Hematopoietic Stem Cell Homeostasis Research Articles

Stem cell homeostasis by integral feedback through the niche

Hematopoiesis is a paradigm for tissue development and renewal from stem cells. Experiments show that the maintenance of hematopoietic stem cells (HSCs) relies on signals from niche cells. However, it is not known how the size of the HSC compartment is set. Competition by HSCs for niche access has been suggested, yet niche cells in the bone marrow outnumber HSCs. Here we propose a cooperative model of HSC homeostasis in which stem and niche cells mutually interact such that niche cells function as negative feedback regulators of HSC proliferation. This model explains puzzling experimental findings, including homeostatic recovery of the HSC compartment after irradiation versus apparent lack of recovery after HSC ablation. We show that bidirectional niche-stem cell regulation has properties of a proportional-integral feedback controller. Moreover, we predict that the outflux of differentiated cells from HSCs can be regulated by the affinity of HSCs for niche cells. Much effort has been devoted to elucidating niche cell signaling to stem cells; our theoretical insights indicate that studying the effect of stem cells on the niche may be equally important for understanding stem cell homeostasis.

Microenvironment Induced Myelophthisis Caused By CYP26 Deficiency

Hematopoietic stem cells (HSCs) reside in a complex microenvironment that enforces the balance between self-renewal and differentiation. The exact physiologic mechanisms by which the niche controls HSC fate remain elusive. Retinoic acid (RA) is a powerful morphogen that controls stem cell behavior across a variety of systems. In bone marrow (BM), mesenchymal stroma cells (MSCs) express cytochrome P450 (CYP)26 enzymes and can inactivate endogenous and pharmacological retinoids (Ghiaur G et al PNAS 2013, Su M et al. PlosOne 2015, Alonso S et al. 2016). Stromal CYP26 activity is required to maintain human HSCs ex vivo (Ghiaur G et al PNAS 2013). Here we set to study the role of CYP26 in HSC homeostasis in vivo. For this, we induced CYP26 knockout via injection of Tamoxifen in ROSA26CreERT CYP26A1loxP/loxPCYP26B1loxP/loxPmice (CYP26KO) and ROSA26CreERT wildtype mice (CTR). After 5 daily tamoxifen injections, the knockout was confirmed at DNA and RNA level in multiple tissues of CYP26KO mice. Within 4 weeks, CYP26KO mice showed profound leukocytosis (5.97 ±0.37 vs. 21.12 ±3.81 k/mm3, n=4, p<0.01 CTR vs. CYP26KO) with neutrophilia and monocytosis compared to CTR mice. By 6 weeks, they experience profound weight loss, became moribund and had to be sacrificed. At that time, they had massive splenomegaly and lymphadenopathy as well as brittle/pale bones compared to CTR (Figure A). Histological analysis revealed presence of extramedullary hematopoiesis with a predominantly myeloid infiltrate in the spleen (confirmed by flow cytometry analysis) and lymph nodes but also multiple clusters megakaryocytes (Figure B). The mice also displayed decreased BM cellularity (11.25 ±1.01 vs. 5.62 ±0.36 x106/femur, n=5, p<0.01, CTR vs. CYP26KO), increased frequency of CFU-C (44.33 ±3.72 vs. 69.83 ±8.10 CFU/25000 BM mononuclear cells (MNCs), n=10, p=0.02, CTR vs. CYP26KO) with a myeloid bias (26.76±3.89 vs. 44.57 ±3.21 %CFU-GM/G/M, n=10, p<0.01, CTR vs. CYP26KO). When cellularity was taken into account, total CFU-C per femur was comparable between the two groups. The mice also had increased frequency of LSK cells in the BM (0.26 ±0.12 vs 0.73 ±0.18 % LSK of MNCs, n=2, CTR vs. CYP26KO) and an increased frequency of circulating CFU-C in the peripheral blood (37 ±4.04 vs. 179.5 ±43.06 per 200 ml of blood, n=4, p=0.01, CTR vs. CYP26KO). On histological analysis, the BM is dominated by a myeloid infiltrate and shows a striking decrease in radial bone diameter (Figure C,D). MSCs derived from CYP26KO mice have lower levels of CXCL12 and SCF and have impaired osteoblast differentiation potential compared to MSCs derived from control mice (Figure E). These findings were confirmed with ex vivo generated CYP26KO MSCs via retroviral mediated Cre recombination of CYP26A1loxP/loxPCYP26B1loxP/loxPstroma cells. More so, preliminary studies suggest that the hematopoietic phenotype observed depends on cell extrinsic presence of CYP26 activity as transplantation of CYP26A1loxP/loxPCYP26B1loxP/loxP BM cells into wildtype BoyJ recipients did not reproduce the phenotype upon injection with Tamoxifen of the recipient mice in spite of 100% donor derived hematopoiesis. Alternatively, transplant of wildtype cells into CYP26A1loxP/loxPCYP26B1loxP/loxPrecipients, did reproduce the phenotype after injection of Tamoxifen. In conclusion, we show here that dysregulated RA homeostasis in the BM impairs MSCs function and result in egress of hematopoiesis to extramedullary sites. These results come to complement data from RARγKO (Walkley CR et al Cell 2007) and SMRTmRIDmice models (Hong S-H et al PNAS 2013) and suggest a pivotal role of RA signaling in pathogenesis of myeloproliferative neoplasm and myelofibrosis. To what extent these findings correlate with human primary myelofibrosis is currently under investigation. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

The Trithorax-Group Protein ASH1L Regulates Hematopoietic Stem Cell Homeostasis Independently of Its Histone Methyltransferase Activity

Absent, small or homeotic discs 1-like (Ash1l) encodes the mammalian homolog of a Trithorax-group protein with a conserved SET domain that harbors histone H3 lysine 36 dimethyltransferase activity. Using mice with constitutive knockdown of Ash1l due to the insertion of a “gene trap” cassette in the first intron (Ash1lGT/GT mice), we previously reported that Ash1l is a critical regulator of quiescence and self-renewal in adult hematopoietic stem cells (HSCs). While wild-type HSCs could readily establish long-term bone marrow reconstitution after transplantation into irradiated recipients, Ash1l-deficient HSCs had markedly decreased quiescence and failed to establish long-term bone marrow reconstitution (Jones, Chase et al., JCI 2015). Here, we addressed three important questions to better understand the role of Ash1l in hematopoiesis: 1) What is the impact of complete, as opposed to partial, Ash1l loss on adult hematopoiesis? 2) Does Ash1l regulate HSC homeostasis in a cell-autonomous manner? 3) Is the catalytic activity of ASH1L required for its function?To move beyond the limitations of the constitutive knockdown Ash1lGT allele and address the first two questions, we studied newly generated mice carrying conditional knockout (cKO) Ash1l alleles (exon 13 flanked by loxP sites) along with the Mx1-Cre transgene. Induction of Mx1-Cre expression with poly(I:C) in Mx1-Cre+Ash1lf/f mice resulted in nonsense-mediated decay of Ash1l mRNA in hematopoietic cells, thereby completely knocking out Ash1l in adult hematopoietic stem and progenitor cells. Four weeks after inducing Ash1l inactivation in the hematopoietic compartment, we observed a profound depletion of HSCs and multipotent progenitors in Ash1l cKO mice similar to the phenotype of Ash1lGT/GTmice, indicating that conditional Ash1l knockout has a comparable impact on adult hematopoiesis to that of constitutive Ash1l knockdown. Of note, overt hematopoietic failure in steady-state conditions was not observed in either model. To address the second question, we transplanted a mixture of wild-type and Mx1-Cre+Ash1lf/f bone marrow into irradiated wild-type hosts, allowed donor bone marrow to occupy the wild-type niche and establish hematopoiesis, then induced Cre-mediated excision to inactivate Ash1l in Mx1-Cre+Ash1lf/f hematopoietic cells. Upon Cre induction, donor-derived Ash1lΔ/Δ HSCs and myeloid progeny were depleted or outcompeted by wild-type cells, consistent with the model that Ash1l regulates HSC homeostasis in a cell-autonomous manner. Given that Ash1l encodes a SET domain, we next sought to directly examine whether its catalytic activity is required for its role in regulating HSCs. We studied an Ash1l allele with an in-frame deletion of exon 11 and 12, resulting in preserved expression of ASH1L with internally deleted SET domain (ΔSET) (Miyazaki et al., PLOS Genetics 2013). Homozygous ΔSET mice were viable, and phenotypic analysis of adult ΔSET mice revealed normal frequencies of HSCs and multipotent progenitors, in contrast to our observations in Ash1lGT/GTand Ash1l cKO mice. Furthermore, transplanting ΔSET donor bone marrow into irradiated wild-type hosts resulted in sustained long-term reconstitution throughout primary, secondary and tertiary competitive transplantation assays, consistent with preserved HSC function and no alterations in cell cycle regulation. These findings establish that Ash1l regulates HSC homeostasis independently of its SET domain and histone methyltransferase activity. As ASH1L is a very large protein encoding multiple chromatin binding domains, we speculate that ASH1L may serve as a platform to recruit other partners to form novel protein complex(es) that regulate genes critical for HSC homeostasis. DisclosuresNo relevant conflicts of interest to declare.

Engraftment and Reconstitution of Hematopoiesis Is Dependent on Endothelial Cell Expressed Prostaglandin EP4 Receptor Signaling Mediated Regeneration of Vascular Niche

Defining the cellular and molecular components of the bone marrow (BM) niche that regulate the homeostasis and regeneration of hematopoietic stem and progenitor cells (HSPCs) can facilitate targeted interventions to improve hematopoietic recovery after injury and enhance stem cell engraftment after transplantation. We have recently shown that BM niche prostaglandin E2 (PGE2) signaling via the EP4 receptor is crucial for hematopoietic reconstitution, as transplantation of wild-type donor cells into EP4 receptor deficient recipient mice shows reduced stem cell engraftment. However, the specific contribution of EP4 signaling on different niche constituents in hematopoietic regeneration is unknown. To investigate which niche constituent/stromal population EP4 signaling functionally contributes to hematopoietic regeneration, we utilized genetic mouse models, in which EP4 could be deleted in specific stromal cell types.We conditionally deleted EP4 expression in nestin expressing mesenchymal stem cell (MSC), leptin receptor expressing mesenchymal stromal cell or tie 2 expressing endothelial cell (EC) invivo using a cre-recombinase approach. At steady-state, selective deletion of EP4 from nestin+ MSC (nestin-cre EP4flox/flox mice), leptin receptor+stromal cell (leptin R-cre EP4flox/flox mice) or tie 2+EC (tie 2-cre EP4flox/flox mice) showed no change in BM MSC (CD45-Ter119-CD31-CD51+PDGFR+), leptin receptor+stromal cells (CD45-Ter119-leptin R+) and EC (CD45-ter119-CD31+VE-cadherin+) number and survival. We next determined whether loss of EP4 expression/signaling in stromal cell specific genetic knockout mouse models affects BM niche constituents after exposure to ionization radiation. Interestingly, loss of EC but not nestin/leptin EP4 expression severely disrupted the BM vasculature and reduced EC and MSC survival at 24 hours post total body lethal (TBI) irradiation, compared to wild-type irradiated mice, and was associated with reduced expression of Survivin and increased expression of Bax, anti-apoptotic and pro-apoptotic factors respectively. In addition, niche EC and MSC regeneration at 2-month post irradiation was substantially reduced in tie 2-cre EP4flox/flox mice compared to wild-type mice. Nestin-cre EP4flox/flox mice and leptin R-cre EP4flox/flox niche restoration was equivalent to wild-type mice. To determine whether loss of EP4 receptor in different niche cells differentially regulates hematopoietic regeneration, we transplanted wild-type donor cells into nestin-cre EP4flox/flox or tie 2-cre EP4flox/flox recipient mice. Chimeric mice made with EC-specific EP4 deficient stroma showed reduced HSPC engraftment compared to chimeric mice made with wild-type stroma. In contrast, stem cell engraftment in chimeric mice made with MSC-specific EP4 deficient stroma was not reduced. In addition, in vivo administration of PGE2 into wild-type recipient mice at six-hour post irradiation enhanced donor cell engraftment. However, PGE2 treatment failed to enhance donor cell engraftment into tie 2-cre EP4flox/flox recipient mice. These data suggest that EC EP4 expression is important for niche restoration and hematopoietic regeneration after transplantation. To identify the mechanism involved in EP4 signaling mediated niche restoration, we measured angiocrine factors in the BM niche of lethally irradiated wild-type and tie 2-cre EP4flox/flox mice. Interestingly, we found that vascular endothelial growth factor (VEGF) expression was substantially reduced in the BM of tie 2-cre EP4flox/flox mice compared to wild-type mice. To investigate whether EP4 signaling regulates niche restoration by stimulating VEGF expression/production, we treated lethally irradiated tie 2-cre EP4flox/flox mice with recombinant VEGF (20 ng/mouse/day) for 3 days. In vivo administration of recombinant VEGF substantially enhanced niche restoration in tie 2-cre EP4flox/floxmice compared to control tie 2-cre EP4flox/flox. In conclusion, our study suggests that EC specific EP4 receptor signaling-mediated increased expression of VEGF promotes vascular niche restoration after myelosuppression and supports hematopoietic regeneration. DisclosuresNo relevant conflicts of interest to declare.

Regulation of Mitophagy By O-Linked N-Acetylglucosamine Transferase Is Essential for Hematopoietic Stem Cell Maintenance

Hematopoietic stem cells (HSCs) reside in hypoxic niche in the bone marrow (BM), where they are maintained in quiescent state. To adapt to this hypoxic microenvironment, HSCs generate ATP mainly from glycolysis, while suppressing oxidative phosphorylation in mitochondria. Through this metabolic regulation, HSCs protect themselves from oxidative stress caused by reactive oxygen species (ROS). ROS levels are controlled by various mechanisms including autophagy, which is one of the critical mechanisms regulating HSC function mainly through maintaining mitochondrial homeostasis. Glycosylation of serine or threonine residues with O-linked N-acetylglucosamine (O-GlcNAcylation) by O-linked N-acetylglucosamine transferase (OGT) is crucial for regulating various protein functions, and dysregulation of O-GlcNAcylation is critically linked to metabolic or degenerative diseases (i.e. diabetes, Alzheimer disease), tumorigenesis and aging. In hematopoietic system, OGT is essential for differentiation and proliferation of T and B cells. However, a role for OGT in the function of hematopoietic stem and progenitor cells (HSPCs) remains elusive. To elucidate a role for OGT in HSPCs, we analyzed the effect of Ogt loss on HSPCs using Ogt-conditional knockout mice. Conditional disruption of Ogt in adult mice led to pancytopenia and significantly reduced numbers of HSPCs in the BM. In addition, Ogt-deficient HSCs exhibited loss of quiescence, increased apoptosis and impaired long-term repopulation as well as self-renewal capacities by serial competitive repopulation assays. Interestingly, ROS levels were significantly increased in Ogt-deficient HSPCs, and the treatment of mice with N-acetyl cysteine (NAC), a scavenger for ROS, at least partially rescued the phenotype of Ogt-deficient hematopoietic cells in in vivo settings. MitoSOX analysis showed that excessive ROS was generated mainly from mitochondria in Ogt-deficient HSCs. In addition, mitochondrial mass was significantly increased in Ogt-deficient HSCs. Assessment of mitochondrial quality by its membrane potential and extracellular flux analysis revealed that mitochondria of Ogt-deficient HSPCs had significantly lower membrane potential and lower spare respiratory capacity. Morphologically, mitochondria in Ogt-deficient HSPCs were enlarged and swollen with disorganized cristae. These findings suggest that inactivation of Ogt leads to loss of HSPCs and impaired HSC homeostasis, which is due, at least in part, to elevated ROS levels with accumulation of defective mitochondria. Since mitophagy (mitochondria-specific autophagy) plays an important role in quality control of mitochondria, we examined gene expressions of mitophagy regulators by RNA sequencing. This revealed that disruption of Ogt led to down-regulation of key mitophagy inducers, Pink1 and Bnip3. These findings together with the accumulation of defective mitochondria indicated that inactivation of Ogt impairs mitophagy probably due to reduced expression of Pink1 and Bnip3. OGT is known to regulate gene expressions by modifying trimethylation of lysine 4 on the histone H3 (H3K4me3) through proteolytic activation of HCF-1, which is a critical component of SET1/COMPASS complex. Chromatin immunoprecipitation (ChIP) assay showed significantly reduced levels of H3K4me3 at the transcriptional start sites of Pink1 and Bnip3 in Ogt-deficient HSPCs. Next we asked whether defective HSPC homeostasis caused by Ogt disruption could be rescued by restoring mitophagy in Ogt-deficient HSPCs through overexpressing Pink1, a critical initiator of mitophagy. As expected, Pink1 overexpression efficiently restored mitophagy in Ogt-deficient HSCs as shown by normalization of mitochondrial mass. Interestingly, a number and apoptosis of Ogt-deficient HSCs were restored to levels similar to those of wild-type HSCs. These data strongly indicate that Pink1 functions as a key downstream effector of OGT in HSC maintenance. In summary, our results revealed that OGT plays an essential role in HSC maintenance by assuring mitochondrial quality through mitophagy. DisclosuresNo relevant conflicts of interest to declare.

Purine Metabolism and Hematopoietic Stem and Progenitor Stem Cells Under Stress

Hematopoietic stem cells (HSCs) play a key role in the lifelong maintenance of hematopoiesis through self-renewal and multi-lineage differentiation. Adult HSCs reside within a specialized microenvironment of the bone marrow (BM), called “niche”, in which they are maintained in a quiescent state in cell cycle. Most of HSCs within BM show quiescence under the hypoxic niche. Since the loss of HSC quiescence leads to the exhaustion or aging of stem cells through excess cell division, the regulation of quiescence in HSCs is essential for hematopoietic homeostasis. On the other hand, cellular metabolism has been suggested to play a critical role in many biological processes including the regulation of stem cell properties and functions. However, the metabolic condition and adaptation of stem cells remain largely unaddressed.First, we have analyzed HSC metabolism using metabolomics approaches. With step-wise differentiation of stem cells, the cell metabolism associated with each differentiation stage may be different. A feature of quiescent HSCs is their low baseline energy production; quiescent HSCs rely on glycolysis and exhibit low mitochondrial membrane potential (ΔΨm). Likewise, HSCs with a low ΔΨm show higher reconstitution activity in BM hematopoiesis, compared to cells with high ΔΨm. By contrast, upon stress hematopoiesis, HSCs actively divide and proliferate. However, the underlying mechanism for the initiation of HSC division still remains unclear. In order to elucidate the mechanism underlying the transition of cell cycle state in HSCs, we analyzed the change of mitochondria activity in HSCs after BM suppression induced by 5-fluoruracil (5-FU). Upon 5-FU treatment, cycling progenitors are depleted and then quiescent HSCs start to divide. We found that HSCs initiate cell division after exhibiting enhanced ΔΨm, as a result of increased intracellular Ca2+ level. We hypothesize that extracellular adenosine, derived from hematopoietic progenitors, inhibits the calcium influx and mitochondrial metabolism. While further activation of Ca2+-mitochondria pathway led to loss of the stem cell function after cell division, the appropriate suppression of intracellular Ca2+ level by nifedipine, a blocker of L-type voltage-gated Ca2+ channels, prolonged cell division interval in HSCs, and simultaneously achieved both cell division and HSC maintenance (self-renewal division). Thus, our results indicate that the adenosine-Ca2+-mitochondria pathway induces HSC division critically to determine HSC cell fate. Next, to examine the mitochondria oxidative metabolism and purinergic pathways, we introduced the study on a tumor suppressor, Folliculin (FLCN). Conditional deletion of FLCN in HSC compartment using the Mx1-Cre or Vav-iCre system disrupted HSC quiescence and BM homeostasis dependently on the lysosomal stress response induced by TFE3. Together all, we propose that the change in cellular metabolism involving mitochondria is crucial for HSC homeostasis in the stress settings. DisclosuresNo relevant conflicts of interest to declare.

Membrane-potential compensation reveals mitochondrial volume expansion during HSC commitment

Proper control of mitochondrial function is a key factor in the maintenance of hematopoietic stem cells (HSCs). Mitochondrial content is commonly measured by staining with fluorescent cationic dyes. However, dye staining can be affected, not only by xenobiotic efflux pumps, but also by dye intake, which is dependent on the negative charge of mitochondria. Therefore, mitochondrial membrane potential (ΔΨmt) must be considered in these measurements because a high ΔΨmt due to respiratory chain activity can enhance dye intake, leading to the overestimation of mitochondrial volume. Here, we show that HSCs exhibit the highest ΔΨmt of the hematopoietic lineages and, as a result, ΔΨmt-independent methods most accurately assess the relatively low mitochondrial volumes and DNA amounts of HSC mitochondria. Multipotent progenitor stage or active HSCs display expanded mitochondrial volumes, which decline again with further maturation. Further characterization of the controlled remodeling of the mitochondrial landscape at each hematopoietic stage will contribute to a deeper understanding of the mitochondrial role in HSC homeostasis.

Chronic kidney failure mineral bone disorder leads to a permanent loss of hematopoietic stem cells through dysfunction of the stem cell niche

In chronic kidney disease (CKD), endothelial injury, is associated with disease progression and an increased risk for cardiovascular complications. Circulating cells with vascular reparative functions are hematopoietic and also reduced in CKD. To explore the mechanistic basis behind these observations, we have investigated hematopoietic stem cell (HSC) homeostasis in a mouse model for non-progressive CKD-mineral and bone disorder with experimentally induced chronic renal failure (CRF). In mice subjected to 12 weeks of CRF, bone marrow HSC frequencies were decreased and transplantation of bone marrow cells from CRF donors showed a decrease in long-term HSC repopulation compared to controls. This loss was directly associated with a CRF-induced defect in the HSC niche affecting the cell cycle status of HSC and could not be restored by the PTH-reducing agent cinacalcet. In CRF, frequencies of quiescent (G0) HSC were decreased coinciding with an increase in hematopoietic progenitor cells (HPC) in the S-and G2-phases of cell cycle. Moreover, in CRF mice, HSC-niche supporting macrophages were decreased compared to controls concomitant to impaired B lymphopoiesis. Our data point to a permanent loss of HSC and may provide insight into the root cause of the loss of homeostatic potential in CKD.

SRC-3 is involved in maintaining hematopoietic stem cell quiescence by regulation of mitochondrial metabolism in mice

AbstractQuiescence maintenance is an important property of hematopoietic stem cells (HSCs), whereas the regulatory factors and underlying mechanisms involved in HSC quiescence maintenance are not fully uncovered. Here, we show that steroid receptor coactivator 3 (SRC-3) is highly expressed in HSCs, and SRC-3–deficient HSCs are less quiescent and more proliferative, resulting in increased sensitivity to chemotherapy and irradiation. Moreover, the long-term reconstituting ability of HSCs is markedly impaired in the absence of SRC-3, and SRC-3 knockout (SRC-3−/−) mice exhibit a significant disruption of hematopoietic stem and progenitor cell homeostasis. Further investigations show that SRC-3 deficiency leads to enhanced mitochondrial metabolism, accompanied by overproduction of reactive oxygen species (ROS) in HSCs. Notably, the downstream target genes of peroxisome proliferator–activated receptor-coactivators 1α (PGC-1α) involved in the regulation of mitochondrial metabolism are significantly upregulated in SRC-3–deficient HSCs. Meanwhile, a significant decrease in the expression of histone acetyltransferase GCN5 accompanied by downregulation of PGC-1α acetylation is observed in SRC-3–null HSCs. Conversely, overexpression of GCN5 can inhibit SRC-3 deficiency-induced mitochondrial metabolism enhancement and ROS overproduction, thereby evidently rescuing the impairment of HSCs in SRC-3−/− mice. Collectively, our findings demonstrate that SRC-3 plays an important role in HSC quiescence maintenance by regulating mitochondrial metabolism.

The histone chaperone NAP1L3 is required for haematopoietic stem cell maintenance and differentiation

Nucleosome assembly proteins (NAPs) are histone chaperones with an important role in chromatin structure and epigenetic regulation of gene expression. We find that high gene expression levels of mouse Nap1l3 are restricted to haematopoietic stem cells (HSCs) in mice. Importantly, with shRNA or CRISPR-Cas9 mediated loss of function of mouse Nap1l3 and with overexpression of the gene, the number of colony-forming cells and myeloid progenitor cells in vitro are reduced. This manifests as a striking decrease in the number of HSCs, which reduces their reconstituting activities in vivo. Downregulation of human NAP1L3 in umbilical cord blood (UCB) HSCs impairs the maintenance and proliferation of HSCs both in vitro and in vivo. NAP1L3 downregulation in UCB HSCs causes an arrest in the G0 phase of cell cycle progression and induces gene expression signatures that significantly correlate with downregulation of gene sets involved in cell cycle regulation, including E2F and MYC target genes. Moreover, we demonstrate that HOXA3 and HOXA5 genes are markedly upregulated when NAP1L3 is suppressed in UCB HSCs. Taken together, our findings establish an important role for NAP1L3 in HSC homeostasis and haematopoietic differentiation.

Luteinizing hormone signaling restricts hematopoietic stem cell expansion during puberty.

The number and self-renewal capacity of hematopoietic stem cells (HSCs) are tightly regulated at different developmental stages. Many pathways have been implicated in regulating HSC development in cell autonomous manners; however, it remains unclear how HSCs sense and integrate developmental cues. In this study, we identified an extrinsic mechanism by which HSC number and functions are regulated during mouse puberty. We found that the HSC number in postnatal bone marrow reached homeostasis at 4weeks after birth. Luteinizing hormone, but not downstream sex hormones, was involved in regulating HSC homeostasis during this period. Expression of luteinizing hormone receptor (Lhcgr) is highly restricted in HSCs and multipotent progenitor cells in the hematopoietic hierarchy. When Lhcgr was deleted, HSCs continued to expand even after 4weeks after birth, leading to abnormally elevated hematopoiesis and leukocytosis. In a murine acute myeloid leukemia model, leukemia development was significantly accelerated upon Lhcgr deletion. Together, our work reveals an extrinsic counting mechanism that restricts HSC expansion during development and is physiologically important for maintaining normal hematopoiesis and inhibiting leukemogenesis.

Epithelial-mesenchymal transition in haematopoietic stem cell development and homeostasis.

Epithelial-mesenchymal transition (EMT) is a morphogenetic process of cells that adopt an epithelial organization in their developmental ontogeny or homeostatic maintenance. Abnormalities in EMT regulation result in many malignant tumours in the human body. Tumours associated with the haematopoietic system, however, are traditionally not considered to involve EMT and haematopoietic stem cells (HSCs) are generally not associated with epithelial characteristics. In this review, we discuss the ontogeny and homeostasis of adult HSCs in the context of EMT intermediate states. We provide evidence that cell polarity regulation is critical for both HSC formation from embryonic dorsal aorta and HSC self-renewal and differentiation in adult bone marrow. HSC polarity is controlled by the same set of surface and transcriptional regulators as those described in canonical EMT processes. With an emphasis on partial EMT, we propose that the concept of EMT can be similarly applied in the study of HSC generation, maintenance and pathogenesis.

The Transcription Factor Zfx Regulates Peripheral T Cell Self-Renewal and Proliferation.

Peripheral T lymphocytes share many functional properties with hematopoietic stem cells (HSCs), including long-term maintenance, quiescence, and latent proliferative potential. In addition, peripheral T cells retain the capacity for further differentiation into a variety of subsets, much like HSCs. While the similarities between T cells and HSC have long been hypothesized, the potential common genetic regulation of HSCs and T cells has not been widely explored. We have studied the T cell-intrinsic role of Zfx, a transcription factor specifically required for HSC maintenance. We report that T cell-specific deletion of Zfx caused age-dependent depletion of naïve peripheral T cells. Zfx-deficient T cells also failed to undergo homeostatic proliferation in a lymphopenic environment, and showed impaired antigen-specific expansion and memory response. In addition, the invariant natural killer T cell compartment was severely reduced. RNA-Seq analysis revealed that the most dysregulated genes in Zfx-deficient T cells were similar to those observed in Zfx-deficient HSC and B cells. These studies identify Zfx as an important regulator of peripheral T cell maintenance and expansion and highlight the common molecular basis of HSC and lymphocyte homeostasis.

The H3K4 methyltransferase Setd1b is essential for hematopoietic stem and progenitor cell homeostasis in mice.

Hematopoietic stem cells require MLL1, which is one of six Set1/Trithorax-type histone 3 lysine 4 (H3K4) methyltransferases in mammals and clinically the most important leukemia gene. Here, we add to emerging evidence that all six H3K4 methyltransferases play essential roles in the hematopoietic system by showing that conditional mutagenesis of Setd1b in adult mice provoked aberrant homeostasis of hematopoietic stem and progenitor cells (HSPCs). Using both ubiquitous and hematopoietic-specific deletion strategies, the loss of Setd1b resulted in peripheral thrombo- and lymphocytopenia, multilineage dysplasia, myeloid-biased extramedullary hematopoiesis in the spleen, and lethality. By transplantation experiments and expression profiling, we determined that Setd1b is autonomously required in the hematopoietic lineages where it regulates key lineage specification components, including Cebpa, Gata1, and Klf1. Altogether, these data imply that the Set1/Trithorax-type epigenetic machinery sustains different aspects of hematopoiesis and constitutes a second framework additional to the transcription factor hierarchy of hematopoietic homeostasis.

Gene dosage effect of CUX1 in a murine model disrupts HSC homeostasis and controls the severity and mortality of MDS

AbstractMonosomy 7 (−7) and del(7q) are high-risk cytogenetic abnormalities common in myeloid malignancies. We previously reported that CUX1, a homeodomain-containing transcription factor encoded on 7q22, is frequently inactivated in myeloid neoplasms, and CUX1 myeloid tumor suppressor activity is conserved from humans to Drosophila. CUX1-inactivating mutations are recurrent in clonal hematopoiesis of indeterminate potential as well as myeloid malignancies, in which they independently carry a poor prognosis. To determine the role for CUX1 in hematopoiesis, we generated 2 short hairpin RNA-based mouse models with ∼54% (Cux1mid) or ∼12% (Cux1low) residual CUX1 protein. Cux1mid mice develop myelodysplastic syndrome (MDS) with anemia and trilineage dysplasia, whereas CUX1low mice developed MDS/myeloproliferative neoplasms and anemia. In diseased mice, restoration of CUX1 expression was sufficient to reverse the disease. CUX1 knockdown bone marrow transplant recipients exhibited a transient hematopoietic expansion, followed by a reduction of hematopoietic stem cells (HSCs), and fatal bone marrow failure, in a dose-dependent manner. RNA-sequencing after CUX1 knockdown in human CD34+ cells identified a −7/del(7q) MDS gene signature and altered differentiation, proliferative, and phosphatidylinositol 3-kinase (PI3K) signaling pathways. In functional assays, CUX1 maintained HSC quiescence and repressed proliferation. These homeostatic changes occurred in parallel with decreased expression of the PI3K inhibitor, Pik3ip1, and elevated PI3K/AKT signaling upon CUX1 knockdown. Our data support a model wherein CUX1 knockdown promotes PI3K signaling, drives HSC exit from quiescence and proliferation, and results in HSC exhaustion. Our results also demonstrate that reduction of a single 7q gene, Cux1, is sufficient to cause MDS in mice.

Regulation of Hematopoietic Cell Development and Function Through Phosphoinositides.

One of the most paramount receptor-induced signal transduction mechanisms in hematopoietic cells is production of the lipid second messenger phosphatidylinositol(3,4,5)trisphosphate (PIP3) by class I phosphoinositide 3 kinases (PI3K). Defective PIP3 signaling impairs almost every aspect of hematopoiesis, including T cell development and function. Limiting PIP3 signaling is particularly important, because excessive PIP3 function in lymphocytes can transform them and cause blood cancers. Here, we review the key functions of PIP3 and related phosphoinositides in hematopoietic cells, with a special focus on those mechanisms dampening PIP3 production, turnover, or function. Recent studies have shown that beyond “canonical” turnover by the PIP3 phosphatases and tumor suppressors phosphatase and tensin homolog (PTEN) and SH2 domain-containing inositol-5-phosphatase-1 (SHIP-1/2), PIP3 function in hematopoietic cells can also be dampened through antagonism with the soluble PIP3 analogs inositol(1,3,4,5)tetrakisphosphate (IP4) and inositol-heptakisphosphate (IP7). Other evidence suggests that IP4 can promote PIP3 function in thymocytes. Moreover, IP4 or the kinases producing it limit store-operated Ca2+ entry through Orai channels in B cells, T cells, and neutrophils to control cell survival and function. We discuss current models for how soluble inositol phosphates can have such diverse functions and can govern as distinct processes as hematopoietic stem cell homeostasis, neutrophil macrophage and NK cell function, and development and function of B cells and T cells. Finally, we will review the pathological consequences of dysregulated IP4 activity in immune cells and highlight contributions of impaired inositol phosphate functions in disorders such as Kawasaki disease, common variable immunodeficiency, or blood cancer.

CCL4 enhances preosteoclast migration and its receptor CCR5 downregulation by RANKL promotes osteoclastogenesis

Chemokine CCL4 (MIP-1β) is released from osteoblast cells to restore the homeostasis of hematopoietic stem cells during the activation of bone marrow. In this study, we investigated the function of CCL4 and its receptor CCR5 during osteoclastogenesis. CCL4 promoted the migration and viability of preosteoclast cells. However, CCL4 had no direct effect on the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation in mouse preosteoclast cells. In addition, CCR5 expression was rapidly reduced by RANKL treatment, which was recovered by IFN-γ during osteoclastogenesis. CCR5 downregulation by RANKL was mediated by MEK and JNK in preosteoclast cells and promoted osteoclastogenesis. These results suggest that CCL4 can enhance the recruitment of preosteoclasts to bone in the early stage, and the reduction of CCR5 promotes osteoclastogenesis when RANKL is prevalent.

Spred1 Safeguards Hematopoietic Homeostasis against Diet-Induced Systemic Stress

Stem cell self-renewal is critical for tissue homeostasis, and its dysregulation can lead to organ failure or tumorigenesis. While obesity can induce varied abnormalities in bone marrow components, it is unclear how diet might affect hematopoietic stem cell (HSC) self-renewal. Here, we show that Spred1, a negative regulator of RAS-MAPK signaling, safeguards HSC homeostasis in animals fed a high-fat diet (HFD). Under steady-state conditions, Spred1 negatively regulates HSC self-renewal and fitness, in part through Rho kinase activity. Spred1 deficiency mitigates HSC failure induced by infection mimetics and prolongs HSC lifespan, but it does not initiate leukemogenesisdue to compensatory upregulation of Spred2.In contrast, HFD induces ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in functional HSC failure, severe anemia, and myeloproliferative neoplasm-like disease. HFD-induced hematopoietic abnormalities are mediated partly through alterations to the gut microbiota. Together, these findings reveal thatdiet-induced stress disrupts fine-tuningof Spred1-mediated signals to govern HSC homeostasis.

NR4A1 and NR4A3 restrict HSC proliferation via reciprocal regulation of C/EBPα and inflammatory signaling

AbstractMembers of the NR4A subfamily of nuclear receptors have complex, overlapping roles during hematopoietic cell development and also function as tumor suppressors of hematologic malignancies. We previously identified NR4A1 and NR4A3 (NR4A1/3) as functionally redundant suppressors of acute myeloid leukemia (AML) development. However, their role in hematopoietic stem cell (HSC) homeostasis remains to be disclosed. Using a conditional Nr4a1/Nr4a3 knockout mouse (CDKO), we show that codepletion of NR4A1/3 promotes acute changes in HSC homeostasis including loss of HSC quiescence, accumulation of oxidative stress, and DNA damage while maintaining stem cell regenerative and differentiation capacity. Molecular profiling of CDKO HSCs revealed widespread upregulation of genetic programs governing cell cycle and inflammation and an aberrant activation of the interferon and NF-κB signaling pathways in the absence of stimuli. Mechanistically, we demonstrate that NR4A1/3 restrict HSC proliferation in part through activation of a C/EBPα-driven antiproliferative network by directly binding to a hematopoietic-specific Cebpa enhancer and activating Cebpa transcription. In addition, NR4A1/3 occupy the regulatory regions of NF-κB-regulated inflammatory cytokines, antagonizing the activation of NF-κB signaling. Taken together, our results reveal a novel coordinate control of HSC quiescence by NR4A1/3 through direct activation of C/EBPα and suppression of activation of NF-κB-driven proliferative inflammatory responses.

Regulation of hematopoietic stem cell homeostasis by Spred1

Various types of stresses account for the dysregulation of the self-renewal activity of stem cells, resulting in the functional failure of tissues or tumorigenesis promotion. Although diets also affect our health, the effect of harmful dietary stresses on the tissue or stem cell homeostasis remains unclear. Recent research has revealed that Spred1, which negatively regulates RAS-MAPK signaling, protects hematopoietic stem cell (HSC) homeostasis against high-fat diet (HFD) -induced systemic stress. In steady-state conditions, Spred1 negatively regulates HSC self-renewal in a manner supported by the Rho kinase (ROCK) activity. In addition, Spred1 deficiency in mice mitigates HSC dysfunction induced by aging or lipopolysaccharide treatment, enhances the HSC self-renewal capacity, and prolongs HSC lifespan, but does not induce leukemia because of the compensatory upregulation of Spred2-the other Spred family member. Conversely, HFD triggers ERK hyperactivation and aberrant self-renewal in Spred1-deficient HSCs, resulting in HSC dysfunction, severe anemia, and the development of lethal myeloproliferative neoplasm-like disease. The depletion of the gut microbiota by antibiotics restored myeloproliferation, anemia, and HSC reconstitution ability in HFD-fed Spred1-deficient mice, suggesting that HFD-induced hematopoietic abnormalities were partially because of alterations in the gut microbiota composition. Thus, HFD-induced systemic stress affects the regulation of HSC self-renewal, and Spred1 safeguards HSC homeostasis against the diet-induced systemic stress.