Wild-type Bone Marrow Cells Research Articles

HDAC7 is a potential therapeutic target in acute erythroid leukemia

Acute erythroleukemia (AEL) is a rare subtype of acute myeloid leukemia with a poor prognosis. In this study, we established a novel murine AEL model with Trp53 depletion and ERG overexpression. ERG overexpression in Trp53-deficient mouse bone marrow cells, but not in wild-type bone marrow cells, leads to AEL development within two months after transplantation with 100% penetrance. The established mouse AEL cells expressing Cas9 can be cultured in vitro, induce AEL in vivo even in unirradiated recipient mice, and enable efficient gene ablation using the CRISPR/Cas9 system. We also confirmed the cooperation between ERG overexpression and TP53 inactivation in promoting the growth of immature erythroid cells in human cord blood cells. Mechanistically, ERG antagonizes KLF1 and inhibits erythroid maturation, whereas TP53 deficiency promotes proliferation of erythroid progenitors. Furthermore, we identified HDAC7 as a specific susceptibility in AEL by the DepMap-based two-group comparison analysis. HDAC7 promotes the growth of human and mouse AEL cells both in vitro and in vivo through its non-enzymatic functions. Our study provides experimental evidence that TP53 deficiency and ERG overexpression are necessary and sufficient for the development of AEL and highlights HDAC7 as a promising therapeutic target for this disease.

Significance of Nitric Oxide Derived from the Nitric Oxide Synthases System in Cardiovascular Inter-Organ Cross-Talk.

Inter-organ cross-talk contributes to the pathogenesis of various disorders, and drug development based on inter-organ cross-talk is attracting attention. The roles of nitric oxide (NO) derived from the NO synthases system (NOSs) in inter-organ cross-talk remain unclear. We have investigated this issue by using our mice deficient in all three NOSs (triple n/i/eNOSs-/- mice). We reported that 2/3 nephrectomized triple n/i/eNOSs-/- mice die suddenly because of early onset of myocardial infarction, suggesting the protective role of NO derived from NOSs in the cross-talk between the kidney and the heart. We studied the role of NO derived from NOSs expressed in the bone marrow in vascular lesion formation. Constrictive arterial remodeling and neointimal formation following unilateral carotid artery ligation were prominently aggravated in wild-type mice transplanted with triple n/i/eNOSs-/- bone marrow cells as compared with those with wild-type bone marrow cells, suggesting the protective role of NO derived from NOSs in the cross-talk between the bone marrow and the blood vessel. We further investigated the role of NO derived from NOSs expressed in the bone marrow in pulmonary hypertension. The extent of pulmonary hypertension after chronic hypoxic exposure was markedly exacerbated in wild-type mice underwent triple n/i/eNOSs-/- bone marrow transplantation as compared with those underwent wild-type bone marrow transplantation, suggesting the protective role of NO derived from NOSs in the cross-talk between the bone marrow and the lung. These lines of evidence demonstrate that systemic and myelocytic NOSs could be novel therapeutic targets for myocardial infarction, vascular disease, and pulmonary hypertension. Significance Statement We demonstrated in studies with triple n/i/eNOSs-/- mice that partial nephrectomy accelerates the occurrence of myocardial infarction induced by systemic NOSs deficiency, that myelocytic NOSs deficiency aggravates vascular lesion formation after unilateral carotid artery ligation, and that myelocytic NOSs deficiency exacerbates chronic hypoxia-induced pulmonary hypertension. These results suggest that NO derived from NOSs plays a protective role in cardiovascular inter-organ cross-talk, indicating that systemic and myelocytic NOSs could be important therapeutic targets for myocardial infarction, vascular disease, and pulmonary hypertension.

Relative contributions of osteal macrophages and osteoclasts to postnatal bone development in CSF1R-deficient rats and phenotype rescue following wild-type bone marrow cell transfer.

Macrophage and osteoclast proliferation, differentiation and survival are regulated by colony-stimulating factor-1 receptor (CSF1R) signaling. Osteopetrosis associated with Csf1 and Csf1r mutations has been attributed to the loss of osteoclasts and deficiency in bone resorption. Here we demonstrate that homozygous Csf1r mutation in rat leads to delayed postnatal skeletal ossification associated with substantial loss of osteal macrophages (osteomacs) in addition to osteoclasts. Osteosclerosis and site-specific skeletal abnormalities were reversed by intraperitoneal transfer of wild-type bone marrow cells (BMT) at weaning. Following BMT, IBA1+ macrophages were detected before TRAP+ osteoclasts at sites of ossification restoration. These observations extend evidence that osteomacs independently contribute to bone anabolism and are required for normal postnatal bone growth and morphogenesis.

Myelodysplastic Syndromes Benefit from Iron-Restricted Bone Marrow Transplant in Preclinical Mouse Model

Background: Hematopoietic stem cell transplantation (HSCT) remains the only fully curative therapy for diseases such as b-thalassemia, sickle cell disease, MDS and AML and represents a relevant procedure for gene therapy .Tissue iron overload and non-transferrin bound iron (NTBI) have emerged as adverse prognostic factors for post-transplant survival. Recently, we showed that - in line with observations in transplanted individuals - preparatory chemotherapy conditioning with a combination of busulfan and fludarabine in wild-type mice resulted in elevated serum iron and transferrin (Tf) saturation and elicited NTBI formation. Tf saturation and NTBI levels increased proportionally with conditioning intensity. Interestingly, we found that pre-conditioning TMPRSS6 silencing through a GalNAc-conjugated siRNA - by increasing hepcidin levels - reduced the peri-transplant elevation of Tf saturation and limited the appearance of NTBI. These results proved that NTBI formation is induced by conditioning chemotherapy in the peri-trasplant period independently of the presence of pre-existing iron overload. Aims & Methods: Here we asked whether these observations hold true in conditions hallmarked by pre-existing iron overload and elevated NTBI and how iron restriction impacts on bone marrow transplant (BMT) outcome. To this aim, we applied the GalNAc TMPRSS6 siRNA as iron restriction strategy in a mouse model of myelodysplastic syndromes (MDS) and assessed its ability to reduce conditioning-elicited NTBI formation as well as its potential benefit on BMT. After TMPRSS6 siRNA administration, CD45.2 + MDS mice received conditioning chemotherapy and transplant of CD45.1 + wild-type bone marrow cells. Systemic iron parameters, engraftment and chimerism were then analyzed. Results: Importantly, conditioning exacerbated the elevated systemic iron levels in MDS mice, which was prevented by pre-conditioning TMPRSS6 silencing. Similar to our observation in wild-type mice, recipient CD45.2 + MDS mice showed a superior blood chimerism, resulting from increased bone marrow and spleen engraftment of CD45.1 + donor cells. Multilineage engraftment in bone marrow, spleen and peripheral blood was superior in iron-restricted recipient MDS mice compared to controls. Finally, donor chimerism of hematopoietic stem and progenitor cells (HSPCs) was higher in iron-restricted versus control MDS mice undergoing BMT. Conclusion: These findings demonstrate that pre-transplant conditioning profoundly alters iron homeostasis and restricting iron improves BMT outcome by increasing engraftment efficiency. Overall, these data suggest that conditioning-elicited NTBI is a critical mediator of peri-transplant toxicity and adversely impacts transplant outcome. Specifically, our observations prove that iron restriction improves donor HSPC engraftment in the recipient bone marrow, likely resulting in more efficient reconstitution of the HSPC pool and improved multilineage engraftment. Lastly, our results indicate that the peri-transplant application of novel iron restriction strategies aimed at reducing conditioning-induced NTBI - by positively affecting BMT outcome - is of potential benefit for patients undergoing BMT or gene therapy, presenting either with or without pre-transplant iron overload.

Transcription Factor GATA2 Is Inevitable for the Survival and Proper Differentiation of Thymic Multipotent Progenitor Cells By Gene Expression Orchestration

Proper differentiation of T cells in the thymus is inevitable for the establishment of acquired immunity. Lymphoid-primed multipotent progenitors (LMPPs) emigrate from the bone marrow (BM) to the thymus for becoming earliest T cell progenitors, so-called early T cell progenitors (ETPs). Previous studies have shown that ETPs retain not only T cell but also innate immune cell (i.e., myeloid cells) differentiation potentials. Therefore, ETPs might be the bifurcation point of innate and adaptive immunity in thymus. However, still little is known about the mechanism by which the differentiation fates are determined in ETPs and the cellular heterogeneity in ETP subset. Since transcription factors that possess important roles in hematopoietic stem and progenitor cells (HSPCs) may play significant roles in the early differentiation of T cells as legacy stem cell genes, we sought to investigate the roles for GATA2, which is known to regulate the HSPC maintenance and myeloid differentiation, in ETPs. First, we revealed the GATA2 expression is preserved in ETPs and sharply disappear with their differentiation (GATA2 expressions were almost undetectable from double-negative 2 cells even by qPCR). To reveal the roles for GATA2 in T cell hematopoiesis, we analyzed Gata2 fl/fl; Mx1-Cre mice in which GATA2 was deletable in hematopoietic cells by polyI:C administration ( Gata2 KO). Gata2 KO mice showed a smaller thymus and a lower number of thymic T cells, including ETPs and double-positive (DP; CD4 +CD8 +) cells, than control. To rule out contributions of potential GATA2 deletions in non-hematopoietic cells, CD45.1 + wild-type unfractionated BM cells were transplanted into Gata2 KO or control mice. There were no particular differences in T cell hematopoiesis from CD45.1 + donor cells between them, supporting the notion that hematopoietic cell intrinsic GATA2 are inevitable for the proper T cell hematopoiesis. To further reveal the roles for GATA2 in ETPs, we performed global transcriptomic analysis of ETPs (RNA-seq; Gata2 KO vs control). Gene set enrichment analysis (GSEA) revealed that signatures associated with cell cycle regulation, hematopoietic progenitor cells and myeloid cells were down-regulated in Gata2 KO ETPs, suggesting that GATA2 is involved in the survival and myeloid differentiation potentials in ETPs. By taking the advantage of novel GATA2 reporter mice (GATA2-VENUS mice) in which GATA2 protein levels are detectable by flow cytometry, we revealed that the ETP fraction contained a mixture of cells with high or low GATA2 expression. To be noted, the protein expression intensity of GATA2 in GATA2-high ETPs was almost equivalent to that of LMPPs. These observations suggest that GATA2-high ETPs might be a thymic multipotent progenitors (T-MPPs), the earliest thymic progenitors in which T or myeloid cell differentiation commitment is defined. To elucidate the mechanism by which GATA2 regulates the survival and differentiation of T-MPPs, we integrated publicly available chromatin immunoprecipitation followed by sequencing data targeting GATA2 (GATA2 ChIP-seq) and our ETP RNA-seq data. We found several key genes in HSPCs maintenance (i.e. Lyl1) or myeloid differentiation (i.e. Itga2b) as potential targets of GATA2. Furthermore, we sought to reveal the effect of GATA2 deletion in progenitor cells to their progenies. To this end, we performed RNA-seq of DP cells ( Gata2 KO vs control). GSEA revealed that cell cycle related gene signature was repressed in Gata2 KO DP cells compared to control. To be noted, in CD4-Cre dependent GATA2 deletion model ( Gata2 fl/fl:CD4-Cre mice), there were almost no particular cell number and transcriptomic differences, which was also analyzed by RNA-seq, in DP cells compared to control. These results suggest that GATA2 in GATA2-high ETPs, but not in DP cells, may affect the feature of DP cells, potentially by epigenetic memory. Collectively, this study revealed that GATA2 contributes to T cell hematopoiesis. GATA2 expression was maintained in a part of the ETP subset (GATA2-high ETPs) which might be equivalent to T-MPPs. GATA2 may affect the differentiation trajectory and function from GATA2-high ETPs to their progenies. Further study of this mechanism will help us to understand how the acquired and innate immune bifurcation is defined, which might pave the way to overcome T cell development related disorders, such as T-cell/myeloid mixed-phenotype acute leukemia.

Mechanisms and Therapeutic Strategies to Reverse TET2 Mutant Clonal Hematopoiesis and the Risk of MDS, AML, and Atherosclerotic Cardiovascular Disease

Inactivating mutations of the epigenetic modifier TET2 are frequent in myeloid malignancies and clonal hematopoiesis of indeterminate potential (CHIP). These mutations in hematopoietic stem and progenitor cells (HSPCs) impart a clonal advantage with increased self-renewal. CHIP is associated with an increased risk of hematological malignancies and, surprisingly, with atherosclerotic cardiovascular disease, supported by studies in mice and humans. The heightened cardiovascular risk in CHIP patients may result from a pro-inflammatory slant of the mutant HSPC progeny, particularly monocytes and macrophages, independent from the risk imparted by elevated LDL cholesterol levels. Targeting molecules like IL-1β may provide a strategy to lower the risk of cardiovascular morbidity in patients with CHIP. However, as an alternative strategy, administration of drugs that selectively suppress the growth of mutant CHIP clones in the bone marrow (BM) might reduce the risk of both i) progression to MDS or hematologic malignancy and of ii) atherosclerotic cardiovascular disease. This study focused on identifying drugs that reverse the inflammatory and atherosclerotic properties of TET2-mutant macrophages. Our previous investigations demonstrated that the nuclear export inhibitor eltanexor selectively kills Tet2-mutant HSPCs in zebrafish embryos and reduces the number of mutant colonies in murine colony formation assays. A murine competitive repopulation model of CHIP tested the in vivo efficacy of eltanexor. We found that eltanexor selectively reduces Tet2-mutant circulating monocytes while having no effect on overall total white blood cell counts. Moreover, a second trial using Ldlr-/- atherosclerosis-prone mice consuming a high-cholesterol diet, showed that treatment with eltanexor selectively reduces the amount of aortic atherosclerotic plaque formation as well as spleen weight in mice injected with Tet2+/- (mutant) BM cells, whereas eltanexor had no significant effect in mice injected with Tet2+/+ (wild-type) BM cells. Single-cell CITE-seq analysis of Ldlr-/- mice injected with a mix of Tet2+/- and Tet2+/+ BM cells showed that eltanexor selectively reduces the percentage of Tet2+/- HSPCs. Additionally, CITE-seq analysis revealed a significant reduction in pro-inflammatory macrophages in the arterial wall after eltanexor treatment, along with a decrease in IL-1β expression by these cells. As TET2 functions in mechanistically regulating gene regulation through epigenetic DNA methylation, we employed ChIP-seq and CUT&RUN-Seq technologies to investigate the mechanisms underlying enhancer dysregulation caused by loss of Tet2. Enhancer profiling of bone marrow-derived macrophages showed that the negative regulator of the macrophage inflammatory response, ATF3, is associated with a super-enhancer. Notably, the ATF3 super-enhancer emerged as the largest super-enhancer in wild-type macrophages but was reduced in Tet2-mutant macrophages. Our investigation also revealed that ATF3 binds with IL-1β at H3K27ac modified regions, and this binding was significantly diminished in Tet2-mutant macrophages. Remarkably, treatment with eltanexor restored the binding of ATF3 to IL1β, providing a mechanism to explain the anti-inflammatory effect of eltanexor concordant with our in vivo results. In summary, this study proposes eltanexor treatment as a novel therapeutic approach to specifically target TET2-mutant cells in individuals with CHIP and sheds light on the underlying mechanisms of the pro-inflammatory slant of TET2-mutant macrophages. These findings offer potential new avenues for precise therapies in humans with CHIP and associated cardiovascular risks from atherosclerosis.

NLRP3 inflammasome-produced pro-inflammatory cytokines IL-1b and IL-18 are critical exacerbating factors of septic cardiomyopathy

Abstract Background Sepsis is life-threatening multiple organ failure due to systemic infection, and frequently causes septic cardiomyopathy (SCM). Recent evidence has established that cytokine storm, an aberrant increase in various cytokines leading to severe inflammation and tissue damage, plays a crucial role in SCM. However, it is unknown which cytokines are the most critical initiators and exacerbating factors of SCM. Purpose To investigate how IL-1β and IL-18 driven by NLRP3 inflammasome contribute to the pathophysiology of SCM, and to study whether blockage of these cytokines can ameliorate cardiac dysfunction in a murine model of lipopolysaccharide (LPS)-induced SCM. Methods Wild-type, Nlrp3-/-, Casp1/11-/-, and Il1b-/- mice were injected intraperitoneally with 6 mg/kg of LPS to induce SCM. To suppress IL-18, the adeno-associated virus (AAV) vectors encoding IL-18 binding protein (IL-18BP) were injected intramuscularly 14 days before LPS. Bone marrow transplantation (5 Gy x2) was performed 6 weeks prior to LPS. Results While 40% of wild-type mice died 48 h after LPS injection in our SCM model, none of Nlrp3-/- mice died. Cardiac function, as determined by echocardiography and BNP, was significantly decreased 6 h after LPS injection in WT mice but not in Nlrp3-/- or Casp1/11-/- mice. Serum CK-MB increase was also ameliorated in Nlrp3-/- or Casp1/11-/- mice. Serum levels of various pro-inflammatory cytokines including TNFα, IL-1β, IL-6, IL-18, and IFNγ were suppressed in Nlrp3-/- mice. These findings collectively indicated a critical role of NLRP3 inflammasome in LPS-induced SCM. However, unexpectedly, genetic deletion of IL-1β, the canonical effector molecule of NLRP3 inflammasome, in Il1b-/- mice failed to alleviate cardiac dysfunction and damage caused by LPS. In addition, IL-18 suppression by AAV-mediated overexpression of IL-18BP, a high-affinity IL-18 decoy receptor, only partially prevented LPS-induced SCM. On the other hand, intriguingly, dual inhibition of IL-1β and IL-18 successfully protected mice from LPS-induced SCM, demonstrating that concomitant blockage of two NLRP3 inflammasome products, IL-1β and IL-18, was necessary and sufficient to prevent SCM. Finally, while bone marrow transplantation experiments showed that cardiac dysfunction after LPS injection was suppressed in recipient Nlrp3-/- mice transplanted with wild-type bone marrow cells, cardiomyocyte-specific deletion of NLRP3 in αMHC-Cre;Nlrp3-/- mice did not prevent LPS-induced SCM. These data suggest that NLRP3 inflammasome in resident cells other than bone marrow-derived cells and cardiomyocytes is important in the pathophysiology of SCM. Conclusion The present study indicates that concomitant blockage of IL-1β and IL-18 driven by NLRP3 inflammasome is necessary and sufficient to prevent SCM. Our data provide fundamental evidence that combination therapy targeting both IL-1β and IL-18 could serve as a new approach to the treatment of SCM.

Clearing and replacing tissue-resident myeloid cells with an anti-CD45 antibody–drug conjugate

AbstractTissue-resident myeloid (TRM) cells in adults have highly variable lifespans, and may be derived from early embryonic yolk sac, fetal liver, or bone marrow. Some of these TRM cells are known pathogenic participants in congenital and acquired diseases. Myeloablative conditioning and hematopoietic stem cell transplantation can replace long-lived brain TRM cells, resulting in clinical improvements in metabolic storage diseases. With the advent of antibody–drug conjugate (ADC)-targeted cell killing as a cell-selective means of transplant conditioning, we assessed the impact of anti-CD45–ADC on TRM cells in multiple tissues. Replacement of TRM cells ranged from 40% to 95% efficiencies in liver, lung, and skin tissues, after a single anti-CD45–ADC dose and bone marrow hematopoietic cell transfer. Of note, the population size of TRM cells in tissues returned to pretreatment levels, suggesting a regulated control of TRM cell abundance. As expected, brain microglia were not affected, but brain monocytes and macrophages were 50% replaced. Anti-CD45–ADC and adoptive cell transfer were then tested in the chronic acquired condition, atherosclerosis exacerbated by Tet2 mutant clonal hematopoiesis. Plaque-resident myeloid cells were efficiently replaced with anti-CD45–ADC and wild-type bone marrow cells. Notably, this reduced existent atherosclerotic plaque burden. Overall, these results indicate that the anti-CD45–ADC clears both hematopoietic stem and TRM cells from their niches, enabling cell replacement to achieve disease modification in a resident myeloid cell–driven disease.

Important Role of Endogenous Nerve Growth Factor Receptor in the Pathogenesis of Hypoxia-Induced Pulmonary Hypertension in Mice.

Pulmonary arterial hypertension (PAH) remains a disease with poor prognosis; thus, a new mechanism for PAH treatment is necessary. Circulating nerve growth factor receptor (Ngfr)-positive cells in peripheral blood mononuclear cells are associated with disease severity and the prognosis of PAH patients; however, the role of Ngfr in PAH is unknown. In this study, we evaluated the function of Ngfr using Ngfr gene-deletion (Ngfr-/-) mice. To elucidate the role of Ngfr in pulmonary hypertension (PH), we used Ngfr-/- mice that were exposed to chronic hypoxic conditions (10% O2) for 3 weeks. The development of hypoxia-induced PH was accelerated in Ngfr-/- mice compared to littermate controls. In contrast, the reconstitution of bone marrow (BM) in Ngfr-/- mice transplanted with wild-type BM cells improved PH. Notably, the exacerbation of PH in Ngfr-/- mice was accompanied by the upregulation of pulmonary vascular remodeling-related genes in lung tissue. In a hypoxia-induced PH model, Ngfr gene deletion resulted in PH exacerbation. This suggests that Ngfr may be a key molecule involved in the pathogenesis of PAH.

Novel Evidence That NADPH Oxidase 2 (Nox-2) Enhances Trafficking of Hematopoietic Stem Progenitor Cells (HSPCs) By Involving ROS Mediated Activation of Nlrp3 Inflammasome

Background. NADPH oxidase 2 (Nox-2) is a superoxide-generating enzyme that forms reactive oxygen species (ROS) to regulate the redox state involved in the self-renewal of hematopoietic stem/progenitor cells (HSPCs). Moreover, ROS are released from Nox-2 expressed by cell surface and mitochondria membranes and are potent activators of intracellular pattern recognition receptor Nlrp3 inflammasome, which regulates several aspects of HSPCs biology, including their trafficking, proliferation, and metabolism. Our previous research demonstrated a novel role of Nlrp3 inflammasome in the migration, mobilization, homing, and engraftment of HSPCs (Leukemia 2022;36(1):23-32;Stem Cell Rev Rep. 2020 16(5):954-967). The role of ROS was extensively studied in the proliferation of hematopoietic cells; however, its role in the migration of HSPCs is not very well understood. Aim of the study.Based on the role of Nox-2 in activating Nlrp3 inflammasome, we hypothesized that activation of Nox-2-ROS-Nlrp3 inflammasome axis in both HSPCs and BM microenvironment orchestrates trafficking of HSPCs.Materials and Methods. First, we studied in Transwells chemotaxis of Nox-2 deficient cells to bone marrow (BM) chemoattractants, including SDF-1, S1P, and eATP. Next, to shed more light on the role of Nox2 in the trafficking of HSPCs, we performed in Nox-2-KO animals G-CSF- and AMD3100-induced pharmacological mobilization. We also studied homing and engraftment of Nox-2-KO cells in BM of wild-type (WT) animals and homing and engraftment o WT BM cells in Nox-2-KO recipients. Nox-2-KO mice were also sublethal irradiated, and we followed the recovery of peripheral blood counts in these animals. Activation of Nlrp3 inflammasome in Nox-2-KO murine Sca-1+c-kit+lineage- HSPCs was evaluated by Glow assay. To ''bypass'' Nox-2-ROS activation in control experiments, nigericin has been employed as a direct activator of Nlrp3 inflammasome. Since membrane lipid rafts (MLRs) play a crucial role optimal migration of HSPCs, we evaluated the formation of membrane lipid rafts by employing confocal analysis in Nox-2-KO cells stimulated with SDF-1. We also studied the expression of enzymes (SREBP2, HMGCs, and HMGCR) involved in providing lipid components for MLRs assemble in activated Nox-2-KO cells. Results. We noticed that Nox-2-KO animals have a profound defect in the trafficking of HSPCs as evidenced by impaired chemotaxis to BM chemoattractants, decrease in pharmacological mobilization, and defect in homing and engraftment after transplantation. Nox-2-KO mice have also defect in recovery from sublethal irradiation. At the molecular level, HSPCs from Nox-2-KO mice displayed a defect in activation of Nlrp3 inflammasome and in cholesterol and sphingolipids synthesizing enzymes, which provide lipid components required for optimal assemble of MLRs. These defects were partially ameliorated by using direct activator of Nlrp3 inflammasome that is nigericin. Conclusions. We provide for the first time evidence that Nox-2-ROS-Nlrp3 inflammasome axis operates in normal HSPCs and is crucial for regulating trafficking and post-irradiation recovery of these cells. These effects are mediated by providing components for MLRs required for optimal migration and signaling in HSPCs

Transcriptional differences between JAK2-V617F and wild-type bone marrow cells in patients with myeloproliferative neoplasms

The JAK2-V617F mutation is the most common cause of myeloproliferative neoplasms. Although experiments have revealed that this gain-of-function mutation is associated with myeloid blood cell expansion and increased production of white cells, red cells, and platelets, the transcriptional consequences of the JAK2-V617F mutation in different cellular compartments of the bone marrow have not yet been fully elucidated. To study the direct effects of JAK2-V617F on bone marrow cells in patients with myeloproliferative neoplasms, we performed joint single-cell RNA sequencing and JAK2 genotyping on CD34+-enriched cells from eight patients with newly diagnosed essential thrombocythemia or polycythemia vera. We found that the JAK2-V617F mutation increases the expression of interferon-response genes (e.g., HLAs) and the leptin receptor in hematopoietic progenitor cells. Furthermore, we sequenced a population of CD34- bone marrow monocytes and found that the JAK2 mutation increased expression of intermediate monocyte genes and the fibrocyte-associated surface protein SLAMF7 in these cells.

Txnip enhances Fitness of Dnmt3a-Mutant Hematopoietic Stem Cells Via p21

Clonal hematopoiesis (CH) refers to the age-related expansion of specific clones in the blood system and manifests from somatic mutations acquired in hematopoietic stem cells (HSCs). Approximately 50% of CH variants occur in the gene DNMT3A. While DNMT3A-mutant CH becomes almost ubiquitous in aging humans, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations increase HSC fitness under recurrent hematopoietic stress.To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated; Dnmt3a heterozygous (Vav-Cre; Dnmt3afl/+ = Dnmt3aHET) and homozygous (Vav-Cre; Dnmt3afl/fl = Dnmt3aKO) hematopoietic loss-of-function, and a knock-in model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre; Dnmt3aR878H/+ = Dnmt3aR878).When Dnmt3a-mutant cells were competitively transplanted with wild-type (WT) competitor bone marrow (BM) cells and challenged with different inflammatory and proliferative stressors, Dnmt3aKO and Dnmt3aR878 HSCs were specifically resistant to the deleterious effects of IFNg on HSC self-renewal and clonal expansion. This insensitivity was also confirmed in a humanized mouse model where human CD34 + cord blood cells edited with DNMT3A-targeting gRNAs were xenografted into recipient mice and episodically exposed to human recombinant IFNg. DNMT3A mutant cells maintained their clone size, whereas AAVS1-targeted cells (control) were depleted over serial transplantation. These data suggest that Dnmt3a-mutant HSCs, mimicked DNMT3A-mutated human HSCs and are specifically resistant to IFNg-mediated depletion. One explanation for the observed resistance is that Dnmt3a-mutant HSCs have a fitness advantage under IFNg challenge. Therefore, we generated a novel mouse model to directly quantify the competition between Dnmt3a-mutant and WT HSCs. 10% donor BM cells (CD45.2; WT or Dnmt3a-mutant), 10% WT competitor BM cells (CD45.1/2; Ubc-GFP+) and 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA-IFNg) that express IFNg by doxycycline were transplanted into CD45.1 recipient mice. To normalize the effect of doxycycline, chimera made with 80% BM cells (CD45.1; IFNgr1KO; Rosa-M2-rtTA) were also transplanted into recipients. Our result from this transplantation experiment showed Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Genetic ablation of IFNgr1 from Dnmt3a-mutant mice revealed that IFNg signaling is cell-intrinsically required by clonal expansion of Dnmt3a-mutant HSCs. In parallel, when HSCs were transplanted into IFNg-deficient recipient mice, clonal expansion of Dnmt3aKO HSCs but not WT HSCs was significantly compromised, suggesting IFNg signaling is also cell-extrinsically crucial for the clonal expansion of Dnmt3a-mutant HSCs in vivo. Mechanistically, DNA hypomethylation-associated over-expression of Thioredoxin-interacting protein (Txnip) in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs led to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence and resistance to apoptosis in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not WT 32D murine myeloid cell line. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 re-sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation and apoptosis. In vivo, down-regulation of p21 had no effect on WT HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-induced exhaustion in a transplantation experiment.Taken together, our data highlighted a Txnip-p53-p21 pathway that preserves the functional potential of Dnmt3a-mutant HSCs under conditions of inflammatory stress, which suggests a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

DNMT3A with Leukemia-Associated R882 Mutations Promotes Fitness Advantage through Functional Heterogeneity within HSCs

Mutations in DNA damage regulators and epigenetic modifier genes including those in DNA methyltransferase 3A (DNMT3A) are common in pre-malignant clonal hematopoiesis (CH) and are recurrent in myeloid malignancies such as myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). While loss-of-function (LOF) mutations in DNMT3A are predominant in CH, single amino-acid substitutions at residue 882, usually from an arginine to a histidine or cysteine (DNMT3A R882H / DNMT3A R882C), are strongly enriched in AML, suggesting a role in promoting pre-malignant clonal evolution. In this study, we compared and contrasted the effects of DNMT3A R882 and LOF on hematopoietic stem cell (HSC) function by investigating self-renewal capacity in vitro and bone marrow repopulation potential in vivo in a genetic mouse model.Bone marrow (BM) from mice conditionally expressing Dnmt3a R878H (equivalent to human DNMT3A R882H) from the endogenous locus, denoted as +/mut, was subjected to colony forming unit (CFU) assays in semisolid media (MethoCult GF M3434, Stem Cell Technologies) to detect hematopoietic progenitors with serial replating every 14 days as a surrogate measure of stem cell self-renewal. Dnmt3a +/mut cells were compared to bone marrow derived from mice with heterozygous or homozygous Dnmt3a knockout (+/- and -/-, respectively), or wild-type control (+/+). Dnmt3a +/- BM cells showed a trend towards a slight increase in the number of replatings compared to wild-type controls (2.83±0.41 in +/+ vs 3.75±0.89 in +/-, n=6,8, Mann-Whitney test p=0.053). In contrast, Dnmt3a +/mut BM occasionally resulted in a significant gain of self-renewal with replating for 6+ passages observed in 4/10 independent trials (6.00±2.06 in +/mut, n=10, p=0.001 vs +/+), yet failed to reach continuous replating of complete Dnmt3a loss that served as a positive control (terminated at passage 10, n=5). Substantial heterogeneity in the Dnmt3a +/mut self-renewal capacity ex vivo was further reflected by increased statistical variance (Welch's F-test p=0.037 compared to +/-).To extend these studies to the in vivo setting, we performed gold-standard serial competitive bone marrow repopulation assays. Here, 250 FACS-sorted Lineage -Sca1 +cKit +CD48 -CD150 + (LSK-SLAM) test cells marked with CD45.2 were mixed with 0.25x10 6 unfractionated wild-type competitor bone marrow cells marked with CD45.1 and engrafted into pre-conditioned congenic recipients (CD45.1). Peripheral blood CD45.2/CD45.1 chimerism is used as a readout for the ability of test cells to reconstitute hematopoiesis; self-renewal potential is assessed via serial retransplantation. In this setting, Dnmt3a +/+ cells showed a decline in their ability to reconstitute hematopoiesis in secondary transplant recipients, while Dnmt3a haploinsufficiency resulted in a competitive advantage over wild-type (CD45.2 chimerism 2.4±2.7% in +/+ vs 75.6±13.0% in +/-, n=4, Welch's t-test p=0.001). In contrast, Dnmt3a +/mut cells displayed a high degree of variability in their long-term stem cell function in some cases yielding high levels of chimerism (CD45.2 >40% in 3/9 recipients) and in other cases not (26.0±21.0%, n=9, p=0.01 vs +/+), in agreement with CFU assays.Collectively, these data suggest that the DNMT3A R882 mutations endow normal HSCs with phenotypic heterogeneity yielding a population of cells with variable stem cell function, some of which may have higher fitness thus accelerating clonal evolution towards malignancy. These findings will be built upon in future experiments aiming to identify underlying molecular mechanisms and to improve our understanding of the pathophysiology and prognostication of clonal hematopoiesis. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Myeloid-Derived Growth Factor Protects Against Pressure Overload–Induced Heart Failure by Preserving Sarco/Endoplasmic Reticulum Ca 2+ -ATPase Expression in Cardiomyocytes

Background: Inflammation contributes to the pathogenesis of heart failure, but there is limited understanding of inflammation’s potential benefits. Inflammatory cells secrete MYDGF (myeloid-derived growth factor) to promote tissue repair after acute myocardial infarction. We hypothesized that MYDGF has a role in cardiac adaptation to persistent pressure overload. Methods: We defined the cellular sources and function of MYDGF in wild-type (WT), Mydgf -deficient ( Mydgf −/− ), and Mydgf bone marrow–chimeric or bone marrow–conditional transgenic mice with pressure overload–induced heart failure after transverse aortic constriction surgery. We measured MYDGF plasma concentrations by targeted liquid chromatography–mass spectrometry. We identified MYDGF signaling targets by phosphoproteomics and substrate-based kinase activity inference. We recorded Ca 2+ transients and sarcomere contractions in isolated cardiomyocytes. Additionally, we explored the therapeutic potential of recombinant MYDGF. Results: MYDGF protein abundance increased in the left ventricular myocardium and in blood plasma of pressure-overloaded mice. Patients with severe aortic stenosis also had elevated MYDGF plasma concentrations, which declined after transcatheter aortic valve implantation. Monocytes and macrophages emerged as the main MYDGF sources in the pressure-overloaded murine heart. While Mydgf −/− mice had no apparent phenotype at baseline, they developed more severe left ventricular hypertrophy and contractile dysfunction during pressure overload than WT mice. Conversely, conditional transgenic overexpression of MYDGF in bone marrow–derived inflammatory cells attenuated pressure overload–induced hypertrophy and dysfunction. Mechanistically, MYDGF inhibited G protein–coupled receptor agonist–induced hypertrophy and augmented SERCA2a (sarco/endoplasmic reticulum Ca 2+ -ATPase 2a) expression in cultured neonatal rat ventricular cardiomyocytes by enhancing PIM1 (Pim-1 proto-oncogene, serine/threonine kinase) expression and activity. Along this line, cardiomyocytes from pressure-overloaded Mydgf −/− mice displayed reduced PIM1 and SERCA2a expression, greater hypertrophy, and impaired Ca 2+ cycling and sarcomere function compared with cardiomyocytes from pressure-overloaded WT mice. Transplanting Mydgf −/− mice with WT bone marrow cells augmented cardiac PIM1 and SERCA2a levels and ameliorated pressure overload–induced hypertrophy and dysfunction. Pressure-overloaded Mydgf −/− mice were similarly rescued by adenoviral Serca2a gene transfer. Treating pressure-overloaded WT mice subcutaneously with recombinant MYDGF enhanced SERCA2a expression, attenuated left ventricular hypertrophy and dysfunction, and improved survival. Conclusions: These findings establish a MYDGF-based adaptive crosstalk between inflammatory cells and cardiomyocytes that protects against pressure overload–induced heart failure.

Functional Impairment of Hematopoietic Stem/Progenitor Cells By Loss of Uhrf2 in Mice

A growing body of evidence has underlined the involvement of epigenetic regulations that include histone modification in hematopoiesis. We previously identified Ubiquitin Like with PHD and Ring Finger Domains 2 (UHRF2) as a crucial multi-domain E3 ubiquitin ligase that facilitates coordination of the cell-cycle machinery and the epigenetic regulations via interaction with cyclins, TP53, DNA methyltransferases, and G9A H3K9 methyltransferase in non-hematopoietic cells (Mori et al, FEBS Letters, 2012). In fact, H3K9 trimethylation (H3K9me3) is reduced in Uhrf2-/- embryonic stem (ES) cells (Pichler et al, J Cell Biochem, 2011). Recently, Lu et al. have also reported that UHRF2 is highly expressed in common lymphoid progenitors and mature lymphocytes rather than myeloid cells, and expression of UHRF2 is reduced in some acute myeloid leukemia (AML) cells and a variety of cancers (Oncotarget, 2016). However, the association of UHRF2 with hematopoiesis has not yet been studied. Here, to clarify the role of UHRF2 in hematopoiesis, we investigated hematopoietic cells and organs in Uhrf2-/- mice.We did not find a significant difference in peripheral blood leukocyte and platelet counts and hemoglobin values, and spleen sizes between wild-type (WT) and Uhrf2-/- mice at young (12 weeks) and old (50 weeks) ages. The total cell counts and proportions of hematopoietic stem/progenitor cell (HSPC)-enriched lineage-Sca1+Kit+ (LSK) cells, common myeloid progenitors, granulocyte-macrophage progenitors and megakaryocyte-erythrocyte progenitors in bone marrow (BM) were also similar between WT and Uhrf2-/- mice. We next performed competitive repopulation assay with BM transplants (BMT) by infusing the same numbers of Ly5.1+ WT BM cells and Ly5.2+Uhrf2-/- BM cells into lethally irradiated Ly5.1+ WT recipients (n = 4). Of note, at 12 weeks after the first BMT, the proportions of Ly5.2+Uhrf2-/- cells were significantly less in all lineages compared with control BMT that used Ly5.1+ WT BM cells and Ly5.2+Uhrf2+/+ BM cells. After the second BMT, Uhrf2-/- cells were almost rejected in neutrophils while 20-30% Uhrf2-/- T cells and B cells were still detected. After the third BMT, however, no Uhrf2-/- myeloid or lymphoid cells were found in peripheral blood of recipients. These findings indicate functional impairment of Uhrf2-/- HSPCs with defective self-renewal capacity.We then investigated the mechanism of impairment in the function of HSPCs in Uhrf2-/- mice. As reported in Uhrf2-/- ES cells, we found reduced H3K9me3 in Uhrf2-/- BM cells. We also performed RNA sequencing for LSK cells, which showed downregulations of the genes that are crucial for maintenance and proliferation of HSPCs, such as Flt3 (2.2-fold) and Stat5a (2.3-fold). GSEA (gene set enrichment analysis) revealed significant enrichment of targets of H3K9me3 (FDRq value = 0.045). Furthermore, the pathway analysis identified global reductions in the canonical immunological pathways associated with differentiation of B cells, including Role of NFAT in Regulation of the Immune Response (Z score = -3.258, P = 0.009), B Cell Receptor Signaling (Z score = -2.197, P = 0.021), and PI3K Signaling in B Lymphocytes (Z score = -2.041, P = 0.024) in Uhrf2-/- LSK cells. In agreement, it has been recently reported that reduction of H3K9me3 in HSPCs results in impairments of their abilities to differentiate and generate B cells (Ugarte et al, Stem Cell Reports, 2015; Djeghloul et al, Stem Cell Reports, 2016).Collectively, Uhrf2 possibly play some roles in self-renewal and differentiation of HSPCs in part through the trimethylation of H3K9. Defect of Uhrf2 might contribute to the impairment of differentiation rather than growth advantage in some cancer or AML cells. DisclosuresNo relevant conflicts of interest to declare.

RUNX3 Promotes the Development of MDS/MPN Overlap Syndrome Via Enhancing Expression of Myc in the Absence of Tet2

The RUNX transcription factors have a critical role in normal and malignant hematopoiesis including myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS). RUNX3 is expressed in various hematopoietic cells, and the reduced expression of RUNX3 due to promoter DNA hyper-methylation is seen in patients with acute lymphoid leukemia, indicating a tumor suppressive role of RUNX3. The loss of Runx3 promotes the development of MPN-like disease in mice. However, loss-of-function mutations and/or a deletion of RUNX3 are rarely observed in patients with myeloid malignancies. In fact, given that enhanced levels of RUNX3 expression are found in a part of patients with AML and MDS, RUNX3 may play an oncogenic role for myeloid malignancies.To determine how RUNX3 over-expression promotes the development of myeloid malignancies, we generated a mouse model of RUNX3 -induced MDS/MPN by utilizing Tet2 -deficient hematopoietic stem cells (HSCs). Since TET2 loss-of-function mutations initiate the transformation of HSCs, we harvested bone marrow CD150+CD34-LSK HSCs from tamoxifen-treated Cre-ERT2 (WT) and Cre-ERT2;Tet2Δ/Δ (Tet2 KO) mice, and transduced expression of 3xFLAG-tagged RUNX3 by using a retrovirus vector in vitro. Then, we transplanted them into lethally-irradiated Ly5.1+ recipient mice together with wild-type competitor cells. We observed that Tet2 KO mice died dominantly due to MPN-like disease, whereas RUNX3 -expressing Tet2 wild-type (RUNX3/WT) HSCs neither competed wild-type bone marrow cells nor developed hematological malignancies in mice by 12 months post transplantation. In contrast, RUNX3 -expressing Tet2 KO (RUNX3/Tet2 KO) HSCs preferentially generated myeloid cells, but not lymphoid cells, and subsequently competed wild-type cells in the PB and the BM. We found that 7 out of 18 RUNX3/Tet2 KO mice developed lethal MDS/MPN diseases despite a longer latency. At the time of sacrifice, RUNX3-Tet2 KO mice showed severe pancytopenia accompanied with dyplastic blood cells including pseudo-Pelger neutrophils and micro megakaryocytes. In addition, these moribund mice significantly enlarged spleen due to the expansion of myeloid cells. FACS analysis demonstrated that the expansion of LSK hematopoietic stem/progenitor cells (HSPCs) in the BM and the spleen, consistent with the features of extramedullary hematopoiesis. Thus, RUNX3/Tet2 KO mice recapitulated the phenotypic features of human MDS/MPN overlap syndrome, indicating that RUNX3 over-expression is prerequisite to the development of MDS/MPN in the absence of Tet2, although it has an adverse effect on the function of Tet2 -present HSCs, reminiscent with a tumor suppressive function of RUNX3 as seen in Runx3 KO mice.To understand the underlying molecular mechanism in the pathogenesis of RUNX3 -induced MDS/MPN, we performed gene expression analysis in HSPCs isolated from WT, RUNX3/WT, Tet2 KO and RUNX3/Tet2 KO mice at pre-disease and MDS/MPN stages. Gene Set Enrichment Analysis (GSEA) demonstrated that RUNX3/Tet2 KO MDS/MPN cells significantly enhanced expression of c-Myc target genes, compared to either WT cells or RUNX3/Tet2 KO cells at the pre-disease stage. While RUNX3/Tet2 KO cells showed negative enrichments of differentiation-related pathway genes, compared to WT cells, we found that those MDS/MPN cells enhanced expression of cell cycle, ribosome, and mitochondria-related biological pathways, in which c-Myc may regulate target genes and contribute to the transformation. In order to determine whether RUNX3 directly activates expression of c-Myc, we performed ChIP-sequencing by using an anti-FLAG antibody in RUNX3/Tet2 KO bone marrow cells, and found that c-Myc 3' distal enhancer regions showed conserved binding sites for Runx transcription factors. In addition, we found that RUNX3 -expressing human AML cells showed higher sensitivity to JQ1, a BRD4 inhibitor, following the significant reduction of both RUNX3 and C-MYC expression. The growth inhibition by JQ1 was rescued by ectopic expression of RUNX3 .In conclusion, RUNX3 and c-Myc collaborated to activate the function of oncogenic enhancers, in which Tet2 deficiency may alter the epigenetic status for allowing the bindings of these transcription factors, to an extent not seen in RUNX3-expressing alone. We demonstrated that RUNX3 functions as either an oncogene or a tumor suppressor for myeloid malignancies in the context dependent manner. DisclosuresHarada:Celgene: Research Funding; NIPPON SHINYAKU CO.: Speakers Bureau; NOVARTIS: Research Funding.

Bone marrow transplantation without myeloablative conditioning in a mouse model for Diamond-Blackfan anemia corrects the disease phenotype

Diamond-Blackfan anemia (DBA) is a congenital erythroid hypoplasia caused by a functional haploinsufficiency of genes coding for ribosomal proteins. Among these genes, the ribosomal protein S19 (RPS19) gene is the most frequently mutated. Previously, a mouse model deficient in RPS19 was developed by our laboratory, which recapitulates the hematopoietic disease phenotype by manifesting pathologic features and clinical symptoms of DBA. Characterization of this model revealed that chronic RPS19 deficiency leads to exhaustion of hematopoietic stem cells and subsequent bone marrow (BM) failure. In this study, we evaluated a nonmyeloablative conditioning protocol for BM transplants in RPS19-deficient mice by transplanting wild-type BM cells to RPS19-deficient recipients given no conditioning or sublethal doses of irradiation before transplant. We describe full correction of the hematopoietic phenotype in mice given sublethal doses of irradiation, as well as in animals completely devoid of any preceding irradiation. In comparison, wild-type animals receiving the same preconditioning regimen and number of transplanted cells exhibited significantly lower engraftment levels. Thus, robust engraftment and repopulation of transplanted cells can be achieved in reduced-intensity conditioned RPS19-deficient recipients. As gene therapy studies with autologous gene-corrected hematopoietic stem cells are emerging, we propose the results described here can guide determination of the level of conditioning for such a protocol in RPS19-deficient DBA. On the basis of our findings, a relatively mild conditioning strategy would plausibly be sufficient to achieve sufficient levels of engraftment and clinical success.

CSF1R-dependent macrophages control postnatal somatic growth and organ maturation.

Homozygous mutation of the Csf1r locus (Csf1rko) in mice, rats and humans leads to multiple postnatal developmental abnormalities. To enable analysis of the mechanisms underlying the phenotypic impacts of Csf1r mutation, we bred a rat Csf1rko allele to the inbred dark agouti (DA) genetic background and to a Csf1r-mApple reporter transgene. The Csf1rko led to almost complete loss of embryonic macrophages and ablation of most adult tissue macrophage populations. We extended previous analysis of the Csf1rko phenotype to early postnatal development to reveal impacts on musculoskeletal development and proliferation and morphogenesis in multiple organs. Expression profiling of 3-week old wild-type (WT) and Csf1rko livers identified 2760 differentially expressed genes associated with the loss of macrophages, severe hypoplasia, delayed hepatocyte maturation, disrupted lipid metabolism and the IGF1/IGF binding protein system. Older Csf1rko rats developed severe hepatic steatosis. Consistent with the developmental delay in the liver Csf1rko rats had greatly-reduced circulating IGF1. Transfer of WT bone marrow (BM) cells at weaning without conditioning repopulated resident macrophages in all organs, including microglia in the brain, and reversed the mutant phenotypes enabling long term survival and fertility. WT BM transfer restored osteoclasts, eliminated osteopetrosis, restored bone marrow cellularity and architecture and reversed granulocytosis and B cell deficiency. Csf1rko rats had an elevated circulating CSF1 concentration which was rapidly reduced to WT levels following BM transfer. However, CD43hi non-classical monocytes, absent in the Csf1rko, were not rescued and bone marrow progenitors remained unresponsive to CSF1. The results demonstrate that the Csf1rko phenotype is autonomous to BM-derived cells and indicate that BM contains a progenitor of tissue macrophages distinct from hematopoietic stem cells. The model provides a unique system in which to define the pathways of development of resident tissue macrophages and their local and systemic roles in growth and organ maturation.

Bone marrow transplantation into Abcd1-deficient mice: Distribution of donor derived-cells and biological characterization of the brain of the recipient mice.

X-linked adrenoleukodystrophy (X-ALD) is a severe inherited metabolic disease with cerebral inflammatory demyelination and abnormal accumulation of very long chain fatty acid (VLCFA) in tissues, especially the brain. At present, bone marrow transplantation (BMT) at an early stage of the disease is the only effective treatment for halting disease progression, but the underlying mechanism of the treatment has remained unclear. Here, we transplanted GFP-expressing wild-type (WT) or Abcd1-deficient (KO) bone marrow cells into recipient KO mice, which enabled tracking of the donor GFP+ cells in the recipient mice. Both the WT and KO donor cells were equally distributed throughout the brain parenchyma, and displayed an Iba1-positive, GFAP- and Olig2-negative phenotype, indicating that most of the donor cells were engrafted as microglia-like cells. They constituted approximately 40% of the Iba1-positive cells. Unexpectedly, no decrease of VLCFA in the cerebrum was observed when WT bone marrow cells were transplanted into KO mice. Taken together, murine study suggests that bone marrow-derived microglia-like cells engrafted in the cerebrum of X-ALD patients suppress disease progression without evidently reducing the amount of VLCFA in the cerebrum.

Loss of homeostatic microglia in rare neurological disorders: implications for cell transplantation

Microglia originating from yolk sac exert various functions to maintain the homeostasis in the brain, and their functional breakdown appears to be involved in the pathophysiology of various neurological diseases. In this review article, loss of homeostatic microglia and new therapeutic approaches for rare neurological disorders are discussed. ASLP (adult-onset leukoencephalopathy with axonal spheroids and pigmented glia) known as a primary microgliopathy is an adult-onset leukoencephalopathy caused by CSF1R mutation. CSF1 receptor encoded by CSF1R plays an important role in the function of microglia. In brain of ALSP patients, homeostatic microglia are significantly reduced. The biallelic mutations for CSF1R cause childhood-onset severe phenotype and elimination of microglia from the brain parenchyma. Since microglia also almost disappear in CSF1R-deficient mice and rats, CSF1R deficiency and loss of microglia appear to be tightly associated across species. Based on the underlying mechanism of homeostatic microglia loss, novel approaches using cell transplantation of normal microglia-like cells have been attempted. Transplantation of wild-type bone marrow cells into Csf1r-/- mice results in replacement by donor-derived microglial-like cells in the recipient's brain. The concept of "microglial niche" may explain the rationale behind the microglial cell transplantation in disease condition(s). Hematopoietic stem cell transplantation (HSCT) has been attempted in 4 patients with ALSP. Beneficial effects by showing stabilization of the disease course have been observed. Although the effectiveness of HSCT for ALSP patients warrants further investigation, the approach of cell transplantation that replaces ruptured homeostatic microglia with normal microglia-like cells seems to be promising.