scholarly journals IFNy Stimulation Induces Hematopoietic Stem Cell Homing and Niche Relocalization

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3726-3726
Author(s):  
Marcus A Florez ◽  
Katie A Matatall ◽  
Laura Ortinau ◽  
Roman Jaksik ◽  
Marek Kimmel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Terminal Differentiation ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
The Impact

Interferon gamma (IFNy) is a pro-inflammatory cytokine that is upregulated during chronic infections and chronic diseases, such as aplastic anemia, and has been associated with pancytopenia and diminished hematopoiesis. Studies have shown that IFNy negatively regulates hematopoietic stem cell (HSC) homeostasis by decreasing self-renewal and promoting terminal differentiation. The tight regulation of HSC homeostasis is dependent upon the bone marrow (BM) microenvironment, or BM niche. The BM niche is composed of a network of cell types that provide elaborate cell-cell interactions, cellular metabolites, transcriptional regulators, and local and distant humoral and neural signals that allow for hematopoietic homeostasis. In particular, CXCL12-abundant reticular (CAR) cells are vital to HSC maintenance, as depletion of CXCL12, or its receptor, leads to HSC depletion. However, the mechanism which IFNy activates HSCs and influences its interaction with the BM niche is unknown. We hypothesize that IFNy promotes HSC terminal differentiation and loss of quiescence by altering HSC interactions with the BM niche. To assess changes in HSC interactions with the BM niche upon IFNy stimulation, we performed intravital imaging using CXCL12 GFP reporter mice before and after administration of recombinant IFNy. We found that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. There was no change in distance with IFNy-receptor deficient HSCs, suggesting that movement away from the CAR cells was due to a cell autonomous IFNy-dependent mechanism. We performed gene expression analysis and transwell migration assays on HSCs from IFNy treated mice, and determined that there was no change in CXCL12 receptor (CXCR4) expression upon IFNy treatment, and IFNy did not alter migration towards CXCL12. These results suggest that HSC re-localization upon IFNy is independent of CXCL12 signaling. To explore the mechanism by which IFNy induces re-localization of HSCs, we first performed microarray analysis on HSCs from IFNy stimulated mice to assess what surface proteins were changed upon IFNy treatment. While there was no change in common HSC receptors thought to influence HSC homeostasis (cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1), we observed an increase in expression of bone marrow stromal antigen 2 (BST2). To explore the impact of BST2 on HSC homeostasis, quantification and proliferation analysis was performed on HSCs from Bst2-/- mice. Interestingly, Bst2-/- HSCs were significantly less proliferative and more abundant compared to controls. These studies suggest that BST2 may play a role in maintaining HSC homeostasis. The functional role of BST2 in cellular movement and adhesion has been studied in cancer. Increased BST2 expression has been associated with promoting the migration, adhesion and metastasis of various cancer cells. Since migration and adhesion is important for HSC homing, we assessed the effects of IFNy on HSC homing. Hematopoietic progenitors from IFNy-treated mice homed to the bone marrow with greater efficiency than PBS-treated controls, whereas progenitors from IFNy-receptor-deficientmice showed a decrease in homing. Additionally, WBM from IFNyR-/- had reduced engraftment than wildtype, consistent with a role for IFNy signaling in promoting HSC homing. The impact of BST2 on homing is currently being explored. In summary, we show that IFNy induces re-localization of HSCs away from quiescence-promoting CAR cells within the bone marrow niche via a mechanism that is independent of CXCL12 signaling. We further show that IFNy promotes HSC homing. The increased expression of BST2 on IFNy-stimulated HSCs appears to impact HSC proliferation and abundance in the bone marrow. Thus, BST2 may play a role in HSC activation and exit from quiescence. Expanding our understanding of the mechanism that drives HSC activation and terminal differentiation has important implications for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Activation of Non-Canonical Immune Signalling Pathways Drives Marrow Failure in a Murine Model of MDS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3246-3246
Author(s):  
Rawa Ibrahim ◽  
Joanna Wegrzyn ◽  
Linda Ya-Ting Chang ◽  
Patricia Umlandt ◽  
Jeff Lam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Signalling Pathway ◽  
Gene Set Enrichment Analysis ◽  
Mouse Bone Marrow ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem

Abstract The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

KIT Blockade Is Sufficient to Sustain Donor Hematopoietic Stem Cell Engraftment in Fanconi Anemia Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1206-1206
Author(s):  
Shanmuganathan Chandrakasan ◽  
Rajeswari Jayavaradhan ◽  
Ernst John ◽  
Archana Shrestha ◽  
Phillip Dexheimer ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Conditioning Regimen ◽  
Cell Transplant ◽  
Hematopoietic Stem ◽  
Increased Risk

Abstract Background: Fanconi anemia (FA) is the most common cause of inherited bone marrow failure (BMF). Currently, the only curative option for the BMF in FA is an allogenic hematopoietic stem cell transplant (HSCT). However, due to the underlying DNA repair defect, FA patients poorly tolerate alkylating chemotherapy or irradiation based conditioning, which is necessary for donor engraftment. However, this results in significant short and long term morbidity/mortality and augments the inherent increased risk of malignancies in FA patients. To overcome the adverse effects associated with alkylating conditioning agents, alternate experimental approaches exploiting the inherent hematopoietic stem cell (HSC) defect in FA are of utmost clinical necessity. Objective: To develop a safe KIT blocking antibody (KIT-Ab) based HSCT conditioning regimen for FA that does not involve chemotherapy or irradiation. Method: High purity KIT-Ab was made from the ACK2 hybridoma and its specificity to KIT binding was validated using mast cell assay. Baseline peripheral blood cells and the bone marrow hematopoietic stem and progenitor cell (HSPC) compartment (Lin-Kit+Sca+ and Lin-Kit+Sca+CD150+CD48- cells) of FANCA-/- and FANCD2-/- murine models were analyzed. Mechanistic studies using sorted FA bone marrow HSPC were performed ex vivo. This was followed by definitive primary and secondary transplants experiments following injection of KIT-Ab. Results: Several features of FA hematopoietic stem/progenitor cells (HSPC) suggested their susceptibility to KIT-Ab blockade-mediated killing: (a) Expression of KIT was significantly lower in FANCA-/- HSPC, while expression of its ligand was higher in bone marrow stroma; (b) Moreover, genes associated with apoptosis/senescence, stress and inflammatory signaling that were upregulated in WT-HSPC following KIT-Ab blockade, were upregulated in FANCA-/- HSPC at baseline; (c) Furthermore, FANCA-/- HSPC demonstrated increased susceptibility to KIT-Ab mediated apoptosis and had a reduced proliferative capacity. In-vivo studies following ACK2 injection showed a marked reduction of colony-forming units (CFU-C) from both FANCA-/- and FANCD2-/- mice one week following injection, when compared to WT mice (48% and 76% decrease in CFU-C, respectively). Based on these findings, we evaluated the role of ACK2 as a sole HSCT conditioning regimen in FANCA-/- and FANCD2-/- mice. Indeed, definitive HSCT in both FANCA-/- and FANCD2-/- mice using KIT-Ab based conditioning resulted in donor HSC engraftment with multi-lineage chimerism, which progressively increased to 22-24% by 4-months, and was sustained in secondary transplants. Overall, we show that KIT-blockade alone is an adequate non-genotoxic HSPC-targeted conditioning in FA mice, and its clinical translation could circumvent the extensive transplant-related morbidity/mortality in this disease. Disclosures No relevant conflicts of interest to declare.

The Zebrafish Provides Mechanistic Insights into the Role of Poly(A)-Specific Ribonuclease (PARN) in Hematopoietic Stem Cell (HSC) Homeostasis and Bone Marrow Failure

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 668-668
Author(s):  
Adam P Deveau ◽  
Andrew J Coombs ◽  
Santhosh Dhanraj ◽  
Gretchen Wagner ◽  
Yigal Dror ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Phosphohistone H3 ◽  
Biallelic Mutations ◽  
Insight Into

Abstract Development of tissues during embryogenesis and their homeostasis after formation are highly regulated by expression of coding and non-coding RNAs. Deadenylation is a core mechanism that regulates RNA function and fate by controlling turnover, abundance and maturation of RNA. Factors that promote or inhibit deadenylation control hematopoietic stem cell (HSC) homeostasis, and inhibition of deadenylation limits differentiation of the HSCs. Importantly, RNA biogenesis has emerged as a mechanism underlying several inherited bone marrow failure syndromes (IBMFSs), such as Diamond Blackfan anemia, dyskeratosis congenita (DC) and Shwachman-Diamond syndrome. Poly(A)-specific ribonuclease (PARN) is a major deadenylation factor and demonstrates high specificity for single-stranded poly (A) tails of various RNA species. We recently identified biallelic mutations in PARN as a cause of hematopoietic failure and profound hypomyelination, similar to the severe form of DC, Hoyeraal-Hreidersson syndrome. We developed a zebrafish model to characterize the hematopoietic phenotype of a patient identified to have severe inherited bone marrow failure resulting from a combined deletion of PARN on one allele and missense mutation in the other. Zebrafish posses a single parn ortholog. Zebrafish parn protein shares homology and high sequence identity (~64%) to its human counterpart. Embryos were injected with either translation start-site or splice-site-blocking morpholino at the one-cell stage. Both morpholino injections resulted in anemic embryos at 48 hours post fertilization (hpf), as evidenced by reduced o-dianisidine staining and gata1 expression by whole-mount in situ hybrization and GFP+ red cell numbers by fluorescence-activated cell sorting (FACS). Morphant embryos also demonstrated reduced expression of myeloid cell markers including l-plastin, myeloperoxidase, and macrophage expressed gene 1 and were leukopenic as evidenced by reduced number of GFP+ myeloid cells. FACS analysis revealed that fluorescently labeled HSCs were increased in parn morphants. Early hematopoietic markers, lmo2 and fli1, expressed in hemogenic and vascular tissue respectively, were also overexpressed in parn morphants. Furthermore, there was reduced global cell proliferation in morphant embryos as determined by phosphohistone H3 antibody staining. These findings suggest that the absence of parn results in a developmental arrest at the HSC stage with an inability to differentiate into leukocyte or erythroid lineages. Similarly, human cell culture data from PARN-deficient HSC/progenitor cells demonstrated markedly reduced colony forming capacity. By modeling parn deficiency in the zebrafish, we validate for the first time an IBMFS that results from biallelic mutations in a major deadenylating protein. Moreover, our zebrafish studies provide insight into the role of parn in maintaining HSC homeostasis/differentiation as the origin of the pancytopenia observed in this patient. Permanent knockouts in the zebrafish using CRISPR/Cas9 technology are underway, which will enable tracking the hematopoietic phenotype into adulthood. These studies have set the stage for critical translational research in a rare form of bone marrow failure as well as new insight into HSC regulation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Oxymetholone Therapy of Fanconi Anemia Induces Hematopoietic Stem Cell Cycling By Suppressing Osteopontin Transcription

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2949-2949
Author(s):  
Qingshuo Zhang ◽  
Eric Benedetti ◽  
Matthew Deater ◽  
Kathryn Schubert ◽  
Angela Major ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Proliferation ◽  
Stem Cell ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematological Parameters ◽  
Hematopoietic Stem ◽  
Stem Cell Proliferation

Abstract Androgens are widely used for treating Fanconi anemia and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2-/- mice were used to assess the therapeutic efficacy of oxymetholone and its mechanism of action. 18-month old Fancd2-/- mice recapitulated key human Fanconi anemia phenotypes including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic oxymetholone treatment significantly improved these hematological parameters by stimulating the proliferation of hematopoietic stem and progenitor cells. RNAseq analysis implicated down-regulation of osteopontin as an important mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic oxymetholone therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. Disclosures No relevant conflicts of interest to declare.

scholarly journals ALLOGENEIC HEMATOPOIETIC STEM CELL TRANSPLANTATION FOR ADULT PATIENTS WITH FANCONI ANEMIA.

2016 ◽  
Vol 8 ◽  
pp. 2016054 ◽  
Author(s):  
Hosein Kamranzadeh fumani ◽  
Mohammad Zokaasadi ◽  
Amir Kasaeian ◽  
Kamran Alimoghaddam ◽  
Asadollah Mousavi ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cell Transplantation ◽  
Hematopoietic Stem Cell ◽  
Fanconi Anemia ◽  
Bone Marrow Failure ◽  
Term Survival ◽  
Hematopoietic Stem

Background & objectives: Fanconi anemia (FA) is a rare genetic disorder caused by an impaired DNA repair mechanism which leads to an increased tendency toward malignancies and progressive bone marrow failure. The only curative management available for hematologic abnormalities in FA patients is hematopoietic stem cell transplantation (HSCT). This study aimed to evaluate the role of HSCT in FA patients.Methods: Twenty FA patients with ages of 16 or more who underwent HSCT between 2002 and 2015 enrolled in this study. All transplants were allogeneic and the stem cell source was peripheral blood and all patients had a full HLA-matched donor.Results: Eleven patients were female and 9 male (55% and 45%). Mean age was 24.05 years. Mortality rate was 50% (n=10) and the main cause of death was GVHD. Survival analysis showed an overall 5-year survival of 53.63% and 13 year survival of 45.96 % among patients.Conclusion: HSCT is the only curative management for bone marrow failure in FA patients and despite high rate of mortality and morbidity it seems to be an appropriate treatment with an acceptable long term survival rate for adolescent and adult group.

scholarly journals Detection of Mutations in Inherited Bone Marrow Failure and Myelodysplastic Syndrome Genes Using Genomic Capture and Massively Parallel Sequencing in Clinical Diagnostics

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1507-1507
Author(s):  
Siobán B. Keel ◽  
Tom Walsh ◽  
Colin Pritchard ◽  
Akiko Shimamura ◽  
Mary-Claire King ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Genetic Testing ◽  
Hematopoietic Stem Cell ◽  
Copy Number ◽  
Bone Marrow Failure ◽  
Copy Number Variants ◽  
Donor Selection ◽  
Hematopoietic Stem ◽  
Pathogenic Mutations

Abstract Accurate and timely diagnosis of inherited bone marrow failure (BMF) and myelodysplastic syndromes (MDS) ensures appropriate clinical management. The correct diagnosis allows appropriate monitoring for both hematopoietic (i.e. clonal evolution and progressive marrow failure) and extra-hematopoietic complications, informs the timing of hematopoietic stem cell transplant, donor selection and transplant regimen planning, and ensures appropriate genetic counseling of family members. Substantial phenotypic overlap among these disorders and the variable expressivity within syndromes complicate their diagnosis based purely on physical exam and standard laboratory testing and provide the rationale for comprehensive genetic diagnostic testing. We report here our initial one-year experience utilizing a targeted capture assay of known inherited BMF/MDS genes for clinical diagnostic purposes at the University of Washington. The assay sequences all exons and 20 base pairs of intronic sequence flanking each exon, as well as several regulatory and intronic regions of specific genes containing known pathogenic variants of 85 known inherited BMF/MDS genes (Zhang M. et al. Haematologica 2016). Between June 2015 and July 2016, 81 individual patients were referred for clinical testing (median age: 15 years-old, range: 0.6-76 years-old). For all samples evaluated, median coverage across the 383kb targeted region was 1887X. This depth of coverage enabled identification of all classes of mutations, including point mutations, small indels, copy number variants, and genomic rearrangements. Pathologic mutations in known inherited BMF/MDS genes were identified in 12 of 82 (14.6%) individuals (median age 13 years-old, range: 1.25-43 years-old) across a broad number of genes and of multiple classes including copy number variants (Table). Among the twelve patients with pathogenic mutations in inherited BMF/MDS genes, genetic testing was consistent with the prior clinical diagnoses of eight patients, including two Fanconi anemia patients subtyped as complementation group A, one of whom demonstrated reversion to wild-type resulting in mosaicism in the peripheral blood. Importantly, four patients carried no specific inherited BMF/MDS diagnosis prior to testing and were found to have pathogenic mutations in RPS10, RTEL1 and RUNX1 (ID 005, 008, 009, 010), suggesting additional diagnostic value to a multiplexed genetic approach in the clinical setting. Detailed clinical information was available for nine of the patients diagnosed with pathogenic mutations, two of whom have or will undergo a sibling or haploidentical hematopoietic stem cell transplantation (009 and 012, respectively) and thus genetic testing informed donor selection. To improve diagnostic accuracy, we are now updating the capture design to include newly discovered inherited BMF/MDS genes and intronic regions to optimize copy number variant detection. We are additionally pursuing CLIA-certified RNA analyses to characterize whether several variants bioinformatically predicted to affect splicing are functionally deleterious. Next-generation sequencing for mutations involved in hereditary marrow failure and MDS may also become increasingly important in the context of precision-medicine in which germline mutations are unexpectedly identified in somatic testing. Disclosures No relevant conflicts of interest to declare.

scholarly journals Inducible SBDS Deficiency Impairs Bone Marrow Niche Function to Engraft Donor Hematopoietic Stem Cell after Transplantation

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3199-3199
Author(s):  
Ji Zha ◽  
Lori Kunselman ◽  
Hongbo Michael Xie ◽  
Brian Ennis ◽  
Jian-Meng Fan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Mouse Model ◽  
Board Of Directors ◽  
Hematopoietic Stem Cell ◽  
Graft Failure ◽  
Advisory Committees ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Deficient Mice

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.

scholarly journals Bone Marrow Cell Aggregates: A Way of Collecting the Adult Human Hematopoietic Stem Cell Niche

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4363-4363
Author(s):  
Alexandre Janel ◽  
Nathalie Boiret-Dupré ◽  
Juliette Berger ◽  
Céline Bourgne ◽  
Richard Lemal ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Confocal Microscopy ◽  
Hematopoietic Stem Cell ◽  
Biological Samples ◽  
Mesenchymal Cells ◽  
Hematopoietic Stem ◽  
Bm Niche ◽  
Cd34 Cells

Abstract Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p<0.01). Also, each hematon contains CFU-GM, BFU-E, CFU-Mk and CFU-F cells. Mesenchymal cells are located mainly on the periphery and seem to participate in supporting the structure. The majority of mesenchymal cells isolated from hematons (21/24) sustain in vitro hematopoiesis. Interestingly, more than 90% of the hematons studied contained LTC-ICs. Furthermore, when studied using confocal microscopy, a co-localization of CD34+ cells with STRO1+ mesenchymal cells was frequently observed (75% under 10 µm of the nearest STRO-1+ cell, association statistically highly significant; p <1.10-16). These results indicate the presence of one or several stem cell niches housing highly primitive progenitor cells. We are confirming these in vitro data with an in vivo xenotransplantation model. These structures represent the elementary functional units of adult hematopoietic tissue and are a particularly attractive model for studying homeostasis of the BM niche and the pathological changes occurring during disease. Disclosures No relevant conflicts of interest to declare.

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