Hematopoietic Stem And Progenitor Cells Niche Research Articles

Inducible CXCL12/CXCR4–dependent extramedullary hematopoietic niches in the adrenal gland

AbstractAdult hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) hematopoietic niche, which regulates HSPC quiescence, self-renewal, and commitment in a demand-adapted manner. Although the complex BM niche is responsible for adult hematopoiesis, evidence exists for simpler, albeit functional and more accessible, extramedullary hematopoietic niches. Inspired by the anecdotal description of retroperitoneal hematopoietic masses occurring at higher frequency upon hormonal dysregulation within the adrenal gland, we hypothesized that the adult adrenal gland could be induced into a hematopoietic-supportive environment in a systematic manner, thus revealing mechanisms underlying de novo niche formation in the adult. Here, we show that upon splenectomy and hormonal stimulation, the adult adrenal gland of mice can be induced to recruit and host functional HSPCs, capable of serial transplantation, and that this phenomenon is associated with de novo formation of platelet-derived growth factor receptor α/leptin receptor (PDGFRα+/LEPR+/–)–expressing stromal nodules. We further show in CXCL12–green fluorescent protein reporter mice that adrenal glands contain a stromal population reminiscent of the CXCL12-abundant reticular cells, which compose the BM HSPC niche. Mechanistically, HSPC homing to hormonally induced adrenal glands was found dependent on the CXCR4–CXCL12 axis. Mirroring our findings in mice, we found reticular CXCL12+ cells coexpressing master niche regulator FOXC1 in primary samples from human adrenal myelolipomas, a benign tumor composed of adipose and hematopoietic tissue. Our findings reignite long-standing questions regarding hormonal regulation of hematopoiesis and provide a novel model to facilitate the study of adult-specific inducible hematopoietic niches, which may pave the way to therapeutic applications.

In Vivo Reprogramming of Adult Liver Sinusoidal Vascular Endothelial Cells into a Hematopoietic Stem and Progenitor Cell Niche

Hematopoietic stem and progenitor cells (HSPCs) reside in specialized microenvironments known as the HSPC niche. Vascular endothelial cells (ECs) are one of the most abundant and significant components of the niche, providing known supportive factors such as E-selectin, CXCL12, and SCF. During marrow stress, extramedullary hematopoiesis can occur in the liver or spleen, but the process is inefficient. The sinusoidal vessels of the liver share many similarities with marrow sinusoids. Here, we aim to identify the transcription factor (TF) code that specifies the vascular EC fate in HSPC marrow niche by conducting differential gene expression analysis with the non-niche supportive ECs of the liver. We performed scRNAseq analysis on FACS-isolated ECs from adult zebrafish kidney marrow and liver. We found that these ECs express conserved sinusoidal genes, such as sele, flt4 and gpr182. However, marrow ECs have significantly higher expression of HSPC niche supportive genes, including mrc1a, lyve1b and cxcl12a. We further identified putative TFs that are uniquely upregulated in the marrow ECs, namely tfec, mafbb, foxp4, irf8 and hoxb8a. The expression of these candidate TFs was conserved in the mammalian bone marrow ECs, as shown by relevant mouse bone marrow scRNA-seq and human bone marrow RNA-seq analyses. To determine whether these candidate TFs can functionally specify a HSPC niche EC fate, we selectively overexpressed them using a zebrafish liver sinusoidal EC-specific enhancer in vivo. Upon the overexpression of tfec and mafbb together, we found adult zebrafish liver sinusoidal ECs significantly upregulated key genes known for HSPC niche supportive functions in both embryonic and adult zebrafish HSPC niches, including mrc1a (log2FC=4.9, p<0.005) and lyve1b (log2FC=3.6, p<0.005) and dab2 (log2FC=5.0, p<0.005). Overexpression of TFEC and MAFB in human iPSC-derived ECs showed upregulation of HPSC niche supportive genes that we previously identified from zebrafish in vivo studies, including LYVE1 (log2FC=5.6, p<0.005) and DAB2 (log2FC=1.1, p<0.005) . Notably, previously published HSPC niche genes, including CXCL12(log2FC=5.1 p< 0.005) , PTN (log2FC=2.6 p<0.005) and JAG1 (log2FC=1.7 p<0.005), are also upregulated upon TFEC and MAFB overexpression. Furthermore, gene ontology analysis revealed an enrichment of genes related to cell adhesion (p<0.005), migration (p<0.005), and extravasation pathways (p=0.014). A colony-forming unit (CFU) assay of CD34+CD45+ HSPCs co-cultured with TFEC and MAFB overexpressing hiPSC-derived ECs had significantly greater numbers of CFUs compared to those co-cultured with hiPSC-derived ECs without the TF overexpression: CFU-GEMM (23.3±2.4 vs. 10.0±2.1, p=0.004), CFU-G (89.3±13.4 vs. 46.0±4.2, p=0.027), and CFU-E (42.3±3.3 vs. 26.7±0.9, p=0.021). Our findings revealed a TF code that specifies the HSPC niche supportive fate of vascular ECs and distinguishes vascular ECs in the hematopoietic niche vs. non-niche organs. These results may translate into novel therapies targeting the sinusoidal vascular niches to better stimulate and support human HSPCs.

Transcription factor induction of vascular blood stem cell niches invivo.

The hematopoietic niche is a supportive microenvironment composed of distinct cell types, including specialized vascular endothelial cells that directly interact with hematopoietic stem and progenitor cells (HSPCs). The molecular factors that specify niche endothelial cells and orchestrate HSPC homeostasis remain largely unknown. Using multi-dimensional gene expression and chromatin accessibility analyses in zebrafish, we define a conserved gene expression signature and cis-regulatory landscape that are unique to sinusoidal endothelial cells in the HSPC niche. Using enhancer mutagenesis and transcription factor overexpression, we elucidate a transcriptional code that involves members of the Ets, Sox, and nuclear hormone receptor families and is sufficient to induce ectopic niche endothelial cells that associate with mesenchymal stromal cells and support the recruitment, maintenance, and division of HSPCs invivo. These studies set forth an approach for generating synthetic HSPC niches, invitro or invivo, and for effective therapies to modulate the endogenous niche.

Endoderm-derived islet1-expressing cells differentiate into endothelial cells to function as the vascular HSPC niche in zebrafish.

Endothelial cells (ECs) line blood vessels and serve as a niche for hematopoietic stem and progenitor cells (HSPCs). Recent data point to tissue-specific EC specialization as well as heterogeneity; however, it remains unclear how ECs acquire these properties. Here, by combining live-imaging-based lineage-tracing and single-cell transcriptomics in zebrafish embryos, we identify an unexpected origin for part of the vascular HSPC niche. We find that islet1 (isl1)-expressing cells are the progenitors of the venous ECs that constitute the majority of the HSPC niche. These isl1-expressing cells surprisingly originate from the endoderm and differentiate into ECs in a process dependent on Bmp-Smad signaling and subsequently requiring npas4l (cloche) function. Single-cell RNA sequencing analyses show that isl1-derived ECs express a set of genes that reflect their distinct origin. This study demonstrates that endothelial specialization in the HSPC niche is determined at least in part by the origin of the ECs.

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.

Extracellular vesicles of stromal origin target and support hematopoietic stem and progenitor cells.

Extracellular vesicles (EVs) have been recently reported as crucial mediators in cell-to-cell communication in development and disease. In this study, we investigate whether mesenchymal stromal cells that constitute a supportive microenvironment for hematopoietic stem and progenitor cells (HSPCs) released EVs that could affect the gene expression and function of HSPCs. By taking advantage of two fetal liver-derived stromal lines with widely differing abilities to maintain HSPCs ex vivo, we demonstrate that stromal EVs play a critical role in the regulation of HSPCs. Both supportive and nonsupportive stromal lines secreted EVs, but only those delivered by the supportive line were taken up by HSPCs ex vivo and in vivo. These EVs harbored a specific molecular signature, modulated the gene expression in HSPCs after uptake, and maintained the survival and clonogenic potential of HSPCs, presumably by preventing apoptosis. In conclusion, our study reveals that EVs are an important component of the HSPC niche, which may have major applications in regenerative medicine.

Continuous blockade of CXCR4 results in dramatic mobilization and expansion of hematopoietic stem and progenitor cells

AbstractInteraction between the chemokine receptor CXCR4 and its chief ligand CXCL12 plays a critical role in the retention and migration of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM) microenvironment. In this study, qualitative and quantitative effects of long-term pharmacologic inhibition of the CXCR4/CXCL12 axis on the HSPC compartment were investigated by using 3 structurally unrelated small molecule CXCR4 antagonists. A >10-fold increase in mobilization efficiency was achieved by administering the antagonists as a subcutaneous continuous infusion for 2 weeks compared to a single bolus injection. A concurrent increase in self-renewing proliferation leading to a twofold to fourfold expansion of the HSPC pool in the BM was observed. The expanded BM showed a distinct repopulating advantage when tested in serial competitive transplantation experiments. Furthermore, major changes within the HSPC niche associated with previously described HSPC expansion strategies were not detected in bones treated with a CXCR4 antagonist infusion. Our data suggest that prolonged but reversible pharmacologic blockade of the CXCR4/CXCL12 axis represents an approach that releases HSPC with efficiency superior to any other known mobilization strategy and may also serve as an effective method to expand the BM HSPC pool.

Glucose-Dependent Insulinotropic Polypeptide Receptor Deficiency Leads to Impaired Bone Marrow Hematopoiesis.

The bone marrow (BM) contains controlled specialized microenvironments, or niches, that regulate the quiescence, proliferation, and differentiation of hematopoietic stem and progenitor cells (HSPC). The glucose-dependent insulinotropic polypeptide (GIP) is a gut-derived incretin hormone that mediates postprandial insulin secretion and has anabolic effects on adipose tissue. Previous studies demonstrated altered bone microarchitecture in mice deficient for GIP receptor (Gipr-/- ), as well as the expression of high-affinity GIP receptor by distinct cells constructing the BM HSPC niche. Nevertheless, the involvement of GIP in the process of BM hematopoiesis remains elusive. In this article, we show significantly reduced representation and proliferation of HSPC and myeloid progenitors in the BM of Gipr-/- mice. This was further manifested by reduced levels of BM and circulating differentiated immune cells in young and old adult mice. Moreover, GIP signaling was required for the establishment of supportive BM HSPC niches during HSPC repopulation in radioablated BM chimera mice. Finally, molecular profiling of various factors involved in retention, survival, and expansion of HSPC revealed significantly lower expression of the Notch-receptor ligands Jagged 1 and Jagged 2 in osteoblast-enriched bone extracts from Gipr-/- mice, which are important for HSPC expansion. In addition, there was increased expression of CXCL12, a factor important for HSPC retention and quiescence, in whole-BM extracts from Gipr-/- mice. Collectively, our data suggest that the metabolic hormone GIP plays an important role in BM hematopoiesis.

EphA5 and EphA7 forward signaling enhances human hematopoietic stem and progenitor cell maintenance, migration, and adhesion via Rac1 activation.

The proliferation, differentiation, adhesion, and migration of hematopoietic stem and progenitor cells (HSPCs) are dependent upon bone marrow stromal cells (BMSCs). In this study, we found that human primitive HSPCs (CD34+CD38-), but not lineage-committed hematopoietic cell populations, express the tyrosine kinase receptors EphA5 and EphA7. Moreover, we found that the ephrinA5 ligand, the high-affinity binding partner of EphA5 and EphA7, is highly expressed by primary human BMSCs. Previous studies have reported that interactions between EphA and ephrinA play important roles in hematopoietic cell trafficking; however, their role in BMSC support of hematopoiesis had not been described previously. Herein, we show that stimulating EphA5 and/or EphA7 forward signaling in HSPCs using soluble ephrinA5-Fc molecules promoted human HSPC-derived colony formation significantly and was associated with increased expression of granulocyte macrophage colony-stimulating factor receptor on HSPCs. Studies using functional blocking peptides to EphA5/7 found that disruption of EphA5/ephrinA5 and/or EphA7/ephrinA5 interactions inhibited HSPC function in BMSC-dependent long-term culture-initiating cell assays. Furthermore, the adhesion and migration of HSPCs was increased significantly in the presence of ephrinA5-Fc molecules compared with human immunoglobulin G-treated controls. Conversely, blocking EphA5 activation led to a reduction of HSPC adhesion, whereas inhibiting EphA5 and/or EphA7 activation hindered HSPC migration. Analysis of HSPC cultured in the presence of ephrinA5-Fc showed that EphA forward signaling stimulated Rac1 gene and protein expression and the Rac1 target molecule WAVE1. Moreover, a significant reduction of ephrinA5-mediated HSPC adhesion and migration was observed in the presence of Rac1 inhibitor. These findings suggest that interactions between EphA and ephrinA5 are important in maintaining the HSPC niche mediated in part by activation of Rac1 signaling.

Protection from UV-Light Is an Evolutionarily Conserved Feature of the Hematopoietic Niche

Hematopoietic stem and progenitor cells (HSPC) reside in the bone marrow cavity in all mammals, but other locations are used in non-mammalian animals, such as the kidney marrow in zebrafish. The niche location is thought to be selected based on unique advantages for HSPC function and protection in each organism. Studying the zebrafish HSPC niches during development and adulthood, we observed that melanocytes occupy the location above the kidney prior to the arrival of HSPCs. To elucidate whether these particular melanocytes play a role for HSPCs, we compared HSPCs in wildtype and nacre-mutant embryos (that lack all melanocytes) by cmyb in situ at different time-points. There were no obvious differences, suggesting that melanocytes are dispensable for steady state hematopoiesis. Since the position of the melanocytes above the HSPCs was very consistent in different strains and at various ages, we asked whether the melanocytes protect the HSPC niche from UV light. Indeed, after irradiation with UV light, levels of UV-induced DNA damage were approximately 50% higher in HSPCs isolated from kidneys of unpigmented embryos compared to their pigmented siblings as assessed by immunocytochemistry with a cyclobutane pyrimidine dimer antibody (TDM-2). In addition to acquiring more DNA damage, the unpigmented embryos also showed a significantly more pronounced decrease of HSPCs as assessed by cmyb in situ: 55% of control embryos had high cmyb staining. Upon irradiation 44% of irradiated pigmented embryos retained high c-myb staining compared to only 28% of irradiated unpigmented embryos (total embryos analyzed: n= 29, 27 and 36 respectively; p=0.01 between the latter two groups). The reduction of HSPCs could not be rescued in unpigmented p53-mutant embryos, indicating a p53-independent mechanism of HSPC loss after UV irradiation. The protective effect of melanocytes could either be due to a physical shielding of the HSPCs or due to a paracrine mechanism, such as secretion of cytokines or uptake of reactive oxygen species. The paracrine mechanism could be dependent on melanin. To address this question, we focused the path of light at a different angle in which the melanocytes would not be able to block the UV light from reaching the kidney marrow. This abrogated the protective effect of pigment containing melanocytes and caused a reduction of HSPCs by cmyb in situ to the levels of unpigmented siblings: pigmented embryos irradiated at an angle now also had high cmyb staining in only 28% of the embryos (n= 25). This suggests that the melanocytes only provide shade for the HSPCs. We then asked if this protective pigmentation pattern is specific to zebrafish and traced the origin of this trait by comparative histology. We found that other teleost fish but also evolutionarily older fish such as sturgeon and polypterus have melanocytes covering the kidney marrow. Even in lamprey, an ancient jawless vertebrate, the HSPC niche (located in the supraspinal organ) is covered by melanocytes, which allows us to date the evolutionary origin of UV protection back to at least 500 million years. We performed histological studies in frogs and found that the HSPCs reside under a melanocyte shield in the tadpole stage, while it is known that in adult frogs the HSPCs move into the bone marrow cavity. Considering that all terrestrial animals such as reptiles, birds and mammals host hematopoiesis in the bone marrow, we suggest that during the transition from aquatic to terrestrial life, the HSPCs were driven out of their melanocyte covered niche due to the high level of irradiation and relocated into a more protective niche, the bone marrow. DisclosuresZon:Scholar Rock: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder; Marauder Therapeutics: Equity Ownership, Other: Founder; Fate, Inc.: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Founder.

The niche for hematopoietic stem and progenitor cells in bone marrow

It has been hypothesized that the special microenvironments known as niches in the bone marrow play an essential role in maintaining hematopoietic stem and progenitor cells(HSPCs), and the identification of the HSPC niche has been a subject of long-standing argument. Recent studies identified candidate cells meeting the criteria for HSPC niches and the critical transcription factor of their development and maintenance.

A Three-Dimensional Scaffold-Based System for Modeling the Bone Marrow Tissue

Hematopoietic stem and progenitor cells (HPC) niche, consisting of HPC and their surrounding stromal components, is the fundamental unit for bone marrow (BM) tissue engineering. Previously, mouse BM-derived cell complexes with HPC niche unit properties called "niche-like units" were isolated and characterized. This study was aimed to evaluate the possibility of bioengineering marrow tissue in heterotypic sites using niche-like units in combination with three-dimensional scaffolds. BM niche-like units were isolated from GFP-transgenic C57BL/6 mice and seeded on electrospun poly (L-lactide) nanofiber scaffolds, which were then roll-folded and aseptically implanted into the peritoneal cavity of irradiated wild-type mice. One month after implantation, donor-derived cells were detected in peripheral blood of the recipients and contributed to restoration of all blood lineages. The transplanted bioengineered tissue histologically resembled native BM structure and was connected to the mouse systemic circulation. Long-term self-renewal was confirmed by serial transplantation into tertiary recipients. In conclusion, this study establishes a novel system for BM tissue engineering, which can be used to improve the HPC transplantation outcomes especially in cases where HPC niche is damaged and also as an in vivo model to test the effects of different factors on hematopoiesis.

The critical and specific transcriptional regulator of the microenvironmental niche for hematopoietic stem and progenitor cells.

It has been assumed that the special microenvironments known as niches in the marrow play an essential role in maintaining hematopoietic stem and progenitor cells (HSPCs), and the identity of the HSPC niche has been a subject of long-standing debate. Recent studies identified cells, which create microenvironments meeting the criteria for HSPC niches and the critical transcriptional regulators of their development and maintenance. Osterix as well as Ebf2 and Bmi1 are critical but not specific transcriptional regulators of HSPC niche development. The transcription factor Foxc1 is expressed preferentially in a population of adipo-osteogenic progenitors, termed CXCL12-abundant reticular (CAR) cells, which create HSPC niches and are largely equivalent to stem cell factor and Lepr-expressing cells, in developing and adult bone marrow. Foxc1 is essential for CAR cell development and maintenance of bone marrow niches for HSPCs upregulating CXCL12 and SCF expression and inhibition of adipogenic processes in CAR cell progenitors. Foxc1 is the first critical and specific transcriptional regulator that is required for development and maintenance of cells creating HSPC niches, including a specialized population of adipo-osteogenic progenitors in bone marrow.

Interaction of Tumor Cells with the Hematopoietic Stem and Progenitor Cell Niche

IntroductionThe bone marrow microenvironment regulates the self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs), through a network dependent on cell-cell interaction. This interaction is mediated by morphogens, the extracellular matrix and cell adhesion molecules expressed and secreted by various cell types in the HSPC niche. Mesenchymal stromal cells (MSCs), as the major cellular component, maintain the stemness properties of the niche. The microenvironment thus becomes conducive for HSPCs to remain quiescent, thereby enabling long term self-renewal. Therefore, the safe haven in the bone marrow microenvironment and its constituent cell types can be targeted during tumorigenesis, thus making the niche neoplastic. Dissemination of breast cancer cells into the bone marrow has been described even in the early stages of the disease. The present study focuses on the influence of breast carcinomas on the genetic and functional profile of mesenchymal and hematopoietic progenitor cells of the bone marrow niche.MethodsIn vitro coculture models of breast cancer cell lines- MDA-MB231, MCF-7 and primary MSCs derived from the bone marrow of healthy donors were used in the study. Atomic- force microscopy based single-cell force spectroscopy (AFM-SCFS) and fluorescence based assays were used for cell adhesion experiments. Hydrogel based culture systems were used for 3-dimensional cocultures of breast cancer cells and MSCs. Hypoxic and normoxic culture conditions (0.5% and 20% oxygen respectively) were used for the experiments.ResultsThe breast cancer cell lines caused a significant reduction in HSPC adhesion to MSCs (88% by MDA-MB 231 cells; p<0.005 and 73% by MCF-7 cells; p<0.005). AFM-SCFS studies also indicated a higher binding force between breast cancer cells and MSCs, as compared to HSPCs (MDA-MB231 cells-0.13nN, MCF-7 cells-0.074nN and HSPCs-0.05nN). MDA-MB231and MCF-7 cells express Intercellular adhesion molecule-1(ICAM-1), which has been shown to promote breast cancer metastasis (Hanlon et al, 2002; Rosette et al, 2005; Schröder C. et al, 2011). There was a significant difference in reduction of HSPC adhesion towards MSCs by ICAM-1 knockdown (ICAM-1 KD) tumor cells as compared to MDA-MB231 cells (84.83% by MDA-MB231 cells versus 28.11% by ICAM-1KD tumor cells, p<0.001). AFM-SCFS studies also showed a reduced binding force between ICAM-1 KD tumor cells and MSCs as compared to MDA-MB231cells (MDA-MB231 cells-0.14nN versus ICAM-1-KD tumor cells-0.05nN, p value<0.001). ICAM-1 KD studies thus showed that reduction in HSPC adhesion to MSCs by breast cancer cells was mediated through ICAM-1 signaling. A cytokine array was performed to investigate if breast cancer cell lines affect the cytokine profile of MSCs. The array showed altered expression of growth factors- Basic fibroblast growth factor (bFGF) and Platelet derived growth factor–beta (PDGF-BB) (2.2 fold upregulation and 0.5 fold downregulation in breast cancer cells- MSC cocultures respectively). Based on the array, a bFGF-mediated increase in the proliferation of MSCs and breast cancer cells in coculture was observed. The bFGF upregulation also caused an increased migration of MDA-MB231 cells towards MSCs in a transwell migration assay. An upregulation in the phosphorylation status of Akt was observed in breast cancer cells – MSC cocultures, as a downstream effect of upregulated bFGF levels. The bFGF-mediated increase in the proliferation of breast cancer cells and MSCs in coculture was shown to be dependent on the activation of PI3K-Akt pathway. The bFGF- mediated increase in the migration of MDA-MB231 cells towards MSCs was also inhibited upon addition of the PI3K blocker. Interestingly, the breast cancer cells caused a reduction in osteoblastic differentiation of MSCs by downregulation of PDGF-BB. Studies with 3-dimensional cocultures of breast cancer cells and MSCs also showed a reduction in osteoblastic differentiation of MSCs. Furthermore, long-term cocultures of breast cancer cells, HSPCs and MSCs showed reduced support for primitive HSPCs in the neoplastic niche.ConclusionsThese findings indicate a perturbed HSPC niche upon tumor invasion. The possible role of altered cytokine expression, consecutive downstream signaling in niche activation and bone turnover will be further studied using in vitro and in vivo approaches to recapitulate tumor micrometastases to the HSPC niche. DisclosuresNo relevant conflicts of interest to declare.

Loss of Notch Receptor-Ligand Engagement Leads to Increased Hematopoietic Stem and Progenitor Cell Egress and Mobilization

BACKGROUNDNotch has long been recognized as an important molecule that regulates stem cell self-renewal and differentiation. Despite that Notch is required for the embryonic hematopoietic stem cell (HSC) generation, and that Notch is expressed in the adult HSC, the role of Notch pathway in adult HSC remains unclear. Recently it has been shown that Notch activation and the ability of interacting with Notch ligand Jagged1 is a signature of human primitive HSC and supports HSC regenerative potential. Here we study the physiological significance of Notch in the adult HSC population focusing on how Notch-ligand engagement regulates HSC quiescence and niche retention. METHODSTo better understand the role of Notch in adult HSC homeostasis, we examined HSC frequency, quiescence maintenance, niche occupancy, and HSC mobilization in mice with either conditional lack of RBP-JK, which mediates the canonical Notch signaling activity, or in mice with conditional lack of Pofut1 that catalyzes O-fucosylation of Notch EGF-like repeats and the generation of O-fucose glycans important for the binding of Notch ligands. We also tested Notch ligand neutralizing antibodies and Notch1 and Notch2 inhibitory antibodies to examine the effect of blocking Notch receptor-ligand engagement or the block of Notch signaling activation on HSC homeostasis. RESULTSWe report here that Pofut1-deficient hematopoietic stem and progenitor cells (HSPCs) display enhanced cell cycling and proliferation cell autonomously. These changes are accompanied by G-CSF-independent increased HSPC egress from the marrow to the periphery and other hematopoietic organs, and their enhanced sensitivity to mobilizing stimuli of G-CSF plus the CXCR4 antagonist, AMD3100. This phenotype is caused by reduced adhesion of Pofut1-deficient HSPC to Notch ligand-expressing stromal cells and the osteoblastic lineage cells. Adhesion to these cells by wild type but not Pofut1-deficient HSPCs can be blocked by recombinant Notch ligand Dll1 or Dll4. In addition, adhesion to these cells inhibits wild type but not Pofut1-deficient HSPC cycling, independent of RBP-Jk-mediated Notch signaling. Further, Pofut1-deficient HSPCs exhibit normal expression of key adhesion molecules and normal SDF-1-mediated chemotaxis, but show scattered and distal occupancy in the endosteal or osteoblastic niche. In support of roles for Notch-ligand engagement in facilitating HSPC niche retention, we show that mice receiving 4 doses of neutralizing antibodies to the Notch ligand Dll4 or Jagged1, but not Dll1, display ~2-3-fold increased HSC and progenitor egress when compared to mice receiving isotype control antibody, and further display 60% increased HSC mobilization when compared to mice receiving control antibody and treated similarly with G-CSF and AMD3100. Dll4 blockade also increases the sinusoidal endothelial cell population and HSPC cell cycling. In comparison, only a mild HSPC proliferation and egress is found in RBP-JK-deficient mice, or in mice receiving Notch2 inhibitory antibody. However, Notch2 blockade but not Notch1 blockade induces unique features of HSC and myeloid progenitor mobilization responding to G-CSF plus AMD3100. CONCLUSIONSBased on these findings, we conclude that HSPC quiescence and retention in the marrow niche is facilitated by the interaction between Notch-expressing HSPCs and Jagged1- or Dll4-expressing niche cells, and is likely also contributed by Notch signaling activation. In addition, Notch receptor-ligand engagement in this process is strengthened by O-fucose modification of Notch receptors. Finally, the observations from our studies may provide a therapeutic indication. Inadequate mobilization in HSPC transplantation remains a clinical problem. Our findings that targeting Notch receptor-ligand interaction and/or inhibiting Notch2 activation increase HSPC emigration suggests a novel approach for enhancing mobilization of stem and progenitor cells for those patients who respond poorly to current mobilizing regimes. DisclosuresShim:Genentech: Employment, Equity Ownership. Yan:Genentech: Employment, Equity Ownership. Lowe:Genentech: Employment, Equity Ownership. Siebel:Genentech: Employment, Equity Ownership.

Osteoblasts as leukemia-initiating cells.

Osteoblasts as leukemia-initiating cells.

Myelodysplastic Cells in Patients Reprogram Mesenchymal Stromal Cells to Establish a Transplantable Stem Cell Niche Disease Unit

Myelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin(-)CD34(+)CD38(-) stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

Granulocyte-Macrophage Progenitors (GMPs) Express Low Adhesive Potential and High CXCR-4 Levels

Hematopoietic stem and progenitor cells (HSPCs) reside in a particular and specialized microenvironmental niche. Interaction of HSPCs with their niche is fundamental for cell proliferation, differentiation and quiescence. While alterations of HSPC-niche interactions have been shown to play an important role in animal models, methods to study the human HSPC niche are lacking. In order to characterize the adhesion of various HSPC subsets to various extracellular matrix proteins found in the bone marrow, we used high-throughput fluorescent microscopy to study cell adhesion by quantifying the ratio of adherent versus non-adherent cells being detached by gravity. This allowed the analysis of low cell numbers making the study of clinical samples feasible. Multiparameter flow cytometry was used to analyze both the adherent and non-adherent fractions to study the major subsets of CD34+ cells, evaluating CD34, CD38, CD45RA, CD123 and CD90 surface levels.More than 80% of plated CD34+ cells adhered to fibronectin, 70% to VCAM-1, while only a minor proportion adhered to laminin, osteopontin and collagen-1 (11%-19%). Inhibitory antibodies against α4 and α5 integrin were used to inhibit adhesion of CD34+ cells to fibronectin and VCAM-1. α4 integrin antibody significantly decrease the number of adherent CD34+ cells to fibronectin from 65% to 38% (p< 0.0001) and on VCAM-1 from 51% to 12% (p< 0.0001). No effect was obtained by α5 integrin antibody. When comparing the major subsets of CD34+ cells, megakaryocyte-erythroid progenitors (MEPs) were the subpopulation that adhered strongest to each substrate followed by common myeloid progenitors (CMPs) and hematopoietic stem cells (HSCs). By contrast, granulocyte-macrophage progenitors (GMPs) adhered less to each of the studied matrix proteins (Fig. 1a-1b). Expression levels of CD34 were higher in adherent than in non-adherent cells, while CD45RA+ cells showed the opposite pattern (Fig. 1c-1d). These data suggest that the level of differentiation influences the adhesion properties and that more immature cells have a higher affinity to niche elements. We then analyzed the expression of adhesion markers in the different CD34+ cells subsets. Unexpectedly, GMPs, the least adhesive population, showed higher expression of CXCR4, a key chemokine receptor involved in HSPCs' homing and differentiation. GMPs have been described to constitute the leukemic stem cell population in AML. GMPs also showed a high expression of CD44, a glycoprotein required for migration, cell interactions and adhesion. HSCs showed higher levels of α2 integrin while MEPs expressed more α4 integrin. The other subpopulations, common myeloid progenitors (CMPs), multipotent progenitors (MPPs) and lymphoid-primed multipotent progenitors (LMPPs) showed lower levels of all studied adhesion markers.In conclusion, we present a method to quantify the adhesion of different human HPSC subpopulations. The assay is suitable for analysis of human myeloid neoplasms and may elucidate aberrant patterns as well as reveal potential targets for anti-adhesive therapy inhibiting the niche«s ability to sustain the leukemic stem cell population. [Display omitted] MEPs show more adhesiveness to all the substrates studied, followed by CMPs and HSCs. HSCs exhibits a preference for fibronectin compared to the other proteins tested. The less adhesive seems to be GMPs (a,b,c). Difference between GMPs and MEPs adhesiveness is statistically validated (p< 0.01). CD34 expression is higher on adherent cells; on the contrary, CD45RA is more expressed on non adherent cells (d,e). [Display omitted] GMPs express more CXCR-4 and CD44 than the other subsets, MEPs exhibit high levels of α4 integrin, explaining why this population seems more adhesive to all the substrates. α4 and α5 integrins are important also for GMPs (a).Expression of CXCR-4 in all the subpopulations (b). Disclosures:No relevant conflicts of interest to declare.

Myeloid Dendritic Cells Regulate HSPC Trafficking In The Bone Marrow

Granulocyte-colony stimulating factor (G-CSF) is the prototypic agent used to mobilize hematopoietic stem and progenitor cells (HSPCs) into the blood where they can then be harvested for stem cell transplantation. G-CSF acts in a non-cell-intrinsic fashion to induce HSPC mobilization. We recently showed that G-CSF signaling in a CD68+ monocyte/macrophage lineage cell within the bone marrow initiates the HSPC mobilization cascade (Christopher et al., 2011). CD68 marks a heterogeneous cell population that includes monocytes, macrophages, myeloid dendritic cells, and osteoclasts. Within the bone marrow, myeloid dendritic cells (MDCs) are found perivascularly and in close association with CXCL12-abundant reticular (CAR) cells, suggesting a role for MDCs in maintaining HSPC niche function. We previously reported that G-CSF treatment (250 µg/kg per day for 5 days) suppresses macrophage (11.8 ± 3.6-fold) and myeloid dendritic cell (MDCs; 5.5 ± 1.2-fold) numbers in the bone marrow (Supakorndej et al., ASH abstract #2319, 2012). Moreover, we showed that CD11c-DTR mediated MDC ablation results in a modest mobilization of HSPCs. However, CD11c-DTR ablates bone marrow macrophages, as well as MDCs, so a definitive role for MDCs in G-CSF-induced HSPC mobilization could not be established.To address this concern, we used transgenic mice expressing the diphtheria toxin receptor under the control of the Zbtb46 promoter (Zbtb46-DTR). A prior study demonstrated that Zbtb46 is expressed specifically in MDCs but not macrophages nor other immune cell lineages in peripheral lymphoid tissues (Satpathy et al., 2012). Using Zbtb46gfp/+ mice, we likewise found that Zbtb46 is expressed in bone marrow MDCs but not bone marrow macrophages. Finally, a recent study showed that Zbtb46-DTR specifically ablates MDCs (Meredith et al., 2012). To avoid systemic toxicity, we transplanted Zbtb46-DTR bone marrow into congenic wild-type recipients. The resulting bone marrow chimeras were treated with diphtheria toxin (DT; 400 ng per day for 6 days), which resulted in an 82% reduction of MDCs in the bone marrow. MDC ablation resulted in significant mobilization of colony-forming cells (figure 1A) and c-Kit+lineage-Sca-1+ (KLS) cells (figure 1B) into the blood and spleen. Moreover, MDC ablation enhanced mobilization of these cells by G-CSF (figures 1C and 1D). Together with the CD11c-DTR mice, the Zbtb46-DTR studies provide strong evidence that MDCs contribute to G-CSF-induced HSPC mobilization. [Display omitted] We previously showed that G-CSF mobilizes HSPCs, at least in part, by decreasing CXCL12 expression in bone marrow stromal cells. We found that MDC ablation (using CD11c-DTR mice) also suppresses CXCL12 expression in the bone marrow (35.2 ± 18.1% reduction). We recently reported that CXCL12 expression from perivascular stromal cells (including mesenchymal progenitors, CAR cells, and endothelial cells) is required for HSC maintenance (Greenbaum et al., 2013). Here, we show that G-CSF suppresses CXCL12 mRNA expression in both CAR cells and endothelial cells. Surprisingly, preliminary data suggest that MDC ablation does not affect CAR cell number nor CXCL12 expression in these cells. Studies are in progress to assess the effect of MDC ablation on endothelial CXCL12 expression. Collectively, these data suggest that MDC-derived signals contribute to HSPC maintenance by modulating stromal cells that comprise the perivascular niche. Disclosures:No relevant conflicts of interest to declare.

Hypoxic Hematopoietic Stem and Progenitor Cells Reside in Structurally Diverse Perivascular Niches in the Bone Marrow,

Abstract 3417The identification of specific microenvironments, in which Hematopoietic Stem and Progenitor Cells (HSPCs), reside within the BM is a major challenge in stem cell biology. Yet the extreme rarity of HSCs, their dynamic nature, and the lack of unique specific markers to identify them, have precluded an accurate definition of HSC niches to date. Using Laser Scanning Cytometry, a powerful emerging quantitative imaging technology that enables analysis of whole femoral sections at the single cell level, we have mapped the global distribution of hematopoietic stem and progenitor cells within femoral bone marrow cavities, and analyzed their inmediate surrounding microenvironment.Systematic mapping of the global distribution of endogenous HSPC-enriched populations in the BM, revealed an accumulation of these cells inside endosteal regions (ER <100μm from inner bone surface), but not necessarily in contact with endosteal surfaces. Interestingly, the vast majority of HSPCs were found in direct association with BM micrrovessels, further supporting previous work, which suggested bone marrow endothelium as a major component of HSPC niches. By employing a novel imaging approach, we provide a three-dimensional (3D) microscopic overview of the unique BM vascular network found in endosteal zones, which contain the transition of bone-lining arterioles and capillaries to the sinusoidal network. Of note, HSPC association to vascular structures is not restricted to sinusoids. A significant fraction of HSPCs lied adjacent to non-sinusoidal endothelium. Using five-color imaging cytometry and pimonidazole incorporation, we have assessed the hypoxic state of HSPCs in different BM microenvironments. Our in situ analysis reveals that intracellular hypoxia is a hallmark of HSPCs, independent of their distance to bone surfaces, and more importantly, regardless of their perivascular localization. These studies provide unequivocal anatomical evidence for the intrinsic rather than environmental regulation of intracellular hypoxia in HSPCs and challenge the hypothesis of a “super hypoxic” HSPC niche. Disclosures:No relevant conflicts of interest to declare.