scholarly journals Human Cytomegalovirus Selectively Suppresses the Megakaryo/Thrombopoiesis of PDGFR+ and αvβ3+ Megakaryocytes Via the TPO/c-Mpl Pathway after Allo-HSCT

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 25-25
Author(s):  
Feng-qi Liu ◽  
Fei-er Feng ◽  
Gao-chao Zhang ◽  
Yan Su ◽  
Xue-yan Sun ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Human Cytomegalovirus ◽  
Hcmv Infection ◽  
Conflicts Of Interest ◽  
Antiviral Treatment ◽  
Infection Model ◽  
Hematopoietic Stem ◽  
The Impact

Introduction Virus-induced thrombocytopenia is a severe complication in immunocompromised hosts. Among patients following allogeneic hematopoietic stem cell transplantation (allo-HSCT), human cytomegalovirus (HCMV) infection contributes to a variety of end-organ diseases and hematological complications, leading to increased mortality. Even with antiviral treatment, HCMV remains a potentially lethal infection due to the lack of understanding of the underlying mechanisms of host-virus interactions. The key to solving this problem is to identify the factors that predispose patients to HCMV infection and carry out targeted therapy. Here, we investigated the megakaryo/thrombopoiesis process, including the thrombopoietin (TPO)/c-Mpl pathway, after HCMV infection in vivo and in vitro, screened for susceptible subsets of megakaryocytes (MKs) and explored novel therapeutic targets for HCMV infection. Methods To test whether thrombocytopenia induced by HCMV results from an impaired megakaryo/thrombopoiesis process, we studied the impact of HCMV in an in vivo model of HCMV DNAemia patients following allo-HSCT and an in vitro model of bone marrow CD34+-derived MKs infected with serum from HCMV DNAemia patients. Forty patients who had received allo-HSCT were enrolled in this study, among whom 18 recipients had HCMV DNAemia and 22 were HCMV negative, and bone marrow-derived mononuclear cells (MNCs) from patients were tested for CD41, vWF, pp65, c-Mpl, PDGFR, αvβ3 and TLR2 using flow cytometry (FCM). Transmission electron microscopy (TEM) was used to detect HCMV capsids inside MKs. Cell apoptosis was measured by Annexin V. MK ploidy was determined by FCM for propidium iodide (PI) staining. Finally, inhibitors of PDGFR (IMC-3G3 and Gleevec), αvβ3 and TLR2 were cocultured with MKs. Results Our data showed that pp65+ cells accounted for 40.59±6.12% of total CD41+vWF+ MKs from HCMV DNAemia patients, and there was a significant increase in the expression of αvβ3, PDGFR and TLR2 in pp65+ MKs compared with that in control patients. Furthermore, the percentage of PDGFR+αvβ3+ MKs emerged as an independent factor associated with HCMV infection in multivariate analysis (p = 0.008). MKs in HCMV-infected patients showed increased apoptosis and necrosis and different patterns of MK ploidy distribution compared with those in HCMV-negative patients, with a decreased proportion from 16N to 64N and a peak at 8N. Meanwhile, the expression of TPO receptor c-Mpl was lower in pp65+ MKs from HCMV DNAemia patients (0.77±0.38% in pp65+ MKs from HCMV DNAemia patients, 1.75±0.40% in pp65- MKs from HCMV DNAemia patients, 1.97±0.67% in MKs from HCMV-negative patients, and 2.06±0.29% in MKs from healthy controls, p<0.01) while the TPO level in serum was increased compared with that in controls. Next, we established an in vitro HCMV infection model of CD34+-derived MKs with serum from HCMV DNAemia patients, and the laboratory HCMV strain Towne was used as a positive control. After 9 days of coculturing, the viral capsids of HCMV were observed in the nuclei of MKs (Figure 1A), and HCMV infection increased the apoptosis of MKs and shifted them to low ploidy, with a significant decrease in platelet release. As with the in vivo results, c-Mpl was downregulated in HCMV-infected MKs. The expression levels of PDGFR, TLR2 and αvβ3 on MKs were increased in coculture with HCMV DNAemia serum, and pp65-positive MKs were decreased compared with the control after treatment with inhibitors of PDGFR and αvβ3 (Figure 1B). However, neither Gleevec nor anti-TLR2 altered the HCMV infection rate. Conclusions Our study showed that HCMV could impair megakaryopoiesis throughout maturation, apoptosis, and platelet generation via the TPO/c-Mpl pathway both in vivo and in vitro. MKs with PDGFR+ and αvβ3+ phenotypes are susceptible to HCMV infection and we proposed PDGFR and αvβ3 inhibitors as potential therapeutic alternatives for allo-HSCT patients with HCMV infection. Disclosures No relevant conflicts of interest to declare.

scholarly journals Endothelial JAK3 Expression Enhances Pro-Hematopoietic Angiocrine Function of Sinusoidal Endothelial Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2488-2488 ◽  
Author(s):  
José Gabriel Barcia Durán
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Nk Cell ◽  
Conflicts Of Interest ◽  
Fluorescent Microscopy ◽  
Hematopoietic Stem ◽  
Interleukin Receptors ◽  
Sinusoidal Endothelium

Unlike Jak1, Jak2, and Tyk2, Jak3 is the only member of the Jak family of secondary messengers that signals exclusively by binding the common gamma chain of interleukin receptors IL2, IL4, IL7, IL9, IL15, and IL21. Jak3-null mice display defective T and NK cell development, which results in a mild SCID phenotype. Still, functional Jak3 expression outside the hematopoietic system remains unreported. Our data show that Jak3 is expressed in endothelial cells across hematopoietic and non-hematopoietic organs, with heightened expression in the bone marrow and spleen. Increased arterial zonation in the bone marrow of Jak3-null mice further suggests that Jak3 is a marker of sinusoidal endothelium, which is confirmed by fluorescent microscopy staining and single-cell RNA-sequencing. We also show that the Jak3-null niche is deleterious for the maintenance of long-term repopulating hematopoietic stem and progenitor cells (LT-HSCs) and that Jak3-overexpressing endothelial cells have increased potential to expand LT-HSCs in vitro. In addition, we identify the soluble factors downstream of Jak3 that provide endothelial cells with this functional advantage and show their localization to the bone marrow sinusoids in vivo. Our work serves to identify a novel function for a non-promiscuous tyrosine kinase in the bone marrow vascular niche and further characterize the hematopoietic stem cell niche of sinusoidal endothelium. Disclosures No relevant conflicts of interest to declare.

An In Vivo Functional Screen Identifies miRNA-150 As a Regulator of Hematopoietic Regeneration Post Chemotherapeutic Injury

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2333-2333
Author(s):  
Brian D. Adams ◽  
Shangqin Guo ◽  
Haitao Bai ◽  
Changchun Xiao ◽  
E. Premkumar Reddy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Negative Regulator ◽  
Post Transplantation ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery ◽  
Expression Construct

Abstract Abstract 2333 . MicroRNAs are important regulators of many hematopoietic processes, yet little is known with regard to the role of microRNAs in controlling normal hematopoietic regeneration. The most common methodology for in vivo microRNA studies follows a hypothesis-driven candidate approach. Here, we report the establishment of an unbiased, in vivo, microRNA gain-of-function screen, and the identification of miR-150 as a negative regulator of hematopoietic recovery post chemotherapeutic challenge. Specifically, a retroviral-library consisting of 135 hematopoietic-expressed microRNAs was generated, with each expression construct containing a barcode sequence that can be specifically recognized using a novel bead-based platform. Hematopoietic-stem-and-progenitor-cell (HSPC)-enriched wild-type bone marrow was transduced with this library and transplanted into lethally-irradiated recipients. Analysis of peripheral blood samples from each recipient up to 11 weeks post transplantation revealed that 87% of the library barcodes are reliably detected. To identify microRNAs that regulate hematopoietic regeneration after chemotherapy-induced injury, we measured the change in barcode abundance for specific microRNA constructs after 5-fluorouracil (5-FU) challenge. Notably, a small number of barcodes were consistently depleted in multiple recipient mice after treatment. Among the top hits was the miR-150-associated barcode, which was selected for further experimentation. Indeed, overexpression of miR-150 in a competitive environment resulted in significantly lower recovery rates for peripheral myeloid and platelet populations after 5-FU treatment, whereas the effects on B- and T-cells were milder. Furthermore, full recovery of these cell populations did not occur until ∼12 weeks after treatment, suggesting the involvement of HSPCs and/or common lineage progenitors. Conversely, knocking out miR-150 led to an opposite phenotype, with platelets and myeloid cells displaying faster recovery in both competitive and non-competitive settings. Interestingly, we could not observe the described effects of miR-150 in bone marrow primary cell cultures, suggesting that such effects cannot be recapitulated in vitro. Overall, these data indicate that miR-150 is a novel regulator of hematopoietic recovery after chemotherapeutic-induced injury, and highlight the important role of microRNAs in the intrinsic wiring of the hematopoietic regeneration program. Our experiments also demonstrate the feasibility and power of functional in vivo screens for studying normal hematopoietic functions, which can become an important tool in the hematology field. Disclosures: No relevant conflicts of interest to declare.

Primary Bone Marrow-Derived MSC Subsets Have Distinct Wnt-Signatures Compared to Conventionally Cultured MSC and Differ in Their Capacity to Support Hematopoiesis in Vitro,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3414-3414 ◽  
Author(s):  
Marijke W Maijenburg ◽  
Marion Kleijer ◽  
Kim Vermeul ◽  
Erik P.J. Mul ◽  
Floris P.J. van Alphen ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Marrow ◽  
Wnt Signaling ◽  
Mononuclear Cells ◽  
Conflicts Of Interest ◽  
Cell Types ◽  
Hematopoietic Stem ◽  
Wnt Proteins

Abstract Abstract 3414 Mesenchymal stromal cells (MSC) are of promising therapeutic use to suppress immunogenic responses following transplantation, and to support expansion of hematopoietic stem- and progenitors cells (HSPC) from small transplants derived for instance from cord blood. Culture-expanded MSC produce a wide variety and quantity of Wnt-proteins and the crucial role of Wnt-signaling in the hematopoietic stem cell niche is well established. However, studies addressing Wnt-signaling in MSC have (i) only focused on culture-expanded MSC and (ii) did not discriminate between phenotypically distinct subpopulations which are present in bulk cultures of expanded MSC. Recently we identified three new subpopulations of MSC in human bone marrow (BM) based on expression of CD271 and CD146: CD271brightCD146−, CD271brightCD146+, CD271−CD146+. These fractions co-express the “classical” MSC markers CD90 and CD105 and lack expression of CD45 and CD34 (Maijenburg et al, Blood 2010, 116, 2590). We and others demonstrated that the adult BM-derived CD271brightCD146− and CD271brightCD146+ cells contain all colony forming units-fibroblasts (Maijenburg et al, Blood 2010, 116, 2590; Tormin et al, Blood 2010, 116, 2594). To investigate how these primary subsets functionally compare to conventional, culture-expanded MSC, we investigated their Wnt-signature and hematopoietic support capacity. To this end, we sorted CD271brightCD146− and CD271brightCD146+ cells from human adult BM (n=3) and compared their Wnt-signatures obtained by Wnt-PCR array to the profiles from cultured MSC from the same donors. Fifteen genes were consistently differentially expressed in the two sorted uncultured subsets compared to their conventionally cultured counterparts. Expression of CCND1, WISP1 and WNT5B was strongly increased, and WNT5A was only detected in the conventionally cultured MSC. In contrast, WNT3A was exclusively expressed by sorted primary CD271brightCD146− and CD271brightCD146+ cells, that also expressed higher levels of JUN, LEF1 and WIF1. The differences in Wnt (target)-gene expression between CD271brightCD146− and CD271brightCD146+ cells were more subtle. The Wnt-receptors LRP6 and FZD7 were significantly higher expressed in CD271brightCD146+ cells, and a trend towards increased expression in the same subset was observed for CTNNB1, WNT11 and MYC. When the sorted subsets were cultured for 14 days (one passage), the differences in Wnt-related gene expression between the subsets was lost and the expanded sorted cells acquired an almost similar Wnt-signature as the MSC cultured from BM mononuclear cells from the same donors. The cultured subsets lost the expression of Wnt3a and gained the expression of Wnt5a, similar to the unsorted MSC cultured from the same donors in parallel. Despite the loss of a distinct Wnt-signature, co-culture experiments combining the sorted MSC subsets with human HSPC revealed that CD271brightCD146+ cells have a significantly increased capacity to support HSPC in short-term co-cultures (2 weeks) compared to CD271brightCD146− cells (p<0.021, n=3), which was analyzed in hematopoietic colony assays following co-culture. In contrast, a trend towards better long-term hematopoietic support (co-culture for 6 weeks) was observed on CD271brightCD146− cells. In conclusion, we demonstrate for the first time that primary sorted uncultured MSC subsets have a distinct Wnt-signature compared to cultured unsorted MSC and display differences in hematopoietic support. As it was recently shown that CD271brightCD146− and CD271brightCD146+ MSC localize to separate niches in vivo (Tormin et al, Blood 2011), our data indicate that the two MSC subsets are not necessarily distinct cell types and that the different Wnt-signature may be a reflection of these distinct microenvironments. Cell culturing for only one passage dramatically changed the Wnt-signature of the sorted MSC subsets, indicating that Wnt-signaling in in vitro expanded MSC does not resemble the Wnt-signature in their tissue resident counterparts in vivo. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Vasculopathy in the Bone Marrow Microenvironment of Humanized Sickle Cell Mice Is Reversible By Blood Transfusion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2256-2256
Author(s):  
Shin-Young Park ◽  
Alessandro Mattè ◽  
Yookyung Jung ◽  
Jina Ryu ◽  
Wilson Anand ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Blood Transfusion ◽  
Sickle Cell ◽  
Conflicts Of Interest ◽  
Vascular Dysfunction ◽  
High Morbidity ◽  
Hematopoietic Stem ◽  
Vascular Niche ◽  
The Impact

Sickle cell disease (SCD) is a monogenic red cell disorder with high morbidity and mortality due to significant end-organ disease. Here we investigated for the first time the impact of SCD on the bone marrow (BM) vascular niche, which is critical for hematopoiesis. In SCD mice, we find a disorganized and structurally abnormal BM vasculature with highly tortuous arterioles and fragmented sinusoidal vessels, the latter filled with aggregates of sickle erythroid and myeloid cells. By in vivo imaging, sickle and control red blood cells exhibit significantly slow intravascular flow speeds in sickle cell BM but not in control BM. These abnormalities lead to increased ROS production in expanded erythroblast populations, and to up-regulation in the BM of HIF-1a and key growth factors required to support angiogenesis, e.g. VEGF-A, Ang1, Ang2. Moreover, increased levels of VCAM-1, a marker for vascular dysfunction, are detected. SCD also impacts the BM perivascular niche, i.e. the number of CXCL12-abundant reticular cells, is markedly decreased, and is associated with mobilization of hematopoietic stem/progenitor cells as well as mature leukocytes to the periphery. Strikingly, the distorted vascular network is completely reversed by blood transfusion, which highlights the plasticity of the BM vascular niche. Thus, we provide molecular and cellular features of the pathologic BM microenvironment, which suggest that the local hypoxia-induced angiogenic network combined with inflammatory vascular dysfunction is responsible for the vasculopathy. In addition, the findings may serve to develop new biomarkers and therapeutic targets of BM end-organ damage. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals Leukemic Stress Targets the mTOR Pathway to Suppress Residual HSC in the BM Microenvironment

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3730-3730
Author(s):  
John T Butler ◽  
Sherif Abdelhamed ◽  
Lina Gao ◽  
Jeong Lim ◽  
Terzah M Horton ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Conflicts Of Interest ◽  
Chemotherapy Resistance ◽  
Hematopoietic Cells ◽  
Tumor Burden ◽  
Mtor Pathway ◽  
Hematopoietic Stem ◽  
The Impact

The remodeling of the bone marrow (BM) microenvironment that occurs along with the progressive spread of acute myeloid leukemia (AML) cells can be considered a constitutive aspect of leukemogenesis. To date most studies have focused on the functional and in part inflammatory adaptation of stroma, and its potential role in extrinsic chemotherapy resistance. Much less is known about the impact of leukemic stress on residual hematopoietic cells. We previously identified the trafficking of select microRNAs (miRs-) in extracellular vesicles (EVs) between AML cells and hematopoietic progenitor cells (HPC). These studies revealed the mechanism underlying the suppression of HPC function in the AML niche (Hornick, Doron et. al., Science Signaling 2016). Several groups, including ours also noted the relative resistance of residual hematopoietic stem cells (HSC) to elimination from the BM of xenografted animals. In the current study we set out to understand how leukemic stress in the AML xenograft niche shapes HSC fate and function. Using AML cell lines (Molm14, U937, HL60) we established NSG xenografts, systematically tracking peripheral blood AML chimerism to recover murine hematopoietic cells at low or negative tumor burden, and replicating key assays using purified EV for intrafemoral injections. In immunofluorescent studies we initially confirmed the uptake of GFP labeled xenograft-derived EVs across the spectrum of HPC and HSC (KSL/CD150+/CD48-), as well as the successive loss and peripheral displacement of HPCs, and gains in HSC frequency in the leukemic niche. These HSC were found to be enriched for G0 cell cycle status with an increase in phospho- p53, but showed no evidence of apoptosis or senescence. To understand the mechanism underlying their apparent quiescence, we performed in vitro proteomics studies of AML EV exposed HPSC identified downregulation of ribosomal biogenesis pathways. We then confirmed in vivo that residual HSC from AML xenografts experienced a loss of protein synthesis (OPP assay). We next reasoned that deficits in ribosome dysfunction and protein synthesis may reflect deregulation by specific miRNAs highly abundant in AML EV. Here, we had an opportunity to profile EV miRNA from the plasma of 12 unselected AML patients at diagnosis versus 12 control samples, and we confirmed a significant enrichment for specific miRNAs, including miR-1246. Raptor is a component of the mTOR pathway and an annotated target of miR-1246. We demonstrated in a series of experiments that miR-1246 translationally suppresses Raptor and downregulates protein synthesis in residual HSC from AML xenografts. The transfection of synthetic anti-miR1246 sequences on the other hand reversed the effects of AML EV in murine HSC. In aggregate we show that direct crosstalk between AML and hematopoietic cells adds to the adaptive changes that occur in the AML niche. Our experiments suggest a functional significance for EV miRNA that can be detected in AML patient plasma in the regulation of residual BM HSC. More broadly, the mechanisms by which leukemic stress alters hematopoietic function remain underexplored, but our observations suggest that leukemia derived EV contribute to changes in competitive fitness of residual HSC. Disclosures No relevant conflicts of interest to declare.

Interferon-Gamma Impairs the Self-Renewal of Hematopoietic Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2351-2351
Author(s):  
Alexander M. de Bruin ◽  
Berend Hooibrink ◽  
Martijn A. Nolte
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Interferon Gamma ◽  
Chimeric Mice ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
The Impact

Abstract Abstract 2351 Regulation of hematopoiesis during stress situations, such as bacterial or viral infections, is crucial for the maintenance of sufficient numbers of cells in the blood. It has become clear that activated immune cells provide such feedback signals to the bone marrow. An important mediator in this respect is the pro-inflammatory cytokine Interferon-gamma (IFNγ), which is produced in the bone marrow by activated T cells during the course of an infection. As such, we have previously shown that T cell-derived IFNγ can directly influence the output of myeloid and erythroid cells. To address whether IFNγ can also influence the function of hematopoietic stem cells (HSCs), we cultured highly purified HSCs from murine bone marrow with or without IFNγ and found that IFNγ strongly reduced the absolute number of HSCs in these cultures, both phenotypically and functionally. We confirmed that the functional impact of IFNγ was due to a direct effect on HSCs and not mediated by more differentiated progenitors. In addition, IFNγ does not directly influence the quiescent state of purified HSC, nor their cell cycle entry. By labeling HSCs with CFSE, we found that IFNγ reduces HSC expansion in vitro by decreasing their proliferative capacity, but not their ability to differentiate. To investigate the impact of IFNγ on HSCs in vivo, we infected WT and IFNγ−/− mice with lymphocytic choriomeningitis virus (LCMV) and found that IFNγ severely impaired HSC recovery upon infection. Finally, to exclude indirect effects of IFNγ on other cell types we generated chimeric mice with bone marrow from both WT and IFNγR−/− mice. Infection of these mixed-chimeric mice with LCMV resulted in decreased recovery of WT HSCs, but not of IFNγR−/− HSCs in the same mouse, which formally demonstrates that IFNγ directly impairs the proliferation of HSCs in vivo. Based on these experiments we conclude that IFNγ reduces HSC self renewal both in vitro and in vivo. Importantly, we thereby challenge the current concept in literature that IFNγ would induce the proliferation of HSCs (Baldridge et al, Nature 2010). Our findings thus provide challenging new insight regarding the impact of immune activation on hematopoiesis and will contribute significantly to the scientific discussion concerning this process. Moreover, our data also provide an explanation for the occurrence of anemia and bone marrow failure in several human diseases in which IFNγ is chronically produced. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Tsc1 Regulates the Proliferation Capacity of Bone-Marrow Derived Mesenchymal Stem Cells

Cells ◽  
2020 ◽  
Vol 9 (9) ◽  
pp. 2072 ◽  
Author(s):  
Maria V. Guijarro ◽  
Laura S. Danielson ◽  
Marta Cañamero ◽  
Akbar Nawab ◽  
Carolina Abrahan ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Myogenic Differentiation ◽  
Negative Regulator ◽  
Benign Tumors ◽  
Hematopoietic Stem ◽  
The Impact

TSC1 is a tumor suppressor that inhibits cell growth via negative regulation of the mammalian target of rapamycin complex (mTORC1). TSC1 mutations are associated with Tuberous Sclerosis Complex (TSC), characterized by multiple benign tumors of mesenchymal and epithelial origin. TSC1 modulates self-renewal and differentiation in hematopoietic stem cells; however, its effects on mesenchymal stem cells (MSCs) are unknown. We investigated the impact of Tsc1 inactivation in murine bone marrow (BM)-MSCs, using tissue-specific, transgelin (Tagln)-mediated cre-recombination, targeting both BM-MSCs and smooth muscle cells. Tsc1 mutants were viable, but homozygous inactivation led to a dwarfed appearance with TSC-like pathologies in multiple organs and reduced survival. In young (28 day old) mice, Tsc1 deficiency-induced significant cell expansion of non-hematopoietic BM in vivo, and MSC colony-forming potential in vitro, that was normalized upon treatment with the mTOR inhibitor, everolimus. The hyperproliferative BM-MSC phenotype was lost in aged (1.5 yr) mice, and Tsc1 inactivation was also accompanied by elevated ROS and increased senescence. ShRNA-mediated knockdown of Tsc1 in BM-MSCs replicated the hyperproliferative BM-MSC phenotype and led to impaired adipogenic and myogenic differentiation. Our data show that Tsc1 is a negative regulator of BM-MSC proliferation and support a pivotal role for the Tsc1-mTOR axis in the maintenance of the mesenchymal progenitor pool.

Overexpression of the TGF-Beta Modulator .Bambi Promotes Hematopoetic Stem Cell Proliferation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1446-1446
Author(s):  
Rui Mao ◽  
Olga Sirin ◽  
Margaret Goodell
Keyword(s):  
Cell Cycle ◽  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Receptor Activation ◽  
Lymphoid Cells ◽  
Quiescent State ◽  
Tgf Beta ◽  
Hematopoietic Stem

Abstract Abstract 1446 Poster Board I-469 Hematopoietic stem cells (HSC) normally reside in a quiescent state in the bone marrow. During times of stress, HSCs are activated to begin differentiation and self-renewal, replenishing the supply of myeloid and lymphoid cells present in the blood. The mechanisms regulating this rapid activation have not been fully elucidated. We previously identified the TGF-beta modulator Bambi (BMP and activin membrane-bound inhibitor) to be upregulated four-fold in HSCs compared to differentiated cells. Bambi codes for a transmembrane pseudoreceptor that inhibits TGF-beta receptor activation. Since TGF-beta signaling has been established to be important for induction of HSC quiescence as well as cell-cycle inhibition in long-term progenitors, we hypothesize that Bambi may play an important role in the regulation of HSCs. Using a retroviral vector, we overexpressed Bambi in bone marrow cells. Overexpression of Bambi resulted in increased colony-formation in vitro when compared to control cells. Furthermore, transduced cells expressed higher levels of the cell-cycle marker Ki-67, indicating a greater proportion of cells in active stages of the cell cycle. To verify the results of these assays in vivo, bone marrow overexpressing Bambi was transplanted into lethally irradiated recipient mice. Bambi-overexpressing cells demonstrated a higher level of engraftment in all lineages than control cells at several time points, which confirms the previous in vitro data suggesting greater cell cycle activity. Moreover, we identified the pathway through which Bambi acts by monitoring the levels of phosphorylated Smad2 (pSmad2), a downstream target of TGF-beta. Overexpression of Bambi resulted in a distinctly lower level of pSmad2, which explains the cell-cycle effects seen in vivo and in vitro. These studies show that Bambi functions to promote HSC proliferation and a probable mode of action in HSCs is through decreased pSmad2 levels from inhibition of the TGF-beta pathway. Bambi has been shown to be upregulated in certain leukemias, and a more complete understanding of the mechanism through which Bambi acts will provide better opportunities for therapeutic innovation. This research was graciously funded by an NIH grant and the ASH Trainee Research Award. Disclosures No relevant conflicts of interest to declare.

A Stable and Reproducible Xenotransplant Model for AML.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1005-1005
Author(s):  
Muriel Malaise ◽  
Konstanze Doehner ◽  
Dirk Reinhardt ◽  
Klaus-Michael Debatin ◽  
Selim Corbacioglu
Keyword(s):  
Bone Marrow ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Chromosome 17 ◽  
Organ Distribution ◽  
Positive Staining ◽  
Hematopoietic Stem ◽  
Median Delay

Abstract Abstract 1005 Poster Board I-27 Background: Xenotransplant models are invaluable tools to generate an unlimited source for in vivo propagation and extensive in vitro studies through consecutive passages of reproducibly stable supply. In vivo analyses of the pathogenetic relevance of these and other unidentified targets is of importance for the development of molecular targeted drug regimens. Whereas in ALL NOD/SCID based xenotransplant models are well established in AML only in rare subsets and animals with additional immunogenic deficiencies the diseases could be established and propagated because of age-dependant leakiness of functional immunity, residual innate immunity and short life span of the immunodeficient animals despite several strategies to enhance engraftment were applied. Over the years several mouse models with a variety if immunodeficient phenotypes were generated to alleviate this problem. Recently the NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mouse model with an IL-2R common gamma-chain deficiency was established and demonstrated stable engraftment rates with mobilized human hematopoietic stem cells. Methods: In this study 6 to 10 weeks old NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) animals were used for xenotransplant experiments. Fresh and frozen samples from adult and pediatric patients with newly diagnosed AML were transplanted via intramedullary injection. Animals were neither irradiated nor were accessory strategies used to enhance engraftment. Primary AML samples were adjusted to 2×107 cells per animal. Animals were anesthetized and samples were equally distributed between both femurs. All procedures were carried out in accordance with national laws and policies. Blood samples were collected weekly. A complete blood count (CBC) was performed and the samples were analyzed for human cells via FACS staining with fluorescence-labeled human anti-CD45 monoclonal antibodies (hCD45). PCR of the alpha-satellite region of human chromosome 17 was performed for confirmation. Animals were sacrificed when hCD45 was >5% or earliest 18 weeks post-injection. Organ distribution of hCD45 positive cells was assessed via FACS analysis of samples from liver, spleen, bone marrow and peripheral blood. Re-transplantion was performed either directly with fresh or from frozen samples. Results: 20 human samples (16 adult and 4 pediatric) were transplanted. The engraftment rate was 80% (16/20) with a median delay of 43.5 days. All pediatric samples engrafted between 30 to 38 days (median 31 days) post-transplant. hCD45 staining in the blood was positive from 13% to 64%, in the liver 0.1% to 54.6%, in the spleen 0.6% to 60.8% and in the bone marrow 0.6% to 71.4%. Adult samples engrafted from 30 to 142 days (median 45 days) post-transplanted with a human CD45 positive staining between 1.5% to 55.7% in the blood, 0.1% to 54.6% in the liver, 0.6% to 60.8% in the spleen and 0.6% to 71.4% in the bone marrow. The percentage of hCD45 in the peripheral blood did not reflect organ infiltration. Second transplants engrafted with a rate of 57.2%, (8/14) with a median delay of 27 days and with human CD45 positive staining between 0.9 to 81.4%. Thrombocytopenia was observed with a median platelet count of 94.500 PLT/μl in engrafted animals compared to control animals with 484.000 PLT/μl (p<0.05). Conclusion: The NSG xenotransplant model demonstrates to be a stable and reproducible tool for the establishment of primary human AML and it is therefore feasible for in vitro and in vivo studies. Engraftment can be predicted via hCD45 analysis and decreasing PLT counts. Engraftment rates of over 80% and a median time to engraftment of 43 days open the possibility to establish individual xenotransplant models in order to assess aberrant mechanisms and molecular rescue strategies for patients who relapsed after treatment. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Alteration of HSC Functions in Thalassemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4752-4752 ◽  
Author(s):  
Annamaria Aprile ◽  
Maria Rosa Lidonnici ◽  
Alessandro Gulino ◽  
Claudio Tripodo ◽  
Giacomo Mandelli ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Conflicts Of Interest ◽  
Pathological Condition ◽  
Autologous Transplantation ◽  
Beta Thalassemia ◽  
Allogeneic Bone ◽  
Erythroid Progenitors ◽  
Hematopoietic Stem

Abstract Beta-thalassemia represents one of the most globally widespread monogenic disorders and is characterized by significantly reduced or absent synthesis of hemoglobin beta-chains. In its severe form the insufficient production of adult hemoglobin results in altered erythropoiesis, hemolytic anemia, bone marrow (BM) hematopoietic hyperplasia and splenomegaly often associated with extramedullary hematopoiesis, requiring regular blood transfusions and iron chelation treatment. Over the last two decades many progresses were made in the field of allogeneic bone marrow (BM) transplantation to definitively cure beta-thalassemia. In parallel, experimental autologous transplantation protocols were developed to correct the disease by gene therapy also in patients lacking a compatible donor. Both in the allogeneic and autologous setting, thalassemic hematopoietic stem cells (HSCs) and the BM niche represent central elements. Although many aspects of the pathophysiology of thalassemia have been extensively investigated, the HSC and its niche have never been explored. In thalassemia, the BM is a stressed environment, characterized by the compensatory expansion of erythroid progenitors secondary to ineffective erythropoiesis. Whether other hematopoietic subpopulations, such as primitive progenitors and/or HSCs, might be affected by such an altered hematopoietic microenvironment is unknown. We investigated the frequency of hematopoietic progenitors in a murine model of severe beta-thalassemia intermedia. Immunophenotypic analyses revealed no differences in MEP, GMP, CMP, LMPP and MPP committed precursor subpopulations, whereas a significantly lower frequency of HSCs (Lin- Sca-1+ c-kit+ CD48- CD150+) was observed in thalassemic mice, as compared to age-matched wild-type controls. Competitive transplantation experiments revealed a disadvantage in the engraftment capacity of thalassemic HSCs, which was substantiated by the preliminary results from in vitro and in vivo cell cycle analyses suggesting an accelerated HSC exhaustion. Analyses of other cellular components, such as BM stroma and differentiated hematopoietic cells, revealed that additional elements are altered in the thalassemic BM microenvironment. The cellular and molecular bases of HSC-niche interaction in this pathological condition are under investigation. Our results uncover a previously ignored defect of HSCs in beta-thalassemia. The investigation of cellular and molecular players that might affect in trans HSC functions in the complexity of this altered microenvironment is ongoing. Disclosures No relevant conflicts of interest to declare.

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