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.

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.

scholarly journals Microrna 199b Regulates Mouse Hematopoietic Stem Cells Maintenance

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3704-3704
Author(s):  
Aldona A Karaczyn ◽  
Edward Jachimowicz ◽  
Jaspreet S Kohli ◽  
Pradeep Sathyanarayana
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Quiescent State ◽  
Differentiation Potential ◽  
Self Renewal ◽  
Hematopoietic Stem

The preservation of hematopoietic stem cell pool in bone marrow (BM) is crucial for sustained hematopoiesis in adults. Studies assessing adult hematopoietic stem cells functionality had been shown that for example loss of quiescence impairs hematopoietic stem cells maintenance. Although, miR-199b is frequently down-regulated in acute myeloid leukemia, its role in hematopoietic stem cells quiescence, self-renewal and differentiation is poorly understood. Our laboratory investigated the role of miR-199b in hematopoietic stem and progenitor cells (HSPCs) fate using miR-199b-5p global deletion mouse model. Characterization of miR-199b expression pattern among normal HSPC populations revealed that miR-199b is enriched in LT-HSCs and reduced upon myeloablative stress, suggesting its role in HSCs maintenance. Indeed, our results reveal that loss of miR-199b-5p results in imbalance between long-term hematopoietic stem cells (LT-HSCs), short-term hematopoietic stem cells (ST-HSCs) and multipotent progenitors (MMPs) pool. We found that during homeostasis, miR-199b-null HSCs have reduced capacity to maintain quiescent state and exhibit cell-cycle deregulation. Cell cycle analyses showed that attenuation of miR-199b controls HSCs pool, causing defects in G1-S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT-HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS-sorted LT-HSCs (LineagenegSca1posc-Kitpos CD48neg CD150pos) lacking miR-199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS-sorted LSK population of 52 weeks old miR-199b KO mice revealed that loss of miR-199b promotes accumulation of GMP-like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age-related pathway. We used non-competitive repopulation studies to show that overall BM donor cellularity was markedly elevated in the absence of miR-199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR-199b-null LT-HSC render enhanced self-renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self-renewal potential of miR-199b-null LT-HSC by a secondary BM transplantation of unfractionated BM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCs populations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT-HSC were similar between recipients reconstituted with wild-type and miR-199b-KO chimeras, whereas miR-199b-null HSCs contributed relatively more into MPPs. Our data identify that attenuation of miR-199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR-199b promotes signals that govern differentiation of LT-HSC to MPP leading to accumulation of highly proliferative progenitors during long-term reconstitution. Hematopoietic regeneration via repopulation studies also revealed that miR-199b-deficient HSPCs have a lineage skewing potential toward myeloid lineage or clonal myeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR-199b in hematopoietic aging. 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.

IFNs Upregulate Sca-1 and Block Proliferation in Murine Hematopoietic Stem and Progenitor Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3394-3394
Author(s):  
Kaitlyn Shank ◽  
Yusup Shin ◽  
Carson Wills ◽  
Nicole Cunningham ◽  
Alevtina Domashenko ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Caspase 3 ◽  
Inflammatory Responses ◽  
Conflicts Of Interest ◽  
Brdu Incorporation ◽  
Apparent Paradox ◽  
Hematopoietic Stem

Abstract Abstract 3394 Hematopoietic stem cells (HSC) replenish the cellular components of the blood throughout life by a homeostatic process in which the majority of HSCs remain quiescent while a small percentage enter the cell cycle to either self-review or differentiate. During inflammatory responses to infections, Interferons (IFNa, IFNg) perturb HSC homeostasis, presumably in response to the demand for increased numbers of inflammatory cells. Previous studies have highlighted an apparent paradox, i.e. IFNs suppress the proliferation of normally cycling murine hematopoietic progenitor cells (HPCs), yet increase the fraction of normally quiescent Sca+ HSCs that proliferate. To investigate the mechanisms underlying this paradox, we dissected the dynamics of cell surface phenotypes, cell cycle kinetics, pro- and anti-apoptotic pathways within the HSC and HPC compartments in response to pIpC and IFNs both in vivo and in vitro. Forty-eight hours after pIpC injection, bone marrow (BM) cellularity declined by 60%, the proportion of Sca- kit+ HPCs fell from 0.45% to 0.05%, while the proportion of BM cells with the Sca+ kit+ HSC phenotype increased from 0.17 to 0.26%. To determine whether the increase in Sca+kit+ cells was due to proliferation of HSCs or upregulation of Sca-1 on HPCs, we cultured purified CD150+ Sca-Kit+ HPCs and CD150+Sca+kit+ HSCs in vitro with IFNa, IFNg, or PBS. Sca expression was induced on previously Sca- HPCs, and the level of Sca expression on HSCs was also increased. This induction was detectable as early as 6 hours after treatment and accompanied by an increase in Sca mRNA. BrdU incorporation into both HPC and HSC populations decreased from pre-treatment baselines, further indicating that the increase in cells with the HSC phenotype was not due to HSC proliferation, but rather the appearance of cycling HPCs within the HSC staining gate following IFN-induced upregulation of Sca. Staining with FITC-DEVD-FMK identified active cleaved capase-3 in pIpC- or IFN-treated cells, suggesting that the reduced cellularity following IFN reflected a cellular stress that killed Lin+ precursors cells and some HPCs, but spared HSCs. In contrast to lin+kit- precursors, all kit + HPCs and HSCs expressed bcl-2, suggesting that expression of anti-apoptotic proteins may prevent IFN-induced stress from resulting in HSC/HPC apoptosis despite the initial triggering of caspase-3 cleavage. In summary, acute treatment with IFNs has anti-proliferative effects on all hematopoietic cells, including precursors, HPCs and HSCs, with the apparent increase in HSC proliferation the result of HPCs masquerading as Sca+HSCs after exposure to IFN. Unlike precursors, HSCs and some HPCs survive treatment to IFNs despite activation of cleaved caspase-3, possibly due to their expression of bcl-2, and likely related anti-apoptotic regulators. The previously observed increase in HSC proliferation days and weeks following IFN treatment is most likely due to the homeostatic response of HSCs to the depopulation of the precursor and HPCs caused by acute IFN exposure. Disclosures: No relevant conflicts of interest to declare.

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&lt;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.

JAK2V617F Promotes Stem Cell Amplification Driving MPN Clonal Dominance in Mice and Treatment by IFNa Prevents This Effect

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 616-616 ◽  
Author(s):  
Caroline Marty ◽  
Catherine Lacout ◽  
Marie Cuingnet ◽  
Salma Hasan ◽  
Eric Solary ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Conflicts Of Interest ◽  
Myeloproliferative Neoplasm ◽  
Jak2 V617f ◽  
Lymphoid Cells ◽  
Hematopoietic Stem ◽  
Endogenous Expression ◽  
Long Term Treatment

Abstract Abstract 616 JAK2V617F is the major mutation involved in classic myeloproliferative neoplasm (MPN). It promotes growth factor independent cell growth and is able to recapitulate MPN features in retroviral, transgenic (TG) or knock-in (KI) mouse models. Several mutations implicated in epigenetic modifications or leukemic transformations have been also identified in MPN and several reports have questioned the particular role of JAK2V617F on hematopoietic stem cells (HSC) proliferation thus as a driver of MPN emergence. Therefore, we investigated the in vivo effect of an endogenous expression of JAK2V617F on early stages of differentiation and their ability to compete for normal cells in a repopulation assay. For this study, we develop a novel mouse conditional JAK2V617F KI model based on the “FLEX switch” strategy. These KI mice were crossed with TG mice expressing the Cre recombinase under the control of the vav promoter in order to restrict JAK2V617F expression to hematopoietic and some endothelial tissues. VavCre/JAK2+/V617F KI mice developed high hematocrit (70 ± 2 %, control values 49 ± 1 % n=13), platelet (2.3 ± 0.1 × 109 / mL, control values 0.84 ± 0.04 × 109 / mL n=20) and white blood cell (20-40 × 106/mL, control values between 6–10 × 106 / mL) values and a splenomegaly at 2–3 months of age but after 6 months of age an anemia and a thrombocytopenia appeared. This model mimics human polycythemia vera with secondary myelofibrosis. At 2–3 months of age, cumulative numbers in bone marrow (BM) and spleen of CFU-E, BFU-E and GM-CFC were increased 15-, 3-, 1.2–fold, respectively, compared to control. Most CFU-E grew without the addition of erythropoietin. A 6-fold amplification of total early progenitors LSK and a tendency toward SLAM (LSK/CD48−/CD150+) cell amplification, mainly due to a significant 9-fold increase in the spleen, were also observed. Competitive repopulation assays using 30% KI and 70% WT bone marrow cells demonstrated 17 weeks after BM transplantation (BMT) a rapid and strong amplification, from 30% to > 80%, of blood myeloid cells (Gr-1+/Mac1+) from KI origin. Late after transplantation (35 weeks), Lin-, LSK and SLAM cell compartments from KI origin raised from the initial 30% to almost 100% in the BM and even KI blood lymphoid cells (B220+ and CD3+) demonstrated a significant amplification compared to control. This shows that endogenous expression of JAK2V617F gives an advantage to HSC, promoting clonal dominance in mice. Then, we analyzed at which levels of differentiation acts IFNα, a drug promoting cycling of dormant cells and proven efficacious in PV treatment in human. In a chimeric model, we demonstrated that IFNα could prevent the development of MPN induced in vavCre/JAK2+/V617F KI recipient mice by inhibiting the amplification of KI cells. Secondary BMT from treated animals demonstrated the eradication of disease-initiating cells after long-term treatment. This study shows that IFNα acts at the level of the disease-initating cell by reverting the HSC promoting clonal dominance induced by JAK2V617F. Disclosures: No relevant conflicts of interest to declare.

scholarly journals β7 Integrin Regulates Intra-Marrow Trafficking of Hematopoietic Stem Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2899-2899
Author(s):  
Jodi Murakami ◽  
Baohui Xu ◽  
Christopher B. Franco ◽  
Xingbin Hu ◽  
Stephen J. Galli ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Cell Adhesion ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Hematopoietic Stem ◽  
Effector Cells ◽  
Cell Cycle Status

Abstract α4β7 integrin is a cell adhesion receptor that is crucial for the migration of hematopoietic progenitors and mature effector cells in the periphery, but its role in adult hematopoiesis remains controversial. To investigate this, we conducted studies using a mouse model in which β7 integrin is absent. Hematopoietic stem cells (HSCs) that lacked β7 integrin (β7KO) had significantly reduced engraftment potential. Intriguingly, the survival of β7KO mice was enhanced and their hematopoietic recovery after 5-fluorouracil-induced myeloablative stress was better compared to wild type (WT) mice, indicating that the decreased engraftment of β7KO HSCs was not caused by a defect in HSC hematopoietic activity. Next we examined the homing abilities of HSCs and we observed that β7KO HSCs had impaired migration abilities in vitro and BM homing capabilities in vivo. Lethal irradiation induced expression of the α4β7 integrin ligand - mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on bone marrow (BM) endothelial cells. Moreover, blocking MAdCAM-1 reduced the homing of HSCs and impaired the survival of recipient mice. Altogether, these data indicate that β7 integrin, when expressed by HSCs, interacted with MAdCAM-1 in the BM microenvironment, thereby promoting HSC homing and engraftment. Interestingly, we also found that β7KO HSCs were retained in the BM, suggesting that β7 integrin may influence the localization of HSCs within different stem cell niches through interaction with MAdCAM-1. To examine the localization of HSCs within the BM, we used the hypoxyprobe pimonidazole to correlate oxygen status with niche localization. We observed that both β7KO and MAdCAM-1KO HSCs were more hypoxic compared to WT HSCs, demonstrating that the absence of either β7 integrin or MAdCAM-1 in mice causes HSCs to be localized in a more hypoxic region of the BM. To confirm these findings, we performed single-cell RT-PCR using Fluidigm Dynamic Array Chips and we discovered that β7KO HSCs differentially expressed genes associated with niche localization and cell cycle status compared to WT HSCs. Since hypoxia correlates with quiescence, we next assessed the cell cycle status of HSCs using Ki67 staining and in vivo BrdU assay and we found that β7KO HSCs may have reduced cell cycle activity. Collectively, these studies suggest that expression of β7 integrin on HSCs may promote exit from quiescence and influence HSC localization within the BM niche. Disclosures No relevant conflicts of interest to declare.

scholarly journals PTPRU, a quiescence-induced receptor tyrosine phosphatase negatively regulates osteogenic differentiation of human mesenchymal stem cells

2021 ◽  
Author(s):  
Mohammad Rumman ◽  
Jyotsna Dhawan
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Osteogenic Differentiation ◽  
Tyrosine Phosphatase ◽  
Quiescent State ◽  
Differentiation Potential

Bone marrow mesenchymal stem cells (MSCs) are heterogeneous osteo-progenitors that are mainly responsible for bone regeneration and homeostasis. In vivo, a subpopulation of bone marrow MSCs persists in a quiescent state, providing a source of new cells for repair. Previously, we reported that induction of quiescence in hMSCs in vitro skews their differentiation potential in favour of osteogenesis while suppressing adipogenesis. Here, we uncover a new role for a protein tyrosine phosphatase, receptor type U (PTPRU) in repressing osteogenesis during quiescence. A 75 kD PTPRU protein isoform was found to be specifically induced during quiescence and down-regulated during cell cycle reactivation. Using siRNA-mediated knockdown, we report that in proliferating hMSC, PTPRU preserves self-renewal, while in quiescent hMSC, PTPRU not only maintains reversibility of cell cycle arrest but also suppresses expression of osteogenic lineage genes. Knockdown of PTPRU in proliferating or quiescent hMSC de-represses osteogenic markers, and enhances induced osteogenic differentiation. We also show that PTPRU positively regulates a β-catenin-TCF transcriptional reporter. Taken together, our study suggests a role for a quiescence-induced 75kD PTPRU isoform in modulating bone differentiation in hMSC, potentially involving the Wnt pathway.

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.

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