Aging profoundly impacts mesenchymal and hematopoietic lineage cells, including their progenitors-the skeletal stem cells (SSCs) and hematopoietic stem cells (HSCs), respectively. SSCs are crucial for skeletal development, homeostasis, and regeneration, maintaining bone integrity by differentiating into osteoblasts, adipocytes, and other lineages that contribute to the bone marrow (BM) microenvironment. Meanwhile, HSCs sustain hematopoiesis and immune function. With aging, SSCs and HSCs undergo significant functional decline, partly driven by cellular senescence-a hallmark of aging characterized by irreversible growth arrest, secretion of pro-inflammatory factors (senescence associated secretory phenotype, SASP), and impaired regenerative potential. In SSCs, senescence skews lineage commitment toward adipogenesis at the expense of osteogenesis, contributing to increased bone marrow adiposity (BMAd), reduced bone quality, and osteoporosis. Similarly, aged HSCs exhibit diminished self-renewal, biased differentiation, and heightened inflammation, compromising hematopoietic output and immune function. In this review, we examine the age-related cellular and molecular changes in SSCs and HSCs, their lineage decisions in the aging microenvironment, and the interplay between skeletal and hematopoietic compartments. We also discuss the role of senescence-driven alterations in BM homeostasis and how targeting cellular aging mechanisms may offer therapeutic strategies for mitigating age-related skeletal and hematopoietic decline.

Megakaryocytes are large, polyploid cells that produce platelets and originate from hematopoietic stem cells (HSCs) in the bone marrow. While in the classical paradigm, megakaryocytes are generated in a stepwise fashion through increasingly committed progenitor stages, studies using in-vivo barcoding, transplantation, and in-vitro culture have suggested that, in addition, a more direct pathway existed. The relevance of this direct pathway and its functional and phenotypic characteristics were unclear, however.

Recent publications using fate-mapping and single-cell transplantation now unequivocally demonstrate the existence of a direct megakaryocyte differentiation pathway, provide molecular characterization, and indicate distinct roles and regulation of both pathways. The direct pathway originates from a separate subset of 'top' HSCs, is enhanced by hematopoietic stress, inflammation and aging, bypasses multipotential progenitors, may be more active in myeloproliferative neoplasms, and generates phenotypically distinct megakaryocyte progenitors and more reactive platelets.

Novel insights into the direct megakaryocyte differentiation pathway provide a deeper understanding of HSC biology, hematological recovery after myeloablation, and aging of the hematopoietic system, and suggest that this pathway may contribute to the increase in thrombotic incidents with age and in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are clonal stem cell disorders characterized by dysregulated megakaryopoiesis and neoplastic hematopoietic stem cell (HSC) expansion. Using a murine model with MK-specific JAK2V617F expression, we establish an MPN aging model where mutant MKs drive HSC expansion and a progressive decline in wild-type HSC function. Compared to wild-type MKs, JAK2V617F MKs exhibit heightened inflammation and innate immune activation with aging, including increased antigen presentation, elevated pro-inflammatory cytokines, skewed T cell populations, and impaired T cell functions in the marrow niche. Enhanced MK immunomodulatory function is linked to mutant cell expansion and MPN progression in a chimeric murine model with co-existing wild-type and JAK2V617F mutant HSCs. LINE-1 (long-interspersed element-1), a retrotransposon linked to innate immune activation and aging, is upregulated in mutant MKs during aging in murine models. We validated that LINE-1-encoded protein ORF1p is expressed in marrow MKs in 12 of 13 MPN patients but absent in control samples from patients undergoing orthopedic surgery (n=5). These findings suggest that MKs reprogram the marrow immune microenvironment, impairing normal HSC function while promoting neoplastic expansion in MPNs. LINE-1 activation in mutant MKs may be a key driver of immune dysregulation in MPNs.

JAK2V617F mutant MKs reprogram the marrow immune microenvironment to promote neoplastic HSC expansion in MPNs.LINE-1 activation in diseased MKs triggers chronic inflammation and immune dysfunction in MPNs.

Platelets are crucial players in hemostasis and thrombosis but also contribute to immune regulation and host defense, using different receptors, signaling pathways, and effector functions, respectively. Whether distinct subsets of platelets specialize in these diverse tasks is insufficiently understood. Here, we used a pulse-labeling method in Mus musculus models for tracking in vivo platelet aging and its functional implications. Using in vitro and in vivo assays, we reveal that young, reticulated platelets show heightened responses in the setting of clot formation, with corresponding, increased responses to agonists, adhesion, and retractile function. Unexpectedly, aged platelets lose their hemostatic proficiency but are more prone to react to inflammatory challenge: compared with reticulated platelets, this cohort was more likely to form platelet-leukocyte aggregates and showed increased adhesion to neutrophils in vitro, as well as enhanced bactericidal function. In vivo, this was reflected in increased pulmonary recruitment of aged platelets in an acute lung injury model. Proteomic analyses confirmed the upregulation of immune pathways in this cohort, including enhanced procoagulant function. In mouse models of prolonged platelet half-life, this resulted in increased pulmonary leukocyte infiltration and inflammation upon acute lung injury. Similarly, human platelet concentrates decreased their hemostatic function and elevated their putative immunomodulatory potential in vitro over time, and in a mouse model of platelet transfusion, aged platelet concentrates resulted in augmented inflammation. In summary, we show that platelets exhibit age-dependent phenotypic shifts, allowing them to fulfill their diverse tasks in the vasculature. Because functional alterations of aging platelets extend to platelet concentrates, this may hold important implications for transfusion medicine.

C3G, a Rap1 GEF, promotes megakaryopoiesis and platelet function. Using transgenic and knock-out mouse models targeting C3G in megakaryocytes, we investigated whether C3G also affects the niche function of megakaryocytes during bone marrow (BM) recovery after myeloablation induced by 5-fluorouracil (5-FU), or total body irradiation (TBI) followed by bone marrow transplantation. C3G promoted megakaryocyte maturation and platelet production during recovery, along with increased white and red blood cell counts and enhanced survival of female mice after repeated doses of 5-FU. Additionally, megakaryocytes favored adipocyte differentiation through a C3G-mediated mechanism, likely involving Fgf1. Changes in the number or behavior of BM megakaryocytes and adipocytes influenced the hematopoietic stem cell pool, with C3G promoting its bias towards the myeloid-megakaryocytic lineage in both 5-FU- and TBI-ablated models. Therefore, C3G could be a potential target in therapies aimed at enhancing hematopoiesis in patients undergoing chemotherapy and/or BM transplantation.

Platelet factor 4 (PF4), also referred to as CXCL4, is a significant component of the C-X-C chemokine family, predominantly localized within the alpha granules of platelets. It is recognized for its anti-heparin and anti-angiogenic properties. However, the involvement of PF4 in inflammatory processes has not been extensively investigated. This article aims to explore the diverse functions of PF4 in the context of inflammatory diseases, emphasizing its potential dual regulatory roles across various immune cell types and pathological conditions. Recent research has enhanced our comprehension of PF4, revealing its production not only in platelets but also in macrophages and activated T cells, thereby extending its functional repertoire beyond its conventional roles. Consequently, this review provides a thorough analysis of PF4's influence on inflammatory diseases and offers perspectives and recommendations for future research endeavors.

Lineage tracing is a valuable technique that has greatly facilitated the exploration of cell origins and behavior. With the continuous development of single-cell sequencing technology, lineage tracing technology based on the single-cell level has become an important method to study biological development. Single-cell Lineage tracing technology plays an important role in the hematological system. It can help to answer many important questions, such as the heterogeneity of hematopoietic stem cell function and structure, and the heterogeneity of malignant tumor cells in the hematological system. Many studies have been conducted to explore the field of hematology by applying this technology. This review focuses on the superiority of the emerging single-cell lineage tracing technologies of Integration barcodes, CRISPR barcoding, and base editors, and summarizes their applications in the hematology system. These studies have suggested the vast potential in unraveling complex cellular behaviors and lineage dynamics in both normal and pathological contexts.

The tissue microenvironment refers to a localised tissue area where a complex combination of cells, structural components, and signalling molecules work together to support specific biological activities. A prime example is the bone marrow microenvironment, particularly the hematopoietic stem cell (HSC) niche, which is of immense interest due to its critical role in supporting lifelong blood cell production and the growth of malignant cells. In this review, we summarise the current understanding of HSC niche biology, highlighting insights gained from advanced imaging and genomic techniques. We also discuss the potential of emerging technologies such as spatial multi-omics to unravel bone marrow architecture in unprecedented detail.

Bone marrow hematopoietic injury encompasses a range of pathological conditions that disrupt the normal function of the hematopoietic system, primarily through the impaired production and differentiation of bone marrow hematopoietic cells. Key pathogenic mechanisms include aging, radiation damage, chemical induction, infection and inflammation, and cross-talk with non-hematopoietic diseases. These pathological factors often lead to myelosuppression and myeloid skewing. Furthermore, we explored the potential and application prospects of natural products in the treatment of bone marrow hematopoietic injury. Natural products, particularly those derived from Chinese herbal medicines and other natural sources, have emerged as promising therapeutic options due to their distinctive mechanisms and minimal side effects. A deeper understanding of the underlying mechanisms of bone marrow hematopoietic injury could illuminate how natural products exert their effects, thereby optimizing treatment strategies and offering safer, more effective options for patients. Future research should leverage emerging technologies to further elucidate the composition and interactions within the bone marrow microenvironment, as well as the specific pathways through which natural products modulate hematopoietic dysfunction.

Megakaryocytes (MKs) produce platelets, and similar to other hematopoietic progenitors, they are involved in homeostatic aspects of their bone marrow niche. MKs release and endocytose various factors, such as platelet factor 4 (PF4)/CXCL4. Here, we show that the intra-α-granular proteoglycan, serglycin (SRGN), plays a key role in this process by retaining PF4, and perhaps other factors, during MK maturation. Immature, SRGN-/- MKs released ∼80% of their PF4, and conditioned media from these cells negatively affected wild-type MK differentiation in vitro. This was replicated in wild-type MKs by treatment with the polycation surfen, a known inhibitor of glycosaminoglycan (GAG)/protein interactions. In vivo, SRGN-/- mice had an interstitial accumulation of PF4, transforming growth factor β1, interleukin-1β, and tumor necrosis factor α in their bone marrow and increased numbers of immature MKs, consistent with their mild thrombocytopenia. SRGN-/- mice also had reduced numbers of hematopoietic stem cells and multipotent progenitors, reduced laminin, and increased collagen I deposition. These findings demonstrate that MKs depend on SRGN and its charged GAGs to balance the distribution of PF4 and perhaps other factors between their α-granules and their adjacent extracellular spaces. Disrupting this balance negatively affects MK development and bone marrow microenvironment homeostasis.

Haematological malignancies comprise a diverse group of life-threatening systemic diseases, including leukaemia, lymphoma, and multiple myeloma. Currently available therapies, including chemotherapy, immunotherapy, and CAR-T cells, are often associated with important side effects and with the development of drug resistance and, consequently, disease relapse. In the last decades, it was largely demonstrated that the tumor microenvironment significantly affects cancer cell proliferation and tumor response to treatment. The development of biomimetic, in vitro models may promote the investigation of the interactions between cancer cells and the tumor microenvironment and may help to better understand the mechanisms leading to drug resistance. Although advanced in vitro models have been largely explored in the field of solid tumors, due to the complex nature of the blood cancer tumor microenvironment, the mimicking of haematological malignancies mostly relies on simpler systems, often limited to two-dimensional cell culture, which intrinsically excludes the microenvironmental niche, or to ethically debated animal models. This review aims at reporting an updated overview of state-of-the-art hematological malignancies 3D in vitro models, emphasizing the key features and limitations of existing systems to inspire further research in this underexplored field.

Megakaryocytes are classified into several subtypes including LSP1-positive immune-skewed, MYLK4-positive hematopoietic stem cell (HSC)-regulating, and BMAL1-positive platelet-producing megakaryocytes. Podoplanin (PDPN)-expressing stromal cells generate a microenvironment that promotes megakaryopoiesis in the bone marrow. In this context, PDPN interacts with C-type lectin-like receptor-2 (CLEC-2) on megakaryocyte progenitors, which induces megakaryocyte proliferation. However, the megakaryocyte subtypes developed by the regulation of the PDPN/CLEC-2 axis have not yet been elucidated.

We established an immortalized bone marrow PDPN-expressing stromal cell line and a PDPN-knockout line (PDPN WT and KO feeder cells, respectively). Bone marrow hematopoietic progenitors were committed to megakaryocytes in co-culture with PDPN WT or KO feeder cells. The number and ploidy of megakaryocytes, resultant platelets, and the polarization of megakaryocyte subtypes were investigated.

The number of megakaryocytes was significantly increased in the co-culture with PDPN WT feeder cells compared to that with PDPN KO feeder cells. The megakaryocytes on the PDPN WT and KO feeders showed their main ploidy at 16 N∼32 N and 8 N∼16 N, respectively. The number of platelets was decreased in the co-culture with the PDPN WT feeder compared to that in the co-culture with the PDPN KO feeder. For each megakaryocyte subtype, the percentage of MYLK4-positive megakaryocytes significantly increased and the percentage of BMAL1-positive megakaryocytes significantly decreased when co-cultured with the PDPN WT feeder. These results were also confirmed in the co-culture of CLEC-2 conditional KO megakaryocytes with PDPN WT feeder cells.

The PDPN/CLEC-2 axis modulates megakaryocyte subtype differentiation, with a predominance of HSC-regulating megakaryocytes.

Hematopoietic stem cells (HSCs) reside in a milieu that supports their functions, differentiation, and survival. This niche consists of several types of cells, including mesenchymal stem/stromal cells, endothelial cells, osteoblasts, megakaryocytes, macrophages, adipocytes, lymphoid cells, and nerve fibers. The interactions between these cells and HSCs have a role in HSC fate. Several studies have focused on identification of the biological and cellular mechanisms contributing to the establishment of this niche. However, the exact mechanisms of the interaction between HSCs and the bone marrow niche have not been elucidated yet. Unraveling these mechanisms would help in the design of effective methods for maintenance and multiplication of HSCs in clinical settings, in addition to establishment of novel therapies for hematopoietic diseases. The current review summarizes the effects of the niche cells on HSC function and underlying mechanisms of interplay between HSCs and their niche.

Brown adipose tissue (BAT) plays an important role in maintaining body temperature in newborn mammals; however, its mechanisms remain poorly understood. Here, we report the identification of a special population of brown adipose tissue-derived stromal cells (ASCs) in neonatal mice that highly express CD45 and can be differentiated into adipocytes with lower thermogenic ability. These CD45+ adipocytes also characteristically contained complement C5a receptor 1(C5aR1) on the cell membrane. C5ar1 deficiency in BAT resulted in an apparent immaturity of adipocytes and cold intolerance in neonatal mice. Mechanistically, loss of C5aR1 in these CD45+ brown adipocytes caused an increase in the secretion of plate factor four (PF4) from these cells, suppressing the maturity of neighboring brown adipocytes. Overall, our results indicated that the accumulation of C5aR1 positive brown adipocyte in neonatal BAT is essential for thermoregulation in newborn mice, which unveiled the regulatory mechanism of BAT-mediated thermogenesis in newborns.

Megakaryocytes (MKs) are large, polyploid cells that contribute to bone marrow homeostasis through the secretion of cytokines such as transforming growth factor β1 (TGFβ1). During neoplastic transformation, immature MKs accumulate in the bone marrow where they induce fibrotic remodeling ultimately resulting in myelofibrosis. Current treatment strategies aim to prevent MK hyperproliferation, however, little is understood about the potential of targeting dysregulated cytokine secretion from neoplastic MKs as a novel therapeutic avenue. Unconventional secretion of TGFβ1 as well as interleukin 1β (IL1β) via secretory autophagy occurs in cells other than MKs, which prompted us to investigate whether similar mechanisms are utilized by MKs. Here, we identified that TGFβ1 strongly co-localized with the autophagy marker light chain 3B in native MKs. Disrupting secretory autophagy by inhibiting the small GTPase RhoA or its downstream effector Rho kinase (ROCK) markedly reduced TGFβ1 and IL1β secretion in vitro . In vivo , conditional deletion of the essential autophagy gene Atg5 from the hematopoietic system limited megakaryocytosis and aberrant cytokine secretion in an MPL W515L -driven transplant model. Similarly, mice with a selective deletion of Rhoa from the MK and platelet lineage were protected from progressive fibrosis. Finally, disease hallmarks in MPL W515L -transplanted mice were attenuated upon treatment with the autophagy inhibitor hydroxychloroquine or the ROCK inhibitor Y27632, either as monotherapy or in combination with the JAK2 inhibitor ruxolitinib. Overall, our data indicate that aberrant cytokine secretion is dependent on secretory autophagy downstream of RhoA, targeting of which represents a novel therapeutic avenue in the treatment of myelofibrosis.

TGFβ1 is released from megakaryocytes via RhoA-mediated secretory autophagy, and targeting this process can alleviate fibrosis progression in a preclinical mouse model of myelofibrosis.

The bone marrow niche is a special microenvironment that comprises elements, including hematopoietic stem cells, osteoblasts, and endothelial cells, and helps maintain their characteristic functions. Here, we elaborate on the crosstalk between various cellular components, hematopoietic stem cells, and other cells in the bone marrow niche. We further explain the mechanism of preserving equilibrium in the bone marrow niche, which is crucial for the directional regulation of bone reconstruction and repair. Additionally, we elucidate the intercommunication among osteocytes, the regulation of osteoblast maturation and activation by lymphocytes, the deficiency of megakaryocytes that can markedly impair osteoblast formation, and the mechanism of interaction between macrophages and mesenchymal stem cells in the bone marrow niche. Finally, we discussed the new immunotherapies for bone tumors in the BM niche. In this review, we aimed to provide a candid overview of the crosstalk among bone marrow niche cells and to highlight new concepts underlying the unknown mechanisms of hematopoiesis and bone reconstruction. Thus, this review may provide a more comprehensive understanding of the role of these niche cells in improving hematopoietic function and help identify their therapeutic potential for different diseases in the future.

Megakaryocytes (MKs), which are traditionally known for their role in platelet production, are now emerging as unique immune cells with diverse capabilities. They express immune receptors, participate in pathogen recognition and response, phagocytose pathogens, contribute to antigen presentation, and interact with various immune cell types. When encountering inflammatory challenges, MKs exhibit intricate immune functions that can either promote or inhibit inflammation. These responses are mediated through mechanisms, such as the secretion of either anti-inflammatory or pro-inflammatory cytokines and release of immunomodulatory platelets according to specific conditions. This intricate array of responses necessitates a detailed exploration to determine whether the immune functions of MKs are carried out by the entire MK population or by a specific subpopulation. Breakthroughs in single-cell RNA sequencing have uncovered a unique "immune MK" subpopulation, revealing its distinct characteristics and immunoregulatory functions. This review provides latest insights into MKs' immune attributes and their roles in physiological and pathological contexts and emphasizes the discovery and functions of "immune MKs".

Hematopoietic stem cells (HSCs) responsible for blood cell production and their bone marrow regulatory niches undergo age-related changes, impacting immune responses and predisposing individuals to hematologic malignancies. Here, we show that the age-related alterations of the megakaryocytic niche and associated downregulation of Platelet Factor 4 (PF4) are pivotal mechanisms driving HSC aging. PF4-deficient mice display several phenotypes reminiscent of accelerated HSC aging, including lymphopenia, increased myeloid output, and DNA damage, mimicking physiologically aged HSCs. Remarkably, recombinant PF4 administration restored old HSCs to youthful functional phenotypes characterized by improved cell polarity, reduced DNA damage, enhanced in vivo reconstitution capacity, and balanced lineage output. Mechanistically, we identified LDLR and CXCR3 as the HSC receptors transmitting the PF4 signal, with double knockout mice showing exacerbated HSC aging phenotypes similar to PF4-deficient mice. Furthermore, human HSCs across various age groups also respond to the youthful PF4 signaling, highlighting its potential for rejuvenating aged hematopoietic systems. These findings pave the way for targeted therapies aimed at reversing age-related HSC decline with potential implications in the prevention or improvement of the course of age-related hematopoietic diseases.

Age-related attrition of the megakaryocytic niche and associated PF4 downregulation is a central mechanism in HSC aging.PF4 supplementation, acting on LDLR and CXCR3 receptors, rejuvenates the function of aged HSCs.

Myeloproliferative neoplasms, polycythemia vera, essential thrombocytosis, and primary myelofibrosis are a unique group of clonal hematopoietic stem cell neoplasms that share somatic, gain-in-function driver mutations in JAK2, CALR, and MPL. As a consequence, these disorders exhibit similar phenotypic features, the most common of which are the ceaseless production of normal erythrocytes, myeloid cells, platelets alone or in combination, extramedullary hematopoiesis, myelofibrosis, and a potential for leukemic transformation. In the case of polycythemia vera and essential thrombocytosis, however, prolonged survival is possible. With an incidence value in the range of 0.5-2.0/100,000, myeloproliferative neoplasms are rare disorders, but they are not new disorders, and after a century of scrutiny, their clinical features and natural histories are well-defined, though their individual management continues to be controversial. With respect to polycythemia vera, there has been a long-standing dispute between those who believe that the suppression of red blood cell production by chemotherapy is superior to phlebotomy to prevent thrombosis, and those who do not. With respect to essential thrombocytosis, there is a similar dispute about the role of platelets in veinous thrombosis, and the role of chemotherapy in preventing thrombosis by suppressing platelet production. Linked to these disputes is another: whether therapy with hydroxyurea promotes acute leukemia in disorders with a substantial possibility of longevity. The 21st century revealed new insights into myeloproliferative neoplasms with the discovery of their three somatic, gain-of-function driver mutations. Almost immediately, this triggered changes in the diagnostic criteria for myeloproliferative neoplasms and their therapy. Most of these changes, however, conflicted with prior well-validated, phenotypically driven diagnostic criteria and the management of these disorders. The aim of this review is to examine these conflicts and demonstrate how genomic discoveries in myeloproliferative neoplasms can be used to effectively complement the known phenotypic features of these disorders for their diagnosis and management.

Lung megakaryocytes (Mks) are a unique subset of Mks that are distinct from their bone marrow counterparts. Recent evidence suggests that lung Mks favor an immune phenotype, but have unclear contributions to the total platelet mass. In this issue of the JCI, Livada et al. used an array of complementary in vivo labeling and tracing models in mice to investigate a longstanding question of where lung Mks are derived. By combining these models with stressed conditions, the authors assessed the contribution of lung Mks to total platelet counts in a homeostatic and thrombocytopenic state. Mks were minor contributors to the circulating pool of platelets during homeostasis but increased output during thrombocytopenia. These findings add critical understanding to the development of lung Mks and demonstrate the dynamic potential of these specialized cells to respond to thrombocytopenia.

Certain immune cells, including neutrophils, macrophages, dendritic cells, B cells, Breg cells, CD4+ T cells, CD8+ T cells, and Treg cells, establish enduring residency within the bone marrow. Their distinctive interactions with hematopoiesis and the bone marrow microenvironment are becoming increasingly recognized alongside their multifaceted immune functions. These cells play a dual role in shaping hematopoiesis. They directly influence the quiescence, self-renewal, and multi-lineage differentiation of hematopoietic stem and progenitor cells through either direct cell-to-cell interactions or the secretion of various factors known for their immunological functions. Additionally, they actively engage with the cellular constituents of the bone marrow niche, particularly mesenchymal stem cells, endothelial cells, osteoblasts, and osteoclasts, to promote their survival and contribute to tissue repair, thereby fostering a supportive environment for hematopoietic stem and progenitor cells. Importantly, these bone marrow immune cells function synergistically, both locally and functionally, rather than in isolation. In summary, immune cells residing in the bone marrow are pivotal components of a sophisticated network of regulating hematopoiesis.

Mammalian hematopoietic stem cells (HSCs) emerge from the hemogenic endothelium in the major embryonic arteries. HSCs undergo a complex journey first migrating to the fetal liver (FL) and from there to the fetal bone marrow (FBM), where they mostly remain during adult life. In this process, a pool of adult HSCs is produced, which sustains lifelong hematopoiesis. Multiple cellular components support HSC maturation and expansion and modulate their response to environmental and developmental cues. While the adult HSC niche has been extensively studied over the last two decades, the niches present in the major embryonic arteries, FL, FBM and perinatal bone marrow (BM) are poorly described. Recent investigations highlight important differences among FL, FBM and adult BM niches and emphasize the important role that inflammation, microbiota and hormonal factors play regulating HSCs and their niches. We provide a review on our current understanding of these important cellular microenvironments across ontogeny. We mainly focused on mice, as the most widely used research model, and, when possible, include relevant insights from other vertebrates including birds, zebrafish, and human. Developing a comprehensive picture on these processes is critical to understand the earliest origins of childhood leukemia and to achieve multiple goals in regenerative medicine, such as mimicking HSC development in vitro to produce HSCs for broad transplantation purposes in leukemia, following chemotherapy, bone marrow failure, and in HSC-based gene therapy.

As the demographic structure shifts towards an aging society, strategies aimed at slowing down or reversing the aging process become increasingly essential. Aging is a major predisposing factor for many chronic diseases in humans. The hematopoietic system, comprising blood cells and their associated bone marrow microenvironment, intricately participates in hematopoiesis, coagulation, immune regulation and other physiological phenomena. The aging process triggers various alterations within the hematopoietic system, serving as a spectrum of risk factors for hematopoietic disorders, including clonal hematopoiesis, immune senescence, myeloproliferative neoplasms and leukemia. The emerging single-cell technologies provide novel insights into age-related changes in the hematopoietic system. In this review, we summarize recent studies dissecting hematopoietic system aging using single-cell technologies. We discuss cellular changes occurring during aging in the hematopoietic system at the levels of the genomics, transcriptomics, epigenomics, proteomics, metabolomics and spatial multi-omics. Finally, we contemplate the future prospects of single-cell technologies, emphasizing the impact they may bring to the field of hematopoietic system aging research.

Hematopoietic stem cells (HSCs) are supported by the bone marrow microenvironment to maintain normal production of blood cells. The niche may be considered an "ecosystem" that support the function of HSCs and other supportive cells. Alterations in the bone marrow niche are commonly observed in hematologic malignancies. Here, we review recent insights into the location and the molecular and cellular components of the bone marrow niche. Moreover, we discuss how the niche interacts with HSCs to drive the pathogenesis of hematopoietic malignancies. Overall, a better understanding of the influences on the HSC niche may drive therapeutic development targeting defective and aberrant hematopoiesis.

Recent advances in imaging suggested that spatial organization of hematopoietic cells in their bone marrow microenvironment (niche) regulates cell expansion, governing progression, and leukemic transformation of hematological clonal disorders. However, our ability to interrogate the niche in pre-malignant conditions has been limited, as standard murine models of these diseases rely largely on transplantation of the mutant clones into conditioned mice where the marrow microenvironment is compromised. Here, we leveraged live-animal microscopy and ultralow dose whole body or focal irradiation to capture single cells and early expansion of benign/pre-malignant clones in the functionally preserved microenvironment. 0.5 Gy whole body irradiation (WBI) allowed steady engraftment of cells beyond 30 weeks compared to non-conditioned controls. In-vivo tracking and functional analyses of the microenvironment showed no change in vessel integrity, cell viability, and HSC-supportive functions of the stromal cells, suggesting minimal inflammation after the radiation insult. The approach enabled in vivo imaging of Tet2+/- and its healthy counterpart, showing preferential localization within a shared microenvironment while forming discrete micro-niches. Notably, stationary association with the niche only occurred in a subset of cells and would not be identified without live imaging. This strategy may be broadly applied to study clonal disorders in a spatial context.

Despite increased understanding of the genomic landscape of Myeloproliferative Neoplasms (MPNs), the pathological mechanisms underlying abnormal megakaryocyte (Mk)-stromal crosstalk and fibrotic progression in MPNs remain unclear. We conducted mass spectrometry-based proteomics on mice with Romiplostim-dependent myelofibrosis to reveal alterations in signaling pathways and protein changes in Mks, platelets, and bone marrow (BM) cells. The chemokine Platelet Factor 4 (PF4)/Cxcl4 was up-regulated in all proteomes and increased in plasma and BM fluids of fibrotic mice. High TPO concentrations sustained in vitro PF4 synthesis and secretion in cultured Mks, while Ruxolitinib restrains the abnormal PF4 expression in vivo. We discovered that PF4 is rapidly internalized by stromal cells through surface glycosaminoglycans (GAGs) to promote myofibroblast differentiation. Cxcl4 gene silencing in Mks mitigated the profibrotic phenotype of stromal cells in TPO-saturated co-culture conditions. Consistently, extensive stromal PF4 uptake and altered GAGs deposition were detected in Romiplostim-treated, JAK2V617F mice and BM biopsies of MPN patients. BM PF4 levels and Mk/platelet CXCL4 expression were elevated in patients, exclusively in overt fibrosis. Finally, pharmacological inhibition of GAGs ameliorated in vivo fibrosis in Romiplostim-treated mice. Thus, our findings highlight the critical role of PF4 in the fibrosis progression of MPNs and substantiate the potential therapeutic strategy of neutralizing PF4-GAGs interaction.

Many lung immune cells are known to respond to inhaled particulate matter. However, current known responses cannot explain how particles induce thrombosis in the lung and how they translocate to distant organs. Here, we demonstrate that lung megakaryocytes (MKs) in the alveolar and interstitial regions display location-determined characteristics and act as crucial responders to inhaled particles. They move rapidly to engulf particles and become activated with upregulation in inflammatory responses and thrombopoiesis. Comprehensive in vivo, in vitro and ex vivo results unraveled that MKs were involved in particle-induced lung damages and shed particle-containing platelets into blood circulation. Moreover, MK-derived platelets exhibited faster clotting, stronger adhesion than normal resting platelets, and inherited the engulfed particles from parent MKs to assist in extrapulmonary particle transportation. Our findings collectively highlight that the specific responses of MKs towards inhaled particles and their roles in facilitating the translocation of particles from the lungs to extrapulmonary organs for clearance.

Hematopoietic differentiation is controlled by intrinsic regulators and the extrinsic hematopoietic niche. Activating transcription factor 4 (ATF4) plays a crucial role in the function of fetal and adult hematopoietic stem cell maintenance. However, the precise function of ATF4 in the bone marrow (BM) niche and the mechanism by which ATF4 regulates adult hematopoiesis remain largely unknown. Here, we used 4 cell-type-specific mouse Cre lines to achieve conditional knockout of Atf4 in Cdh5+ endothelial cells, Prx1+ BM stromal cells, Osx+ osteoprogenitor cells, and Mx1+ hematopoietic cells and uncovered the role of Atf4 in niche cells and hematopoiesis. Intriguingly, depletion of Atf4 in niche cells did not affect hematopoiesis; however, Atf4-deficient hematopoietic cells exhibited erythroid differentiation defects, leading to hypoplastic anemia. Mechanistically, ATF4 mediated direct regulation of Rps19bp1 transcription, which is, in turn, involved in 40 S ribosomal subunit assembly to coordinate ribosome biogenesis and promote erythropoiesis. Finally, we demonstrate that under conditions of 5-fluorouracil-induced stress, Atf4 depletion impedes the recovery of hematopoietic lineages, which requires efficient ribosome biogenesis. Taken together, our findings highlight the indispensable role of the ATF4-RPS19BP1 axis in the regulation of erythropoiesis.

The bone marrow microenvironment (BMM) has highly specialized anatomical characteristics that provide a sanctuary place for hematopoietic stem cells (HSCs) that allow appropriate proliferation, maintenance, and self-renewal capacity. Several cell types contribute to the constitution and function of the bone marrow niche. Interestingly, uncovering the secrets of BMM and its interaction with HSCs in health paved the road for research aiming at better understanding the concept of leukemic stem cells (LSCs) and their altered niche. In fact, they share many signals that are responsible for interactions between LSCs and the bone marrow niche, due to several biological similarities between LSCs and HSCs. On the other hand, LSCs differ from HSCs in their abnormal activation of important signaling pathways that regulate survival, proliferation, drug resistance, invasion, and spread. Targeting these altered niches can help in better treatment choices for hematological malignancies and bone marrow disorders in general and acute myeloid leukemia (AML) in particular. Moreover, targeting those niches may help in decreasing the emergence of drug resistance and lower the relapse rate. In this article, the authors reviewed the most recent literature on bone marrow niches and their relations with either normal HSCs and AML cells/LSC, by focusing on pathogenetic and therapeutic implications.

Hematopoietic stem cell transplantation (HSCT) remains an indispensable therapeutic strategy for various hematological diseases. This review discusses the pivotal role of bone marrow (BM) niches in influencing the efficacy of HSCT and evaluates the current animal models, emphasizing their limitations and the need for alternative models. Traditional animal models, mainly murine xenograft, have provided significant insights, but due to species-specific differences, are often constrained from accurately mimicking human physiological responses. These limitations highlight the importance of developing alternative models that can more realistically replicate human hematopoiesis. Emerging models that include BM organoids and BM-on-a-chip microfluidic systems promise enhanced understanding of HSCT dynamics. These models aim to provide more accurate simulations of the human BM microenvironment, potentially leading to improved preclinical assessments and therapeutic outcomes. This review highlights the complexities of the BM niche, discusses the limitations of current models, and suggests directions for future research using advanced model systems. [BMB Reports 2024; 57(8): 352-362].

Hematopoietic stem cells (HSCs) are the cornerstone of the hematopoietic system. HSCs sustain the continuous generation of mature blood derivatives while self-renewing to preserve a relatively constant pool of progenitors throughout life. Yet, long-term maintenance of functional HSCs exclusively takes place in association with their native tissue microenvironment of the bone marrow (BM). HSCs have been long proposed to reside in fixed and identifiable anatomical units found in the complex BM tissue landscape, which control their identity and fate in a deterministic manner. In the last decades, tremendous progress has been made in the dissection of the cellular and molecular fabric of the BM, the structural organization governing tissue function, and the plethora of interactions established by HSCs. Nonetheless, a holistic model of the mechanisms controlling HSC regulation in their niche is lacking to date. Here, we provide an overview of our current understanding of BM anatomy, HSC localization, and crosstalk within local cellular neighborhoods in murine and human tissues, and highlight fundamental open questions on how HSCs functionally integrate in the BM microenvironment.

Hematopoiesis requires a complex interplay between the hematopoietic stem and progenitor cells and the cells of the bone marrow microenvironment (BMM). The BMM is heterogeneous, with different regions having distinct cellular, molecular, and metabolic composition and function. Studies have shown that this niche is disrupted in patients with acute myeloid leukemia (AML), which plays a crucial role in disease progression. This review provides a comprehensive overview of the components of vascular and endosteal niches and the molecular mechanisms by which they regulate normal hematopoiesis. We also discuss how these niches are modified in the context of AML, into a disease-promoting niche and how the modified niches in turn regulate AML blast survival and proliferation. We focus on mechanisms of modifications in structural and cellular components of the bone marrow (BM) niche by the AML cells and its impact on leukemic progression and patient outcome. Finally, we also discuss mechanisms by which the altered BM niche protects AML blasts from treatment agents, thereby causing therapy resistance in AML patients. We also summarize ongoing clinical trials that target various BM niche components in the treatment of AML patients. Hence, the BM niche represents a promising target to treat AML and promote normal hematopoiesis.

Background Tyrosine kinase inhibitors have revolutionized the treatment of chronic myeloid leukemia (CML) since the beginning of the century. However, resistance to therapy and the progression of disease tend to occur in certain patients. The bone marrow microenvironment may play a role in the disease outcome. Megakaryocytes have multiple roles in the regulation and maintenance of the hematopoietic stem cell microenvironment. In the current study, we evaluated the association of megakaryocyte morphology, morphometry, and microenvironment with disease progression and therapy resistance in CML. Methodology Megakaryocyte morphology and morphometry were analyzed and compared between the different phases (chronic and advanced) at diagnosis in 150 cases of BCR-ABL-positive CML. All CML-CP patients (n = 119) were followed up on tyrosine kinase inhibitor therapy for a minimum of 15 months and classified based on their treatment outcome as a response, resistance to therapy, or progression of disease based on standard criteria. Immunohistochemistry on a bone marrow trephine biopsy was done for vascular endothelial growth factor (VEGF), FOXP3, CD150, CD48, CD44, osteopontin, CXCL12, N-cadherin, PDL-1, and IL-7, and their expression on megakaryocytes and their association with treatment outcome was evaluated. Results The morphology and morphometry of megakaryocytes showed a heterogeneous population in CML. Morphology and morphometric parameters, when compared between the chronic and advanced phases of disease at diagnosis, did not show any statistical difference. Megakaryocytes were variably positive for VEGF, FOXP3, CD150, CD48, osteopontin, N-cadherin, CXCL12, CD44, PDL-1, and IL-7. However, only CD44-positive megakaryocytes were statistically associated with the treatment outcome. The patients with a higher expression of CD44 megakaryocytes progressed to the advanced phase of the disease during therapy compared to those who responded. Conclusion Megakaryocyte morphology and morphometry were heterogeneous in CML; however, they did not show any significant difference with either the phase of the disease or with treatment outcomes. Among the various immunohistochemical markers of the microenvironment, only CD44-positivity on megakaryocytes was associated with poor treatment outcomes.

Previous studies of hematopoietic stem cells (HSCs) primarily focused on single cell-based niche models, yielding fruitful but conflicting findings 1-5 . Here we report our investigation on the fetal liver (FL) as the primary fetal hematopoietic site using spatial transcriptomics. Our study reveals two distinct niches: the portal-vessel (PV) niche and the sinusoidal niche. The PV niche, composing N-cadherin (N-cad) Hi Pdgfrα + mesenchymal stromal cells (MSCs), endothelial cells (ECs), and N-cad Lo Albumin + hepatoblasts, maintains quiescent and multipotential FL-HSCs. Conversely, the sinusoidal niche, comprising ECs, hepatoblasts and hepatocytes, as well as potential macrophages and megakaryocytes, supports proliferative FL-HSCs biased towards myeloid lineages. Unlike prior reports on the role of Cxcl12, with its depletion from vessel-associated stromal cells leading to 80% of HSCs' reduction in the adult bone marrow (BM) 6,7 , depletion of Cxcl12 via Cdh2 CreERT (encoding N-cad) induces altered localization of HSCs from the PV to the sinusoidal niches, resulting in an increase of HSC number but with myeloid-bias. Similarly, we discovered that adult BM encompasses two niches within different zones, each composed of multi-cellular components: trabecular bone area (TBA, or metaphysis) supporting deep-quiescent HSCs, and central marrow (CM, or diaphysis) fostering heterogenous proliferative HSCs. This study transforms our understanding of niches by shifting from single cell-based to multicellular components within distinct zones, illuminating the intricate regulation of HSCs tailored to their different cycling states.

Hematopoietic stem cells (HSCs) are instrumental for organismal survival because they are responsible for lifelong production of mature blood lineages in homeostasis and response to external stress. To fulfill their function, HSCs rely on reciprocal interactions with specialized tissue microenvironments, termed HSC niches. From embryonic development to advanced aging, HSCs transition through several hematopoietic organs in which they are supported by distinct extrinsic cues. Here, we describe recent discoveries on how HSC niches collectively adapt to ensure robust hematopoietic function during biological aging and after exposure to acute stress. We also discuss the latest strategies leveraging niche-derived signals to revert aging-associated phenotypes and enhance hematopoietic recovery after myeloablation.

Hematopoiesis occurs in the bone marrow (BM), within a specialized microenvironment referred to as the stem cell niche, where the hematopoietic stem cells (HSCs) reside and are regulated for quiescence, self-renewal and differentiation through intrinsic and extrinsic mechanisms. The BM contains at least two distinctive HSC-supportive niches: an endosteal osteoblastic niche that supports quiescence and self-renewal and a more vascular/perisinusoidal niche that promotes proliferation and differentiation. Both associate with supporting mesenchymal stromal cells. Within the more hypoxic osteoblastic niche, HSCs specifically interact with the osteoblasts that line the endosteal surface, which secrete several important HSC quiescence and maintenance regulatory factors. In vivo imaging indicates that the HSCs and progenitors located further away, in the vicinity of sinusoidal endothelial cells, are more proliferative. Here, HSCs interact with endothelial cells via specific cell adhesion molecules. Endothelial cells also secrete several factors important for HSC homeostasis and proliferation. In addition, HSCs and mesenchymal stromal cells are embedded within the extracellular matrix (ECM), an important network of proteins such as collagen, elastin, laminin, proteoglycans, vitronectin, and fibronectin. The ECM provides mechanical characteristics such as stiffness and elasticity important for cell behavior regulation. ECM proteins are also able to bind, sequester, display, and distribute growth factors across the BM, thus directly affecting stem cell fate and regulation of hematopoiesis. These important physical and chemical features of the BM require careful consideration when creating three-dimensional models of the BM.

Platelet homeostasis is essential for vascular integrity and immune defence1,2. Although the process of platelet formation by fragmenting megakaryocytes (MKs; thrombopoiesis) has been extensively studied, the cellular and molecular mechanisms required to constantly replenish the pool of MKs by their progenitor cells (megakaryopoiesis) remains unclear3,4. Here we use intravital imaging to track the cellular dynamics of megakaryopoiesis over days. We identify plasmacytoid dendritic cells (pDCs) as homeostatic sensors that monitor the bone marrow for apoptotic MKs and deliver IFNα to the MK niche triggering local on-demand proliferation and maturation of MK progenitors. This pDC-dependent feedback loop is crucial for MK and platelet homeostasis at steady state and under stress. pDCs are best known for their ability to function as vigilant detectors of viral infection5. We show that virus-induced activation of pDCs interferes with their function as homeostatic sensors of megakaryopoiesis. Consequently, activation of pDCs by SARS-CoV-2 leads to excessive megakaryopoiesis. Together, we identify a pDC-dependent homeostatic circuit that involves innate immune sensing and demand-adapted release of inflammatory mediators to maintain homeostasis of the megakaryocytic lineage.

Extensive research has explored the functional correlation between stem cells and progenitor cells, particularly in blood. Hematopoietic stem cells (HSCs) can self-renew and regenerate tissues within the bone marrow, while stromal cells regulate tissue function. Recent studies have validated the role of mammalian stem cells within specific environments, providing initial empirical proof of this functional phenomenon. The interaction between bone and blood has always been vital to the function of the human body. It was initially proposed that during evolution, mammalian stem cells formed a complex relationship with the surrounding microenvironment, known as the niche. Researchers are currently debating the significance of molecular-level data to identify individual stromal cell types due to incomplete stromal cell mapping. Obtaining these data can help determine the specific activities of HSCs in bone marrow. This review summarizes key topics from previous studies on HSCs and their environment, discussing current and developing concepts related to HSCs and their niche in the bone marrow.

Megakaryocytes (MKs), integral to platelet production, predominantly reside in the bone marrow (BM) and undergo regulated fragmentation within sinusoid vessels to release platelets into the bloodstream. Inflammatory states and infections influence MK transcription, potentially affecting platelet functionality. Notably, COVID-19 has been associated with altered platelet transcriptomes. In this study, we investigated the hypothesis that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection could affect the transcriptome of BM MKs. Using spatial transcriptomics to discriminate subpopulations of MKs based on proximity to BM sinusoids, we identified ∼19 000 genes in MKs. Machine learning techniques revealed that the transcriptome of healthy murine BM MKs exhibited minimal differences based on proximity to sinusoid vessels. Furthermore, at peak SARS-CoV-2 viremia, when the disease primarily affected the lungs, MKs were not significantly different from those from healthy mice. Conversely, a significant divergence in the MK transcriptome was observed during systemic inflammation, although SARS-CoV-2 RNA was never detected in the BM, and it was no longer detectable in the lungs. Under these conditions, the MK transcriptional landscape was enriched in pathways associated with histone modifications, MK differentiation, NETosis, and autoimmunity, which could not be explained by cell proximity to sinusoid vessels. Notably, the type I interferon signature and calprotectin (S100A8/A9) were not induced in MKs under any condition. However, inflammatory cytokines induced in the blood and lungs of COVID-19 mice were different from those found in the BM, suggesting a discriminating impact of inflammation on this specific subset of cells. Collectively, our data indicate that a new population of BM MKs may emerge through COVID-19-related pathogenesis.

Platelet dysregulation is drastically increased with advanced age and contributes to making cardiovascular disorders the leading cause of death of elderly humans. Here, we reveal a direct differentiation pathway from hematopoietic stem cells into platelets that is progressively propagated upon aging. Remarkably, the aging-enriched platelet path is decoupled from all other hematopoietic lineages, including erythropoiesis, and operates as an additional layer in parallel with canonical platelet production. This results in two molecularly and functionally distinct populations of megakaryocyte progenitors. The age-induced megakaryocyte progenitors have a profoundly enhanced capacity to engraft, expand, restore, and reconstitute platelets in situ and upon transplantation and produce an additional platelet population in old mice. The two pools of co-existing platelets cause age-related thrombocytosis and dramatically increased thrombosis in vivo. Strikingly, aging-enriched platelets are functionally hyper-reactive compared with the canonical platelet populations. These findings reveal stem cell-based aging as a mechanism for platelet dysregulation and age-induced thrombosis.

Hematopoietic stem cells (HSCs) generate all blood cell lineages responsible for tissue oxygenation, life-long hematopoietic homeostasis and immune protection. In adulthood, HSCs primarily reside in the bone marrow (BM) microenvironment, consisting of diverse cell types that constitute the stem cell 'niche'. The adaptability of the hematopoietic system is required to respond to the needs of the host, whether to maintain normal physiology or during periods of physical, psychosocial or environmental stress. Hematopoietic homeostasis is achieved by intricate coordination of systemic and local factors that orchestrate the function of HSCs throughout life. However, homeostasis is not a static process; it modulates HSC and progenitor activity in response to circadian rhythms coordinated by the central and peripheral nervous systems, inflammatory cues, metabolites and pathologic conditions. Here, we review local and systemic factors that impact hematopoiesis, focusing on the implications of aging, stress and cardiovascular disease.