. These differences were not observed with Jam2-HSCs, indicating that Jam2/Jam1 interaction amplifies Notch signal transduction and is crucial for the subsequent T cell specification of Jam2+HSCs.To elucidate the molecular signature of Jam2+HSCs, gene expression profiling was performed using a microarray analysis. Gene set enrichment analysis (GSEA) observed that Jam2+HSCs were significantly enriched for common lymphoid progenitor (CLP) and early T cell gene expression. Of note, Jam2+HSCs were also enriched for E2F target genes, G2M checkpoint genes and glycolysis related genes, which potentially explains the reason why Jam2+HSCs display a bivalent phenotype: being more dormant compared to Jam2-HSCs at the steady state but at the same time having the capacity to reconstitute more actively upon engraftment.Since Jam2 positivity correlates to T cell potential, we asked if altered T lymphopoietic environment affects the proportion of Jam2+HSCs. In vivo T cell depletion resulted in significantly higher frequency of Jam2+HSCs but not upon other stress inducers, such as 5-FU treatment, suggesting that the increase in Jam2+HSC pool was specifically due to the T cell deficiency. These findings indicate that the lack of T cells, which also means a requirement for immediate T cell replenishment, leads to an increase of Jam2+HSC fraction.Our findings suggest that Jam2 is the key protein that controls T lymphopoiesis by enhancing the Notch/Delta signal transduction via interaction with Jam1. It also means that the lineage balance particularly towards T lymphopoiesis might be regulated at a higher stage of hematopoietic hierarchy than currently understood. Thus, Jam2 is a new marker representing the T lymphocyte potential of HSCs, as the frequency of Jam2+HSCs sensitively reflects the state of the T cell environment. DisclosuresNo relevant conflicts of interest to declare.

Stag2 regulates Hematopoietic Differentiation and Self-Renewal

Cohesin is a multimeric protein complex, which has been initially implicated in the cohesion of replicated sister chromatids but more recently shown to be involved in the long-range regulation of gene expression by stabilizing 3-dememsional structure of the genomic DNA. Recently, multiple components of the cohesin complex has been shown to be a recurrent target of somatic mutations in various myeloid malignancies, in which STAG2 is most frequently mutated and undergoes loss-of-function. However, the leukemogenic mechanism of mutated- STAG2 has not been fully understood, although recent studies have reported deregulated differentiation and enhanced self-renewal of STAG2 -mutated stem cells.To investigate the functional role of STAG2 in leukemogenesis as well as normal hematopoiesis, we generated Stag2 conditional knockout (cKO) mice with an Mx1 -cre allele, in which Stag2 deletion was induced by polyIC injection. When assessed in the peripheral blood of 12 to 20 week-old mice, the complete blood count showed no significant changes between Stag2 cKO mice and controls. No morphological alterations were observed in the peripheral blood as well as in the bone marrow. However, in the spleen, the extramedullary hematopoiesis was evident, exhibiting increased myeloid progenitors and erythroblasts. In repeated methylcellulose cultures, Stag2 deficient BM cells showed an enhanced serial replating capacity, suggesting an increased self-renewal potential in Stag2 deficient hematopoietic stem cells (HSCs) in vitro.Flow cytometry of bone marrow cells revealed increased numbers of hematopoietic stem and progenitor cells (HSPCs) defined as Lineage−Sca-1+Kit+(LSK) cells in Stag2 cKO mice compared with controls. Within the myeloid progenitor (MP) compartment, we observed increased common myeloid progenitors (CMPs), while decreased megakaryocyte/erythroid (MEPs) and common lymphoid progenitors (CLPs), compared to controls. Moreover, CD11b+Gr-1+ mature myeloid cells were significantly increased in the bone marrow of Stag2 cKO mice. These results suggest that Stag2 deficiency causes myeloid skewing.In competitive bone marrow transplantation assays assessing the reconstitution potential, Stag2 cKO-derived cells showed reduced chimerism in the peripheral blood compared to wild-type mice-derived cells. However, the reduced chimerism of Stag2 cKO-derived cells was largely confined to B-lymphocytes showing a severe reduction, while the chimerism of Stag2 cKO-derived myeloid cells was not affected compared to controls. In the bone marrow, by contrast, the chimerism of Stag2 cKO cells was not significantly changed, but rather tended to show increased numbers in the LSK, CMP and granulocyte/macrophage progenitor (GMP) fractions, while the MEP and CLP fractions were reduced. These results suggest that Stag2 deficiency could enhance the self-renewal capacity of HSCs in vivo, but Stag2 -deleted cells may not uniformly contribute to all hematopoietic cell fractions probably due to the impaired differentiation of these HSCs.Next, we assessed the effect of Stag2 -deficiency on gene expression, where RNA sequencing of Stag2 -deleted HSPCs revealed a subset of genes differentially expressed between Stag2 WT and cKO cells. Including key myeloid-specific regulators, these genes were considered to be potential gene targets of Stag2, deregulation of which is implicated in the abnormal hematopoiesis of Stag2 cKO mice. Given that the cohesin complex is known to be involved in the establishing and maintaining DNA accessibility, we also assessed the global chromatin accessibility by assays for transposase accessible chromatin with sequencing (ATAC-seq) of Stag2 -deleted HSPCs. Of interest, chromatin accessibility in Stag2 -deleted cells was increased for genes implicated in myeloid differentiation, whereas reduced for those in lymphoid differentiation. Motif analysis of more accessible regions in Stag2 -deleted cells revealed an enrichment of the binding site for the transcription factor Runx1, which is known to regulate HSC differentiation.Our results demonstrate that Stag2 loss leads to the impaired hematopoietic differentiation and enhances the self-renewal potential of HSCs through the modulation of chromatin accessibility and consequent abnormal gene expression, possibly contributing to leukemogenesis. DisclosuresTakaori-Kondo:Novartis: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Research Funding; Janssen Pharma: Honoraria; Pfizer​: Honoraria.

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells.

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis.These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3β inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Directed Differentiation of Primitive and Definitive Hematopoietic Progenitors from Human Pluripotent Stem Cells

One of the major goals for regenerative medicine is the generation and maintenance of hematopoietic stem cells (HSCs) derived from human pluripotent stem cells (hPSCs). Until recently, efforts to differentiate hPSCs into HSCs have predominantly generated hematopoietic progenitors that lack HSC potential, and instead resemble yolk sac hematopoiesis. These resulting hematopoietic progenitors may have limited utility for in vitro disease modeling of various adult hematopoietic disorders, particularly those of the lymphoid lineages. However, we have recently described methods to generate erythro-myelo-lymphoid multilineage definitive hematopoietic progenitors from hPSCs using a stage-specific directed differentiation protocol, which we outline here. Through enzymatic dissociation of hPSCs on basement membrane matrix-coated plasticware, embryoid bodies (EBs) are formed. EBs are differentiated to mesoderm by recombinant BMP4, which is subsequently specified to the definitive hematopoietic program by the GSK3β inhibitor, CHIR99021. Alternatively, primitive hematopoiesis is specified by the PORCN inhibitor, IWP2. Hematopoiesis is further driven through the addition of recombinant VEGF and supportive hematopoietic cytokines. The resulting hematopoietic progenitors generated using this method have the potential to be used for disease and developmental modeling, in vitro.

Frequencies of gene variant CCR5-Δ32 in 87 countries based on next-generation sequencing of 1.3 million individuals sampled from 3 national DKMS donor centers

Homozygous carriers of CCR5-Δ32, a gene variant of CC-type chemokine receptor 5 (CCR5), are highly resistant to infections with human immunodeficiency virus type 1 (HIV-1) and therefore preferred stem cell donors for HIV-infected patients. We analyzed CCR5 typing data of 1,333,035 potential hematopoietic stem cell donors enlisted with three national DKMS donor centers. Allele and genotype frequencies were determined for 87 countries of origin as self-assessed by the donors. CCR5-Δ32 allele frequencies ranged from 16.4% in the Norwegian sample to 0 in donors from Ethiopia. The highest CCR5-Δ32/Δ32 genotype frequency was found in the sample from the Faroe Islands (2.3%), whereas in 27 samples, predominantly of donors from Africa, Asia and South America, none of the individuals carried this genotype. The characteristic CCR5-Δ32 allele frequency decline from Northern to Southeastern Eurasia supports findings of earlier studies. With available HLA haplotype frequency information for the patient’s ethnicity, our data allows upfront estimation of the probability that an HLA-matched donor with CCR5-Δ32/Δ32 genotype can be found for a patient in need of hematopoietic stem cell transplantation.

P248 Next generation sequencing reveals a novel hla-a*24 allele associated with hla-dqb1*06:10 in asians

Aim Next Generation Sequencing (NGS) for HLA typing improves not only the resolution of typing results but also aids in the discovery of novel alleles. Here we present a novel HLA-A∗24 allele associated with HLA-DQB1∗06:10, which was found in an Asian family and an unrelated donor. Methods Genomic DNA samples isolated from a patient, family members and potential unrelated donors were tested by reverse sequence specific oligonucleotide probes (rSSOP), sequence based typing (SBT) and NGS technology. In NGS typing, genomic DNA was amplified using the Illumina TruSight HLA v2 Sequencing Panel kit. After normalization, fragmentation/tagmentation and dual-indexing procedures, the amplicons were pooled onto the MiSeq sequencer to facilitate cluster generation and sequencing by synthesis on the flow cell. The generated data was then analyzed with Illumina TruSight HLA Assign software. Results A patient being evaluated for a potential hematopoietic stem cell transplant (HSCT) was typed by Sanger sequencing and found to be homozygous at HLA-A, B, C and DRB1. By Luminex LabType, the patient was heterozygous at DQA1, DQB1 and DPB1. However, subsequent NGS typing revealed heterozygousity for HLA-A, with an expected A∗24:02:01 and a novel A∗24 with a mutation in exon 6 which resulted in an amino acid change from lysine to glutamic acid. The same mutation was also found in one of his children and one of four unrelated donors. The patient, child and unrelated donor who had the HLA-A∗24:NEW allele all self-identified as Asian. In all three cases, the HLA-A∗24:NEW allele was associated with HLA-DQB1∗06:10; this association is found only in Asians at low frequency. Conclusions This HLA-A∗24:NEW allele may be associated with DQB1∗06:10 in Asians. Implementing NGS technology in clinical practice will not only allow us to choose the best donor from many potentially matched donors but also will fill in gaps in HLA databases to improve our typing.

Age-associated clonal dominance of myeloid-biased HSC is underwritten by unique transcriptional and epigenetic alterations

Age-associated changes of the functional potential of hematopoietic stem cells (HSCs) are believed to underlie a variety of age-dependent blood cell pathophysiologies and contribute to the overall decline of the hematopoietic system. Modifications of the epigenetic landscape, DNA damage accumulation, and changes in cell polarity have all been identified as potential factors contributing to the diminished functional potential of aged HSCs. Additionally, changes in the HSC’s clonal composition, with a predominance of myeloid-biased clones in the aged compartment, have been implicated in the reduction of immune competence and elevated predisposition to myelogenous diseases in the elderly. To explore age-associated changes in the lineage-biased subsets, we analyzed genome-wide expression profiles of the CD150 subsets of HSCs isolated from young and aged mice and defined transcripts with expression patterns unique to each aging subset of hematopoietic stem cells. We also established the epigenetic profiles of the aging CD150 subsets and defined significant alterations in the DNA methylation patterns between the subsets and also during aging. The methylation and transcriptional profiles were then correlated to establish if epigenetic changes contributed to the altered expression in the HSC subsets. We also demonstrated that there is no significant difference in the DNA damage accumulation of the aged lineage-biased subsets when assaying for strand break accrual. Finally, using time-lapse microscopy on cells purified from the HSC reporter mouse (Fgd5ZsGreen-ZsGreen/+), we have examined the lineal hierarchy and defined the symmetric and asymmetric division preferences of the CD150 subsets. Taken together, we have defined the transcriptional and epigenetic profiles that underlie the age-associated dominance of the myeloid-biased HSCs.

Phenotype and Function of Human Gut Hematopoietic Stem Cells and Progenitors

Lin−CD45+CD34+ cells have been reported in human small intestine and liver, but further phenotypic or functional analysis was not described[1][2]. We utilized flow cytometry and cytometry by time of flight (CyTOF) to study the composition and phenotype of human gut hematopoietic stem cells (HSCs) and hematopoietic progenitors (HPs) from deceased donors. Colony-forming-cell (CFC) assays, long-term-culture-initiating-cell (LTC-IC) assays and humanized mouse models were established to further evaluate the differentiation potential of gut HSCs/HPs. HSCs and multiple types of HPs, including multipotent progenitors (MPP), lymphoid-primed multipotent progenitors (LMPP), common lymphoid progenitors (CLP), myeloid progenitors (MP), and unclassified progenitors co-expressing CD56, were detected in human intestinal mucosa and mesenteric lymph nodes (MLNs). Ileum lamina propria lymphocytes (LPL) included significantly higher percentages of HSCs than intraepithelial lymphocytes (IEL). Both IEL and LPL showed significantly lower percentages of CLP compared to MLNs. From jejunum or ileum specimens of ITx patients collected from their stoma revision/closure (POD47- 1544), variable levels of Lin−CD45+CD34+ cells were detected (Figure 1A). However, when gating on HSCs, donor cells (>90%) predominated over recipient cells in four patients with chimerism, regardless of their ages or early clinical outcomes (Figure 1B-C). Lin−CD45+CD34+ cells from ileum LP and bone marrow (BM) demonstrated similar properties in viSNE plots from CyTOF analysis (Figure 2), suggesting similar functional phenotypes. Both ileum LP and BM Lin−CD45+CD34+ cells contained subpopulations expressing the thymus-homing marker CCR9, and T/NK lineage polarized progenitors coexpressing CD45RA and CD7. Additionally, CFC assays showed that both ileum LP and BM Lin−CD34-enriched cells can form myeloid/erythroid colonies, although ileum LP CD34 cells were less efficient than BM CD34 cells. LTC-IC assay using single cell sorted ileum LP HSC co-culture with murine feeder cells suggested myeloid/lymphoid differentiation potential of human gut HSCs. Experiments using human gut Lin−CD45+CD34+ cells to reconstitute humanized mice are ongoing.FIGURE 1: (A) Lin−CD45+CD34+ cells were detected in human intestinal mucosa long-term post-Tx (POD47-1544) (B) Donor-derived hematopoietic stemcells (HSCs) and/or multiple types of hematopoietic progenitors (HPs)were detected in transplanted small bowel specimens. (C) Composition of HSCs/HPs when gating on donor (upper panel) or recipient (lower panel) Lin−CD45+CD34+ cells.FIGURE 2: Lin−CD45+CD34+ cells from bone marrow (BM) and ileum lamina propria (LP) located in the same region of the viSNE plot from CyTOF analysis of individual organ donor, suggesting the similarity of their phenotype and origin.Taken together, our data suggest that human gut HSCs/HPs may be functionally similar to BM HSCs/HPs that have thymus-homing ability and can give rise to multiple lineages, which likely contribute to the long-persistent mixed chimerism post-ITx. Research reported in this publication was performed in the Columbia Center for Translational Immunology (CCTI) Flow Cytometry Core, supported in part by the Office of the Director, National Institutes of Health under awards S10RR027050 and S10OD020056. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. CyTOF study was performed in collaboration with the Human Immune Monitoring Core (HIMC) of Icahn School of Medicine at Mount Sinai.

Distinct Molecular Signature of Murine Fetal Liver and Adult Hematopoietic Stem Cells Identify Novel Regulators of Hematopoietic Stem Cell Function.

During ontogeny, fetal liver (FL) acts as a major site for hematopoietic stem cell (HSC) maturation and expansion, whereas HSCs in the adult bone marrow (ABM) are largely quiescent. HSCs in the FL possess faster repopulation capacity as compared with ABM HSCs. However, the molecular mechanism regulating the greater self-renewal potential of FL HSCs has not yet extensively been assessed. Recently, we published RNA sequencing-based gene expression analysis on FL HSCs from 14.5-day mouse embryo (E14.5) in comparison to the ABM HSCs. We reanalyzed these data to identify key transcriptional regulators that play important roles in the expansion of HSCs during development. The comparison of FL E14.5 with ABM HSCs identified more than 1,400 differentially expressed genes. More than 200 genes were shortlisted based on the gene ontology (GO) annotation term "transcription." By morpholino-based knockdown studies in zebrafish, we assessed the function of 18 of these regulators, previously not associated with HSC proliferation. Our studies identified a previously unknown role for tdg, uhrf1, uchl5, and ncoa1 in the emergence of definitive hematopoiesis in zebrafish. In conclusion, we demonstrate that identification of genes involved in transcriptional regulation differentially expressed between expanding FL HSCs and quiescent ABM HSCs, uncovers novel regulators of HSC function.