Number Of Hematopoietic Progenitors Research Articles

The bone marrow of mouse-rat chimeras contains progenitors of multiple pulmonary cell lineages.

Radiation-induced lung injury (RILI) is a common complication of anti-cancer treatments for thoracic and hematologic malignancies. Bone marrow (BM) transplantation restores hematopoietic cell lineages in cancer patients. However, it is ineffective in improving lung repair after RILI due to the paucity of respiratory progenitors in BM transplants. In the present study, we used blastocyst injection to create mouse-rat chimeras, these are artificial animals in which BM is enriched with mouse-derived progenitor cells. FACS-sorted mouse BM cells from mouse-rat chimeras were transplanted into lethally irradiated syngeneic mice, and the contribution of donor cells to the lung tissue was examined using immunostaining and flow cytometry. Donor BM cells provided long-term contributions to all lung-resident hematopoietic cells which includes alveolar macrophages and dendritic cells. Surprisingly, donor BM cells also contributed up to 8% in pulmonary endothelial cells and stromal cells after RILI. To identify respiratory progenitors in donor BM, we performed single-cell RNA sequencing (scRNAseq). Compared to normal mouse BM, increased numbers of hematopoietic progenitors were found in the BM of mouse-rat chimeras. We also identified unique populations of hemangioblast-like progenitor cells expressing Hes1, Dntt and Ebf1, along with mesenchymal stromal cells expressing Cpox, Blvrb and Ermap that were absent or ultra-rare in the normal mouse BM. In summary, by using rats as "bioreactors", we created a unique mouse BM cell transplant that contributes to multiple respiratory cell types after RILI. Interspecies chimeras have promise for future generations of BM transplants enriched in respiratory progenitor cells.

GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation

Abstract GATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b +myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge. This work was supported by a grant from the National Institute of Allergy and Infectious Diseases, National Institutes of Health (T32 training grant T32AI134646).

Neutrophils Regulate Bone Formation Via IL-1b Secretion

The bone marrow (BM) is surrounded by a shell of cortical bone that protects the hematopoietic stem cells (HSC) from external aggressions. Within the BM, progenitors for bone and blood coexist and, intriguingly, the same mesenchymal progenitors that give rise to bone also form niches that regulate hematopoiesis. Whether, and if so how, the hematopoietic system regulates the formation of its protective osteal shell is poorly understood. Among HSC descendants, innate immune cells have been involved in maintaining homeostasis in multiple organs, and neutrophils in particular have been shown to regulate the BM stroma both at steady-state and during stress, and to contribute to vascular repair. Hence, we examined MRP8cre iDTR mice (iDTR) which allow for inducible neutropenia upon diphtheria toxin injection. We found that acute neutropenia in these mice caused bone mass reduction (48% reduction) as determined by μCT, while no changes in osteoclast numbers. We confirmed this reduction using the calcein double labeling assay, altogether indicating that neutrophils regulate bone formation. We found a similar osteoporotic phenotype in MRP8cre CXCR4fl mice (CXCR4ΔN), in which only the return of circulating neutrophils to the BM is compromised, which suggested that it is peripheral-experienced neutrophils returning to the BM that exert the regulatory effect on the bone-producing mesenchymal cells. Concomitant with bone loss, we found that the number of hematopoietic progenitors were elevated in both in the iDTR and CXCR4ΔN models as assessed by flow cytometry, CFU-C assays and limited dilution reconstitution assays, suggesting that neutrophils counteract natural, age-related alterations in the BM. To identify the mechanisms by which neutrophils regulate bone formation, we searched for factors differentially produced by neutrophils returning to the BM compared with marrow-residing neutrophils. Transcriptomic analyses of neutrophils that migrated to the BM in a parabiotic model identified 8 soluble factors that were not produced by immature neutrophils. Among these factors, we found that interleukin 1 beta (Il1b) was highly expressed in returning neutrophils by qRT-PCR and protein levels, and single-cell RNAseq demonstrated that the production of Il1b was a hallmark of neutrophil maturation and egress into blood. To explore whether this cytokine mediated the osteogenic program of neutrophils in the BM, we generated mice with neutrophil-specific deletion of Il1b ; MRP8cre Il1bfl mice (Il1bΔN). Single-cell RNA-seq analysis of CD45-Ter119-CD31-CXCL12+ stroma cells from control CXCL12GFP mice, iDTR CXCL12GFP and Il1bΔN CXCL12GFP demonstrated marked anomalies in the differentiation of mesenchymal stem cell (MSC) into osteoblast lineage cells (OLC) and, to a lesser extent, adipocytic-lineage cells in the absence of neutrophils or neutrophil-derived Il1b. In vitro cultures confirmed a modulatory effect of Il1b on MSC differentiation. These findings correlated with the close proximity of neutrophils to CXCL12GFP+ MSC over random distribution, and with high expression of interleukin 1 receptor type1 (Il1r1) in OLC, altogether suggesting that neutrophils modulate the osteogenic program of MSC through Il1b secretion. To explore physiological contexts in which neutrophils modulate bone formation, we first examined the circadian transcriptome of total BM cells and found that Il1b was one of the few cytokines whose transcript levels in the BM followed a tight circadian pattern. We found that depletion of neutrophils fully ablated Il1b expression at all time points, raising the possibility that neutrophils enable diurnal differentiation of mesenchymal cells to produce bone. Indeed, the calcein double labeling assay assays revealed marked circadian patterns of bone synthesis that were significantly blunted in neutropenic and Il1bΔN mice. We propose that bone and blood lineages have co-evolved as an interlocked system that ensures both physical protection and cellular output of this organ.

GATA1 controls numbers of hematopoietic progenitors and their response to autoimmune neuroinflammation

AbstractGATA-binding factor 1 (GATA1) is a transcription factor that governs the development and function of multiple hematopoietic cell lineages. GATA1 is expressed in hematopoietic stem and progenitor cells (HSPCs) and is essential for erythroid lineage commitment; however, whether it plays a role in hematopoietic stem cell (HSC) biology and the development of myeloid cells, and what that role might be, remains unclear. We initially set out to test the role of eosinophils in experimental autoimmune encephalomyelitis (EAE), a model of central nervous system autoimmunity, using mice lacking a double GATA-site (ΔdblGATA), which lacks eosinophils due to the deletion of the dblGATA enhancer to Gata1, which alters its expression. ΔdblGATA mice were resistant to EAE, but not because of a lack of eosinophils, suggesting that these mice have an additional defect. ΔdblGATA mice with EAE had fewer inflammatory myeloid cells than the control mice, suggesting that resistance to EAE is caused by a defect in myeloid cells. Naïve ΔdblGATA mice also showed reduced frequency of CD11b+ myeloid cells in the blood, indicating a defect in myeloid cell production. Examination of HSPCs revealed fewer HSCs and myeloid cell progenitors in the ΔdblGATA bone marrow (BM), and competitive BM chimera experiments showed a reduced capacity of the ΔdblGATA BM to reconstitute immune cells, suggesting that reduced numbers of ΔdblGATA HSPCs cause a functional deficit during inflammation. Taken together, our data show that GATA1 regulates the number of HSPCs and that reduced GATA1 expression due to dblGATA deletion results in a diminished immune response following the inflammatory challenge.

The role of P2Y12 in cardiovascular disease beyond atherothrombosis: P2Y12 signaling promotes emergency hematopoiesis after myocardial infarction

Abstract Background Adenosine diphosphate (ADP) plays a pivotal role in platelet activation. The purinergic ADP-receptor P2Y12 has therefore been targeted in the treatment of cardiovascular disease (CVD) to prevent atherothrombosis (1). Beyond P2Y12 expression on platelets, purinergic receptors have also been described on hematopoietic stem and progenitor cells (LSK) (2). After myocardial infarction (MI), accelerated LSK proliferation launches emergency hematopoiesis as the driving force behind the inflammatory response to MI, increasing inflammatory cell production in the bone marrow (BM) and providing leukocyte resupply for local cell recruitment to the infarct (3). The inflammatory cascade after MI covers intricate multilayered interactions between the injured myocardium and the hematopoietic BM that still remain to be fully elucidated and may unearth novel therapeutic strategies. Whereas P2X receptors have recently been found to be involved in cell trafficking (4), the role of P2Y receptors in the hematopoietic BM have not yet been characterized. Purpose This study aims to characterize the influence of P2Y12 signaling on emergency hematopoiesis and cardiac remodeling after MI. Methods Permanent coronary ligation was performed for MI to assess BM activation, inflammatory cell composition, cardiac remodeling and function in murine global and platelet-specific P2Y12 knockout models and under pharmacological P2Y12 inhibition with prasugrel using flow cytometry, qPCR, immunohistochemistry and echocardiography. In vitro studies including colony forming unit (CFU) assays and flow cytometry allowed for investigation of ADP-dependent effects on LSK cells and intracellular pathway analysis. Results We identified ADP as a danger signal for the hematopoietic BM, fueling emergency hematopoiesis by promoting Akt phosphorylation and cell cycle progression. Detection of P2Y12 expression in LSK implicated a direct effect of ADP on LSK via P2Y12 signaling. P2Y12 deficiency and P2Y12 inhibition with prasugrel decelerated emergency hematopoiesis and consecutively reduced the excessive inflammatory response to MI, translating to lower numbers of hematopoietic progenitors and inflammatory cells in the blood and infarct. Ultimately, P2Y12 inhibition ameliorated chronic adverse cardiac remodeling and preserved cardiac function after MI. Conclusion ADP-dependent P2Y12-mediated activation of hematopoietic stem and progenitor cells in the BM promotes emergency hematopoiesis after MI and fuels post-ischemic inflammation, proposing a novel role of P2Y12 antagonists in CVD beyond atherothrombosis. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Deutsche Forschungsgemeinschaft (DFG)

Elevated 2HG does not cause features of tumorigenesis

Abstract Aims Gliomas are the most frequent brain tumours, representing 75% of all primary malignant brain tumours in adults. IDH1 (and IDH2) driver mutations occur in >80% of low grade gliomas and secondary GBMs, in <10% of primary GBMs and other cancers. How IDH1/2 mutations contribute to tumorigenesis is mostly unknown. IDH1/2 convert isocitrate to α-ketoglutarate, but when mutated possess a novel enzymatic function that reduces α-ketoglutarate to D2-hydroxyglutarate (2HG). Indeed 2HG accumulates in IDH1/2-mutant tumours, and this discovery suggested that 2HG may have a role in IDH1/2-mutant tumours onset and progression, possibly by causing dysregulations of various enzymes in the cells. Studies are undergoing to clarify the causative role of 2HG in IDH1/2-mutant tumours, but it is still not clear whether 2HG is the driver/oncometabolite. Our aim is to understand the role of 2HG in developing and adult mouse tissues and whether its accumulation might cause features of gliomagenesis. Method A constitutive D2hgdh Knock-out mouse (D2hgdh KO) was generated and the relative molecular and cellular analysis were performed. Results Brains dissected from D2hgdh KO mice appeared to be histologically normal. No differences were found in the proliferation and labelling retaining capacity of neural stem and progenitors cells (NSC/NPC) of the D2hgdh KO mice compared to controls. A comprehensive metabolites analysis showed that D2hgdh KO mouse accumulated 2HG in various organs and tissues, included total brains and in the NSC/NPC microdissected from the subventricular zone, the site of origin of many human gliomas. The DNA amount of 5mC and 5hmC extracted from brains of D2hgdh KO mice was similar to controls. A normal number of haematopoietic progenitors was also found. Conclusion Although D2hgdh KO mice accumulated 2HG in all tissues analysed, they did not develop any abnormalities and remained completely asymptomatic. This suggests that a mere increment of 2HG in developing and adult tissues may be not sufficient to cause tumorigenesis (and gliomagenesis), leading some doubts on the oncogenic roles of the 2HG in IDH1/2-mutant tumours.

Recapitulative haematopoietic development of human pluripotent stem cells in the absence of exogenous haematopoietic cytokines.

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC‐derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine‐free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC‐derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine‐free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine‐free differentiation a preferred model for studying the early human haematopoietic development.

A Novel View of Umbilical Cord Blood Versus Maternal Peripheral Blood — Evidence for an Increase in the Number of Circulating Stem Cells on Both Sides of the Fetal–Maternal Circulation Barrier.

Background. Umbilical cord blood (UCB) is a rich source of stem cells, including hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs), endothelial progenitors cells (EPCs), and very small embryonic-like stem cells (VSELs). We envision that these cells are mobilized into UCB in response to hypoxia and increases in the levels of pro-mobilizing factors released in response to delivery stress. We have hypothesized that these stem cells may play a role in repairing tissue/organ injuries that occur in the newborn child after delivery and that mobilization of these cells into newborn peripheral blood could be a kind of autologous, physiological stem cell therapy that all newborns experience after delivery. Aim of the study. Interestingly, in contrast to the intensive research on stem cells present in UCB, parallel research on stem cells released into maternal blood has been lacking. This is despite the fact that, like the newborn child, the mother is exposed to several types of injuries related to delivery, such as damage due to passage of the fetus through the reproductive tract, hypoxia, and the release of pro-mobilizing cytokines and bioactive lipids. Hypothesis. We hypothesized that the number of circulating stem cells also increases in the maternal circulation due to stress related to delivery as part of an autologous regeneration mechanism.Materials and Methods. To evaluate the possibility of a parallel increase in the number of circulating stem cells in the maternal circulation, we analyzed the number of circulating HSCs, MSCs, EPCs, and VSELs in maternal peripheral blood 12-24 hours before and 24 hours after normal delivery (n=17). Specifically, we analyzed CD133+/Lin-/CD45+ or CD34+/Lin-/CD45+ cells enriched for hematopoietic SCs (HSCs), the CD34+/CD133+/KDR+ population of endothelial progenitor cells (EPCs), the CD45-/CD105+/CD90+/CD29+ population of mesenchymal SCs (MSCs), and the CD34+ and CD133+ populations of Lin−CD45− very small embryonic-like stem cells (VSELs). We also employed standard clonogenic assays to enumerate the number of hematopoietic progenitors circulating in maternal blood. Results. We observed that the number of HSCs, MSCs, EPCs, VSELs and clonogenic progenitors increases significantly in maternal blood at 24 hours after normal delivery (n=17). Conclusions. Based on this observation, we propose that delivery stress is associated with an increase in the number of circulating stem cells, not only on the fetal side but also on the maternal side of the fetal-maternal circulatory barrier. We propose that, most likely, the increase in the number of circulating stem cells in maternal blood indicates that these cells play a role in repairing small tissue/organ injuries that occur in the mother's body due to delivery. Currently, we are determining whether these changes correspond with other clinical parameters, such as the extent of the mother's internal injuries or the Apgar status of the newborn child, and whether there are changes in the number of circulating stem cells in maternal blood after planned C-sections. We propose that this phenomenon of maternally circulating stem cells could potentially be harnessed to accelerate the recovery of the mother following delivery. DisclosuresNo relevant conflicts of interest to declare.

The JAK2V617F-Bearing Megakaryocytes Promote Hematopoietic Stem/Progenitor Cell Expansion through the Thrombopoietin/MPL Signaling

IntroductionThe myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by hematopoietic stem/progenitor cell (HSPC) expansion and overproduction of mature blood cells. The acquired mutation JAK2V617F plays a central role in these disorders. The mechanisms responsible for MPN HSPC expansion are not fully understood. One hallmark of MPNs is megakaryocytic (MK) hyperplasia. Very recent evidence has implicated MKs in regulating HSPC activity. Since thrombopoietin (TPO) acting through its receptor MPL are key regulators of both megakaryopoiesis and HSPC activity, we hypothesize that JAK2V617F-bearing MKs promote MPN stem cell function via altered TPO/MPL signaling.MethodsJAK2V617F Flip-Flop (FF1) mice and Pf4-Cre mice were crossed to generate a MK cell lineage-specific human JAK2V617F knock-in mouse strain (Pf4/FF1). TPO or MPL was ablated in Pf4/FF1 by crossing Pf4/FF1 mice with TPO knock-out mice (TPOnull) or MPL knockout mice (MPLnull) to generate TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice. Animal experiments were performed in accordance with the Institutional Animal Care and Use Committee guideline.ResultsPreviously, we have demonstrated that JAK2V617F-bearing MKs cause a murine myeloproliferative syndrome with HSPC expansion in a Pf4/FF1 murine model. Here, we studied the effects of the JAK2V617F-bearing MK niche on MPN stem cell function. First, we isolated LineagenegcKit+ (Lin-cKit+) HSPCs from 28wk old Pf4/FF1 mice and their age-matched littermate controls (Pf4-Cre) (n=4 in each group) and analyzed their cell cycle status using propidium iodide staining. We found that the percentage of Lin-cKit+ cells in the G0/G1 phase was moderately increased in the Pf4/FF1 mice compared to control mice (63% vs 56%, P=0.012), suggesting that HSPCs in the JAK2V617F-bearing MK niche are more quiescent than in the WT MK niche. (Figure 1)Next, we performed a competitive repopulation assay in which both CD45.2 marrow cells from Pf4/FF1 mice or control mice were injected intravenously together with competitor CD45.1 WT marrow cells into lethally irradiated (950 cGy) CD45.1 recipients. During 16 weeks follow up post transplantation, recipients of Pf4/FF1 marrow cells displayed a higher peripheral blood donor (CD45.2) chimerism than the control mice. Marrow cell colony assays demonstrated a moderate increase of CD45.2+ hematopoietic progenitors in the Pf4/FF1 marrow recipients compared to control mice (1.5-fold, p = 0.0025). Marrow cell flow cytometry analysis revealed that, although there was no significant difference in the total CD41+ MK cell numbers between the two groups, CD45.2+ MKs were significantly increased (3.3-fold, P=0.012) while CD45.1+ MKs were significantly decreased (3.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Similarly, although the total numbers of marrow CD45+EPCR+CD48-CD150+ (E-SLAM) cells, which is a highly purified long-term repopulating HSPC population, were not changed between the two groups, CD45.2+ E-SLAM cells were significantly increased (3.5-fold, P=0.012) while CD45.1+ E-SLAM cells were significantly decreased (5.4-fold, P=0.024) in the Pf4/FF1 marrow recipient compared to the controls. Therefore, the JAK2V617F-bearing MK niche promoted the expansion of JAK2V617F HSPCs, probably at the expense of suppressing WT hematopoiesis. No significant difference in blood cell count, spleen weight, or total femoral cell count was observed between the Pf4/FF1 marrow recipients and control marrow recipients. (Figure 2)To determine the role(s) of TPO/MPL signaling in the development of JAK2V617F MK-induced myeloproliferation, we ablated TPO or MPL in the Pf4/FF1 mice. At 28 weeks of age, in contrast to the Pf4/FF1 mice which developed thrombocytosis, increased numbers of hematopoietic progenitors, and a substantial expansion of MKs and HSPCs compared to age-matched WT controls, TPOnullPf4+FF1+ mice and MPLnullPf4+FF1+ mice did not display any of these features. (Figure 3) These data suggest that TPO and MPL are required for the development of JAK2V617F MK-induced myeloproliferation.ConclusionsOur study establishes that JAK2V617F-bearing MKs form an important part of the MPN HSPC niche and TPO/MPL signaling is critical for the MK niche function. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Isolation of Mouse Megakaryocyte Progenitors.

Bone marrow megakaryocytes are large polyploid cells that ensure the production of blood platelets. They arise from hematopoietic stem cells through megakaryopoiesis. The final stages of this process are complex and classically involve the bipotent Megakaryocyte-Erythrocyte Progenitors (MEP) and the unipotent Megakaryocyte Progenitors (MKp). These populations precede the formation of bona fide megakaryocytes and, as such, their isolation and characterization could allow for the robust and unbiased analysis of megakaryocyte formation. This protocol presents in detail the procedure to collect hematopoietic cells from mouse bone marrow, the enrichment of hematopoietic progenitors through magnetic depletion and finally a cell sorting strategy that yield highly purified MEP and MKp populations. First, bone marrow cells are collected from the femur, the tibia, and also the iliac crest, a bone that contains a high number of hematopoietic progenitors. The use of iliac crest bones drastically increases the total cell number obtained per mouse and thus contributes to a more ethical use of animals. A magnetic lineage depletion was optimized using 450 nm magnetic beads allowing a very efficient cell sorting by flow cytometry. Finally, the protocol presents the labeling and gating strategy for the sorting of the two highly purified megakaryocyte progenitor populations: MEP (Lin-Sca-1-c-Kit+CD16/32-CD150+CD9dim) and MKp (Lin- Sca-1-c-Kit+CD16/32-CD150+CD9bright). This technique is easy to implement and provides enough cellular material to perform i) molecular characterization for a deeper knowledge of their identity and biology, ii) in vitro differentiation assays, that will provide a better understanding of the mechanisms of maturation of megakaryocytes, or iii) in vitro models of interaction with their microenvironment.

Plerixafor added to G‐CSF allows mobilization of a sufficient number of hematopoietic progenitors without impacting the efficacy of TCR‐alpha/beta depletion in pediatric haploidentical and genoidentical donors failing to mobilize with G‐CSF alone

Collection of a large number of early hematopoietic progenitors is essential for allogeneic apheresis products intended for TCR-alpha/beta depletion. We added plerixafor 0.24 mg/kg body weight (bw) on day 4 of high-dose filgrastim mobilization 10hours prior to apheresis in 16 (30.5%) pediatric allogeneic donors who failed to recover a sufficient number of CD34+ cells. On day 4 of G-CSF, the median CD34+ cell count in peripheral blood was 6 per μL (range 4-9 per μL) in 6 poor mobilizers and 16 per μL (range 12-19 per μL) in insufficient mobilizers. In all donors, the threshold of 50 CD34+ cells/μL was achieved, and the median increase was 14.8-fold in poor mobilizers and 6.5-fold in insufficient mobilizers, whereas it was 3.45-fold increase in those mobilized with G-CSF alone. In all donors, a predefined number of >10 × 106 CD34+ cells/kg of recipient bw before depletion was reached in the apheresis product. The use of plerixafor did not affect the purity of further TCR-alpha/beta depletion. Side effects were mild to moderate and consisted of nausea and vomiting. Thus, the safety and high efficacy of plerixafor was proven in healthy pediatric allogeneic hematopoietic cell donors.

Corrective effects of original bioflavonoid complex in the cyclophosphamide-induced immunity disorders

Our aim was to evaluate immunomodulatory properties of an original bioflavonoid complex in experimental immune disturbances induced by cyclophosphamide (Cy). We have studied morphometric indexes of thymus and spleen, as well as blood leukocyte counts, cell proliferative activity in lymphoid organs, delayed hypersensitivity responses to T cell-dependent antigen, along with differentiation activity of bone marrow stem cells in experimental animals during Cy-induced immune suppression after a course of bioflavonoid treatment. Suspension of the bioflafonoid complex was introduced to the male mice (СВАхC57Bl/6)F1 aged 12- 14 weeks at a daily dose of 2 mg/animal (80 mg/kg), per os, using gastric catheter, over 14 days. Cytostatic immunosuppression was produced by a single intraperitoneal Cy injection. Proliferative activity of spleen and thymic cells was determined by standard method with Н3 -thymidine incorporation in the 72-h cell culture. Cellular immune response was assayed by the degree of delayed-type hypersensitivity development in response to sheep erythrocytes. The number of hematopoietic progenitors was evaluated by culturing bone marrow cells in methylcellulose-based medium. The experiments have shown mitigation of immunosuppressive effects induced by Cy, in the course of bioflavonoid complex treatment, with respect to absolute and relative mass of lymphoid organs and leukocyte numbers in peripheral blood. Moreover, we have demonstrated decreased effects of Cy treatment upon the spontaneous activity of spleen cells, mitogen-induced thymocyte and splenocyte proliferation, intensivity of delayed-type hypersensitivity response that reached the values of intact animals. Following the course of bioflavonoids, we have revealed an increase in early hematopoietic progenitors. Alleviation of Cy-induced suppressive effects upon cellular immune response, proliferation rates of immune cells, as well as stimulation of hematopoietic stem cell functions suggest a sufficient capacity of the original bioflavonoid complex for modulation of immunity and hematopoiesis, thus presenting experimental proofs for its potential usage as an adjuvant treatment of the patients with malignant diseases.

In VitroandIn VivoInteractions between Stromal and Leukemic Cells

Understanding the mechanisms underlying changes in the microenvironment caused by pathological cells is essential to develop new therapeutic strategies. Such modifications allow leukemia cells to modify their microenvironment in order to promote their growth. Here, we studiedin vivoandin vitrointeractions between Primary Myelofibrosis and the microenvironment. This clonal malignant disorder is caused by three major driver mutations: JAK2V617F, MPLW515L and CALR exon 9 mutations. We performed 72h co-cultures of the mesenchymal stromal cell line MS5 and the MO7e human hematopoietic cell line transduced with either the MPLW515L mutation (MO7eMPLW515L) or GFP as a control. As Mo7e cells are dependent on the human growth factor h-GM-CSF, co-cultures were performed with and without it Flow cytometry confirmed that leukemic cells expressing the MPLW515L mutation proliferated actively in the absence of hGM-CSF, unlike the control MO7e GFP cells. In addition, the presence of MS5 stromal cells did not have a major impact on the proliferation of MO7eMPLW515L but promoted MO7e GFP cell survival. Thus the MPLW515L mutation activates signaling pathways that make the cells independent of cytokines and stroma. On the other hand, the number of stromal cells increased upon co-culture with either MO7e GFP or MO7e W515L and in the presence of h-GM-CSF. Thus, either growth factor addition or the MPLW515L mutation allows MO7e cells to enhance the proliferation of stromal cells. To test our observations in primary cells, we also performed 72h co-cultures using isolated murine cKIT+ hematopoietic progenitor cells from JAK2V617F knock-in or wild type mice. The impact of stromal cells was evaluated in the presence or absence of thrombopoietin (TPO). Cells were analyzed by flow cytometry. The results showed that MS5 stromal cells induced proliferation of both primary wild type and JAK2V617F cKIT+ hematopoietic cells independently of TPO. Moreover, the proliferation of MS5 stromal cells was also significantly induced by both wild type and JAK2617F cKIT + primary cells. In vivointeractions were studied using transgenic mice expressing CXCL12HT-dsRED crossed with JAK2V617F mice (CXCL2-HT-JAK2V617F). We followed different parameters, namely the weight and the size of the spleen, the different blood parameters, the number of hematopoietic progenitors in bone marrow, spleen and blood, and the cellularity and the number of fibroblast colonies (CFU-F). Results showed that CXCL12 HT JAK2KI mice developed significant splenomegaly, associated with changes in blood parameters characteristic of JAK2KI mice, i.e. increases in white blood cells, red blood cells, hematocrit, and hemoglobin. There was also an increase in blood, spleenand bone marrow progenitors compared to the CXCL12HT GFP control mice. The number of fibroblast colonies (CFU-F), reflecting the activity of mesenchymal stem cells (MSC) in the microenvironment, was also greatly increased, especially in the marrow of CXCL12HT JAK2KI mice, indicating that the malignant clone modified the microenvironment of the mice. Taken together, our findings show that hematopoietic cells, either normal or leukemic, can modify their own microenvironmentin vitroandin vivo. This offers a new avenue for therapeutic targeting in the treatment of this type of hemopathologies. Disclosures No relevant conflicts of interest to declare.

Plerixafor-aided Mobilization of Peripheral Blood Hematopoietic Stem Cells to Support Subsequent High-dose Chemotherapy After a Prior Autologous Transplant

BackgroundAn appreciable proportion of patients in need of salvage high-dose chemotherapy (HDC) and autologous peripheral blood stem cell (PBSC) transplantation (PBSCT) fail to mobilize adequate numbers of hematopoietic progenitors, and plerixafor is applied for that purpose. Limited data exist on remobilization of PBSCs in patients who have relapsed after prior HDC + PBSCT. Herein, we report on consecutive patients that had undergone successful prior single or tandem HDC for a variety of malignant neoplasms in our institution, and later required re-mobilization of PBSCs in order to support further HDC cycles. Patients and MethodsPlerixafor was administered in combination with granulocyte-colony stimulating factor alone, or after mobilizing chemotherapy. Five patients, 2 B-cell non-Hodgkin lymphomas, 1 multiple myeloma, 1 germ-cell tumor, and 1 Ewing sarcoma, having relapsed after prior HDC + PBSCT, were deemed candidates for further cycle(s) of PBSC-supported HDC. Plerixafor was applied in a “just-in-time” strategy after low CD34+ numbers were measured on the first day of anticipated hematopoietic stem cell collection (non-Hodgkin lymphoma, germ-cell tumor, and Ewing sarcoma), or pre-emptively in multiple myeloma. ResultsSuccessful collection of adequate PBSCs was achieved in all patients, from 1.8 to 3.8 × 106/kg after a median of 2 (range, 1-3) leukaphereses; 4 of 5 patients underwent subsequent HDC + PBSCT and engrafted after a median of 11 days (range, 9-55 days) and 25 days (range, 17-76 days) for neutrophils and platelets, respectively. ConclusionPlerixafor proved effective to mobilize adequate numbers of PBSCs in individual patients with relapsed malignancies after prior single or tandem HDC + PBSCT. These PBSCs could establish sustained multi-lineage hematopoietic engraftment without any sequelae.

An Adapting Bone Marrow Niche Creates a Nurturing Environment for Hematopoiesis during Immune Thrombocytopenia Progression

Immune thrombocytopenia (ITP) is an acquired autoimmune disease characterised by low platelet counts (<100 x 109/L) and manifests as a bleeding tendency. The demand on hematopoiesis is elevated in chronic ITP, where sustained platelet destruction mediated by an activated immune system is likely to cause considerable stress on progenitor populations. Intriguingly, this increased stress does not appear to result in functional exhaustion, as chronic ITP patients do not present with pancytopenia. By using a novel murine model of chronic ITP, generated by injecting mice with anti-CD41 antibody (ITP group) or IgG (control group) every 48hrs for 4 weeks, we aimed to define the effect of chronic ITP on hematopoietic progenitors and to elucidate the mechanisms behind the preservation of hematopoiesis. The relative numbers of hematopoietic progenitors in mice with chronic ITP vs controls were analysed by flow cytometry and their fitness was assessed by measuring their relative ability to reconstitute the hematopoietic system of lethally irradiated recipients. There was a significant increase in all hematopoietic progenitors analysed in ITP: 2.96-fold increase in multipotent progenitors, 4.66-fold increase in short-term hematopoietic stem cells (ST-HSCs) and 4.93-fold increase in long-term hematopoietic stem cells (LT-HSCs), which led to an increased ability of ITP donor bone marrow to reconstitute irradiated recipients. The results indicate that chronic ITP drives LT-HSCs out of quiescence and causes increased differentiation into committed progenitors in order to meet the increased demand in platelet production. In support of this, increased megakaryopoiesis was observed in chronic ITP, with a 60.5% increase in the number of megakaryocytes observed in bone marrow sections. Interestingly, similar to the clinical manifestation of ITP, we observed no change in levels of circulating TPO in our ITP model. Next, the effect of chronic ITP on the bone marrow microenvironment was determined due to its essential role in the support and maintenance of hematopoiesis. Histological analysis of bone marrow from mice with chronic ITP (Figure 1) revealed a 66.7% increase in the numbers of LepR+/ Cxcl12-DsRed stromal cells. LepR+/ Cxcl12-DsRed stromal cells are a well characterised stromal cell subset, known to be essential for maintenance and retention of HSCs in the bone marrow microenvironment. During chronic ITP, this stromal cell subset maintained their classically defined perivascular location and retained their ability to produce high levels of hematosupportive cytokines (Cxcl12 and Kitl). Chronic ITP was associated with a significant increase in total bone marrow expression (Cxcl12=2.39-fold increase; Kitl=1.71-fold increase), pointing to perivascular stromal cell expansion as being the source of increased local hematopoietic support. Analysis of the bone marrow vascular network revealed that the average vessel area was increased in chronic ITP (54.3% increase), whilst the number of vessels remained unchanged implying that the marrow sinusoids are vasodilated. We hypothesise that an increase in blood vessel area would aid the extravasation of circulating HSCs back into the bone marrow microenvironment where they would contribute to hematopoiesis. By developing an accurate mouse model of chronic ITP, we have identified key alterations in HSCs and the bone marrow microenvironment. Our data clearly demonstrates that in chronic ITP, HSCs are driven out of quiescence and expand in number in order to contribute to the increased demand for hematopoiesis. Furthermore, the bone marrow microenvironment adapts to this increased differentiation pressure on HSCs by creating a hematosupportive, quiescence promoting environment through the expansion of bone marrow stromal cells, and an increase in blood vessel area. Disclosures No relevant conflicts of interest to declare.

Mutation of Murine Rpl5 reveals a New Model for Diamond Blackfan Anemia Characterized By Defective Erythropoiesis

Background Ribosome dysfunction is implicated in several abnormal developmental conditions and diseases in humans. Heterozygous germline ribosomal protein (RP) variants are found in the majority of individuals with Diamond Blackfan anemia (DBA) with RPL5 (uL18) being the second most frequently mutated gene. Patients with RPL5 mutations are for unknown reasons at increased risk of congenital defects when compared to other genotypes. There is currently no available Rpl5 mouse model with anemia and therefore hematopoiesis in this genotype has not been well studied. Methods We characterized a novel mutant mouse with a growth and kinky tail defect, and increased mortality. The mouse was generated in a chromosome 11 balancer ENU mutagenesis screen. Following genetic mapping and whole exome sequencing, we identified a heterozygous intronic Rpl5 missense variant, which segregated with all affected mice. We first performed molecular analyses to validate the impact of this variant on RPL5 and ribosome levels, and then examined the development and physiology of multiple tissues by histology, micro-CT, echocardiogram and measurement of erythroid differentiation to determine the etiology of the observed increased mortality in newborn Rpl5 mutant mice. Results This variant led to decreased Rpl5 mRNA and protein, and consequential polysome defects (decreased 60S and 80S ribosome subunits) consistent with RPL5 haploinsufficiency. All Rpl5+/- neonates exhibited deficient terminal erythroid differentiation. A fraction of the mutants progressed to severe anemia leading to increased mortality while others appeared to undergo spontaneous remission. Surviving adult/aged mice exhibited no anemia although preliminary studies indicate reduced numbers of hematopoietic progenitors. Most newborn mutant mice had large ventricular septal defects (VSD) while surviving adult mice had small VSDs on echocardiogram. Rpl5+/- mice were significantly smaller without craniofacial abnormalities as assessed by micro-CT or histology. Bone and cartilage staining of newborn mice revealed that the kinky tail defect resulted from delayed endochondral ossification. Conclusion This model recapitulates the major developmental and erythroid differentiation defects of DBA in an animal model. We propose that this novel Rpl5 mutant provides an opportunity to study the mechanism of the variable penetrance of anemia and the congenital defects normally encountered in DBA. Disclosures Singh: Emmaus Medical, Inc: Consultancy.

PDGFRB SIGNALING IS REQUIRED TO GENERATE AORTIC HAEMATOPOIETIC CELLS IN VIVO

The first hematopoietic stem cells (HSCs) are generated in the aorta-gonads-mesonephros (AGM) region of the mid-gestation mouse embryo. Signals from the microenvironment are required for HSC generation. However, the signals and identity of cells releasing them in vivo remain unknown. The AGM microenvironment includes perivascular cells. Pericytes (PCs) support HSC maintenance in the adult bone marrow, but their involvement in HSC birth has not been studied. PCs are recruited to the developing blood vessel wall via PDGFRβ signaling which was recently shown to mediate HSC specification in zebrafish. We hypothesize that PDGFRβ signaling is required to generate the first HSCs in vivo. To answer this question, we used the PDGFRβ knock-out mice which lack PCs and die perinatally. Results from our hematopoietic progenitor assays (CFU-Cs) and transplantations show that the germline deletion of PDGFRβ affects both hematopoietic progenitor numbers and HSC activity in the E11 AGM. HSPCs in other hematopoietic organs are not affected at this stage. Whether PDGFRβ+ cells are HSPC precursors or the effect seen is only due to a defect in the niche is under investigation. Characterization of E11 wild-type embryos by immunohistochemistry and RNA sequencing show that the dorsal aorta is surrounded by three phenotypically and transcriptionally distinct perivascular cell layers that include PCs (PDGFRβ+NG2+) and PDGFRβ+NG2- adjacent cells. PCs are highly enriched in HSC niche genes described in the adult microenvironment, suggesting a putative role in AGM hematopoiesis. In conclusion, our results define PDGFRβ signalling as key component of the HSC generating niche in the mouse embryo in vivo that could be tested in vitro to derive HSCs from hematopoietic and non-hematopoietic cell sources for therapy.

PS1225 NOVEL PRO‐INFLAMMATORY MACROPHAGES ARE INVOLVED IN HEMATOPOIETIC STEM CELL DEVELOPMENT

Background:Knowledge of the developmental cells and factors that affect the generation of Hemaotpoietic Stem Cells (HSC) in the embryo will inform strategies on ex vivo HSC generation for therapeutic application. HSC are generated from specialized endothelial cells of the embryonic aorta. In the rapidly changing microenvironment surrounding the aorta, the so‐called Aorta‐Gonad‐Mesonephros (AGM) region provides inducting factors for generation of the potent HSC that are responsible for the life‐long production of the blood system. Previously, inflammatory factors have been implicated in regulating HSC development in the mouse embryo, but it is unclear what cells in the AGM microenvironment are the source. Whereas it is known that macrophages play both pro‐ and anti‐inflammatory roles and influence HSCs in the adult, it is as yet unknown whether the macrophages found in the embryo prior to HSC generation are involved in the AGM HSC‐generative microenvironment.Aims:To investigate whether innate immune cell types and macrophages, which are generated prior to HSCs in the mouse embryo, play a role the AGM microenvironment and to characterize those cells phenotypically and functionally to understand whether and how these cells influence the development of the potent adult‐type HSCs.Methods:We examined the cell types in the midgestation mouse AGM by mass cytometry (CyTOF) with 23 hematopoietic markers. Immuno‐histology of embryos was performed to localize macrophages and vital time‐lapse imaging to study cell motility. Quantitative (FACS, developmental time courses) and qualitative analyses (functional hematopoietic progenitor and in vivo hematopoietic cell transplantation assays) were performed to characterize AGM macrophages. Co‐cultures of aortic hemogenic endothelial cells and AGM macrophages on OP9‐DL1 stromal cells tested the induction abilities of macrophages. Mouse genetic models and specific macrophage‐depletion methods were used to eliminate macrophages or prevent mobilization. RNA sequencing and qRT‐PCR identified and validated the transcriptome of AGM macrophages.Results:Our CyTOF results on mouse embryonic day 10 AGM regions indicate two abundant myeloid cell types – mannose‐receptor positive AGM‐associated macrophages (AGM‐aM) and mannose‐receptor negative progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1/CX3CL1 – in the absence of this chemokine signaling pathway, macrophages are not mobilized to the AGM and instead are retained in the yolk sac. Depletion of AGM macrophages by two complementary methods resulted in reduced hematopoietic progenitor numbers and an almost complete absence of HSCs in the AGM. Importantly, AGM‐aM express a pro‐inflammatory signature, localize to the embryonic aorta and dynamically interact with nascent and emerging intra‐aortic hematopoietic cells (IAHC).Summary/Conclusion:Macrophages that are produced prior the generation of HSCs dynamically interact with nascent/emerging hematopoietic cells in the midgestation aorta to affect the generation of HSCs. Mannose receptor positive macrophages specifically localize to intra‐aortic cluster cells. The timely presence of macrophages in the AGM enhances the generation of fully functional hematopoietic progenitor cells and HSCs. Together, our results identify a novel pro‐inflammatory macrophage subset that regulates the embryonic development of adult‐type HSCs.

CRTAM+ NK cells endowed with suppressor properties arise in leukemic bone marrow.

Due to their increasing rates of morbidity and mortality, childhood malignancies are considered a global health priority, with acute lymphoblastic leukemias (ALLs) showing the highest incidence worldwide. Control of malignant clone emergence and the subsequent normal-leukemic hematopoietic cell out-competition require antitumor monitoring mechanisms. Investigation of cancer surveillance innate cells may be critical to understand the mechanisms contributing in either disease progression or relapse, and to promote displacement of leukemic hematopoiesis by the normal counterpart. We report here that NK cell production is less and low hematopoietic progenitor numbers contribute to this defect. By investigating the expression of the activation molecule class I restricted T-cell associated molecule (CRTAM) along the hematopoietic lineage differentiation pathway, we have identified lymphoid precursor populations coexpressing CD34, CD56/CD3/CD19, and CRTAM as the earliest developmental stage where activation may take place in specialized niches that display the ligand nectin-like-2. Of note, bone marrow (BM) from patients with ALL revealed high contents of preactivated CD56high NK cells expressing CRTAM and endowed with an exhaustion-like phenotype and the functional capability of producing IL-10 and TGF-β in vitro. Our findings suggest, for the first time, that the tumor microenvironment in ALL directly contribute to exhaustion of NK cell functions by the CRTAM/Necl-2 interaction, and that the potential regulatory role of exhausted-like NK cells may favor malignant progression at the expense of anti-tumor responses. Phenotypic and functional identity of this unique suppressor-like NK cell population within the leukemic BM would be of special interest for the pathobiology of ALL and development of targeting strategies.

Genetic Ablation of the ABC Transporter Abcc4 Impairs Lymphoid Leukemogenesis

ABCC4 is an ABC transporter of the C sub‐family that is ubiquitously expressed and exports key endogenous compounds including cAMP and prostaglandins, and cancer chemotherapeutic drugs. cAMP and prostaglandins are signaling molecules that, among other functions, regulate thymocyte development and function. Thymocyte progenitors, IL7 receptor positive cells, migrate from the bone marrow to the thymus and give rise to mature T‐cells. The maturation of thymocytes can be followed by monitoring for the presence of surface markers CD4 and CD8. Briefly, thymocytes are initially double negative (DN) cells, with neither CD4 nor CD8; after T‐cell receptor rearrangement, CD4 and CD8 are expressed on double positive cells (DP), which following positive and negative selection, become mature, single positive (SP) cells expressing either CD4 or CD8. Abcc4 is highly expressed in DP and CD4‐positve thymocytes, while being undetectable in DN thymocytes from wild‐type (WT) mice, suggesting a developmental regulation of Abcc4. Therefore, we hypothesized that ablation of Abcc4 from mice would lead to aberrant T‐cell development and function.ABCC4 absence had no measureable impact on the number of hematopoietic progenitors. We hypothesized that lymphoid progenitors in Abcc4‐knockout(KO) mice may have an impaired leukemogenesis potential. To test this hypothesis, hematopoietic progenitors from Abcc4‐KO or WT mice were transduced with a retrovirus harboring a MYCN cDNA, a known driver of lymphoid leukemia, and subsequently transplanted into lethally irradiated congenic WT recipient mice. Mice transplanted with either WT or Abcc4‐KO MYCN‐transduced hematopoietic progenitors developed both B‐ and T‐cell leukemia, with tumors expressing Abcc4 on the plasma membrane. Kaplan‐Meier survival analysis revealed that mice transduced with Abcc4‐KO progenitors had a dramatic increase in median survival from 81.5 days in mice who received WT progenitors to 112 days in mice who received Abcc4‐KO progenitors. These studies suggested the lymphoid progenitors from Abcc4‐KO mice harbored an intrinsic defect manifested by reduced lymphoid leukemia. This was further investigated by performing a competitive bone‐marrow transplantation experiment. Briefly, hematopoietic progenitors from Abcc4‐KO and wildtype mice were isolated and mixed in equal numbers (each harboring a different surface marker to enable identification), then transplanted into congenic wildtype mice following lethal irradiation; subsequently the differentiated lymphoid progeny were identified in the peripheral blood. These studies revealed that the number of CD4+ cells from Abcc4‐KO donors was reduced, thus confirming a defect in Abcc4‐KO lymphoid progenitors. A defect in peripheral thymocyte function is also a potential mechanism, as genes in the arachidonic acid pathway regulate further differentiation and function of mature thymocytes. We discovered altered expression of genes in the arachidonic acid pathway such as Cox‐1 and Ptger4, when compared to age‐matched WT mice. These data suggest that genetic ablation of Abcc4 has an effect on arachidonic acid signaling, which may in turn affect thymocyte function.Overall, the data presented herein suggest that ABCC4 impacts lymphoid progenitors and differentiated thymocytes. One implication of these findings is that deficiency or insufficiency of ABCC4 may reduce the possibility of developing lymphoid malignancies.Support or Funding InformationThis work was funded by NIH and ALSACThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.