Hematopoietic Stem Cell Niche Research Articles

Living Biomaterials to Engineer Hematopoietic Stem Cell Niches.

Living biointerfaces are a new class of biomaterials combining living cells and polymeric matrices that can act as biologically active and instructive materials that host and provide signals to surrounding cells. Here, living biomaterials based on Lactococcus lactis to control hematopoietic stem cells in 2D surfaces and 3D hydrogels are introduced. L. lactis is modified to express C-X-C motif chemokine ligand 12 (CXCL12), thrombopoietin (TPO), vascular cell adhesion protein 1 (VCAM1), and the 7th-10th type III domains of human plasma fibronectin (FN III7-10 ), in an attempt to mimic ex vivo the conditions of the human bone marrow. These results suggest that living biomaterials that incorporate bacteria expressing recombinant CXCL12, TPO, VCAM1, and FN in both 2D systems direct hematopoietic stem and progenitor cells (HSPCs)-bacteria interaction, and in 3D using hydrogels functionalized with full-length human plasma fibronectin allow for a notable expansion of the CD34+ /CD38- /CD90+ HSPC population compared to the initial population. These results provide a strong evidence based on data that suggest the possibility of using living materials based on genetically engineered bacteria for the ex-vivo expansion of HSPC with eventual practical clinical applications in HSPCs transplantation for hematological disorders.

Defining the ultrastructure of the hematopoietic stem cell niche by correlative light and electron microscopy.

The blood system is supported by hematopoietic stem and progenitor cells (HSPCs) found in a specialized microenvironment called the niche. Many different niche cell types support HSPCs, however how they interact and their ultrastructure has been difficult to define. Here, we show that single endogenous HSPCs can be tracked by light microscopy, then identified by serial block-face scanning electron microscopy (SBEM) at multiscale levels. Using the zebrafish larval kidney marrow (KM) niche as a model, we followed single fluorescently labeled HSPCs by light sheet microscopy, then confirmed their exact location in a 3D SBEM dataset. We found a variety of different configurations of HSPCs and surrounding niche cells, suggesting there could be functional heterogeneity in sites of HSPC lodgement. Our approach also allowed us to identify dopamine beta-hydroxylase (dbh) positive ganglion cells as a previously uncharacterized functional cell type in the HSPC niche. By integrating multiple imaging modalities, we could resolve the ultrastructure of single rare cells deep in live tissue and define all contacts between an HSPC and its surrounding niche cell types.

Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells

Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.

Peeling Back the Layers of Lymph Gland Structure and Regulation.

During the past 60 years, the fruit fly, Drosophila melanogaster, has proven to be an excellent model to study the regulation of hematopoiesis. This is not only due to the evolutionarily conserved signalling pathways and transcription factors contributing to blood cell fate, but also to convergent evolution that led to functional similarities in distinct species. An example of convergence is the compartmentalization of blood cells, which ensures the quiescence of hematopoietic stem cells and allows for the rapid reaction of the immune system upon challenges. The lymph gland, a widely studied hematopoietic organ of the Drosophila larva, represents a microenvironment with similar features and functions to classical hematopoietic stem cell niches of vertebrates. Lymph gland studies were effectively supported by the unparalleled toolkit developed in Drosophila, which enabled the high-resolution investigation of the cellular composition and regulatory interaction networks of the lymph gland. In this review, we summarize how our understanding of lymph gland structure and hematopoietic cell-to-cell communication evolved during the past decades and compare their analogous features to those of the vertebrate hematopoietic stem cell niche.

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

SummaryIn this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

S245: ALTERATION OF BONE MARROW NICHE BY ALLOGENEIC IMMUNE REACTION AFTER HSCT

Background: Allogeneic stem cell transplantation (allo-SCT) has been considered as a curable therapy for hematologic diseases. Post-transplant myelosuppression is one of the life-threatening complications. Hematopoietic stem cell (HSC) functions are tightly regulated by a specialized microenvironment called “niche” in the bone marrow (BM) and we have identified differential contributions of perivascular stromal cells as HSC niche (Asada et al. Nat Cell Biol 2017). Recent studies indicate that BM niche can be targeted by allo-immune reaction but it remains unclear how the perivascular niche cells are involved in the mechanism of HSC dysfunction after allo-SCT. Aims: In this study, we aim to investigate the mechanisms of the impairment of the niche cells by allo-immune reactions, and to explore a targetable pathway for myelosuppression after allo-SCT. Methods: To evaluate the alterations in niche cells after allo-SCT, we utilized allo-SCT mice models (C3H/HeJ into C57BL/6, C57BL/6 into B6D2F1). Hematopoietic cells, including HSCs and mature cells, and perivascular stromal cells in BM were analyzed by flow cytometry (FACS) after transplantation. In imaging studies, we visualized perivascular stromal cells using Nestin-GFP (Nes-GFP), Myh11-CreER/tdTomato, LeptinR-Cre/tdTomato transgenic mice and observed them with confocal laser scanning microscopy. The gene expression analysis of niche factors was performed by real-time PCR. To assess the landscape of transcriptional remodeling in BM after allo-SCT, we performed single-cell RNA sequencing (scRNAseq) of BM cells. Results: FACS analyses showed that Nes-GFP+ stromal cells were dramatically reduced, and HSC recovery was severely impaired in allo-SCT mice compared to control mice at day 21 after SCT. Imaging studies also revealed that perivascular stromal cells are morphologically damaged in allo-SCT mice. Nes-GFP+ stromal cells in allo-SCT mice presented a decreased expression of niche factors, including Cxcl12 and Scf, which are essential for HSC maintenance, indicating the dysfunction of niche cells. We confirmed the impaired niche function in allo-SCT mice by tandem transplantation analysis in vivo, in which the recovery of syngeneic HSCs was also impaired in damaged microenvironment in allo-SCT mice. Prevention of allo-reactive T cell expansion by post-transplant cyclophosphamide ameliorated the niche cell reduction along with improved recovery of HSCs. These results indicate that niche impairment caused by allo-immune reaction leads to HSC dysfunction. The scRNAseq of BM highlighted the elevated expression levels of inflammation response genes, including Cxcl9 and Cxcl10, and MHC classⅡ, together with the deceased expression of niche factors in niche cells in allo-SCT mice. These alterations seemed to be induced by proinflammatory cytokines such as interferon-gamma (INF-γ) derived from allo-T cells, as the increased number of T cells and high levels of INF-γ in BM were observed in allo-SCT mice. Additionally, evaluation of T cell localization by using imaging techniques of BM revealed that allo-T cells reside significant closer to LepR+ niche cells in allo-SCT mice. Since activation of CXCR3, which is a receptor for chemokines Cxcl9 and Cxcl10, has been shown to induce chemotaxis of activated T cells, we treated allo-SCT mice with AMG487, an antagonist of CXCR3, resulted in an improved recovery of both perivascular niche cells and hematopoiesis in allo-SCT mice. Summary/Conclusion: Collectively, allo-immune reaction severely damages perivascular HSC niche cells, leading to impaired recovery of hematopoiesis after allo-SCT.

P1414: MARROW ADIPOCYTE-ENRICHED NICHE HOSTS AND INFLUENCES A FRACTION OF HEMATOPOIETIC PROGENITORS IN ADULT HIP BONE

Background: The hematopoietic process is tightly regulated by the bone marrow (BM) microenvironment. Emerging studies recognized a regulatory role of mouse BM adipose tissue (BMAT) on self-renewal and differentiation of hematopoietic stem cells (HSC) and progenitors, while results on human BMAT are still scarce. Aims: Although hip arthroplasty-derived BM serves as an available resource for non-malignant hematopoietic cell research, the BMAT component of this BM and its’ impact on hematopoietic progenitors is not yet revealed. Therefore, our aims were to explore the contribution of human BMAT to the maintenance of hematopoietic progenitors. Methods: BM samples of acetabular bones were obtained from patients undergoing hip arthroplasty (n=13, 65.7±12.5 years, Ethical approval 187/18). Centrifugation was applied to separate the fatty layer containing BMAT from the BM cell suspension. The BM fraction was used for isolation of BM mononuclear (BMC) and expansion of BM mesenchymal stromal cells (BM-MSC). Digestion of BMAT was applied to separate the non-adipocytic cells of BMAT from mature adipocytes, expand them, and to investigate their functions and transcriptional activity by RNA sequencing. Multiparametric flow cytometry was applied to explore surface marker expression. Hematopoietic activity was investigated by short-term colony assays of cells ex vivo and in vitro upon co-culture with BM-MSC or sub-cultured BMAT adipocytes. Results are presented as mean ±SD and statistical significance was estimated by paired tests. Results: We found that the total colony forming cell number in BMC (0.147±0.068) was significantly higher than in BMAT-associated cells (0.0684±0.062%, p=0.0002, n=11). The strongest reduction within BMAT was observed in colony forming unit (CFU)-macrophages and CFU-granulocyte-erythroid-macrophage/monocyte-megakaryocytes. The content of CD45+CD34+ cells was higher in BMC (0.901±1.009%) than in BMAT (0.357±0.505%), while interestingly no differences were found in CD33+ cell frequencies. Both, expanded BMAT and sub-cultured BMAT adipocytes showed stronger adipogenic potential than BM-MSC. When compared to BM-MSC, sub-cultured BMAT adipocytes showed weaker potential to support growth of committed hematopoietic progenitors within BMC ex vivo, favorizing the growth of early and late erythroid progenitors at the expense of other hematopoietic cells. Transcriptomic analyses showed that HSC niche factors (Angptl4, Wnt5b, Il7, Dkk3), but also LepR were upregulated in BM-MSC compared to expanded BMAT cells. Summary/Conclusion: Our findings indicate a yet unrevealed participation of the BMAT niche in compartmentalization of adult hip bone hematopoietic progenitors by hosting a set of hematopoietic cells and potentially, BMAT progenitors in the close proximity of BM adipocytes. Since a behavior of BM populations is dependent on local microenvironmental context and bone region, obtained results might reflect pathologic settings in arthroplasty patients. Further studies have to address the potential coupling of marrow adipogenesis and hematopoietic progenitor maintenance during aging and diseases.

Regulation of Malignant Myeloid Leukemia by Mesenchymal Stem Cells.

Bone marrow microenvironment (BMM) has been proven to have benefits for both normal hematopoietic stem cell niche and pathological leukemic stem cell niche. In fact, the pathological leukemia microenvironment reprograms bone marrow niche cells, especially mesenchymal stem cells for leukemia progression, chemoresistance and relapse. The growth and differentiation of MSCs are modulated by leukemia stem cells. Moreover, chromatin abnormality of mesenchymal stem cells is sufficient for leukemia initiation. Here, we summarize the detailed relationship between MSC and leukemia. MSCs can actively and passively regulate the progression of myelogenous leukemia through cell-to-cell contact, cytokine-receptor interaction, and exosome communication. These behaviors benefit LSCs proliferation and survival and inhibit physiological hematopoiesis. Finally, we describe the recent advances in therapy targeting MSC hoping to provide new perspectives and therapeutic strategies for leukemia.

Haematopoiesis in Zebrafish (Danio Rerio).

Haematopoiesis in fish and mammals is a complex process, and many aspects regarding its model and the differentiation of haematopoietic stem cells (HSCs) still remain enigmatic despite advanced studies. The effects of microenvironmental factors or HSCs niche and signalling pathways on haematopoiesis are also unclear. This review presents Danio rerio as a model organism for studies on haematopoiesis in vertebrates and discusses the development of this process during the embryonic period and in adult fish. It describes the role of the microenvironment of the haematopoietic process in regulating the formation and function of HSCs/HSPCs (hematopoietic stem/progenitor cells) and highlights facts and research areas important for haematopoiesis in fish and mammals.

Runx1 and Runx2 inhibit fibrotic conversion of cellular niches for hematopoietic stem cells

In bone marrow, special microenvironments, known as niches, are essential for the maintenance of hematopoietic stem cells (HSCs). A population of mesenchymal stem cells, termed CXC chemokine ligand 12 (CXCL12)-abundant reticular (CAR) cells or leptin receptor-expressing cells are the major cellular component of HSC niches. The molecular regulation of HSC niche properties is not fully understood. The role of Runx transcription factors, Runx1 and Runx2 in HSC cellular niches remains unclear. Here we show that Runx1 is predominantly expressed in CAR cells and that mice lacking both Runx1 and Runx2 in CAR cells display an increase in fibrosis and bone formation with markedly reduced hematopoietic stem and progenitor cells in bone marrow. In vitro, Runx1 is induced by the transcription factor Foxc1 and decreases fibrotic gene expression in CAR cells. Thus, HSC cellular niches require Runx1 or Runx2 to prevent their fibrotic conversion and maintain HSCs and hematopoiesis in adults.

Targeting the Hematopoietic Stem Cell Niche in β-Thalassemia and Sickle Cell Disease.

In the last decade, research on pathophysiology and therapeutic solutions for β-thalassemia (BThal) and sickle cell disease (SCD) has been mostly focused on the primary erythroid defect, thus neglecting the study of hematopoietic stem cells (HSCs) and bone marrow (BM) microenvironment. The quality and engraftment of HSCs depend on the BM microenvironment, influencing the outcome of HSC transplantation (HSCT) both in allogeneic and in autologous gene therapy settings. In BThal and SCD, the consequences of severe anemia alter erythropoiesis and cause chronic stress in different organs, including the BM. Here, we discuss the recent findings that highlighted multiple alterations of the BM niche in BThal and SCD. We point out the importance of improving our understanding of HSC biology, the status of the BM niche, and their functional crosstalk in these disorders towards the novel concept of combined therapies by not only targeting the genetic defect, but also key players of the HSC–niche interaction in order to improve the clinical outcomes of transplantation.

MDS cells impair osteolineage differentiation of MSCs via extracellular vesicles to suppress normal hematopoiesis.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both invivo and invitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.

In vitro culture of hematopoietic stem cell niche using angiopoietin-1-coupled alginate hydrogel

Stem cells exist and maintain their quiescence and pluripotency in stem cell niche. Here, we hypothesized that regulation of cell-cell interactions using a polymeric scaffold as synthetic extracellular matrix (ECM) could be critical in creating a hematopoietic stem cell (HSC) niche in vitro. Angiopoietin-1 (Ang1) binds to the tyrosine kinase receptor (Tie2), and regulation of the Tie2/Ang1 interaction is important in maintaining the quiescence of HSCs in vivo. Alginate hydrogel was thus modified with Ang1 as a synthetic ECM to mimic the HSC niche. Long-term HSCs (CD34−, CD135−, and CD150+) were isolated from mouse femurs and cultured on Ang1-modified alginate hydrogel. The percentage of LT-HSCs in G0 phase was 46.8 ± 1.8%, which was comparable to that of LT-HSCs co-cultured with osteoblasts (46.8 ± 2.1%). Ang1-coupled alginate gels were useful to provide a niche for HSC quiescence without a co-culture system. Polymeric scaffolds containing biomimetic and cell-instructive characteristics for stem cell phenotype regulation might help create HSC niches in vitro and be useful to engineer tissues and transplant stem cells.

Bone Marrow Niches of Hematopoietic Stem and Progenitor Cells.

The mammalian hematopoietic system is remarkably efficient in meeting an organism’s vital needs, yet is highly sensitive and exquisitely regulated. Much of the organismal control over hematopoiesis comes from the regulation of hematopoietic stem cells (HSCs) by specific microenvironments called niches in bone marrow (BM), where HSCs reside. The experimental studies of the last two decades using the most sophisticated and advanced techniques have provided important data on the identity of the niche cells controlling HSCs functions and some mechanisms underlying niche-HSC interactions. In this review we discuss various aspects of organization and functioning of the HSC cell niche in bone marrow. In particular, we review the anatomy of BM niches, various cell types composing the niche, niches for more differentiated cells, metabolism of HSCs in relation to the niche, niche aging, leukemic transformation of the niche, and the current state of HSC niche modeling in vitro.

Alterations in Hematopoietic and Mesenchymal Stromal Cell Components of the Osteopetrotic Bone Marrow Niche.

Osteopetrosis is a rare inherited disease characterized by impaired osteoclast activity causing defective bone resorption and bone marrow aplasia. It is fatal in early childhood unless hematopoietic stem cell transplantation is performed. But, the transplant course is complicated with engraftment failure. Recently, osteoclasts have been described as the potential regulators of hematopoietic stem cell (HSC) niche. Here we investigated the alterations in the HSC and mesenchymal stromal cell (MSC) components of osteopetrotic niche and their interactions to mimic the stem cell dynamics/trafficking in the BM niche after HSC transplantation. Induced pluripotent stem cells were generated from peripheral blood mononuclear cells of patients with osteopetrosis carrying TCIRG1 mutation. iPSC lines were differentiated into hematopoietic and myeloid progenitors, then into osteoclasts using a step-wise protocol. We first demonstrated a shift toward monocyte-macrophages lineage regarding hematopoietic differentiation potential of osteopetrotic iPSC-derived hematopoietic progenitors (HPCs) and phenotypically normal and functionally defective osteoclast formation. The expression of the genes involved in HSC homing and maintenance (Sdf-1, Jagged-1, Kit-L, and Opn) in osteopetrotic MSCs recovered significantly after coculture with healthy HPCs. Similarly, the restoration of phenotype, impaired differentiation, and migratory potential of osteopetrotic iHPCs were observed upon interaction with healthy MSCs. Our results establish significant alterations in both MSC and HPC compartments of the osteopetrotic niche, and support the impact of functionally impaired osteoclasts in defective niche formation.

The long and winding road: homeostatic and disordered haematopoietic microenvironmental niches: a narrative review.

ABSTRACTHaematopoietic microenvironmental niches have been described as the ‘gatekeepers’ for the blood and immune systems. These niches change during ontogeny, with the bone marrow becoming the predominant site of haematopoiesis in post-natal life under steady state conditions. To determine the structure and function of different haematopoietic microenvironmental niches, it is essential to clearly define specific haematopoietic stem and progenitor cell subsets during ontogeny and to understand their temporal appearance and anatomical positioning. A variety of haematopoietic and non-haematopoietic cells contribute to haematopoietic stem and progenitor cell niches. The latter is reported to include endothelial cells and mesenchymal stromal cells (MSCs), skeletal stem cells and/or C-X-C motif chemokine ligand 12-abundant-reticular cell populations, which form crucial components of these microenvironments under homeostatic conditions. Dysregulation or deterioration of such cells contributes to significant clinical disorders and diseases worldwide and is associated with the ageing process. A critical appraisal of these issues and of the roles of MSC/C-X-C motif chemokine ligand 12-abundant-reticular cells and the more recently identified skeletal stem cell subsets in bone marrow haematopoietic niche function under homeostatic conditions and during ageing will form the basis of this research review. In the context of haematopoiesis, clinical translation will deal with lessons learned from the vast experience garnered from the development and use of MSC therapies to treat graft versus host disease in the context of allogeneic haematopoietic transplants, the recent application of these MSC therapies to treating emerging and severe coronavirus disease 2019 (COVID-19) infections, and, given that skeletal stem cell ageing is one proposed driver for haematopoietic ageing, the potential contributions of these stem cells to haematopoiesis in healthy bone marrow and the benefits and challenges of using this knowledge for rejuvenating the age-compromised bone marrow haematopoietic niches and restoring haematopoiesis.

An old player, the right niche.

The cellular and molecular components of the niche for hematopoietic stem cells (HSCs) are still not well defined. Angiopoietin-like proteins (Angptls) are a group of secreted glycoproteins that have been reported to play various roles, including the regulation of HSC activity.1 Specifically, Angptl2, a member of the Angptl family, was demonstrated to support HSC stemness through binding to inhibitory receptors.2 Angptl2 has also been shown to support HSC activity in exosomes.3 However, whether and how Angptl2 regulates HSC activities in the HSC niche were still unknown. Yu et al used an elegant approach to study these questions.1 Based on the expression pattern of Angptl2 in bone marrow, several conditional knockout (KO) mice were generated to deplete Angptl2 from endothelial, mesenchymal stromal cells, megakaryocytes, and HSCs. Using a number of functional assays, including reconstitution analysis, flow cytometry, and immunofluorescence microscopy, the authors discovered that only endothelial cell-derived Angptl2 but not Angptl2 from other niche cell types supported the repopulation capacity, quiescent status, and niche localization of HSCs. They further demonstrated that Angptl2 enhances peroxisome-proliferator-activated receptor D expression to transactivate G0s2 and sustain the perinuclear localization of nucleolin that prevents HSCs from entering the cell cycle. This study has clarified sustained questions about the physiological role of Angptl2 in regulating HSC activity. It suggests that Angptl2 is indeed a molecular component in the endothelial niche of HSCs that inhibits differentiation and preserves the stemness of stem cells. This study also elicits additional questions. The obvious follow-up questions include what is the detailed mechanism by which the extracellular Angptl2 regulates HSC activity and why does Angptl2, a hormone that can circulate in blood, need to be in the endothelial niche to regulate HSC activities? In addition, since Angptl2 can be expressed by cells in the mouse bone marrow other than endothelial cells, what are the functions of Angptl2 expressed by these other cells? What are the roles of other members of the Angptl family in mouse bone marrow and the human HSC niche? What are the functions of bone marrow Angptls and receptors such as LILRB2 in other physiological and pathological processes? Overall, the study suggests that one molecule, when expressed by different types of cells, may play different roles by regulating different functions. Since Angptl2 and other Angptls were reported to play additional roles such as the regulation of lipid metabolism, angiogenesis, inflammation, and cancer development, further investigations are warranted to understand the diverse functions of Angptls in different types of cells.

New transgenic mouse models enabling pan-hematopoietic or selective hematopoietic stem cell depletion in vivo

Hematopoietic stem cell (HSC) multipotency and self-renewal are typically defined through serial transplantation experiments. Host conditioning is necessary for robust HSC engraftment, likely by reducing immune-mediated rejection and by clearing limited HSC niche space. Because irradiation of the recipient mouse is non-specific and broadly damaging, there is a need to develop alternative models to study HSC performance at steady-state and in the absence of radiation-induced stress. We have generated and characterized two new mouse models where either all hematopoietic cells or only HSCs can be specifically induced to die in vivo or in vitro. Hematopoietic-specific Vav1-mediated expression of a loxP-flanked diphtheria-toxin receptor (DTR) renders all hematopoietic cells sensitive to diphtheria toxin (DT) in “Vav-DTR” mice. Crossing these mice to Flk2-Cre mice results in “HSC-DTR” mice which exhibit HSC-selective DT sensitivity. We demonstrate robust, rapid, and highly selective cell ablation in these models. These new mouse models provide a platform to test whether HSCs are required for long-term hematopoiesis in vivo, for understanding the mechanisms regulating HSC engraftment, and interrogating in vivo hematopoietic differentiation pathways and mechanisms regulating hematopoietic homeostasis.

The extracellular matrix of hematopoietic stem cell niches

Hematopoietic stem cells (HSCs) are the life-long source of all types of blood cells. Their function is controlled by their direct microenvironment, the HSC niche in the bone marrow. Although the importance of the extracellular matrix (ECM) in the niche by orchestrating niche architecture and cellular function is widely acknowledged, it is still underexplored. In this review, we provide a comprehensive overview of the ECM in HSC niches. For this purpose, we first briefly outline HSC niche biology and then review the role of the different classes of ECM molecules in the niche one by one and how they are perceived by cells. Matrix remodeling and the emerging importance of biophysics in HSC niche function are discussed. Finally, the application of the current knowledge of ECM in the niche in form of artificial HSC niches for HSC expansion or targeted differentiation as well as drug testing is reviewed.

Role of SVEP1 in Stroma-Dependent Hematopoiesis In vitro.

Study of the microenvironment that supports hematopoietic stem cell (HSC) development in vivo is very difficult involving small numbers of interacting cells which are usually not well defined. While much is known about HSC niches located within the bone marrow in terms of contributing cell types and signalling molecules, very little is known about equivalent niches within spleen. Extramedullary hematopoiesis in spleen contributes myeloid cells important in the mobilisation of an immune response. As a result, it is important to develop in vitro models to identify the cells which constitute HSC niches in spleen and to identify the regulatory molecules supporting myeloid cell development. Studies described here document a model system to study the maintenance and differentiation of HSC by splenic stromal cells in vitro. The splenic stromal lines 5G3 and 3B5 differ in hematopoietic support capacity. SVEP1 and IGF2 are molecules of interest specifically expressed by 5G3 stroma. Gene knockdown technology using shRNA plasmids has been used to reduce gene expression in 5G3 and to determine specific effects on myeloid cell development following co-culture with overlaid hematopoietic progenitors in vitro. Knockdown of Svep1 gave specific inhibition of a dendritic cell (DC) population described previously in spleen (L-DC). Knockdown of Igf2 resulted in loss of production of a minor subset of conventional (c) DC. SVEP1 is now considered a marker of mesenchymal stromal cells with osteogenic differentiative capacity reflective of perivascular stromal cells. The power of this in vitro model is evidenced by the fact that it has been used to define SVEP1 as a specific adhesion molecule that regulates the hematopoietic process dependent on stromal niche interaction. The identification of stromal cells and molecules that contribute to the hematopoietic process in spleen, brings us closer to the realm of therapeutically regulating hematopoiesis in vivo, and to inhibiting niches which support cancer stem cells.