ABSTRACT:Hematopoietic stem and progenitor cells are regulated by interactions with stromal cells in the bone marrow (BM) cavity, which can be segregated into 2 spatially defined central marrow (CM) and endosteal (Endo) compartments. However, the importance of this spatial compartmentalization for BM responses to complex conditions such as inflammation remains largely unknown. Here, we extensively validate a combination of single-cell RNA sequencing profiling and matching flow cytometry isolation that reproducibly identifies 7 key CM and Endo populations and accurately surveys both niche locations. We demonstrate that inflammatory perturbations exert specific effects on different cellular compartments, with type I interferon responses causing leptin receptor-expressing mesenchymal stromal cells to abandon their normal stromal functions and instead adopt an inflammatory phenotype associated with overproduction of chemokines that modulate local monocyte dynamics in the surrounding microenvironment. Our results provide a comprehensive method for molecular and functional stromal characterization and highlight the importance of altered stomal cell activity in regulating hematopoietic responses to inflammatory challenges.

Objectives: The peripheral blood T cells epigenetic modification and adhesion capacity alterations are the characteristic of SLE. EZH2 induces proinflammatory epigenetic changes in immune regulation. JAM-A widely involves in cell adhesion. However, the molecular regulation mechanisms between EZH2 and JAM-A in T cells of SLE patients remain undefined. Methods: EZH2 and JAM-A expression levels in T cells from SLE patients and healthy controls were evaluated via FCM and RT-qPCR. The regulatory mechanism between miR-26a-5p/EZH2 and JAM-A were investigated via BSP-PCR, ChIP assays, cell adhesion assays in vitro, and intraperitoneal GSK126 administration in MRL/lpr mice in vivo. Results: We found the expression of EZH2 and JAM-A was upregulated in SLE patients’ T cells. Mechanistically, EZH2 epigenetically repressed its target gene DNMT3A, mediated by H3K27me3, which consequently decreased the methylation levels in JAM-A promoter region, resulting in promoting the expression of JAM-A. Subsequently, JAM-A promoted the expression of its functionally relevant target gene Rap1a, a regulator of β1-integrin, involved in T cell adhesion. In addition, we show that both EZH2 and Rap1a are the target genes of miR-26a-5p. Specially, EZH2-mediated H3K27me3 modification in miR-26a-5p promoter region further inhibited the transcription of miR-26a-5p, resulting in the upregulation of EZH2 in T cells of SLE patients, creating a vicious cycle. And intraperitoneal administrating the inhibitor of EZH2 with GSK126 significantly ameliorated the nephritis in MRL/lpr mice. Conclusion: This research reveals a novel epigenetic regulation of JAM-A by EZH2-DNMT3A cascade contributes to T cell adhesion capacity via the activation of Rap1a/β1-integrin in lupus patients.

Self-renewing hematopoietic stem cells (HSCs) generate all blood cells and give rise to long-term reconstitution of the hematopoietic system after transplantation, but the molecular mechanisms that specifically regulate HSCs remain poorly defined. Here, we found that HSCs displayed a distinct messenger RNA alternative splicing pattern and preferentially expressed Rbfox2, an alternative splicing regulator, compared with multipotent progenitors (MPPs). Deletion of Rbfox2 from the hematopoietic compartment specifically depleted HSCs, but not progenitors in the adult bone marrow. Rbfox1 did not function redundantly with Rbfox2 in HSCs. Mechanistically, Rbfox2 loss led to proteostasis stress, including increased protein synthesis rate and accumulated misfolded/unfolded protein contents, in HSCs, but not in progenitors. Small molecules that restore proteostasis rescued HSC defects in Rbfox2-deficient mice. Our work thus reveals that HSCs, but not progenitors, selectively rely on Rbfox2 for their self-renewal and maintenance.

The tumor microenvironment (TME) dictates the outcome of cancer immunotherapy. In this issue of Cell Chemical Biology, Yu et al.1 report that targeting Mettl3 leads to a more inflamed, “hot” TME and effective anti-PD-1 therapy. This study points to a new target in remodeling the TME for improved immunotherapy.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. Whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche is unknown. To address this, we compared perinatal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. MSCs exhibited enrichment in genes mediating m6A mRNA methylation at the perinatal stage and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs but not osteoblasts led to excessive osteogenic differentiation and a severe HSC niche formation defect, which was significantly rescued by deletion of Klf2, an m6A target. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

Aging is underpinned by pronounced metabolic decline; however, the drivers remain obscure. Here, we report that IgG accumulates during aging, particularly in white adipose tissue (WAT), to impair adipose tissue function and metabolic health. Caloric restriction (CR) decreases IgG accumulation in WAT, whereas replenishing IgG counteracts CR's metabolic benefits. IgG activates macrophages via Ras signaling and consequently induces fibrosis in WAT through the TGF-β/SMAD pathway. Consistently, B cell null mice are protected from aging-associated WAT fibrosis, inflammation, and insulin resistance, unless exposed to IgG. Conditional ablation of the IgG recycling receptor, neonatal Fc receptor (FcRn), in macrophages prevents IgG accumulation in aging, resulting in prolonged healthspan and lifespan. Further, targeting FcRn by antisense oligonucleotide restores WAT integrity and metabolic health in aged mice. These findings pinpoint IgG as a hidden culprit in aging and enlighten a novel strategy to rejuvenate metabolic health.

Hematopoietic stem cells (HSCs) regenerate after myeloablation, a procedure that adversely disrupts the bone marrow and drives leptin receptor-expressing cells, a key niche component, to differentiate extensively into adipocytes. Regeneration of the bone marrow niche is associated with the resolution of adipocytes, but the mechanisms remain poorly understood. Using Plin1-creER knock-in mice, we followed the fate of adipocytes in the regenerating niche in vivo. We found that bone marrow adipocytes were highly dynamic and dedifferentiated to leptin receptor-expressing cells during regeneration after myeloablation. Bone marrow adipocytes could give rise to osteolineage cells after skeletal injury. The cellular fate of steady-state bone marrow adipocytes was also plastic. Deletion of adipose triglyceride lipase (Atgl) from bone marrow stromal cells, including adipocytes, obstructed adipocyte dedifferentiation and led to severely compromised regeneration of HSCs as well as impaired B lymphopoiesis after myeloablation, but not in the steady state. Thus, the regeneration of HSCs and their niche depends on the cellular plasticity of bone marrow adipocytes.

Innate lymphoid cells (ILCs) can quickly switch from a quiescent state to an active state and rapidly produce effector molecules that provide critical early immune protection. How the post-transcriptional machinery processes different stimuli and initiates robust gene expression in ILCs is poorly understood. Here, we show that deletion of the N 6 -methyladenosine (m 6 A) writer protein METTL3 has little impact on ILC homeostasis or cytokine-induced ILC1 or ILC3 responses but significantly diminishes ILC2 proliferation, migration and effector cytokine production and results in impaired antihelminth immunity. m 6 A RNA modification supports an increase in cell size and transcriptional activity in activated ILC2s but not in ILC1s or ILC3s. Among other transcripts, the gene encoding the transcription factor GATA3 is highly m 6 A methylated in ILC2s. Targeted m 6 A demethylation destabilizes nascent Gata3 mRNA and abolishes the upregulation of GATA3 and ILC2 activation. Our study suggests a lineage-specific requirement of m 6 A for ILC2 responses.

Remodeling of the tissue niche is often evident in diseases, yet, the stromal alterations and their contribution to pathogenesis are poorly characterized. Bone marrow fibrosis is a maladaptive feature of primary myelofibrosis (PMF). We performed lineage tracing and found that most collagen-expressing myofibroblasts were derived from leptin-receptor-positive (LepR+) mesenchymal cells, whereas a minority were from Gli1-lineage cells. Deletion of Gli1 did not impact PMF. Unbiased single-cell RNA sequencing (scRNA-seq) confirmed that virtually all myofibroblasts originated from LepR-lineage cells, with reduced expression of hematopoietic niche factors and increased expression of fibrogenic factors. Concurrently, endothelial cells upregulated arteriolar-signature genes. Pericytes and Sox10+ glial cells expanded drastically with heightened cell-cell signaling, suggesting important functional roles in PMF. Chemical or genetic ablation of bone marrow glial cells ameliorated fibrosis and improved other pathology in PMF. Thus, PMF involves complex remodeling of the bone marrow microenvironment, and glial cells represent a promising therapeutic target.

The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. It is unknown whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche. To address this, we compared fetal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. Fetal MSCs exhibited an enrichment in genes mediating m6A mRNA methylation regulation and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs led to excessive osteogenic differentiation and a severe HSC niche formation defect which was rescued by deletion of Klf2, a target of m6A. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche or hematopoiesis. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine. The niche is typically considered as a pre-established structure sustaining stem cells. Therefore, the regulation of its formation remains largely unexplored. It is unknown whether distinct molecular mechanisms control the establishment versus maintenance of a stem cell niche. To address this, we compared fetal and adult bone marrow mesenchymal stromal cells (MSCs), a key component of the hematopoietic stem cell (HSC) niche. Fetal MSCs exhibited an enrichment in genes mediating m6A mRNA methylation regulation and downregulated the expression of Mettl3, the m6A methyltransferase, shortly after birth. Deletion of Mettl3 from developing MSCs led to excessive osteogenic differentiation and a severe HSC niche formation defect which was rescued by deletion of Klf2, a target of m6A. In contrast, deletion of Mettl3 from MSCs postnatally did not affect HSC niche or hematopoiesis. Stem cell niche generation and maintenance thus depend on divergent molecular mechanisms, which may be exploited for regenerative medicine.

Purpose of Review Hematopoiesis is the process of generating all blood and immune cells, which is fueled at the root by self-renewing multipotent hematopoietic stem cells (HSCs). Unlike other systems in the body, hematopoiesis occurs in several waves in different organs (yolk sac, AGM, placenta, embryonic head, fetal liver, and fetal spleen) across ontogeny until it settles down in the bone marrow and remains there throughout adult life. Within a given hematopoietic organ, the micro-environmental niche plays critical roles in regulating HSCs and hematopoiesis by elaborating cytokines and other factors. Interestingly, under pathologic conditions in adults, hematopoiesis can be re-initiated in organs that are hematopoietic during fetal stages, such as the spleen and liver. Here we will review recent progresses on the identification of cellular components and mechanisms in these hematopoietic niches. We will also compare and contrast the niches to identify outstanding questions that are still unsolved in hematopoietic niche biology. Recent Findings Over the past several years, cutting-edge technologies have been applied to uncover the nature and mechanisms of the adult and fetal hematopoietic niches across tissues and organs in vivo. A general theme of the hematopoietic niche where endothelial cells and perivascular mesenchymal stromal cells are the core components is emerging. Summary In contrast to the maintenance niche in the adult bone marrow, the fetal hematopoietic niches promote HSC emergence and expansion. Elucidating the fetal hematopoietic niche mechanisms will help devise ways to amplify HSCs for clinical use. The on-and-off nature of the fetal hematopoietic niche suggests that the hematopoietic niche is highly dynamic. Understanding these dynamic changes offers the opportunity to harness the niche to promote hematopoiesis.

Tissue stem cells temporally change intrinsic mechanisms to meet physiological demands. However, little is known whether and how stem cells rely on distinct extrinsic maintenance mechanisms over time. Here, we found that hematopoietic stem cells (HSCs) temporally transition to depend on thrombopoietin (TPO), a key extrinsic factor, from E16.5 onward in the developing liver. Deletion of Tpo reduced mTOR activity, induced differentiation gene expression, and preferentially depleted metabolically active HSCs. Ectopic activation of the JAK2 or MAPK pathway did not rescue HSCs in Tpo−/− mice. Enforced activation of the mTOR pathway by conditionally deleting Tsc1 significantly rescued HSCs and their gene expression in Tpo−/− mice. Lin28b intrinsically promoted mTOR activation in HSCs, and its expression diminished over time. Conditional deletion of Lin28b further reduced mTOR activity and strongly exacerbated HSC depletion in Tpo−/− mice. Therefore, HSCs temporally transition from intrinsic LIN28B-dependent to extrinsic TPO-dependent maintenance in the developing liver.

Glioblastoma (GBM) is the most common and aggressive primary malignant brain tumor and is characterized by high mortality and morbidity rates and unpredictable clinical behavior. The disappointing prognosis for patients with GBM even after surgery and postoperative radiation and chemotherapy has fueled the search for specific targets to provide new insights into the development of modern therapies. MicroRNAs (miRNAs/miRs) act as oncomirs and tumor suppressors to posttranscriptionally regulate the expression of various genes and silence many target genes involved in cell proliferation, the cell cycle, apoptosis, invasion, stem cell behavior, angio-genesis, the microenvironment and chemo-and radiotherapy resistance, which makes them attractive candidates as prognostic biomarkers and therapeutic targets or agents to advance GBM therapeutics. However, one of the major challenges of successful miRNA-based therapy is the need for an effective and safe system to deliver therapeutic compounds to specific tumor cells or tissues in vivo, particularly systems that can cross the blood-brain barrier (BBB). This challenge has shifted gradually as progress has been achieved in identifying novel tumor-related miRNAs and their targets, as well as the development of nanoparticles (NPs) as new carriers to deliver therapeutic compounds. Here, we provide an up-to-date summary (in recent 5 years) of the current knowledge of GBM-related oncomirs, tumor suppressors and microenvironmental miRNAs, with a focus on their potential applications as prognostic biomarkers and therapeutic targets, as well as recent advances in the development of carriers for nontoxic miRNA-based therapy delivery systems and how they can be adapted for therapy.

Skeletal stem cells (SSCs) were originally discovered in the bone marrow stroma. They are capable of self-renewal and multilineage differentiation into osteoblasts, chondrocytes, adipocytes, and stromal cells. Importantly, these bone marrow SSCs localize in the perivascular region and highly express hematopoietic growth factors to create the hematopoietic stem cell (HSC) niche. Thus, bone marrow SSCs play pivotal roles in orchestrating osteogenesis and hematopoiesis. Besides the bone marrow, recent studies have uncovered diverse SSC populations in the growth plate, perichondrium, periosteum, and calvarial suture at different developmental stages, which exhibit distinct differentiation potential under homeostatic and stress conditions. Therefore, the current consensus is that a panel of region-specific SSCs collaborate to regulate skeletal development, maintenance, and regeneration. Here, we will summarize recent advances of SSCs in long bones and calvaria, with a special emphasis on the evolving concept and methodology in the field. We will also look into the future of this fascinating research area that may ultimately lead to effective treatment of skeletal disorders.

The bone marrow niche plays critical roles in hematopoietic recovery and hematopoietic stem cell (HSC) regeneration after myeloablative stress. However, it is not clear whether systemic factors beyond the local niche are required for these essential processes in vivo. Thrombopoietin (THPO) is a key cytokine promoting hematopoietic rebound after myeloablation and its transcripts are expressed by multiple cellular sources. The upregulation of bone marrow-derived THPO has been proposed to be crucial for hematopoietic recovery and HSC regeneration after stress. Nonetheless, the cellular source of THPO in myeloablative stress has never been investigated genetically. We assessed the functional sources of THPO following two common myeloablative perturbations: 5-fluorouracil (5-FU) administration and irradiation. Using a Thpo translational reporter, we found that the liver but not the bone marrow is the major source of THPO protein after myeloablation. Mice with conditional Thpo deletion from osteoblasts and/or bone marrow stromal cells showed normal recovery of HSCs and hematopoiesis after myeloablation. In contrast, mice with conditional Thpo deletion from hepatocytes showed significant defects in HSC regeneration and hematopoietic rebound after myeloablation. Thus, systemic THPO from the liver is necessary for HSC regeneration and hematopoietic recovery in myeloablative stress conditions.

The liver maintains hematopoietic stem cells (HSCs) during development. However, it is not clear what cells are the components of the developing liver niche in vivo. Here, we genetically dissected the developing liver niche by systematically determining the cellular source of a key HSC niche factor, stem cell factor (SCF). Most HSCs were closely associated with sinusoidal vasculature. Using Scf(gfP) knockin mice, we found that Scf was primarily expressed by endothelial and perisinusoidal hepatic stellate cells. Conditional deletion of Scf from hepatocytes, hematopoietic cells, Ng2(+) cells, or endothelial cells did not affect HSC number or function. Deletion of Scf from hepatic stellate cells depleted HSCs. Nearly all HSCs were lost when Scf was deleted from both endothelial and hepatic stellate cells. The expression of several niche factors was down-regulated in stellate cells around birth, when HSCs egress the developing liver. Thus, hepatic stellate and endothelial cells create perisinusoidal vascular HSC niche in the developing liver by producing SCF.

Paucity of the glucose transporter-1 (Glut1) protein resulting from haploinsufficiency of the SLC2A1 gene arrests cerebral angiogenesis and disrupts brain function to cause Glut1 deficiency syndrome (Glut1 DS). Restoring Glut1 to Glut1 DS model mice prevents disease, but the precise cellular sites of action of the transporter, its temporal requirements, and the mechanisms linking scarcity of the protein to brain cell dysfunction remain poorly understood. Here, we show that Glut1 functions in a cell-autonomous manner in the cerebral microvasculature to affect endothelial tip cells and, thus, brain angiogenesis. Moreover, brain endothelial cell-specific Glut1 depletion not only triggers a severe neuroinflammatory response in the Glut1 DS brain, but also reduces levels of brain-derived neurotrophic factor (BDNF) and causes overt disease. Reduced BDNF correlated with fewer neurons in the Glut1 DS brain. Controlled depletion of the protein demonstrated that brain pathology and disease severity was greatest when Glut1 scarcity was induced neonatally, during brain angiogenesis. Reducing Glut1 at later stages had mild or little effect. Our results suggest that targeting brain endothelial cells during early development is important to ensure proper brain angiogenesis, prevent neuroinflammation, maintain BDNF levels, and preserve neuron numbers. This requirement will be essential for any disease-modifying therapeutic strategy for Glut1 DS.

NOD-SCID IL2rγ c null (NSG) mice are widely used to model human immune cell development because they are more permissive for human hematopoietic cell engraftment and reconstitution than NOD-SCID mice. While increased human reconstitution in the blood of NSG mice has been attributed to the absence of mouse NK cells, deletion of the common gamma chain (γc) limits development of lymphoid tissue inducer cells and precludes development of normal secondary lymphoid structures. The disorganized lymphoid tissue leads to compromised human T cell-B cell interactions and results in variable human immune cell function in human immune system (HIS) NSG compared to NOD-SCID mice. We attempted to remove mouse NK cells from NOD-SCID mice while retaining other γ c -dependent cytokine responses by targeted disruption of the mouse genomic IL15RA locus with CRISPR/Cas9. IL15Rα is required for the development, function and survival of NK cells. NOD-SCID IL15Rα -/- mice showed reductions in NK cells and NK cell function. However, NOD-SCID IL15Rα -/- mice demonstrated accelerated thymic lymphomagenesis and showed earlier mortality compared to NOD-SCID mice. This result suggests that mouse NK cells are important to delay lymphoma development in NOD-SCID mice. We transplanted thymectomized NOD-SCID ILl5Rα -/- mice with human fetal liver CD34 + cells and thymus to determine if these mice supported engraftment and development of a transplanted HIS. Surprisingly, we found that peripheral human engraftment was inferior (mean 0.05% of lymphocytes) to that in both NOD-SCID (mean, 10.5% of lymphocytes) and NSG (mean, 54% of lymphocytes) mice. These results indicate that NOD-SCID IL15Rα -/- mice are not permissive for human CD34 + cell engraftment.

Primary myelofibrosis (PMF) is a severe subtype of myeloproliferative neoplasm (MPN) characterized by progressive bone marrow (BM) fibrosis and hematopoietic insufficiency, reflecting profound pathology of the BM microenvironment. However, a comprehensive understanding of the stromal cell changes that drive BM remodeling in PMF remains lacking.