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1
Kandarakov, Oleg, Alexander Belyavsky, and Ekaterina Semenova. "Bone Marrow Niches of Hematopoietic Stem and Progenitor Cells." International Journal of Molecular Sciences 23, no. 8 (April 18, 2022): 4462. http://dx.doi.org/10.3390/ijms23084462.
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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.
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Zha, Ji, Lori Kunselman, Hongbo Michael Xie, Brian Ennis, Jian-Meng Fan, and Timothy S. Olson. "Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation." Blood 134, Supplement_1 (November 13, 2019): 3199. http://dx.doi.org/10.1182/blood-2019-121763.
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Hematopoietic stem cell (HSC) transplantation (HSCT) is required for curative therapy for patients with high-risk hematologic malignancies, and a number of non-malignant disorders including inherited bone marrow failure syndromes (iBMFS). Strategies to enhance bone marrow (BM) niche capacity to engraft donor HSC have the potential to improve HSCT outcome by decreasing graft failure rates and enabling reduction in conditioning intensity and regimen-associated complications. Several studies in animal models of iBMFS have demonstrated that BM niche dysfunction contributes to both the pathogenesis of iBMFS, as well as impaired graft function after HSCT. We hypothesize that such iBMFS mouse models are useful tools for discovering targetable niche elements critical for donor engraftment after HSCT. Here, we report the development of a novel mouse model of Shwachman-Diamond Syndrome (SDS) driven by conditional Sbds deletion, which demonstrates profound impairment of healthy donor hematopoietic engraftment after HSCT due to pathway-specific dysfunctional signaling within SBDS-deficient recipient niches. We first attempted to delete Sbds specifically in mature osteoblasts by crossing Sbdsfl/flmice with Col1a1Cre+mice. However, the Col1a1CreSbdsExc progenies are embryonic lethal at E12-E15 stage due to developmental musculoskeletal abnormalities. Alternatively, we generated an inducible SDS mouse model by crossing Sbdsfl/flmice with Mx1Cre+ mice, and inducing Sbds deletion in Mx1-inducible BM hematopoietic and osteolineage niche cells by polyinosinic-polycytidilic acid (pIpC) administration. Compared with Sbdsfl/flcontrols, Mx1CreSbdsExc mice develop significantly decreased platelet counts, an inverted peripheral blood myeloid/lymphoid cell ratio, and reduced long-term HSC within BM, consistent with stress hematopoiesis seen in BMF and myelodysplastic syndromes. To assess whether inducible SBDS deficiency impacts niche function to engraft donor HSC, we transplanted GFP+ wildtype donor BM into pIpC-treated Mx1CreSbdsExc mice and Sbdsfl/flcontrols after 1100 cGy of total body irradiation (TBI). Following transplantation, Mx1CreSbdsExc recipient mice exhibit significantly higher mortality than controls (Figure 1). The decreased survival was related to primary graft failure, as Mx1CreSbdsExc mice exhibit persistent BM aplasia after HSCT and decreased GFP+ reconstitution in competitive secondary transplantation assays. We next sought to identify the molecular and cellular defects within BM niche cells that contribute to the engraftment deficits in SBDS-deficient mice. We performed RNA-seq analysis on the BM stromal cells from irradiated Mx1CreSbdsExc mice versus controls, and the results revealed that SBDS deficiency in BM niche cells caused disrupted gene expression within osteoclast differentiation, FcγR-mediated phagocytosis, and VEGF signaling pathways. Multiplex ELISA assays showed that the BM niche of irradiated Mx1CreSbdsExc mice expresses lower levels of CXCL12, P-selectin and IGF-1, along with higher levels of G-CSF, CCL3, osteopontin and CCL9 than controls. Together, these results suggest that poor donor HSC engraftment in SBDS-deficient mice is likely caused by alterations in niche-mediated donor HSC homing/retention, bone metabolism, host monocyte survival, signaling within IGF-1 and VEGF pathways, and an increased inflammatory state within BM niches. Moreover, flow cytometry analysis showed that compared to controls, the BM niche of irradiated Mx1CreSbdsExc mice contained far fewer megakaryocytes, a hematopoietic cell component of BM niches that we previously demonstrated to be critical in promoting osteoblastic niche expansion and donor HSC engraftment. Taken together, our data demonstrated that SBDS deficiency in BM niches results in reduced capacity to engraft donor HSC. We have identified multiple molecular and cellular defects in the SBDS-deficient niche contributing to this phenotype. Such niche signaling pathway-specific deficits implicate these pathways as critical for donor engraftment during HSCT, and suggest their potential role as targets of therapeutic approaches to enhance donor engraftment and improve HSCT outcome in any condition for which HSCT is required for cure. Disclosures Olson: Merck: Membership on an entity's Board of Directors or advisory committees; Bluebird Bio: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria.
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Ma, Chao, Matthew T. Witkowski, Jacob Harris, Igor Dolgalev, Sheetal Sreeram, Weiyi Qian, Jie Tong, Xin Chen, Iannis Aifantis, and Weiqiang Chen. "Leukemia-on-a-chip: Dissecting the chemoresistance mechanisms in B cell acute lymphoblastic leukemia bone marrow niche." Science Advances 6, no. 44 (October 2020): eaba5536. http://dx.doi.org/10.1126/sciadv.aba5536.
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B cell acute lymphoblastic leukemia (B-ALL) blasts hijack the bone marrow (BM) microenvironment to form chemoprotective leukemic BM “niches,” facilitating chemoresistance and, ultimately, disease relapse. However, the ability to dissect these evolving, heterogeneous interactions among distinct B-ALL subtypes and their varying BM niches is limited with current in vivo methods. Here, we demonstrated an in vitro organotypic “leukemia-on-a-chip” model to emulate the in vivo B-ALL BM pathology and comparatively studied the spatial and genetic heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. We revealed the heterogeneous chemoresistance mechanisms across various B-ALL cell lines and patient-derived samples. We showed that the leukemic perivascular, endosteal, and hematopoietic niche-derived factors maintain B-ALL survival and quiescence (e.g., CXCL12 cytokine signal, VCAM-1/OPN adhesive signals, and enhanced downstream leukemia-intrinsic NF-κB pathway). Furthermore, we demonstrated the preclinical use of our model to test niche-cotargeting regimens, which may translate to patient-specific therapy screening and response prediction.
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Zha, Ji, Lori K. Kunselman, Hongbo M. Xie, Brian Ennis, Yash B. Shah, Xia Qin, Jian-Meng Fan, Daria V. Babushok, and Timothy S. Olson. "Inducible Sbds deletion impairs bone marrow niche capacity to engraft donor bone marrow after transplantation." Blood Advances 6, no. 1 (January 4, 2022): 108–20. http://dx.doi.org/10.1182/bloodadvances.2021004640.
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Abstract Bone marrow (BM) niche-derived signals are critical for facilitating engraftment after hematopoietic stem cell (HSC) transplantation (HSCT). HSCT is required for restoration of hematopoiesis in patients with inherited BM failure syndromes (iBMFSs). Shwachman-Diamond syndrome (SDS) is a rare iBMFS associated with mutations in SBDS. Previous studies have demonstrated that SBDS deficiency in osteolineage niche cells causes BM dysfunction that promotes leukemia development. However, it is unknown whether BM niche defects caused by SBDS deficiency also impair efficient engraftment of healthy donor HSC after HSCT, a hypothesis that could explain morbidity noted after clinical HSCT for patients with SDS. Here, we report a mouse model with inducible Sbds deletion in hematopoietic and osteolineage cells. Primary and secondary BM transplantation (BMT) studies demonstrated that SBDS deficiency within BM niches caused poor donor hematopoietic recovery and specifically poor HSC engraftment after myeloablative BMT. We have also identified multiple molecular and cellular defects within niche populations that are driven by SBDS deficiency and are accentuated by or develop specifically after myeloablative conditioning. These abnormalities include altered frequencies of multiple niche cell subsets, including mesenchymal lineage cells, macrophages, and endothelial cells; disruption of growth factor signaling, chemokine pathway activation, and adhesion molecule expression; and p53 pathway activation and signals involved in cell cycle arrest. Taken together, this study demonstrates that SBDS deficiency profoundly impacts recipient hematopoietic niche function in the setting of HSCT, suggesting that novel therapeutic strategies targeting host niches could improve clinical HSCT outcomes for patients with SDS.
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Chow, Andrew, Daniel Lucas, Andrés Hidalgo, Simón Méndez-Ferrer, Daigo Hashimoto, Christoph Scheiermann, Michela Battista, et al. "Bone marrow CD169+ macrophages promote the retention of hematopoietic stem and progenitor cells in the mesenchymal stem cell niche." Journal of Experimental Medicine 208, no. 2 (January 31, 2011): 261–71. http://dx.doi.org/10.1084/jem.20101688.
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Hematopoietic stem cells (HSCs) reside in specialized bone marrow (BM) niches regulated by the sympathetic nervous system (SNS). Here, we have examined whether mononuclear phagocytes modulate the HSC niche. We defined three populations of BM mononuclear phagocytes that include Gr-1hi monocytes (MOs), Gr-1lo MOs, and macrophages (MΦ) based on differential expression of Gr-1, CD115, F4/80, and CD169. Using MO and MΦ conditional depletion models, we found that reductions in BM mononuclear phagocytes led to reduced BM CXCL12 levels, the selective down-regulation of HSC retention genes in Nestin+ niche cells, and egress of HSCs/progenitors to the bloodstream. Furthermore, specific depletion of CD169+ MΦ, which spares BM MOs, was sufficient to induce HSC/progenitor egress. MΦ depletion also enhanced mobilization induced by a CXCR4 antagonist or granulocyte colony-stimulating factor. These results highlight two antagonistic, tightly balanced pathways that regulate maintenance of HSCs/progenitors in the niche during homeostasis, in which MΦ cross talk with the Nestin+ niche cell promotes retention, and in contrast, SNS signals enhance egress. Thus, strategies that target BM MΦ hold the potential to augment stem cell yields in patients that mobilize HSCs/progenitors poorly.
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Boyd, Allison L., Clinton J. V. Campbell, Claudia I. Hopkins, Aline Fiebig-Comyn, Jennifer Russell, Jelena Ulemek, Ronan Foley, et al. "Niche displacement of human leukemic stem cells uniquely allows their competitive replacement with healthy HSPCs." Journal of Experimental Medicine 211, no. 10 (September 1, 2014): 1925–35. http://dx.doi.org/10.1084/jem.20140131.
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Allogeneic hematopoietic stem cell (HSC) transplantation (HSCT) is currently the leading strategy to manage acute myeloid leukemia (AML). However, treatment-related morbidity limits the patient generalizability of HSCT use, and the survival of leukemic stem cells (LSCs) within protective areas of the bone marrow (BM) continues to lead to high relapse rates. Despite growing appreciation for the significance of the LSC microenvironment, it has remained unresolved whether LSCs preferentially situate within normal HSC niches or whether their niche requirements are more promiscuous. Here, we provide functional evidence that the spatial localization of phenotypically primitive human AML cells is restricted to niche elements shared with their normal counterparts, and that their intrinsic ability to initiate and retain occupancy of these niches can be rivaled by healthy hematopoietic stem and progenitor cells (HSPCs). When challenged in competitive BM repopulation assays, primary human leukemia-initiating cells (L-ICs) can be consistently outperformed by HSPCs for BM niche occupancy in a cell dose-dependent manner that ultimately compromises long-term L-IC renewal and subsequent leukemia-initiating capacity. The effectiveness of this approach could be demonstrated using cytokine-induced mobilization of established leukemia from the BM that facilitated the replacement of BM niches with transplanted HSPCs. These findings identify a functional vulnerability of primitive leukemia cells, and suggest that clinical development of these novel transplantation techniques should focus on the dissociation of L-IC–niche interactions to improve competitive replacement with healthy HSPCs during HSCT toward increased survival of patients.
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Fielding, Claire, and Simón Méndez-Ferrer. "Neuronal regulation of bone marrow stem cell niches." F1000Research 9 (June 16, 2020): 614. http://dx.doi.org/10.12688/f1000research.22554.1.
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The bone marrow (BM) is the primary site of postnatal hematopoiesis and hematopoietic stem cell (HSC) maintenance. The BM HSC niche is an essential microenvironment which evolves and responds to the physiological demands of HSCs. It is responsible for orchestrating the fate of HSCs and tightly regulates the processes that occur in the BM, including self-renewal, quiescence, engraftment, and lineage differentiation. However, the BM HSC niche is disturbed following hematological stress such as hematological malignancies, ionizing radiation, and chemotherapy, causing the cellular composition to alter and remodeling to occur. Consequently, hematopoietic recovery has been the focus of many recent studies and elucidating these mechanisms has great biological and clinical relevance, namely to exploit these mechanisms as a therapeutic treatment for hematopoietic malignancies and improve regeneration following BM injury. The sympathetic nervous system innervates the BM niche and regulates the migration of HSCs in and out of the BM under steady state. However, recent studies have investigated how sympathetic innervation and signaling are dysregulated under stress and the subsequent effect they have on hematopoiesis. Here, we provide an overview of distinct BM niches and how they contribute to HSC regulatory processes with a particular focus on neuronal regulation of HSCs under steady state and stress hematopoiesis.
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Born, Gordian, Marina Nikolova, Arnaud Scherberich, Barbara Treutlein, Andrés García-García, and Ivan Martin. "Engineering of fully humanized and vascularized 3D bone marrow niches sustaining undifferentiated human cord blood hematopoietic stem and progenitor cells." Journal of Tissue Engineering 12 (January 2021): 204173142110448. http://dx.doi.org/10.1177/20417314211044855.
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Hematopoietic stem and progenitor cells (HSPCs) are frequently located around the bone marrow (BM) vasculature. These so-called perivascular niches regulate HSC function both in health and disease, but they have been poorly studied in humans due to the scarcity of models integrating complete human vascular structures. Herein, we propose the stromal vascular fraction (SVF) derived from human adipose tissue as a cell source to vascularize 3D osteoblastic BM niches engineered in perfusion bioreactors. We show that SVF cells form self-assembled capillary structures, composed by endothelial and perivascular cells, that add to the osteogenic matrix secreted by BM mesenchymal stromal cells in these engineered niches. In comparison to avascular osteoblastic niches, vascularized BM niches better maintain immunophenotypically-defined cord blood (CB) HSCs without affecting cell proliferation. In contrast, HSPCs cultured in vascularized BM niches showed increased CFU-granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM) numbers. The vascularization also contributed to better preserve osteogenic gene expression in the niche, demonstrating that niche vascularization has an influence on both hematopoietic and stromal compartments. In summary, we have engineered a fully humanized and vascularized 3D BM tissue to model native human endosteal perivascular niches and revealed functional implications of this vascularization in sustaining undifferentiated CB HSPCs. This system provides a unique modular platform to explore hemato-vascular interactions in human healthy/pathological hematopoiesis.
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Mansour, Anna, Grazia Abou-Ezzi, Ewa Sitnicka, Sten Eirik W. Jacobsen, Abdelilah Wakkach, and Claudine Blin-Wakkach. "Osteoclasts promote the formation of hematopoietic stem cell niches in the bone marrow." Journal of Experimental Medicine 209, no. 3 (February 20, 2012): 537–49. http://dx.doi.org/10.1084/jem.20110994.
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Formation of the hematopoietic stem cell (HSC) niche in bone marrow (BM) is tightly associated with endochondral ossification, but little is known about the mechanisms involved. We used the oc/oc mouse, a mouse model with impaired endochondral ossification caused by a loss of osteoclast (OCL) activity, to investigate the role of osteoblasts (OBLs) and OCLs in the HSC niche formation. The absence of OCL activity resulted in a defective HSC niche associated with an increased proportion of mesenchymal progenitors but reduced osteoblastic differentiation, leading to impaired HSC homing to the BM. Restoration of OCL activity reversed the defect in HSC niche formation. Our data demonstrate that OBLs are required for establishing HSC niches and that osteoblastic development is induced by OCLs. These findings broaden our knowledge of the HSC niche formation, which is critical for understanding normal and pathological hematopoiesis.
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Balderman, Sophia R., and Laura M. Calvi. "Biology of BM failure syndromes: role of microenvironment and niches." Hematology 2014, no. 1 (December 5, 2014): 71–76. http://dx.doi.org/10.1182/asheducation-2014.1.71.
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Abstract The BM microenvironment and its components regulate hematopoietic stem and progenitor cell (HSC) fate. An abnormality in the BM microenvironment and specific dysfunction of the HSC niche could play a critical role in initiation, disease progression, and response to therapy of BM failure syndromes. Therefore, the identification of changes in the HSC niche in BM failure syndromes should lead to further knowledge of the signals that disrupt the normal microenvironment. In turn, niche disruption may contribute to disease morbidity, resulting in pancytopenia and clonal evolution, and its understanding could suggest new therapeutic targets for these conditions. In this chapter, we briefly review the evidence for the importance of the BM microenvironment as a regulator of normal hematopoiesis, summarize current knowledge regarding the role of dysfunctions in the BM microenvironment in BM failure syndromes, and propose a strategy through which niche stimulation can complement current treatment for myelodysplastic syndrome.
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Hosokawa, Kentaro, Fumio Arai, Hiroki Yoshihara, Hiroko Iwasaki, Yuka Nakamura, Yumiko Gomei, and Toshio Suda. "Knockdown of N-cadherin suppresses the long-term engraftment of hematopoietic stem cells." Blood 116, no. 4 (July 29, 2010): 554–63. http://dx.doi.org/10.1182/blood-2009-05-224857.
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Abstract During postnatal life, the bone marrow (BM) supports both self-renewal and differentiation of hematopoietic stem cells (HSCs) in specialized microenvironments termed stem cell niches. Cell-cell and cell-extracellular matrix interactions between HSCs and their niches are critical for the maintenance of HSC properties. Here, we analyzed the function of N-cadherin in the regulation of the proliferation and long-term repopulation activity of hematopoietic stem/progenitor cells (HSPCs) by the transduction of N-cadherin shRNA. Inhibition of N-cadherin expression accelerated cell division in vitro and reduced the lodgment of donor HSPCs to the endosteal surface, resulting in a significant reduction in long-term engraftment. Cotransduction of N-cadherin shRNA and a mutant N-cadherin that introduced the silent mutations to shRNA target sequences rescued the accelerated cell division and reconstitution phenotypes. In addition, the requirement of N-cadherin for HSPC engraftment appears to be niche specific, as shN-cad–transduced lineage−Sca-1+c-Kit+ cells successfully engrafted in spleen, which lacks an osteoblastic niche. These findings suggest that N-cad–mediated cell adhesion is functionally required for the establishment of hematopoiesis in the BM niche after BM transplantation.
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Kouzi, Farah, Kazem Zibara, Jerome Bourgeais, Frederic Picou, Nathalie Gallay, Julie Brossaud, Hassan Dakik, et al. "Disruption of gap junctions attenuates acute myeloid leukemia chemoresistance induced by bone marrow mesenchymal stromal cells." Oncogene 39, no. 6 (October 24, 2019): 1198–212. http://dx.doi.org/10.1038/s41388-019-1069-y.
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Abstract The bone marrow (BM) niche impacts the progression of acute myeloid leukemia (AML) by favoring the chemoresistance of AML cells. Intimate interactions between leukemic cells and BM mesenchymal stromal cells (BM-MSCs) play key roles in this process. Direct intercellular communications between hematopoietic cells and BM-MSCs involve connexins, components of gap junctions. We postulated that blocking gap junction assembly could modify cell–cell interactions in the leukemic niche and consequently the chemoresistance. The comparison of BM-MSCs from AML patients and healthy donors revealed a specific profile of connexins in BM-MSCs of the leukemic niche and the effects of carbenoxolone (CBX), a gap junction disruptor, were evaluated on AML cells. CBX presents an antileukemic effect without affecting normal BM-CD34+ progenitor cells. The proapoptotic effect of CBX on AML cells is in line with the extinction of energy metabolism. CBX acts synergistically with cytarabine (Ara-C) in vitro and in vivo. Coculture experiments of AML cells with BM-MSCs revealed that CBX neutralizes the protective effect of the niche against the Ara-C-induced apoptosis of leukemic cells. Altogether, these results suggest that CBX could be of therapeutic interest to reduce the chemoresistance favored by the leukemic niche, by targeting gap junctions, without affecting normal hematopoiesis.
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Jing, Zhixin, Zachary L. Benet, and David R. Fooksman. "Plasma cells Dynamics in the Bone Marrow Niche." Journal of Immunology 206, no. 1_Supplement (May 1, 2021): 11.02. http://dx.doi.org/10.4049/jimmunol.206.supp.11.02.
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Abstract Plasma cells (PCs) can become long-lived with age to maintain prophylactic antibody titer for decades in the bone marrow (BM) microenvironment that are enriched in supporting chemokines and pro-survival cytokines. It has been that PCs are sessile in the BM to receive various pro-survival signals, but how they access these signals in the dynamic BM microenvironment remains unknown. Here by establishing long-term intravital imaging of the mouse tibial BM, we show that PCs are overall motile within the BM parenchyma with unique intermittent and heterogenous motility over time. Their motility is reduced when they are in clusters or as they enter a cluster. PC cluster formation and motility requires the pro-survival cytokine APRIL. PCs motility also requires the chemokine receptor CXCR4 and its ligand CXCL12 that are known important for PC homing to BM. CXCR4/CXCL12 signaling-triggered integrin VLA-4 activation negatively regulates PC motility in the BM. Notably, not only do PCs migrate within the BM parenchyma, but they can also egress and recirculate under steady state condition or CXCR4 and VLA4 inhibition. PC motility, clustering, and recirculation are all increased with mouse age. Thus, PC motility and cluster formation underlie a dynamic BM survival niche, contributing to PC longevity and function.
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Psaila, Bethan, Anna Podolanczuk, Simon Lavotshkin, John Lawrence, Rosandra Kaplan, Selena Granitto, James B. Bussel, Naresh Kikkeri, Irene Roberts, and David Lyden. "Interactions Between Megakaryocytes and Tumour Cells at the Bone Marrow Vascular Stem Cell Niche Promote Tumour Growth and Metastasis." Blood 114, no. 22 (November 20, 2009): 470. http://dx.doi.org/10.1182/blood.v114.22.470.470.
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Abstract Abstract 470 Introduction: Specialized microenvironments, or niches, of the bone marrow (BM) maintain and regulate physiological haematopoietic stem/progenitor cells and have been implicated as areas of preferential engraftment and ‘sanctuary sites' for leukaemic cells and metastasizing cells of solid tumours. The cellular and molecular factors that comprise the BM vascular niche have not been well described. Megakaryocytes (MKs) reside in close association with the BM sinusoidal endothelium. The aim of this study was to investigate whether MKs play a role in the homing and engraftment of malignant cells in the BM. Methods: C57Bl/6 wild type and thrombopoietin (TPO)-/- mice (which have <10% of the normal number of MKs and platelets) received flank or tail vein injections of the syngeneic B16-F10 melanoma or EL4 lymphoma cell lines fluorescently labelled with mCherry or GFP. The MK-vascular niche was examined using citrulline, MECA32, and TSP1 immunostaining. MKs were cultured from the Lin-Sca1+cKit+-enriched fraction of BM cells from flushed femurs and tibias, using 50ng/ml TPO + 20ng/ml SCF. Costar transwell co-culture plates and Neuroprobe chemotaxis chambers were used; cellular proliferation was assessed using the CellTitre MTS assay. RNA was extracted from flushed BM cells and in vitro cell cultures using Qiagen RNeasy columns/Trizol and gene expression was analyzed by RT-qPCR. Results: In wild type mice, the majority of BM sinusoids were surrounded by one or more large MKs forming the MK-vascular niche, with MKs tightly abutting the vascular endothelium. In TPO-/- mice, MKs were largely absent from the BM, blood vessels appeared more tortuous and the mean vessel diameter in the BM was significantly larger than in wild type mice (P<0.01). Expression of the angiogenic regulatory proteins platelet factor 4 (PF4) and thrombospondin 1 (TSP1) was markedly lower in BM from TPO-/- mice than wild type; expression of VEGF and TGFb was also reduced. Together these findings suggest that MKs support the integrity of the vascular niche and that homeostasis of the niche may be disrupted in the absence of MKs. Wild type mice injected with either B16-F10 melanoma or EL4 lymphoma had increased numbers of MKs and a larger mean vessel diameter. Although there was no increase in platelet count, the mean platelet volume was significantly increased by day 18 (p=0.002), suggesting increased thrombopoiesis. Furthermore, there was a linear decrease in PF4 in response to tumour, reaching a 3-fold reduction by late-stage tumour growth. This finding was consistent with the increase in MK-vascular niches as PF4 normally acts as an autocrine inhibitor of megakaryopoiesis and inhibits endothelial cell proliferation and migration. Consistent with a modulatory effect of MKs and/or the MK-vascular niche on tumour phenotype, tumour growth in TPO-/- mice was markedly retarded and there was reduced metastasis to the BM and lung. To investigate the mechanism of these effects, MK-conditioned medium (MCM) was added to in vitro cultures of B16 cells. MCM significantly enhanced the proliferation rate of B16 melanoma cells (P<0.001). Further, MCM was highly chemotactic for B16 cells (P<0.001). This effect was found to be mediated by pertussis toxin-sensitive Gi-protein receptors and reduced but not entirely abrogated in the absence of TSP1 (using MCM generated from TSP1-/- mice). To investigate the interactions between tumour cells and MKs, MKs were cocultured with B16 cells. Coculture increased MK expression of proangiogenic factors VEGF and TGFb while cocultured B16 cells displayed increased expression of alpha integrins, a4, a5 and a6. Moreover, coculturing B16 cells with MKs prior to tail vein injection enhanced tumour cell engraftment in the lung. A pilot study of BM trephine biopsies from 8 patients with carcinoma (breast, lung, prostate, bladder and kidney) supported these preclinical findings. MKs in 3/3 patients with BM metastasis and 3/5 patients without BM metastasis showed a variable excess of MKs, some in loose clusters, with abnormal morphology and localization. Conclusions: These findings suggest that MKs contribute to the integrity and functionality of the BM vascular niche in homeostasis and in malignancy, and that cellular/molecular cross-talk between MKs and tumour cells at the vascular niche may promote metastasis. Targeting these interactions may be a useful as adjunctive therapy to prevent dissemination of cancer to the BM. Disclosures: No relevant conflicts of interest to declare.
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Nishida, Chiemi, Heissig Beate, Motoharu Seiki, Hiromitsu Nakauchi, and Koichi Hattori. "MT1-MMP Is Required for Hematopoietic Maturation in the BM Niche." Blood 114, no. 22 (November 20, 2009): 3634. http://dx.doi.org/10.1182/blood.v114.22.3634.3634.
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Abstract Abstract 3634 Poster Board III-570 Specialized niches, in which hematopoietic stem cells (HSCs) reside control the balance between HSC quiescence and self-renewal, yet little is known about the extrinsic signals provided by the niche and how these niche signals regulate such a balance. Activation of the fibrinolytic pathway via matrix metalloproteinase-9 (MMP-9) resulted in the release of kit ligand (KitL) in the BM niche. Membrane type 1-MMP (MT1-MMP) can activate e.g. MMP-9. To investigate the role of MT1-MMP in hematopoiesis, we used MT1-MMP deficient mice. MT1-MMP−/− mice examined 12 days after birth showed pancytopenia and reduced numbers of bone marrow mononuclear cells (BMMCs) and splenocytes. BM cytospins from MT1-MMP−/− mice showed mild perturbations in erythropoiesis and a more severe impairment of myelopoiesis. Although all lineages were present, the ratio of erythroid to myeloid precursors increased from 0.36 in wildtype to 0.60 in MT1-MMP−/− mice. Myeloid and erythroid cell differentiation was impaired in MT1-MMP−/− BMMCs. The numbers of colony forming unit cells (CFU-C) was reduced in MT1-MMP−/− BMMCs. In contrary, the number of immature hematopoietic cells (CFU-S8, KSL cells) was augmented in MT1-MMP−/− BMMCs. FACS analysis of BM cells showed a decrease in the percentage of mature B cells with an increase number of Pro-B and immature B cells in MT1-MMP−/− BMMCs relative to controls. MT1-MMP−/− BM cells showed lower expression of CXCL12 and KitL, typical niche growth factors important for myelopoiesis and lymphopoisis. Thus, MT1-MMP is required for normal hematopoietic differentiation of lymphoid and myeloid lineage cells, most likely due to growth factor defective niche cells. Disclosures: No relevant conflicts of interest to declare.
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Carlesso, Nadia. "Reacting to Inflammatory Signals." Blood 126, no. 23 (December 3, 2015): SCI—30—SCI—30. http://dx.doi.org/10.1182/blood.v126.23.sci-30.sci-30.
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Specialized cellular niches in the bone marrow (BM) modulate critical functions of the hematopoietic stem and progenitor cells, such as self-renewal, cell-fate decisions and the balance between proliferation and differentiation. Constituents of the BM niche, such as osteoblasts, endothelial cells and macrophages, respond to inflammation with secretion of pro-inflammatory cytokines, and serve as effectors of the inflammatory circuitry; thus their excessive activation can potentially impact on the regulation of hematopoietic cells, and on the initiation and progression of myeloid malignancies. Our current understanding of the mechanisms for Bcr/Ab-negative myeloproliferative neoplasia (MPN) involves recognition of driver mutations (targeting i.e. Tet2, Jak2, Mpl, or Asxl) and of an inflammatory microenvironment. However, despite accumulating evidences on the role of inflammation and of the microenvironment in the maintenance and progression of myeloid malignancies, it is still unclear how the inflamed BM niche contributes to disease establishment and progression. Similarly, the causes leading to an inflammatory microenvironment in MPN are not fully understood. We use genetically-controlled animal model of BM inflammation to study the contribution of the inflammatory microenvironment to MPN. We found that loss of Notch signaling in the BM microenvironment results in an inflammatory state of the BM niche promoting myeloproliferation and marrow fibrosis. Using this model, we discovered that loss of Notch/RBPJ signaling leads to transcriptional upregulation of the pro-inflammatory microRNA miR-155, especially in BM endothelial and mesenchymal cells, resulting in miR-155-dependent inhibition of the NF-κB inhibitor κB-Ras1. Decreased levels of kB-Ras1 resulted in the heightened and persistent activation of NF-κB and in the increased production of NF-kB dependent pro-inflammatory cytokines, leading to uncontrolled myeloproliferation. Deletion of miR-155 in the stroma of RBPJ-/- mice prevented the development of the myeloproliferative disease, and patients affected by MPN exhibit elevated expression of miR155 in their BM. Therefore, we hypothesize that the Notch/miR155/NF-kB axis regulates the level of the inflammatory tonus in the BM niche, and that persistent deregulation of this pathway may contribute to the establishment and progression of MPN. In this presentation, we will discuss our findings and ongoing studies in the context of other recent insights on the crosstalk between tumor cells and the BM niche, and their implications for disease initiation, progression and therapeutic approaches. Disclosures No relevant conflicts of interest to declare.
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Xiao, Pingnan, Monika Dolinska, Lakshmi Sandhow, Makoto Kondo, Anne-Sofie Johansson, Thibault Bouderlique, Ying Zhao, et al. "Sipa1 deficiency–induced bone marrow niche alterations lead to the initiation of myeloproliferative neoplasm." Blood Advances 2, no. 5 (March 7, 2018): 534–48. http://dx.doi.org/10.1182/bloodadvances.2017013599.
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Harutyunyan, Karine G., Felix Nwajei, M. Anna Zal, David A. Fruman, Saradhi Mallampati, Xiaoping Sun, Tomasz Zal, and Marina Konopleva. "The Dynamics of Stroma-Leukemia Interaction in the Hypoxic BM Niches in Vivo." Blood 124, no. 21 (December 6, 2014): 2396. http://dx.doi.org/10.1182/blood.v124.21.2396.2396.
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Abstract We and others have previously reported that leukemia progression is associated with vast expansion of the hypoxic niches and stabilization of hypoxia-inducible factor 1 alpha (HIF-1α) in leukemic cells (Frolova et al. Cancer Biol Ther. 2012, 10:858; Benito et al. PLoS One 2011, 6(8); e23108:1). Interactions of leukemia and the bone marrow (BM) microenvironment are known to play a key role in the survival and growth of leukemic cells, and we have shown that HIF-1α stabilization in stromal cells of the microenvironment facilitates leukemia homing and progression (Chen et al. Blood 2012, 119:4971). In this study, we aimed to characterize the time-dependent progression of BM hypoxia involving both leukemia cells and components of the BM niche, using the multiphoton intravital microscopy (MP-IVM) technique. We first generated a transplantable, imageable leukemia model by retrovirally transducing C57Bl6-Ai14 murine BM cells that express red fluorescing tdTomato with the p190-Bcr/Abl oncogene. The resulting p190-Bcr/Abl tdTomato cells caused rapid development of acute lymphocytic leukemia (ALL) in un-irradiated C57Bl6 immunocompetent mice, manifested by infiltration of the spleen, liver, BM within long bones, skull, and central nervous system followed by death within 28 days. Leukemia cells collected from the BM (LBC) of these animals were transplantable into secondary recipients and triggered accelerated ALL development (14-16 days). Time-course analysis of skull and femur bones in the secondary recipients by MP-IVM demonstrated LBC lodging on day 1 after ALL cell injection, followed by rapid accumulation of leukemia cells localized predominantly within the sinusoidal spaces, which were visualized by injecting the vascular fluorescent dye BSA-647 (Fig. 1a). To detect in vivo hypoxia development, we utilized HS680 (HypoxiSense 680), a carbonic anhydrase IX (CAIX)–targeted fluorescent agent that can be used to image overexpression of CAIX, a direct HIF-1α target, in tumors in response to regional hypoxia. C57Bl6 mice were engrafted with 2 x 105 LBC , and HS680 was injected intravenously at serial intervals followed by MP-IVM. In two separate experiments, increased HS680 fluorescence was detected in bone-lining cells in the BM niches of mice harboring ALL on days 8 and 13, but not in their healthy littermates (Fig 1b). To obtain an independent confirmation of hypoxia, additional mice (n=3) at the same stage (day 14) of leukemia development were sacrificed 3 hr after injection of chemical hypoxia probe pimonidazole (Pimo), and hypoxic BM cells that bound the hypoxia probe were detected by immunohistochemistry. Pimo staining demonstrated vastly spread areas of hypoxia that enclosed both leukemia cells and BM niche cells (Fig 1c), consistent with our previously published observations in different leukemia models. In summary, these findings demonstrate rapid development of intra-BM hypoxia that parallels leukemia progression and involves not only leukemia cells, but also BM niche cells. The HS680 probe can detect hypoxia in vivo within niche cells but not in leukemia cells, likely because of differential expression of CAIX. Our ongoing studies will characterize the cellular origin of hypoxic niche cells by utilizing immunohistochemical techniques and Col2.3-GFPemd transgenic mice to visualize osteoblasts. We postulate that the tumor microenvironment altered with hypoxic niche cells will influence leukemia development or responses to therapy. To this end, we have generated mice with conditionally deleted HIF-1α within BM stromal cells and are investigating the differences in leukemia homing, progression, and chemoresistance between these mice and mice whose BM stromal cells express HIF-1a. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.
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Bourgine, Paul E., Thibaut Klein, Anna M. Paczulla, Takafumi Shimizu, Leo Kunz, Konstantinos D. Kokkaliaris, Daniel L. Coutu, et al. "In vitro biomimetic engineering of a human hematopoietic niche with functional properties." Proceedings of the National Academy of Sciences 115, no. 25 (June 4, 2018): E5688—E5695. http://dx.doi.org/10.1073/pnas.1805440115.
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In adults, human hematopoietic stem and progenitor cells (HSPCs) reside in the bone marrow (BM) microenvironment. Our understanding of human hematopoiesis and the associated niche biology remains limited, due to human material accessibility and limits of existing in vitro culture models. The establishment of an in vitro BM system would offer an experimentally accessible and tunable platform to study human hematopoiesis. Here, we develop a 3D engineered human BM analog by recapitulating some of the hematopoietic niche elements. This includes a bone-like scaffold, functionalized by human stromal and osteoblastic cells and by the extracellular matrix they deposited during perfusion culture in bioreactors. The resulting tissue exhibited compositional and structural features of human BM while supporting the maintenance of HSPCs. This was associated with a compartmentalization of phenotypes in the bioreactor system, where committed blood cells are released into the liquid phase and HSPCs preferentially reside within the engineered BM tissue, establishing physical interactions with the stromal compartment. Finally, we demonstrate the possibility to perturb HSPCs’ behavior within our 3D niches by molecular customization or injury simulation. The developed system enables the design of advanced, tunable in vitro BM proxies for the study of human hematopoiesis.
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Bernasconi, Paolo, and Oscar Borsani. "Targeting Leukemia Stem Cell-Niche Dynamics: A New Challenge in AML Treatment." Journal of Oncology 2019 (August 7, 2019): 1–12. http://dx.doi.org/10.1155/2019/8323592.
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One of the most urgent needs in AML is to improve the disease cure rate as relapse still occurs in 60–80% of patients. Recent evidence suggests that dismal clinical outcomes may be improved by a better definition of the tight interaction between the AML cell population and the bone marrow (BM) microenvironment (“the niche”); the latter has been progressively highlighted to have an active role in the disease process. It has now been well established that the leukemic population may misinterpret niche-derived signals and remodel the niche, providing a shelter to AML cells and protecting them from the cytotoxic effects of chemoradiotherapy. Novel imaging technological advances and preclinical disease models have revealed that, due to the finite number of BM niches, leukemic stem cells (LSCs) and normal hematopoietic stem cells (HSCs) compete for the same functional areas. Thus, the removal of LSCs from the BM niche and the promotion of normal HSC engraftment should be the primary goals in antileukemic research. In addition, it is now becoming increasingly clear that AML-niche dynamics are disease stage specific. In AML, the niche has been linked to disease pathogenesis in the preleukemic stage, the niche becomes permissive once leukemic cells are established, and the niche is transformed into a self-reinforcing structure at a later disease stage. These concepts have been fostered by the demonstration that, in unrelated AML types, endosteal vessel loss occurs as a primary AML-induced niche alteration, and additional AML-induced alterations of the niche and normal hematopoiesis evolve focally and in parallel. Obviously, this endosteal vessel loss plays a fundamental role in AML pathogenesis by causing excessive vascular permeability, hypoxia, altered perfusion, and reduced drug delivery. Each of these alterations may be effectively targeted by various therapeutic procedures, but preservation of endosteal vessel integrity might be the best option for any future antileukemic treatment.
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Han, Hyun Ho, Baek Gil Kim, Joo Hyun Lee, Suki Kang, Ji Eun Kim, and Nam Hoon Cho. "Angiopoietin-2 promotes ER+ breast cancer cell survival in bone marrow niche." Endocrine-Related Cancer 23, no. 8 (August 2016): 609–23. http://dx.doi.org/10.1530/erc-16-0086.
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In estrogen receptor-positive (ER+) breast cancer, it is recognized that metastases may develop after a long period of dormancy. Bone marrow (BM) vascular niche is where the dormant tumor cells are most likely to reside. So far, it is not fully understood why the dormant tumor cells become proliferative and eventually generate tumor. We hypothesized that therapeutic or menopause-related estrogen depletion may be the switch behind dormant ER+ tumor cell awakening in BM. We utilized an existing experimental model of BM endothelial niche that can simulate ER+ tumor cell dormancy to test our hypothesis. In results, estrogen depletion paradoxically promoted ER+ tumor cell proliferation in the BM endothelial niche, and their molecular phenotype shifted from dormant to awaken. Following estrogen depletion, the BM niche cells produced angiopoietin-2 (ANGPT2), which destabilized niche endothelium by interfering ANGPT1/Tie2 signaling, and promoted ER+ tumor cell survival under estrogen deficiency via cell surface integrin β1. Knockdown of ANGPT2 completely negated ER+ tumor cell awakening in the niche. Furthermore, ANGPT2 expression in ER+ tumor human samples was associated with increased risk of distant metastasis only in those who underwent adjuvant estrogen depletion therapy, not in those who did not undergo adjuvant therapy. In conclusion, we demonstrate that ANGPT2 signaling activated after estrogen depletion paradoxically triggers ER+ tumor cell awakening from dormancy in their BM niche, partly indirectly via endothelial Tie2 receptor and partly directly via tumor cell surface integrin β1.
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Asada, Noboru, Yuya Kunisaki, Takashi Nagasawa, and Paul S. Frenette. "Distinct Contributions By Perivascular Niche Cells in Hematopoietic Stem Cell Maintenance." Blood 126, no. 23 (December 3, 2015): 661. http://dx.doi.org/10.1182/blood.v126.23.661.661.
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Abstract Hematopoietic stem cells (HSCs) self-renew and differentiate into all blood types in response to various demands through life. HSC functions are tightly and finely tuned by a specialized microenvironment called "niche" in the bone marrow (BM). Using Nestin-GFP transgenic mice, we have identified Nestin-GFP+ perivascular stromal cells exhibiting a mesenchymal stem/progenitor cell activity as niche cells. Furthermore, we found two types of Nestin-GFP+ cells expressing different surface markers, Nerve/glial antigen 2 (NG2) and Leptin receptor (Lepr) that are associated with arterioles and sinusoid, respectively, in the BM (Kunisaki et al. Nature, 2013). Both arteriolar and sinusoidal niche cells have been reported to show high gene expression of cytokines essential for HSC maintenance such as CXCL12 and stem cell factor (SCF), however, it remains unknown how the distinct niche cells differentially regulate HSC functions. To investigate the mechanisms, we utilized genetic mouse models, in which CXCL12 or SCF can be deleted in specific cell types. CXCL12 deletion in sinusoidal niche cells by using Lepr-cre/Cxcl12fl/− mice mobilized HSCs and lineage− Sca-1+ c-kit+ (LSK) progenitors into spleen (HSC, CT: Control/DL: Deleted: 760±165 / 2193±557 / spleen, n=6, p<0.05) and blood (LSK, CT/DL: 177±36 / 668±156 / mL blood, n=5, p<0.05), but had no effect on HSC numbers in the BM (CT/DL: 1435±101 / 1194±75 / femur, n=6, p=0.085), which is consistent with a previous report (Ding and Morrison, Nature, 2013). Furthermore, assessments of endogenous HSC localization using whole-mount 3D imaging technology revealed that the deletion of CXCL12 in Lepr+ niche cells had no impact on HSC location (KS-test: Two-sample Kolmogorov-Smirnov test, p=0.9981). By contrast, deletion of CXCL12 in NG2-cre derived cells, which recombines efficiently in the entire Nestin-GFP+ non-endothelial stromal fraction including both peri-arteriolar and peri-sinusoidal cells (96.9±1.3%), and overlapping with Lepr+ cells (88.5±1.6%) and CXCL12-abundant reticular cells (90.7±1.4%), led to a robust reduction of HSC numbers in the BM (CT/DL: 1487±87 / 179±40 / femur, n=10, p<0.0001) with HSC and progenitor mobilization into spleen (HSC, CT/DL: 705±262 / 3550±540 / spleen, n=6-8, p<0.01) and blood (LSK, CT/DL: 494±178 / 5357±896 / mL blood, n=5-7, p<0.01). In addition, deletion of CXCL12 in NG2-cre targeted cells led to HSC displacement away from arterioles (KS-test: Two-sample Kolmogorov-Smirnov test; p=0.001). To examine further a role of CXCL12 produced by NG2+ arteriolar niches on HSC maintenance, we generated tamoxifen-inducible NG2-creERTM/Cxcl12fl/− mice. Deletion of CXCL12 postnatally in NG2+ arteriolar niche cells significantly reduced the number of HSCs in the BM (CT/DL: 1617±160 / 960±95 / femur, n=10-13, p=0.0013), which was confirmed functionally by a competitive repopulation assay. Moreover, 3D imaging revealed that HSCs were located further away from arterioles in NG2-creERTM/Cxcl12fl/− marrow (KS-test: p<0.0001), suggesting a role for arteriolar niches in CXCL12-mediated HSC maintenance. As niche cells synthesize several factors, we evaluated the contribution of arteriolar niches in SCF synthesis, a cytokine shown to be critical for HSC maintenance. As expected, deletion of SCF in NG2-cre targeted cells led to a significant reduction of HSC numbers in the BM (CT/DL: 606±85 / 96±23 / femur, n=5-7, p<0.0001). To further evaluate functions of SCF produced by distinct vascular niches, we also compared these mice with deletions using Lepr-cre or tamoxifen-inducible NG2-creERTM mice. We found that deletion of SCF in Lepr-cre targeted cells showed a significant reduction of HSC numbers in the BM (CT/DL: 690±84 / 220±83 / femur, n=3-4, p<0.0118), consistent with previous studies (Ding et al., Nature, 2012), whereas there was no significant change observed in NG2-creERTM/SCFfl/− mice, suggesting that Lepr+ vascular niches rather than NG2+ arteriolar niches are the most important source of SCF in the BM. These results highlight distinct contributions of perivascular cells primarily located in separate vascular niches, arteriolar and sinusoidal, in HSC maintenance and mobilization. Disclosures No relevant conflicts of interest to declare.
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Qing, Yulan, Yuan Lin, Youngji Park, and Stanton L. Gerson. "Self-Renewal and BM Niche Occupancy Defects In NHEJ Deficient HSCs." Blood 116, no. 21 (November 19, 2010): 1455. http://dx.doi.org/10.1182/blood.v116.21.1455.1455.
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Abstract Abstract 1455 Maintenance of hematopoietic stem cells (HSC) requires proper interaction between HSC and the bone marrow niche. DNA repair proteins, especially proteins involved in nonhomologous end joining (NHEJ), are critical for HSC maintenance. We hypothesize that NHEJ deficient HSCs have a BM niche occupancy defect. NHEJ pathway is the major mechanism for double strand break repair in mammalian cells, several proteins participate in the process. Ku70 recognizes and binds to the DNA DSB ends with Ku80, recruits DNA activated catalytic polypeptide (Prkdc) to form DNA dependent protein kinase complex, and further recruits Artemis to process the ends and DNA ligase 4 to rejoin the broken ends. NHEJ deficient cells are hypersensitive to irradiation, and NHEJ deficient mice display SCID phenotypes. We have previous shown that Ku70 deficient HSCs are defective in repopulation as well as BM niche occupancy. Prkdc is another key factor in the NHEJ pathway, and mice homologous for a spontaneous nonsense mutation (Prkdcscid, commonly referred to as scid) are characterized by an absence of functional T cells and B cells due to the inability of V(D)J recombination. Though scid mice has been widely used for human HSCs engraftment studies and the nonobese diabetic/severe combined immune deficiency (NOD/scid) xenotransplantation model is now the “gold standard” for assaying human HSC activity, the function and BM niche occupancy capacity of HSCs of scid mice origin has not been characterized. scid mice show comparable HSC frequency and similar apoptosis rates compared to HSC from WT mice. To assess scid HSC function in vivo, single and competitive serial transplantation was performed. BM from scid mice were able to reconstitute the myeloid lineage (Mac1+) but not lymphoid lineages (CD3+ or B220+) in primary recipients due to the deficiency in V(D)J recombination. Serial transplantation was hampered by a high frequency of donor-derived thymic lymphomas in primary recipients. Competitive repopulation assays between WT and scid BM cells showed that when scid BM were mixed with WT competitor BM at a 1:1 ratio, scid BM cells were completely outcompeted by the WT BM cells. To investigate the BM niche occupancy ability of scid HSCs, we used the hematopoiesis niche occupancy assay, in which 5×106 WT congenic BM cells were transplanted into WT and scid recipients without any recipient conditioning and evaluated for blood and marrow cell origins at 16–24 weeks. Remarkably, transplanted WT BM made a long-term multi-lineage (Mac1+, CD3+ and B220+) contribution to hematopoiesis as well as HSC engraftment in scid recipients, (18.4+ 3.8% Mac1+, all the CD3+, B220+ cells, and 8.2+ 2.6% of HSC are donor derived). By contrast, less than 1% stem cell engraftment occurred in WT recipients as expected. Thus, have a long term niche occupancy defect impacting hematopoiesis. NHEJ is involved in both DNA double strand break repair and V(D)J recombination in lymphocytopoiesis. To determine whether lymphocytopoiesis is required for HSC function and BM niche occupancy, RAG1 deficient mice were used in the competitive repopulation assay and the hematopoiesis niche occupancy assay. Our results showed that RAG1-/- HSCs competed with WT HSC did not have defects in competitive repopulation or BM niche occupancy. These data demonstrate that Prkdc, a key component of NHEJ, is required for HSC function. Prkdc deficiency in the HSC compartment results in loss of competitive repopulation ability and loss of long term BM niche occupancy. Together with our previous results obtained from Ku70-/- mice, we conclude that NHEJ is critical for HSC self-renewal and BM niche occupancy, a function independent of its role in lymphogenesis. These data also explains the nature of the conducive marrow niche environment of scid mice for xenotransplantation. Disclosures: No relevant conflicts of interest to declare.
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Prendergast, Áine M., Andrea Kuck, Mieke van Essen, Simon Haas, Sandra Blaszkiewicz, and Marieke A. G. Essers. "Ifnα Mediated Remodeling of the Bone Marrow Stem Cell Vascular Niche." Blood 128, no. 22 (December 2, 2016): 2667. http://dx.doi.org/10.1182/blood.v128.22.2667.2667.
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Abstract In the bone marrow (BM), endothelial cells (ECs) are a major component of the hematopoietic stem cell (HSC) vascular niche and are a first line of defense against inflammatory stress and infection. The primary response of an organism to infection involves the synthesis of immune-modulatory cytokines, including interferon alpha (IFNα). In the BM, IFNα induces rapid cell cycle entry of HSCs in vivo. However, the effect of acute IFNα treatment on BM ECs has not been described. Here, we demonstrate that IFNα leads to rapid stimulation of BM ECs in vivo, resulting in increased BM vascularity and vascular leakage. We find that stimulation of BM ECs involves the expression of key inflammatory and EC-stimulatory markers, including ESAM, VE-Cadherin and Laminin. Using the anti-VEGF antibody, Avastin, we could confirm that IFNα-mediated activation of BM ECs is dependent in part on VEGF signaling in BM hematopoietic cell types, including HSCs. Thus, this implies a role for HSCs in remodeling of the BM niche in vivo followinginflammatory stress. Taken together, these data increase our current understanding of the relationship between HSCs and the BM niche under inflammatory stress. In addition, we have clarified the response of BM niche ECs to acute IFNα treatment in vivo. IFNα treatment has been associated with anti-angiogenic effects in patients and therefore our described acute response of the BM vasculature may additionally inform future regimes of combination therapy. Disclosures No relevant conflicts of interest to declare.
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Olson, Timothy S., Satoru Otsuru, Ted J. Hofmann, Massimo Dominici, and Edwin M. Horwitz. "Expansion of the Endosteal Hematopoietic Stem Cell Niche Following Myeloablative and Reduced Intensity Conditioning Is Triggered By Hematopoietic Cell Loss." Blood 124, no. 21 (December 6, 2014): 1090. http://dx.doi.org/10.1182/blood.v124.21.1090.1090.
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Abstract The efficiency and durability of donor hematopoietic stem cell (HSC) engraftment at specialized niches within recipient bone marrow (BM) are critical determinants of successful clinical stem cell transplant (SCT). Osteolineage elements of BM niches play active, critical roles in regulating HSC engraftment, differentiation, and self-renewal both during homeostasis and post-SCT. We have previously shown that SCT preparative regimens consisting of myeloablative total body irradiation (MB-TBI) result in expansion of osteolineage niche cells at the endosteal surface of metaphyseal bone. This expansion is facilitated by IGF-1 and megakaryocyte-derived PDGF-BB signaling, and is required for optimal HSC engraftment. However, the pathway that integrates these signals to induce expansion remains undefined, and whether expansion is unique to conditioning with MB-TBI due to direct effects on osteolineage cells is also not known. We now present data using chemotherapy-based and reduced intensity conditioning (RIC) models, demonstrating that niche expansion is triggered by hematopoietic cell loss, and not by direct effects of conditioning agents on the niche. In vitro, MB-TBI (1125 cGy) inhibited primary murine osteoblast (OB) growth and increased apoptosis (Annexin V+ OB at 72 hours: TBI 17% versus control 4%, p < 0.001). In contrast, busulfan treatment caused significantly less OB growth inhibition versus MB-TBI, with no increased apoptosis compared to sham-treated controls. We therefore conditioned wildtype (WT) C57BL/6 mice with myeloablative busulfan (Day -7 to -4) and cyclophosphamide (Day -3 to -2) (BuCy) in a schedule similar to clinical SCT regimens. Endosteal niche expansion increased over this time course, correlating with the extent of hematopoietic ablation, with most expansion occurring prior to cyclophosphamide administration. Maximal expansion occurred by the end of the treatment course, and based on a quantitative scoring index, was not significantly different than maximal MB-TBI-induced niche expansion, demonstrating that niche expansion is not specific to radiation-based conditioning. As with MB-TBI treatment, BuCy-treated recipients of GFP+donor BM consistently exhibited >95% donor chimerism. We next investigated whether RIC regimens can mediate niche expansion, using an anti-cKit antibody (clone ACK2) known to enable donor engraftment when administered alone in immunodeficient mice or in combination with low dose irradiation (LD-TBI, 300 cGy) in WT mice. ACK2 treatment alone resulted in modest endosteal cell expansion (33% of MB-TBI induced expansion), correlating with transient reductions in host BM cellularity, but absence of definitive donor engraftment in ACK2-treated WT mice. In contrast, LD-TBI alone or in combination with ACK2 produced 58% and 74%, respectively, of the endosteal expansion seen following MB-TBI. Interestingly, while these regimens reduced total BM cellularity by 85% (LD-TBI) and 93% (LD-TBI + ACK2), and led to clearance of BM Lin-Sca1+cKit+(LSK) progenitors, this lack of full niche expansion correlated with incomplete and inconsistent donor chimerism in SCT recipients. Finally, to prove that endosteal niche expansion results from signals triggered specifically by hematopoietic cell loss, we crossed mice expressing Cre-recombinase under control of the CD45 promoter (CD45Cre) with mice expressing inducible diphtheria toxin receptor (iDTRlox). When treated with diphtheria toxin (DT), BM cellularity in these mice was reduced by 90%, and this targeted ablation of hematopoietic cells was sufficient to induce similar expansion of endosteal mesenchymal cells as seen with radiation or chemotherapy-based conditioning regimens. Taken together, our results demonstrate that endosteal niche expansion occurs in response to both radiation- and chemotherapy-based SCT conditioning, and that the degree of expansion correlates with both conditioning intensity and with the subsequent degree of donor cell engraftment/chimerism in SCT recipients. Importantly, expansion is triggered not by direct effects of the preparative regimen on mesenchymal niche cells, but rather by loss of hematopoietic cells. These findings provide important insights into how SCT conditioning modulates niche function, and suggests that therapeutic strategies to enhance niche function may be effective in improving engraftment outcomes following RIC SCT. Disclosures No relevant conflicts of interest to declare.
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Kumar, Bijender, Mayra Garcia, Lihong Weng, Xiaoman Lewis, Jodi Murakami, Xingbin Hu, Tinisha McDonald, et al. "Acute Myeloid Leukemia-Derived Exosomes Transform Bone Marrow Niche into Leukemic Niche." Blood 124, no. 21 (December 6, 2014): 352. http://dx.doi.org/10.1182/blood.v124.21.352.352.
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Abstract Increasing evidence suggests that leukemia cells take shelter in the bone marrow (BM) niche, where they hide from chemotherapy and continue to divide. As yet, how leukemia cells alter the BM niche to facilitate their growth and assist them in evading chemotherapy is unclear. In this study, we provide compelling evidences that acute myeloid leukemia (AML), through exosome secretion, transformed the BM niche to facilitate their own growth and suppress normal hematopoiesis. Using AML xenograft and MLL-AF9 knock-in mouse model, we show that leukemia cells as well as AML-derived exosomes stimulate the growth of BM stromal progenitors and blocked the osteolineage development in our stromal compartment analysis. Histological analysis and micro-CT examination confirmed loss or thinning of the bone in both leukemia and leukemic exosome-treated animals. Expression of cell adhesion molecules (NCAM1, VCAM1, CD44, OPN & ICAM1) and factors important for angiogenesis (Angpt1, Angpt2 and VEGF) are upregulated, whereas genes important for HSC maintenance (CXCL12 and SCF), osteoblast (OCN, OSX, Notch3 and IGF1) and chondrocyte (ACAN, SOX9) development are suppressed. While we observed increases in phenotypic LT-HSC in AML-derived exosomes treated mice, these mice show reduced multilineage reconstitution ability, increased cell cycle entry and higher sensitivity to myeloablative stress suggesting that HSCs from exosome-treated mice have lower stem cell activity than their counterparts from normal mice.In addition, leukemia-modified stroma cells exhibit marked reduction in ability to support normal HSCs. Pre-treatment of AML-derived exosome “prime” the animal for leukemia cell invasion and accelerate leukemia progression. Conversely,disruption of exosome secretion by targeting Rab27a in AML cells significantly delays leukemia progression. These data strongly support the notion that leukemia-modified niches favor leukemic cell proliferation and suppress normal hematopoiesis. Disclosures No relevant conflicts of interest to declare.
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Fang, Shentong, Shuo Chen, Harri Nurmi, Veli-Matti Leppänen, Michael Jeltsch, David Scadden, Lev Silberstein, Hanna Mikkola, and Kari Alitalo. "VEGF-C protects the integrity of the bone marrow perivascular niche in mice." Blood 136, no. 16 (October 15, 2020): 1871–83. http://dx.doi.org/10.1182/blood.2020005699.
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Abstract Hematopoietic stem cells (HSCs) reside in the bone marrow (BM) stem cell niche, which provides a vital source of HSC regulatory signals. Radiation and chemotherapy disrupt the HSC niche, including its sinusoidal vessels and perivascular cells, contributing to delayed hematopoietic recovery. Thus, identification of factors that can protect the HSC niche during an injury could offer a significant therapeutic opportunity to improve hematopoietic regeneration. In this study, we identified a critical function for vascular endothelial growth factor-C (VEGF-C), that of maintaining the integrity of the BM perivascular niche and improving BM niche recovery after irradiation-induced injury. Both global and conditional deletion of Vegfc in endothelial or leptin receptor–positive (LepR+) cells led to a disruption of the BM perivascular niche. Furthermore, deletion of Vegfc from the microenvironment delayed hematopoietic recovery after transplantation by decreasing endothelial proliferation and LepR+ cell regeneration. Exogenous administration of VEGF-C via an adenoassociated viral vector improved hematopoietic recovery after irradiation by accelerating endothelial and LepR+ cell regeneration and by increasing the expression of hematopoietic regenerative factors. Our results suggest that preservation of the integrity of the perivascular niche via VEGF-C signaling could be exploited therapeutically to enhance hematopoietic regeneration.
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Manier, S., A. Sacco, X. Leleu, I. M. Ghobrial, and A. M. Roccaro. "Bone Marrow Microenvironment in Multiple Myeloma Progression." Journal of Biomedicine and Biotechnology 2012 (2012): 1–5. http://dx.doi.org/10.1155/2012/157496.
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Substantial advances have been made in understanding the biology of multiple myeloma (MM) through the study of the bone marrow (BM) microenvironment. Indeed, the BM niche appears to play an important role in differentiation, migration, proliferation, survival, and drug resistance of the malignant plasma cells. The BM niche is composed of a cellular compartment (stromal cells, osteoblasts, osteoclasts, endothelial cells, and immune cells) and a noncellular compartment including the extracellular matrix (ECM) and the liquid milieu (cytokines, growth factors, and chemokines). In this paper we discuss how the interaction between the malignant plasma cell and the BM microenvironment allowed myeloma progression through cell homing and the new concept of premetastatic niche.
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Hanoun, Maher, Dachuan Zhang, Sandra Pinho, Toshihide Mizoguchi, Julie Lacombe, Scott A. Armstrong, Ulrich Duehrsen, and Paul S. Frenette. "Acute Myeloid Leukemia Alters The Mesenchymal Stem Cell Potential Of The HSC Niche: Evidence For Modulation By β-Adrenergic Signals." Blood 122, no. 21 (November 15, 2013): 342. http://dx.doi.org/10.1182/blood.v122.21.342.342.
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Abstract Hematopoietic stem cells (HSC) reside in specific bone marrow niches comprised of perisinusoidal Nestin-GFP+ (Nes-GFP+) and leptin receptor (LepR)+ stromal cells which highly overlap with each other, as well as osteolineage and endothelial cells. These different cellular constituents regulate HSC maintenance and retention in the bone marrow (BM). Recently, our laboratory further identified rare periarteriolar Nes-GFPbright cells which have been identified to be pericytes and functionally crucial for HSC quiescence and maintenance, from the more abundant reticular Nes-GFPdim population (Kunisaki et al, unpublished data). The function of Nes-GFP+ niche cells is tightly regulated by the sympathetic nervous system (SNS) via β2- and β3-adrenergic receptors. In acute myeloid leukemia (AML), BM infiltration by leukemic blasts is known to lead to hematopoietic failure. Although cytopenias are thought to result from the BM occupation by AML, the mechanisms remain unclear. In this study, we investigated the impact of AML on BM niche constituents, and evaluated the influence of SNS signals in AML progression using a syngeneic murine MLL-AF9 transplantation model. We observed a ∼4.4-fold expansion of Nes-GFP+ stromal cells (p<0.05) in leukemic BM, which positively correlated with the degree of leukemic marrow infiltration (r=0.49, p<0.01). However, while we observed a massive expansion of reticular Nes-GFPdim cells, we detected a ∼2.2-fold reduction in pericytic Nes-GFPbright cells (p<0.05). Phenotypically, Nes-GFPdim cells showed similar expression of LepR, PDGFRα and CD51 to healthy controls, markers enriching for BM mesenchymal stem cells. Interestingly, Nes-GFP+ cells in leukemic BM had a ∼1.5-fold higher colony-forming units-fibroblast (CFU-F) and ∼2.6-fold higher CFU-Osteoblast capacity. The gene expression profile of Nes-GFP+ cells using Affymetrix Gene 1.0 ST microarrays revealed a profound dysregulation when the BM was infiltrated with AML, including most notably activated TGFβ signaling pathways and upregulation in the expression of inflammatory response genes. Nes-GFP+ niche cells were previously shown to be highly enriched for HSC-maintenance gene expression. In leukemic BM, Nes-GFP+ cells showed a significant reduction in the expression of Cxcl12, Scf and Vcam1 (p<0.01), while no significant differences were observed for Opn. In line with these data, we observed a significant reduction of HSC-enriched Lin−Sca1+c-Kit+Flt3− and progenitor cells in the BM (p<0.05, except for granulocyte-macrophage progenitors) and their mobilization into peripheral blood and spleen (p<0.01). To further analyze if Nes-GFP+ niche cells in leukemic BM still depended on adrenergic signals, we performed in vivo chemical sympathetic denervation with 6-hydroxydopamine. Sympathectomized leukemic mice showed a ∼1.5-fold increase in Nes-GFP+ cells, a ∼1.8-fold increase of IL-7R-Lin-c-KithiCD34+FcγRII/IIIhi leukemic stem cells (LSC, p<0.05) which was associated with significantly shorter survival (p=0.017). Denervated mice did not show differences in homing, proliferation or apoptosis of leukemic cells. We confirmed our findings in a xenograft model, in which sympathectomized mice were transplanted with primary human AML samples and showed a higher leukemic BM infiltration (p<0.05). Further analyses using specific adrenergic β2- and β3-receptor antagonists (ICI118,551 hydrochloride and SR59230A) suggested that adrenergic signals were predominantly mediated by the β2-adrenergic receptor. Preliminary data indicate that activation of the β2-adrenergic receptor leads to decreased leukemia burden. In summary, our data identify that leukemic cells severely transform the BM niche by gradually expanding and directing mesenchymal stem cell differentiation into the osteoblastic lineage and simultaneously decreasing numbers of pericytic niche cells. Ultimately, this creates a microenvironment with impaired HSC maintenance capacity and favors myeloid expansion. This transformed niche, however, remains regulated by signals from the SNS which in turn modulates leukemic BM infiltration. These results thus uncover a novel approach for niche-targeted therapeutic strategies in AML. Disclosures: Armstrong: Epizyme Inc.: Has consulted for Epizyme Inc. Other.
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Wang, Min, Ru Feng, Jia-min Zhang, Lin-lin Xu, Fei-er Feng, Chen-cong Wang, Qian-ming Wang, et al. "Dysregulated megakaryocyte distribution associated with nestin+ mesenchymal stem cells in immune thrombocytopenia." Blood Advances 3, no. 9 (May 3, 2019): 1416–28. http://dx.doi.org/10.1182/bloodadvances.2018026690.
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AbstractImpaired megakaryocyte (MK) maturation and reduced platelet production are important causes of immune thrombocytopenia (ITP). However, MK distribution and bone marrow (BM) niche alteration in ITP are unclear. To investigate the maturation and distribution of MKs in the BM niche and examine the components of BM niche regulation of MK migration, BM and peripheral blood were obtained from 30 ITP patients and 28 healthy donors. Nestin+ mesenchymal stem cells (MSCs) and CD41+ MKs were sorted by fluorescence-activated cell sorting. The components of the BM niche and related signaling were analyzed via immunofluorescence, flow cytometry, enzyme-linked immunosorbent assay, reverse transcription polymerase chain reaction, and western blot analysis. The number of MKs in the BM vascular niche was reduced in ITP. Moreover, the concentrations of CXCL12 and CXCR4+ MKs in the BM were decreased in ITP. Further investigation demonstrated that nestin+ MSCs and CXCL12 messenger RNA (mRNA) in nestin+ MSCs were both reduced whereas the apoptosis of nestin+ MSCs was significantly increased in ITP. Sympathetic nerves, Schwann cells, the proportion of β3-adrenoreceptor (β3-AR)+ nestin+ MSCs, and β3-AR mRNA in nestin+ MSCs were all markedly reduced in ITP. Moreover, matrix metalloproteinase 9, vascular endothelial growth factor (VEGF), and VEGF receptor 1 were significantly reduced in ITP. Our data show that impaired MK distribution mediated by an abnormal CXCL12/CXCR4 axis is partially involved in reduced platelet production in ITP. Moreover, sympathetic neuropathy and nestin+ MSC apoptosis may have an effect on the alterations of BM CXCL12 in ITP.
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Aprile, Annamaria, Alessandro Gulino, Mariangela Storto, Isabella Villa, Stefano Beretta, Ivan Merelli, Alessandro Rubinacci, et al. "Hematopoietic stem cell function in β-thalassemia is impaired and is rescued by targeting the bone marrow niche." Blood 136, no. 5 (July 30, 2020): 610–22. http://dx.doi.org/10.1182/blood.2019002721.
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Abstract Hematopoietic stem cells (HSCs) are regulated by signals from the bone marrow (BM) niche that tune hematopoiesis at steady state and in hematologic disorders. To understand HSC-niche interactions in altered nonmalignant homeostasis, we selected β-thalassemia, a hemoglobin disorder, as a paradigm. In this severe congenital anemia, alterations secondary to the primary hemoglobin defect have a potential impact on HSC-niche cross talk. We report that HSCs in thalassemic mice (th3) have an impaired function, caused by the interaction with an altered BM niche. The HSC self-renewal defect is rescued after cell transplantation into a normal microenvironment, thus proving the active role of the BM stroma. Consistent with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. In vivo activation of PTH signaling through the reestablished Jagged1 and osteopontin levels correlated with the rescue of the functional pool of th3 HSCs by correcting HSC-niche cross talk. Reduced HSC quiescence was confirmed in thalassemic patients, along with altered features of the BM stromal niche. Our findings reveal a defect in HSCs in β-thalassemia induced by an altered BM microenvironment and provide novel and relevant insight for improving transplantation and gene therapy approaches.
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Reinisch, Andreas, Nathalie Etchart, Nicole A. Hofmann, Margareta Frühwirth, Anna Ortner, Daniela Thaler, Birgit Feilhauer, et al. "Organotypic Epigenetic Signature Predicts Bone and Marrow Niche Forming Capacity of Stromal Progenitors in a Novel Mouse Model in Vivo." Blood 120, no. 21 (November 16, 2012): 2987. http://dx.doi.org/10.1182/blood.v120.21.2987.2987.
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Abstract Abstract 2987 Mesenchymal stem/progenitor cells (MSPCs) from numerous tissues are currently tested in clinical trials despite a limited understanding of their in vivo behavior. In this study we used MSPCs from adult and fetal tissues to select the appropriate source for clinical application. We asked whether MSPCs derived from human bone marrow (BM), white adipose tissue (WAT) and umbilical cord (UC), compared to skin fibroblasts, bear an equivalent bone and marrow niche formation potential with of in vivo. Furthermore we evaluated attraction and engraftment of murine as well as human hematopoietic stem/progenitor cells (HSPCs) into newly formed MSPC-derived niches. To elucidate potential mechanisms responsible for a tissue-specific MSPC potential after transplantation gene expression profiling and DNA methylation analysis on a novel high resolution 450K-CpG methylation array were employed. MSPCs were transplanted subcutaneously to test for their spontaneous bone and marrow niche formation potential in immune-deficient NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ). BM-derived MSPC transplantation reproducibly led to the development of mature bone (17/17 donors) through an endochondral ossification process leading to subsequent marrow niche formation. Additionally, these newly formed hematopoietic microenvironments attracted complete mouse hematopoiesis including immature lineage negative, Sca-1 positive, c-kit positive (LSK) HSPCs. Non-BM derived MSPCs completely lacked bone and marrow niche-forming potential and did not attract hematopoietic cells (0/9 donors). Induction of human hematopoietic chimerism through transplantation of umbilical cord blood (UCB)-derived human CD34+ HSPCs in advance of subcutaneous ectopic bone and marrow development resulted in immigration of re-transplantable human hematopoiesis into extra-medullary ossicles. Comparative 450K-CpG methylation array profiling of MSPCs revealed a tissue-specific epigenetic signature virtually corresponding to the in vivo differentiation. MSPCs from BM but neither fibroblasts nor MSPCs from WAT or UC showed epigenetically imprinted human bone and marrow niche (HuNiche) formation capacity favoring BM-MSPCs for skeletal regeneration. This novel HuNiche model should be ideally suited for studying normal and malignant hematopoiesis regulation in an ectopic human marrow with subsequent human hematopoietic engraftment that mimics clinical BM transplantation reality. Disclosures: No relevant conflicts of interest to declare.
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Hirata, Yuichi, Kazuhiro Furuhashi, Hiroshi Ishi, Hao-Wei Li, Sandra Pinho, Lei Ding, Paul S. Frenette, and Joji Fujisaki. "Adenosine from Niche-Associated Tregs Maintains Hematopoietic Stem Cell Quiescence." Blood 130, Suppl_1 (December 7, 2017): 91. http://dx.doi.org/10.1182/blood.v130.suppl_1.91.91.
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Abstract A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. For example, specific subsets of Tregs in the adipose tissue control metabolic indices; muscle Tregs potentiate muscle repair, and lung Tregs prevent tissue damage. These studies, together with a previous finding that Tregs are enriched in the primary site for hematopoiesis, the bone marrow (BM), prompted us to examine whether there is a special Treg population which controls hematopoietic stem cells (HSCs). We showed that HSCs within the BM were frequently adjacent to distinctly activated FoxP3+ Tregs which highly expressed an HSC marker, CD150. Moreover, specific reduction of BM Tregs achieved by conditional deletion of CXCR4in Tregs, increased reactive oxygen species (ROSs) in HSCs. The reduction of BM Tregs further induced loss of HSC quiescence and increased HSC numbers in a manner inhibited by anti-oxidant treatment. Additionally, this increase in HSC numbers in mice lacking BM Tregs was reversed by transfer of CD150high BM Tregs but not of CD150low BM Tregs. These results indicate that CD150high niche-associated Tregs maintain HSC quiescence and pool size by preventing oxidative stress. We next sought to identify an effector molecule of niche Tregs which regulates HSCs. Among molecules highly expressed by niche Tregs, we focused on CD39 and CD73, cell surface ecto-enzymes which are required for generation of extracellular adenosine, because 1) CD39highCD73high cells within the BM were prevalent among CD150high Tregs and 2) HSCs highly expressed adenosine 2a receptors (A2AR). We showed that both conditional deletion of CD39 in Tregs and in vivo A2AR antagonist treatment induced loss of HSC quiescence and increased HSC pool size in a ROS-dependent manner, which is consistent with the findings in mice lacking BM Tregs. In addition, transfer of CD150high BM Tregs but not of CD150low BM Tregs reversed the increase in HSC numbers in FoxP3cre CD39flox mice. The data indicate that niche Treg-derived adenosine regulates HSCs. We further investigated the protective role of niche Tregs and adenosine in radiation injury against HSCs. Conditional deletion of CD39 in Tregs increased radiation-induced HSC apoptosis. Conversely, transfer of as few as 15,000 CD150high BM Tregs per B6 mouse (iv; day-1) rescued lethally-irradiated (9.5Gy) mice by preventing hematopoiesis failure. These observations indicate that niche Tregs protect HSCs from radiation stress. Finally, we investigated the role of niche Tregs in allogeneic (allo-) HSC transplantation. Our previous study showed that allo-hematopoietic stem and progenitor cells but not allo-Lin+ cells persisted in the BM of non-conditioned immune-competent recipients without immune suppression in a manner reversed by systemic Treg depletion1. This observation suggests that HSCs have a limited susceptibility to immune attack, as germline and embryonic stem cells are located within immune privileged sites. Because the study employed systemic Treg depletion and non-conditioned recipients, it remains unknown whether niche Tregs play a critical role in immune privilege of HSCs and in allo-HSC engraftment following conditioning. We showed here that the reduction of BM Tregs and conditional deletion of CD39 in Tregs abrogated allo-HSC persistence in non-conditioned immune-competent mice as well as allo-HSC engraftment following nonmyeloablative conditioning. Furthermore, transfer of CD150high BM Tregs but not of other Tregs (15,000 cells/recipient; day -2) significantly improved allo-HSC engraftment. This effect of niche Treg transfer is noteworthy given that 1-5 million Tregs per mouse were required in case of transfer of spleen or lymph node Tregs. These observations suggest that niche Tregs maintain immune privilege of HSCs and promote allo-HSC engraftment. In summary, our studies identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, numbers, stress response, engraftment, and immune privilege, further highlighting potential clinical utility of niche Treg transfer in radiation-induced hematopoiesis failure and in allo-HSC engraftment (under revision in Cell Stem Cell). 1 Fujisaki, J. et al. In vivo imaging of Treg cells providing immune privilege to the haematopoietic stem-cell niche. Nature474, 216-219, doi:10.1038/nature10160 (2011). Disclosures No relevant conflicts of interest to declare.
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Poulos, Michael, David Redmond, Michael Gutkin, Pradeep Ramalingam, and Jason M. Butler. "Single-Cell Characterization of the HSC-Supportive Bone Marrow Vascular Microenvironment." Blood 132, Supplement 1 (November 29, 2018): 2577. http://dx.doi.org/10.1182/blood-2018-99-120339.
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Abstract Hematopoietic stem and progenitor cells (HSPCs) balance the physiological demands of maintaining peripheral leukocytes, erythrocytes, and platelets, while maintaining a potent stem cell reserve. These characteristics have made HSPC transplantation the only curative option for treating many hematological disorders. The regenerative potential of hematopoietic stem cells (HSCs) reside in their ability to home to a supportive niche, allowing for both HSC self-renewal and reconstitution of the hematopoietic system. The bone marrow (BM) microenvironment regulates HSC quiescence, self-renewal, and differentiation. The BM niche is composed of a number of cell types, including Lepr+ cells, Nestin+ cells, and endothelial cells. Collectively, the HSC niche modulates HSC fate decisions through the expression of paracrine factors, including Cxcl12 and Kitl. The BM microenvironment also plays a critical role in the reestablishment of hematopoiesis following myeloablative injury. Vegfr2-mediated vascular repair is critical for hematopoietic reconstitution following chemotherapeutic and radiation-mediated insult, while BM granulocyte production of Tnfa supports the regeneration of sinusoidal endothelium and subsequent hematopoietic recovery. While the importance of an HSC-supportive microenvironment during hematopoietic homeostasis and during regenerative conditions is coming into focus, poorly defined BM niche cells have limited the precise mechanistic insights necessary to elucidate new regenerative factors and strategies. Current methodology used to examine cellular subsets within the BM microenvironment rely on immunophenotypic fractionation, localization, and genetic lineage tracing. Ambiguous BM niche cellular immunophenotypes and gene expression have limited cellular resolution and confounded the interpretation of cre-mediated genetic deletion models. Herein, we aim to resolve the identities of distinct BM endothelial cell (BMEC) subpopulations to ultimately develop genetic tools to elucidate the paracrine requirements of the HSC-supportive endothelial niche. To this end, we sort-purified murine BMECs (VECAD+CD31+CD45-TER119-) for single cell RNA sequencing (scRNA-Seq). scRNA-Seq revealed the emergence of distinct BM arteriole, sinusoidal, and transitional endothelial populations, with arteriole BMECs significantly enriched for Kitl and Cxcl12. To confirm an arteriole enrichment in Kitl and Cxcl12 expression, we performed scRNA-Seq transcriptional analysis of sort-purified BM cells from KitlGFP and Cxcl12DsRed reporter mice. KitlGFP BM cells identified a distinct arteriole, but not sinusoidal, BMEC population. Cxcl12DsRed BM cells identified both arteriole and sinusoidal BMEC cell populations, but confirmed an increase in Cxcl12 expression in arterioles. We next examined candidate genes to generate BMEC subset-specific inducible cre mice. Analysis revealed that Vegfr3 (Flt4) expression was specific to sinusoidal BMECs, while Bmx1 appeared enriched in arterioles. We utilized a previously described Vegfr3YFP transgenic reporter mouse and found sinusoidal BMEC restricted expression. We then generated an inducible Vegfr3creERT2 line that directs efficient recombination to sinusoidal endothelium, with no detectable off-target activity. We next examined a previously described Bmx1creERT2 mouse line by generating Bmx1creERT2;ROSA26tdTomato reporter mice. In contrast to a recent report, Bmx1creERT2 activity was not spatially confined to the BM arteriole niche, but also labeled additional niche components, making it an unsuitable for arteriole-specific deletion. Moreover, previously reported constitutive Eporcre mice used to delete Kitl in sinusoids displays detectable cre activity in both arteriole and erythrocyte populations. More refined genetic models will need to be generated to test current and future candidate factors in the BM niche. Using our transcriptional data set, we have generated and validated a new inducible Vegfr3creERT2 mouse line that displays sinusoidal-restricted expression in the BM. Arteriole-specific creERT2 lines are currently being evaluated. These models will be used to systematically evaluate novel candidate arteriole- and sinusoidal-specific hematopoietic paracrine factors identified in our transcriptional analysis. Disclosures No relevant conflicts of interest to declare.
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Balaian, Larissa, Leslie C. Robertson, Anna Kulidjian, Edward D. Ball, and Catriona Jamieson. "SHH Inhibitor PF-0449913 in Vitro Remodels Leukemic Niche into HSC Supportive Niche." Blood 124, no. 21 (December 6, 2014): 4784. http://dx.doi.org/10.1182/blood.v124.21.4784.4784.
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Abstract Background Acute myelogenous leukemia (AML) is a highly lethal disease with only 20% of 5 years survival. Most AML patients experience relapse usually leading to death. Progression to therapy resistant AML is driven by leukemia stem cells (LSC) harboring enhanced survival, dormancy and self-renewal capacity in supportive niches. Growing evidence indicates that in the development of myeloid malignancies deregulation of stem cells activity is as important as deregulation of the BM microenvironment. Sonic hedgehog (SHH) pathway molecules in general and SMO in particular are well known to maintain the growth of cancer cells. However, it’s role in leukemia niche-stem cell interaction is not well defined. Therefore, here we investigate: 1/ how the small molecule, SMO antagonist, PF-0449913 impacts the AML BM microenvironment and 2/ how in turn, changes in the activity of AML BM niche cells contribute to its remodeling into HSC supportive niche. Methods CD34- cell from bone marrow patients undergoing hip replacement surgery (normal BM) as well as AML bone marrow were utilized for the development of the primary human stromal monolayers. We investigated stromal cultures of primary normal BM (NBM, n=6) and AML (n=6). As a control human normal bone marrow stromal cell line HS-5 was used. Then human CD34+ cells were selected from AML primary samples (n=6). As a normal control, CD34+ cells from cord blood (CB, n=5), or NBM (n=5) were utilized for the co-culture experiments for up to 9 weeks and then plated in survival and self-renewal assays. PF-0449913 was added to stromal co-cultures or, as a pre-treatment, directly to stromal monolayers. Results In order to examine the role of SMO regulation in LSC generation and maintenance, primary AML (n=6) or cord blood (n=3) CD34+ cells were co-cultures with normal BM (n=3) or AML (n=3) stroma in the presence of novel small molecule inhibitor (PF-0449913) for 2 weeks. It significantly reduced LSC survival and self-renewal, while spared HSC. These effects were not observed in the absence of stroma. Importantly, pre-treatment of the AML stroma with PF-0449913 for 1 week prior- to co-culture also resulted in LSC’s inhibition of survival and self-renewal. AML- and normal BM-derived stroma differ in their ability to support HSC and LSC: LSC (n=6) were capable to self-renew after 9 weeks of co-culture with both normal and AML stroma, while cord blood (n=4) as well as normal BM (n=5) HSC lost their self-renewal potential after only 2 weeks of co-culture with the AML stroma. PF-0449913 induced changes in AML stroma. which was previously unsupportive to HSC survival, it became compliant : self-renewal of HSC increased more than 3 times. It reversed also the inhibitory effect of AML stroma-derived conditioned media (CM). Pre-treatment of the HS-5 cells with CM from AML stroma for 4 weeks prior to co-culture experiments led to significant inhibition of the cord blood (n=3) and NBM (n=3) HSC survival and self-renewal. However, in CM obtained from AML stroma treated with PF-0449913 that effect was reversed. Combined treatment of AML cells (n=6) with PF-0449913 and de-methylating agent Vidaza (5-azacytodine) or CXCR4 antagonist AMD3100 resulted in even greater inhibition of LSC survival and self-renewal. However, Vidaza did not mediate changes in AML stroma. Conclusions Together these data indicate that, while both conditioned media and co-culture with AML stroma impaired HSC survival and self-renewal, co-culture conditions resulted in a greater reduction in survival and self-renewal capacity, implicating that cell-cell contact or unstable secreted factors exacerbate the effects. That suggests that microenvironmental cues play a key role in regulating normal HSC versus LSC survival and maintenance, and leukemic stroma exibit severely compromised ability to maintain normal HSCs, but effectively support LSCs. PF-0449913treatment of AML induces not only eradication of LSC, but also mediate changes in AML stroma, supporting HSC generation and maintenance. Simultaneous targeting of both AML niche and LSC could represent a novel avenue for treatment of AML patients. Disclosures No relevant conflicts of interest to declare.
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Agarwal, Puneet, Hui Li, Andrew J. Paterson, Jianbo He, Takashi Nagasawa, and Ravi Bhatia. "Role of CXCL12-Expressing Bone Marrow Populations in Leukemic Stem Cell Regulation." Blood 128, no. 22 (December 2, 2016): 26. http://dx.doi.org/10.1182/blood.v128.22.26.26.
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Abstract CXCL12 is the major chemoattractant for hematopoietic stem cells (HSC) in the bone marrow microenvironment (BMM), and plays a major role in HSC localization to their regulatory niches. Studies using genetic drivers/reporters have shown that CXCL12 deletion from perivascular mesenchymal stem cells (MSC) and CAR cells (using Prx1-Cre line) leads to loss of HSC quiescence, frequency and self-renewal potential, while deletion from endothelial cells (using Tek-Cre line) results in modest loss of HSC long-term repopulating activity. In contrast, deletion of CXCL12 from osteoprogenitors (using Osx-Cre line) resulted in HSC mobilization without any effect on HSC function, while deletion from mature osteoblasts (using OCN-Cre line) had no effect on HSC function (Greenbaum et al. Nature. 2013;495(7440):227-30; Ding et al. Nature. 2013;495(7440):231-5.). These studies have been useful in identifying MSC/CAR cells and endothelial cells as important HSC niche components but the BM niches for LSC remain poorly characterized. In the present study, we examined alterations in CXCL12-producing niche cells in the CML BMM, and their role in regulating LSC growth, using the SCL-tTA BCR-ABL mouse model of CML. Our previous studies indicated that CXCL12 levels are reduced in CML compared to normal BM (Zhang et al. Cancer Cell. 2012; 21(4):577). To evaluate the effect of leukemia development on specific CXCL12-expressing cell populations in the BMM, we crossed CXCL12GFP mice (GFP reporter knocked into the CXCL12 locus) with SCL-tTA-BCR-ABL mice to generate CXCL12GFP-SCL-tTA-BCR-ABL mice. CXCL12-expressing cells in the BM were identified by GFP expression. Mice developing CML after BCR-ABL induction by tet-withdrawal demonstrated significantly increased numbers of GFP+ endothelial cells (CD45-Ter119-CD31+) and reduced numbers of GFP+ BM stromal cells (CD45-Ter119-31-) compared to WT mice. Within the stromal population, the number of GFP+ MSC (PDGFRα+Sca-1+) were decreased. To evaluate the contribution of CXCL12-expressing populations towards LSC regulation, we crossed CXCL12f/f mice (loxP sites flanking exon 2) with Tek-Cre, Prx1-Cre, OCN-Cre and OSX-Cre transgenic lines. CML BM cells (CD45.1/2+; 2*106/mouse) were transplanted into lethally irradiated (8Gy) WT (CD45.2) Cre- or Cre+ CXCL12f/f knockout animals, and followed for CML development. When compared to WT mice, CXCL12f/f-Tek-Cre+ animals exhibited significantly reduced engraftment of CML cells (CD45.1/2+) in the BM, with associated reduction in CML myeloid cells (Gr-1+Mac-1+), MEP (CD16/32- CD34-), CMP (CD16/32lowCD34+), MPP (LSK+CD48+) and LTHSC (CD150+CD48-) numbers. No changes in splenic engraftment were seen. To evaluate long-term reconstitution, BM cells from primary transplanted WT or knockout animals were transplanted into lethally irradiated (8Gy) WT secondary recipients. CML engraftment in secondary mice receiving BM from Tek-Cre+ and WT animals was similiar at 12 weeks, indicating that residual LTHSC retained repopulating potential. In contrast, CXCL12f/f-OCN-Cre and CXCL12f/f-Osx-Cre mice did not demonstrate significant differences in total CML cell or CML LTHSC engraftment, but showed increased LMPP engraftment in the BM. Interestingly CXCL12f/f-Prx1-Cre+ animals exhibited significantly increased leukocytosis and BM cellularity, and increased MEP, CMP, LMPP, MPP, STHSC and LTHSC numbers in the BM compared to WT mice. Increased numbers of CML myeloid cells and LSK were seen in the peripheral blood, but no change in splenic engraftment was seen. CML engraftment in secondary mice receiving BM from Prx1-Cre+ animals was significantly increased at 12 weeks compared to WT animals, indicating that the expanded LTHSC population maintained repopulating potential. These results suggest that loss of endothelial cell expressed CXCL12 reduced CML LTHSC in BM, whereas loss of MSC/CAR cell expressed CXCL12 enhances CML LTHSC numbers in BM, in association with increased mobilization to PB. Collectively, these results reveal important and distinct niche functions for CXCL12 expressing BM endothelial cell and MSC/CAR cells in CML, and indicate significant differences in niche regulation of CML LSC compared with normal HSC. We expect that improved characterization of BM niches in CML will facilitate further dissection of key niche interactions underlying LSC maintenance and expansion. Disclosures No relevant conflicts of interest to declare.
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Krauss, Aviva C., Martin Guimond, Stefania Dobre, and Crystal L. Mackall. "Tyrosine Kinase Inhibition with Sunitinib Facilitates Thymic Engraftment by Modulating Thymic Niche Accessibility." Blood 112, no. 11 (November 16, 2008): 72. http://dx.doi.org/10.1182/blood.v112.11.72.72.
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Abstract Stable mixed chimerism is likely to be sufficient for clinical benefit when HSCT is undertaken for benign disease. However, potential short and long term morbidities of current ablative or non-myeloablative conditioning regimens present an unfavorable risk:benefit ratio when HSCT is undertaken in this setting. We sought to develop an entirely non-cytotoxic approach to achieve stable mixed chimerism by modulating accessibility of thymic niches. Previous work identified occupancy of the double negative (DN)3 niche (CD3-4-8-44-25+) as a primary barrier to thymic engraftment based upon evidence that IL-7Rα−/− and γc−/− mice (who have an accessible DN3 niche) are receptive to thymic engraftment, whereas RAG−/− mice (who have a full DN3 niche) are resistant to thymic engraftment. Our results are consistent with this paradigm, since transfer of 5 × 106 TCD BM cells from WT into IL-7Rα−/− mice vs RAG−/− mice showed higher rates of thymic chimerism (mean donor % 83±8 vs 3±3 respectively, p=0.001). In an attempt to “open” the DN3 niche, we pre-treated RAG−/− recipients of WT BM with PC61, an anti-CD25 MoAb that diminished DN3 cell numbers (pre-44±11 × 105, post-14±1 × 105). Anti-CD25 therapy modestly enhanced donor chimerism (20±9%, n=12 vs 12±6%, n=10, p=NS) and significantly increased thymic engraftment (90±20 vs 41±11 x106 thymocytes, p=0.05) in anti-CD25 treated RAG−/− mice vs controls respectively. Importantly however, modulation of the DN3 niche via anti-CD25 treatment in RAG−/− mice did not increase thymic chimerism to the levels achieved in IL-7Rα−/− mice which have defects in the DN1/DN2 niches, implicating a role for DN1/DN2 niche accessibility as well. Indeed, RAG−/−γc−/− mice were receptive to thymic engraftment, with mean donor chimerism of 81.84±14% and a 13-fold increase in thymic cellularity after transplantation of WT BM cells without any prior conditioning. We postulated therefore that the limited efficacy of anti-CD25 alone when RAG−/− recipients were used was due to the fact that donor thymic engraftment is limited by accessibility to the entire range of DN1, DN2 and DN3 niches, while anti-CD25 selectively modulates the DN3 niche. Current models hold that growth factors critical for DN1 and DN2 thymocytes include SCF, FLT3L, and IL-7, among others. To test whether targeted pharmacotherapy could diminish the competitive advantage of resident DN1 and DN2 thymocytes and render the DN1/DN2 niche accessible, we treated RAG−/− recipients with Sunitinib, a receptor tyrosine-kinase (TK) inhibitor that inhibits c-kit and Flt-3, which signal SCF and FLT3L respectively. Sunitinib treatment of donors (60 mg/kg/d on Days -4, -3, -2, -1) followed by transfer of 5 × 106 TCD WT BM cells on Day 0 and 1 enhanced donor chimerism and thymic engraftment (10±4%, n=9 vs 2±0.7%, n=8 donor chimerism, p=0.01; 43±5 vs 13±1 × 106 thymocytes, p<0.0001) in Sunitinib vs. vehicle treated recipients, respectively. Combined anti-CD25 and Sunitinib therapy was superior to Sunitinib alone prior to transplantation of TCD WT BM (15±4%, n=5 vs 7±2%, n=5 donor chimerism, p=NS; 74±14 vs 25±6 × 106 thymocytes, p=0.02) We postulate that Sunitinib plus anti-CD25 renders DN1-DN3 thymic niches accessible to transferred thymic progenitors. This therapy is virtually non-toxic, and could provide new opportunities for HSCT for benign disease. Ongoing studies are underway to clarify effects of this regimen on marrow stem cell engraftment, durability of engraftment, and activity in a minor-mismatched model.
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Pievani, Alice, Marta Biondi, Chiara Tomasoni, Andrea Biondi, and Marta Serafini. "Location First: Targeting Acute Myeloid Leukemia Within Its Niche." Journal of Clinical Medicine 9, no. 5 (May 18, 2020): 1513. http://dx.doi.org/10.3390/jcm9051513.
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Despite extensive research and development of new treatments, acute myeloid leukemia (AML)-backbone therapy has remained essentially unchanged over the last decades and is frequently associated with poor outcomes. Eradicating the leukemic stem cells (LSCs) is the ultimate challenge in the treatment of AML. Emerging evidence suggests that AML remodels the bone marrow (BM) niche into a leukemia-permissive microenvironment while suppressing normal hematopoiesis. The mechanism of stromal-mediated protection of leukemic cells in the BM is complex and involves many adhesion molecules, chemokines, and cytokines. Targeting these factors may represent a valuable approach to complement existing therapies and overcome microenvironment-mediated drug resistance. Some strategies for dislodging LSCs and leukemic blasts from their protective niche have already been tested in patients and are in different phases of the process of clinical development. Other strategies, such as targeting the stromal cells remodeling processes, remain at pre-clinical stages. Development of humanized xenograft mouse models, which overcome the mismatch between human leukemia cells and the mouse BM niche, is required to generate physiologically relevant, patient-specific human niches in mice that can be used to unravel the role of human AML microenvironment and to carry out preclinical studies for the development of new targeted therapies.
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Wang, Jiwei, Taral Lunavat, Wenjing Zhou, Zichao Feng, Tuyen Hoang, and Frits Thorsen. "TMOD-04. INHIBITION OF MELANOMA BRAIN METASTASIS BY TARGETING miR146a." Neuro-Oncology 22, Supplement_2 (November 2020): ii228. http://dx.doi.org/10.1093/neuonc/noaa215.955.
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Abstract INTRODUCTION It is well known that cancer patients with brain metastasis (BM) have a poor prognosis and current treatments are not effective. It has been shown that BM development is caused by molecular interactions between the tumor cells and the normal brain, constituting the so-called brain metastatic niche. In this respect, miRNAs delivered by exosomes released by the primary tumor cells seem to be an important factor in BM niche establishment yet the mechanisms behind this process is poorly understood. AIM The aim of this project was to determine miRNAs released by exosomes from melanomas that may be important in establishing the BM niche, and to validate these findings in vitro and in vivo. METHODS We performed a miRNA profiling on exosomes collected from 3 human melanoma BM cell lines and from human astrocytes and melanocytes, to determine the most important differently expressed miRNAs. Functional in vitro validation was performed by cell growth and migration assays, cytokine arrays, qPCR and Western blots. Functional in vivo studies were performed after miR-knockdown in BM cell lines, in an experimental animal/tumor model. RESULTS AND DISCUSSION The miRNA profiling showed that miR-146a was by far the most differentially upregulated miRNA in exosomes from melanoma BM cell lines, compared to exosomes from normal cells. Mir-146a mimics activated human astrocytes, shown by increased proliferation and migration, elevated expression of GFAP in vitro and in mouse brain tumor samples, and increased cytokine production. These effects were reversed by adding anti-miR-146a to the experiments. In animal studies, knockdown of miR-146 in melanoma BM cells injected intracardially into nod/scid mice resulted in a significant reduction in BM burden, compared to wild type cells, and increased animal survival. CONCLUSIONS Inhibition of miR-146a reduces activation of the BM niche, leading to inhibition of BM development.
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Yang, Li-Jun, Ilicia L. Schlossman, Samuel E. Myrick, Haoyang Zhuang, Hai Wang, and Westley H. Reeves. "Role Of Tumor Necrosis Factor α In Bone Marrow Niche Dysfunction Of Patients With Systemic Lupus Erythematosus (SLE)." Blood 122, no. 21 (November 15, 2013): 1036. http://dx.doi.org/10.1182/blood.v122.21.1036.1036.
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Abstract Objectives and background SLE is a chronic systemic autoimmune disorder associated with autoantibodies and cytopenias. There are few studies of the pathogenesis of anemia of chronic inflammation in lupus. This study addresses the pathogenesis of the hematological manifestations of lupus. We have shown that SLE bone marrow (BM) exhibits striking death of niche and hematopoietic cells associated with tumor necrosis factor-α (TNFα) over-production. Here, we further examined the pathogenesis of hematological BM niche dysfunction. Methods Pathology records over the past 10 years from the University of Florida were reviewed and BM aspirates/core biopsies from 6 SLE patients were identified for further study. Wright-Giemsa stained BM aspirate smears and cytospin preparations, hematoxylin and eosin (H&E)-stained and reticulin stained BM core biopsies were reviewed. Immunohistochemistry (IHC) for TNFα, cleaved caspase-3, and CD71 was performed on core biopsies and expression levels were quantified morphometrically. BM specimens from individuals undergoing staging for lymphoma were selected as controls. Results Five of 6 SLE patients had nephritis and 3 were direct Coombs+ (one with hemolytic anemia). Mean hemoglobin was 9.1g/dL in SLE patients and 11.9g/dL in controls. Mean WBC was 3800/mm3 in patients and 8200/mm3 in controls, and mean platelet counts were 133,000/mm3 and 258,000/mm3, respectively. BM aspirates exhibited numerous apoptotic cells, erythroid dyspoiesis, plasmacytosis, hemophagocytosis, and phagocytosis of nuclear material by mature neutrophils (LE cells). Numerous LE cells were seen in 5/6 SLE BM aspirates. Compared to normal BM biopsies, SLE BM biopsies exhibited hypocellularity, erythroid dyspoiesis, polyclonal plasmacytosis, mild reticulin fibrosis, BM stromal damage/disorganization and 3 out of 6 with interstitial lymphoid aggregates. IHC with anti-cleaved caspase-3 antibodies, a specific marker of cell death, revealed numerous caspase-3+ cells in a range of 20-40% of total BM cells in contrast to control BMs of <5%. Interestingly, a large majority of osteoblasts or lining cells in BM osteal niches were caspase-3+ in SLE BM, whereas caspase-3+ cells were rare in control BM biopsies. Using double IHC (TUNEL assay plus anti-CD71 or myeloperoxidase antibodies), prominent apoptotic erythroid precursors with less extensive cell death of the myeloid and megakaryocytic lineages was found in SLE BM core biopsies. In SLE, caspase-3+ cells occupied 9-12% of the total BM area vs. 1-2% in control BM (p<0.001). As TNFα promotes Fas-mediated apoptosis and may damage hematopoietic precursors and/or stromal cells, we examined its production in SLE BM. IHC with anti-TNFα antibodies revealed intense intra- and extracellular staining surrounding neutrophils and monocytes, and the extent of TNFα staining was dramatically higher in SLE patients’ BM biopsies than in controls. Staining was particularly intense adjacent to presumptive osteoblasts lining the surface of bone trabeculae, though there also was staining in interstitial regions. There was little TNFα staining of control BM. Morphometric analysis revealed 10-18% of the area in SLE BM intensely stained with anti-TNFα vs. 1-3.5% in controls (P < 0.001, Student t-test), suggesting that TNFα overproduction may cause BM niche dysfunction. Conclusion BM TNFα-mediated niche cell apoptosis is likely to be involved in the pathogenesis of SLE-associated hematological abnormalities. Disclosures: No relevant conflicts of interest to declare.
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Aprile, Annamaria, Silvia Sighinolfi, Laura Raggi, and Giuliana Ferrari. "Targeting the Hematopoietic Stem Cell Niche in β-Thalassemia and Sickle Cell Disease." Pharmaceuticals 15, no. 5 (May 11, 2022): 592. http://dx.doi.org/10.3390/ph15050592.
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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.
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Mizukami, Takuo, Kazuya Takizawa, Madoka Kuramitsu, Haruka Momose, Jumpei Yamazaki, Atsuko Masumi, William W. Hall, Hideki Hasegawa, Kazunari Yamaguchi, and Isao Hamaguchi. "Identification of Leukemic Stem Cells and Their Niche in Adult T Cell Leukemia Using the Tax-Transgenic Mouse Model." Blood 120, no. 21 (November 16, 2012): 1877. http://dx.doi.org/10.1182/blood.v120.21.1877.1877.
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Abstract Abstract 1877 Adult T cell leukemia (ATL) is a lymphoproliferative disorder caused by infection with HTLV-I. Although various chemotherapies have shown significant complete remission rates, most of the treated patients relapse. These data indicate the existence of leukemic stem cells (LSCs) and a specific niche that regulates stemness and protects these cells from chemotherapy. We have reported in previous studies that the ATL-LSCs isolated from a Tax-transgenic (Tax-Tg) mouse are enriched in the CD117+/CD38–/CD71– fraction of the lymphoma, and LSCs have the potential to reproduce the original tumor when transplanted into a NOD/SCID mouse (Yamazaki et al., Blood, 2009). However, the niche of ATL-LSCs in the spleen, bone marrow (BM), thymus and lymph node (LN) is still unclear. To identify the ATL-LSC niche in vivo, we performed a homing assay. Lymphoma cells isolated from a Tax-Tg mouse were GFP transduced by a lentivirus, and then sorted GFP+ cells (2×106) were transplanted intraperitoneally into a non-irradiated NOD/SCID mouse. The homing of GFP+ cells to tissues was traced by flow cytometry (FCM) at 16 hours and 3, 7, 14 and 21 days after transplantation. At 16 hours after transplantation, GFP+ lymphoma cells were detected in the spleen and BM. No GFP+ lymphoma cells were detected in the thymus and LN. Interestingly, more than 60% of first colonized cells in the spleen and BM at 16 hours were AT-LSCs (GFP+/CD117+ cells). From day 3 to 7, more than 40% of proliferating cells in the BM and spleen were ATL-LSCs. At day 3, only a few non-ATL-LSCs (GFP+/CD117–cells) were detected in the thymus, LN and peripheral blood. The number of GFP+ cells was drastically increased at day 14 in the spleen. These data indicate that ATL-LSCs prefer to colonize and proliferate in the spleen and BM. To identify the specific niche of ATL-LSCs in the spleen and BM, we performed imaging analysis of ATL-LSCs. ATL-LSCs (GFP+/CD117+ and CD38–/CD71–/CD117+cells) were mainly localized near the vascular region in the spleen and endosteal region of trabecular bone in the BM. We found that some ATL-LSCs were attached to reticular cells (RC) in the spleen. In the BM, ATL-LSCs cells were localized at the endosteal region of the trabecular bone. Interestingly, similar to the spleen, RCs were observed at the endosteal region and contacted ATL-LSCs in the BM. FCM analysis confirmed that the number of reticular cells and mesenchymal stem cells (MSCs), were increased in the ATL BM and spleen. These data suggest that RCs are a possible candidate for the ATL-LSC niche and may be a new target of therapy. Finally, to characterize the ATL-LSC niche, we isolated osteoblastic cells, blood endothelial cells, lymphatic endothelial cells and reticular cells from normal and ATL BM to compare the gene expression profiles of each niche cell type. Here, together with DNA microarray analysis of ATL-LSCs both in the BM and spleen, we have characterized ATL-LSC niche cells both in the spleen and BM. Disclosures: No relevant conflicts of interest to declare.
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Noort, Willy A., Regina de Jong-Korlaar, Linda Lubbers-Aalders, Huipin Yuan, Joost D. de Bruijn, Constantine S. Mitsiades, Anton CM Martens, and Richard WJ Groen. "Addition of the Vascular Niche Component to the Human Bone Marrow-like Scaffold Model." Blood 126, no. 23 (December 3, 2015): 2402. http://dx.doi.org/10.1182/blood.v126.23.2402.2402.
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Abstract Previously, we have reported that our human bone marrow (BM)-like scaffold xenograft model allows the engraftment and outgrowth of normal and malignant hematopoiesis (e.g. multiple myeloma (MM), acute myelocytic/lymphocytic leukemia (AML/ALL) and MDS (Groen et al. Blood 2012; Gutierrez et al. JCI 2014 and data not shown). Whereas the presence of osteoblasts and bone of human origin mimics a human BM-like niche more closely than the murine BM in standard xenotransplant models (e.g. NOD-SCID/NSG mice), still some essential components of the human BM niche, i.e. human blood vessels, are missing. To this end, in addition to human mesenchymal stromal cells we now incorporated cord blood-derived endothelial progenitor cells (CB-EPCs) in the hybrid scaffold production process, to create a multi-tissue compartment that "maximally humanizes" the BM-like niche of our scaffolds. Towards successful implementation of a human vascular system we compared: i) scaffold material composition (biphasic calcium phosphate (BCP) vs. tricalcium phosphate (TCP)); ii) scaffold shape (particles vs. tubes); iii) different types of matrigel for CB-EPC embedding. Histological analysis of the humanized scaffolds, eight weeks after implantation in mice, showed a large number of functional human blood vessels, as indicated by hCD31+ staining and the presence of erythrocytes within. Comparison of the composition and the shapes of the scaffolds indicated superiority of TCP and tube-shaped scaffolds in supporting the formation of vessels. Further analysis of scaffolds for CD44, CD146, LEPR and nestin-positive cells, revealed the presence of other stromal niche cells besides human osteoblasts and endothelial cells. Irradiation of mice carrying these humanized implants did not have a significant deleterious effect on the established human vessels, allowing their further functional evaluation in xenotransplantation. Additionally, mice carrying tubes with and without human CB-EPC derived vessels (on either flank) were subsequently inoculated with adult BM-derived CD34-positive cells by intracardiac injection. Upon analysis 12 weeks later, all tubes showed multi-lineage hematopoietic outgrowth. Interestingly, CB-EPC embedment resulted in increased numbers of CD45+ (2-fold), CD13+ (4-fold) and CD7+ (2-fold), while CD19+ cell numbers were equal. In contrast, in mouse BM almost only CD19+ cells could be detected. Moreover, we observed that the use of CB-EPCs in our scaffolds provides faster kinetics of in vivo engraftment and growth of both patient-derived MM or AML cells. With the addition of both human CB-EPCs and human BM stromal cells, our scaffold systems now simulate both human endosteal and vascular niches of the BM, thereby more closely recapitulating the human hematopoietic niche. Disclosures Yuan: Xpand Biotechnology BV: Employment. de Bruijn:Xpand Biotechnology BV: Employment. Mitsiades:TEVA: Research Funding; Janssen/Johnson & Johnson: Research Funding; Novartis: Research Funding. Martens:Johnson & Johnson: Research Funding. Groen:Johnson & Johnson: Research Funding.
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Jeong, Seon-Yeong, Jin-A. Kim, and Il-Hoan Oh. "5-Fluorouracil Treatment Leads to Activation of Stem Cell Niche By Reconstructing Mesenchymal Stromal Cells and Exert a Distinct Microenvironmental Impact on Normal and Leukemic Cells." Blood 126, no. 23 (December 3, 2015): 1197. http://dx.doi.org/10.1182/blood.v126.23.1197.1197.
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Abstract Reactivation of endogenous hematopoietic stem cells (HSCs) are initiated by stimulation of bone marrow niche triggered by various injury signals. Here, we show that treatment with 5-fluorouracil (5-FU) leads to reconstruction of bone marrow (BM) microenvironment to establish an activated niche stimulating hematopoietic stem cells (HSCs). First, we show that pre-treatment with 5-FU leads to engraftment of donor cells in non-irradiated recipient mice without affecting the homing efficiency of HSCs into BM. The HSC activation effects were reproduced in-vitro by co-culturing hematopoietic cells with CD45-Ter119- stromal cells derived from 5-FU treated BM, but not by co-culture with CD45+ cells or stromal cells obtained from enzymatic digestion of bone from the same mice. Examination of BM mesenchymal cells after 5-FU treatment revealed a rapid emergence of high-proliferating mesenchymal progenitors exhibiting large size colony (CFU-F) and higher self-renewal of colonogenic cells 3-5 days after 5-FU treatment, which was concomitantly associated with regeneration of CD34+Lin-Sca-1+c-kit+ (LSK) cells in the same BM. The cellular changes in mesenchymal stroma was associated with rapid emergence of characteristic mesenchymal cell populations (PDGFR-a+/Leptin receptor+/SSEA-3+: PLS) with 650-folds increase of the PLS cells in BM in 3 days after 5-FU treatment. However, the increase of these PLS mesenchymal cells were not associated with increase in mitotic activity of mesenchymal cells (<5% BrdU+ cells), indicating phenotypic conversion of subpopulation in BM. Moreover, cellular changes in mesenchymal niche were associated with rapid increase of mesenchymal cells expressing cross-talk molecules such as CXCL-12 (20-folds), Jagged-1 (13-folds) and DLL-1 (15-folds). Furthermore, in-vivo administration of chemicals blocking CXCL-12 and notch signaling during the recovery from the 5-FU treatment led to the significant loss of LSK-SLAM cells in the regenerated BM. Interestingly, the BM niche activated by 5-FU exerted a distinct effect on normal and leukemic cells in a manner that it provide higher support on the primitive state of normal HSCs than for MN-1 induced leukemia cells. Thus, leukemic mice engrafted with MN-1 cells exhibited a decrease in primitive leukemic cell (Lin-c-kit+) and higher survival by 5-FU treatment than those treated by radiation. Taken together, our study reveals the cellular reconstruction of mesenchymal niche in BM during stimulus-induced niche activation and provides an insight on the selective niche targeting as a novel therapeutic strategies for hematological diseases. Disclosures No relevant conflicts of interest to declare.
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Mourcin, Frédéric, Caroline Breton, Julie Tellier, Priyanka Narang, Lionel Chasson, Audrey Jorquera, Mark Coles, Claudine Schiff, and Stéphane J. C. Mancini. "Galectin-1–expressing stromal cells constitute a specific niche for pre-BII cell development in mouse bone marrow." Blood 117, no. 24 (June 16, 2011): 6552–61. http://dx.doi.org/10.1182/blood-2010-12-323113.
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Abstract In the bone marrow (BM), stromal cells constitute a supportive tissue indispensable for the generation of pro-B/pre-BI, pre-BII, and immature B lymphocytes. IL-7–producing stromal cells constitute a cellular niche for pro-B/pre-BI cells, but no specific stromal cell microenvironment was identified for pre-BII cells expressing a functional pre-B cell receptor (pre-BCR). However expression of the pre-BCR represents a crucial checkpoint during B-cell development. We recently demonstrated that the stromal cell derived-galectin1 (GAL1) is a ligand for the pre-BCR, involved in the proliferation and differentiation of normal mouse pre-BII cells. Here we show that nonhematopoietic osteoblasts and reticular cells in the BM express GAL1. We observed that pre-BII cells, unlike the other B-cell subsets, were specifically localized in close contact with GAL1+ reticular cells. We also determined that IL-7+ and GAL1+ cells represent 2 distinct mesenchymal populations with different BM localization. These results demonstrate the existence of a pre-BII specific stromal cell niche and indicate that early B cells move from IL-7+ to GAL1+ supportive BM niches during their development.
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Yin, Xiuxiu, Linping Hu, Yawen Zhang, Caiying Zhu, Hui Cheng, Xiaowei Xie, Ming Shi, et al. "PDGFB-expressing mesenchymal stem cells improve human hematopoietic stem cell engraftment in immunodeficient mice." Bone Marrow Transplantation 55, no. 6 (December 5, 2019): 1029–40. http://dx.doi.org/10.1038/s41409-019-0766-z.
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AbstractThe bone marrow (BM) niche regulates multiple hematopoietic stem cell (HSC) processes. Clinical treatment for hematological malignancies by HSC transplantation often requires preconditioning via total body irradiation, which severely and irreversibly impairs the BM niche and HSC regeneration. Novel strategies are needed to enhance HSC regeneration in irradiated BM. We compared the effects of EGF, FGF2, and PDGFB on HSC regeneration using human mesenchymal stem cells (MSCs) that were transduced with these factors via lentiviral vectors. Among the above niche factors tested, MSCs transduced with PDGFB (PDGFB-MSCs) most significantly improved human HSC engraftment in immunodeficient mice. PDGFB-MSC-treated BM enhanced transplanted human HSC self-renewal in secondary transplantations more efficiently than GFP-transduced MSCs (GFP-MSCs). Gene set enrichment analysis showed increased antiapoptotic signaling in PDGFB-MSCs compared with GFP-MSCs. PDGFB-MSCs exhibited enhanced survival and expansion after transplantation, resulting in an enlarged humanized niche cell pool that provide a better humanized microenvironment to facilitate superior engraftment and proliferation of human hematopoietic cells. Our studies demonstrate the efficacy of PDGFB-MSCs in supporting human HSC engraftment.
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Landspersky, Theresa, Mehmet Saçma, Jennifer Rivière, Judith S. Hecker, Franziska Hettler, Erik Hameister, Katharina Brandstetter, et al. "Autophagy in mesenchymal progenitors protects mice against bone marrow failure after severe intermittent stress." Blood 139, no. 5 (February 3, 2022): 690–703. http://dx.doi.org/10.1182/blood.2021011775.
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Abstract The cellular mechanisms required to ensure homeostasis of the hematopoietic niche and the ability of this niche to support hematopoiesis upon stress remain elusive. We here identify Wnt5a in Osterix+ mesenchymal progenitor and stem cells (MSPCs) as a critical factor for niche-dependent hematopoiesis. Mice lacking Wnt5a in MSPCs suffer from stress-related bone marrow (BM) failure and increased mortality. Niche cells devoid of Wnt5a show defective actin stress fiber orientation due to an elevated activity of the small GTPase CDC42. This results in incorrect positioning of autophagosomes and lysosomes, thus reducing autophagy and increasing oxidative stress. In MSPCs from patients from BM failure states which share features of peripheral cytopenia and hypocellular BM, we find similar defects in actin stress fiber orientation, reduced and incorrect colocalization of autophagosomes and lysosomes, and CDC42 activation. Strikingly, a short pharmacological intervention to attenuate elevated CDC42 activation in vivo in mice prevents defective actin-anchored autophagy in MSPCs, salvages hematopoiesis and protects against lethal cytopenia upon stress. In summary, our study identifies Wnt5a as a restriction factor for niche homeostasis by affecting CDC42-regulated actin stress-fiber orientation and autophagy upon stress. Our data further imply a critical role for autophagy in MSPCs for adequate support of hematopoiesis by the niche upon stress and in human diseases characterized by peripheral cytopenias and hypocellular BM.
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Qing, Yulan, Yuan Lin, and Stanton L. Gerson. "An intrinsic BM hematopoietic niche occupancy defect of HSC in scid mice facilitates exogenous HSC engraftment." Blood 119, no. 7 (February 16, 2012): 1768–71. http://dx.doi.org/10.1182/blood-2011-05-350611.
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Abstract Although scid mice have been widely used for human HSC engraftment studies, the function of HSCs of scid mice has not been characterized. We hypothesized that the DNA repair defect of scid mice results in a stem cell defect that facilitates HSC engraftment. scid BM cells showed severely impaired repopulation potentials in the competitive repopulation assay. To assess the BM hematopoietic niche occupancy ability of scid HSC, WT BM cells were transplanted into scid mice without any conditioning and observed to achieve long-term engraftment. Furthermore, the defects of scid HSCs are independent of their inability to perform lymphopoiesis because a similar defect in hematopoietic niche occupancy was not observed with Rag1−/− recipients. These results demonstrate that scid HSCs are impaired in maintenance within the niche, which may explain the nature of the conducive marrow niche environment of scid mice for xenotransplantation.
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Hosokawa, Kentaro, Fumio Arai, and Toshio Suda. "N-Cadherin Induces Hematopoietic Stem Cells in a Quiescent State in the Bone Marrow Niche." Blood 106, no. 11 (November 16, 2005): 470. http://dx.doi.org/10.1182/blood.v106.11.470.470.
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Abstract Hematopoietic stem cells (HSCs) are responsible for blood cell production throughout the lifetime of individuals. Interaction of HSCs with their particular microenvironments, known as stem cell niches, is critical for maintaining the stem cell properties, including self-renewal capacity and the ability of differentiation into single or multiple lineages. The niche cells produce signaling molecules, extracellular matrix, and cell adhesion molecules, and regulate stem cell fates. Recently, it was clarified that long-term bone marrow (BM) repopulating (LTR) HSCs exist frequently in BM trabecular bone surface, and that N-cadherin + spindle-shaped osteoblasts (OBs) are identified as a major niche component. We found that side-population (SP) in c-Kit +Sca-1 +Lin −(KSL) fraction, which is the quiescent HSC in the OB niche, expressed N-cadherin. Expression of N-cadherin in both of the quiescent HSCs and OBs thought to be essential for an adherens junction between HSCs and OBs in the niche. However, the role of N-cadherin in hematopoiesis is still unclear. In this study, we focused on the function of N-cadherin in the maintenance of the stem cell specific property, such as cell adhesion, quiescence, and LTR-activity. To clarify the function of N-cadherin in hematopoiesis, we prepared the retroviruses expressing wild-type N-cadherin, transfected retroviruses into OP9 stromal cell line and KSL cells, and performed the coculture. After coculture of KSL cells with OP9 cells, long-term culture-initiating cells (LTC-ICs) were maintained on OP9 cells overexpressing WT-N-cadherin (OP9/WT-NCAD). In addition, overexpression of WT-N-cadherin in both of the KSL cells and stromal cells enhanced cobblestone formation. N-cadherin overexpressing KSL cell showed slow-cell division from the single cell, when they cultured on OP9/WT-N-cedherin or N-cadherin-Fc protein coated plates, suggesting that N-cadherin-mediated cell-cell adhesion between HSCs and stromal cells enhances the quiescence of HSCs and keeps HSCs in immature state in in vitro. To clarify the role of N-cadherin in the BM reconstitution ability of HSC, we transfected control-IRES-GFP, WT-N-cadherin-IRES-GFP and N-cedherin/390Δ-IRES-GFP retrovirus into the Ly5.1 BM mononuclear cells and transplanted into lethally irradiated Ly5.2 mice. N-cedherin/390Δ, which is a mutant N-cadherin with a deletion at the extracellular domain, exhibits a dominant negative effect on the activity of endogenous cadherins. Control and WT-N-cadherin expressing cell reconstitute the recipient mice BM, while N-cadherin/390Δ expressing cells did not. It suggests that the adhesion between HSCs and BM niche cell is indispensable for the LTR-activity. In addition, we found that WT-N-cadherin overexpressing HSCs were enriched in the SP fraction after 4 months of BM transplantation, indicating that N-cadherin-mediated cell adhesion induced HSCs in the quiescent and kept quiescent HSCs in the niche. Altogether, these observations suggest that N-cadherin is a critical niche factor for the maintenance of the quiescence and self-renewal activity of HSCs. N-cadherin promotes tight adhesion of HSCs to the niche and keeps HSCs in the quiescent state
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Aprile, Annamaria, Alessandro Gulino, Isabella Villa, Stefano Beretta, Ivan Merelli, Alessandro Rubinacci, Maurilio Ponzoni, et al. "Hematopoietic Stem Cell Function in β-Thalassemia Is Impaired and Is Rescued By Targeting the Bone Marrow Niche." Blood 134, Supplement_1 (November 13, 2019): 967. http://dx.doi.org/10.1182/blood-2019-126829.
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Hematopoietic stem cells (HSC) are regulated by signals from the bone marrow (BM) niche and little is known about their fate in altered hematological conditions associated to non-malignant diseases. In β-thalassemia ineffective erythropoiesis and secondary alterations, as abnormal regulation of bone metabolism, iron overload and hormonal factors, induce changes in the BM homeostasis with a potential impact on HSC-niche interaction. We addressed these unexplored issues in the murine disease model and in patients' cells. We investigated hematopoiesis in thalassemic Hbbth3/+ (th3) mutant mice and we found lower frequency, reduced quiescence and reconstituting potential of HSC. th3 HSC have impaired self-renewal, which is rescued upon transplantation in a normal BM, proving an active role of the niche microenvironment. Both stromal and hematopoietic components of the BM niche are altered in th3 mice. Consistently with the common finding of osteoporosis in patients, we found reduced bone deposition with decreased levels of parathyroid hormone (PTH), which is a key regulator of bone metabolism but also of HSC activity. Low PTH negatively affects bone deposition and expression of the Notch-ligand Jag1 by th3 mesenchymal and osteolineage cells, thus reducing the activation of Notch1 in HSC and consequently impairing their function. In vivo activation of PTH signaling through the reestablished Jag1-Notch1 pathway restores the functional pool of th3 HSC by correcting HSC-niche crosstalk. In addition to the stromal component of the BM, hematopoietic cells with a key role in regulating the fate of HSC, such as megakaryocytes (Mk), were found defective in maturation, possibly due to reduced circulating levels of thrombopoietin (TPO). We are currently investigating the molecular causes of dysmegakaryopoiesis and the Mk-HSC interaction in thalassemic mice. Strikingly, reduced HSC quiescence was confirmed in samples from patients affected by β-thalassemia, along with impaired stromal niche and megakaryopoiesis, thus highlighting the clinical relevance of our findings. Further investigation will unravel the multiple molecular mechanisms that affect in trans HSC functions in the complexity of the stressed thalassemic BM microenvironment. Our results uncover a defect of HSC in β-thalassemia, induced by an altered BM niche and provide new relevant insight for improving transplantation and gene therapy approaches. Disclosures No relevant conflicts of interest to declare.