Quercetin induces autophagy in myelodysplastic bone marrow including hematopoietic stem/progenitor compartment

2020 ◽  
Vol 36 (2) ◽  
pp. 149-167
Author(s):  
Suchismita Daw ◽  
Sujata Law
Keyword(s):  
Bone Marrow ◽  
Hematopoietic Stem ◽  
Progenitor Compartment

Thrombopoietin Administration Alleviates Hematopoietic Stem Cells Intrinsic Long Term Ionizing Radiation Damages. Identification of Preferential Cellular Expansion Sites

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2441-2441
Author(s):  
Diana Tronik-Le Roux ◽  
Johnny Nehme ◽  
Arthur Simonnet ◽  
Pierre Vaigot ◽  
Marie Anne Nicola ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Ionizing Radiation ◽  
Hematopoietic Stem Cells ◽  
Cellular Response ◽  
Side Population ◽  
Hematopoietic Stem ◽  
Functional Changes ◽  
Progenitor Compartment

Abstract Hematopoietic stem cells (HSC) are indispensable for the integrity of complex and long-lived organisms since they can reconstitute the hematopoietic system for life and achieve long term repopulation of lethally irradiated mice. Exposure of an organism to ionizing radiation (IR) causes dose dependant bone marrow suppression and challenge the replenishment capacity of HSC. Yet, the precise damages that are generated remain largely unexplored. To better understand these effects, phenotypic and functional changes in the stem/progenitor compartments of sublethally irradiated mice were monitored over a ten week period after radiation exposure. We report that shortly after sublethal IR-exposure, HSC, defined by their repopulating ability, still segregate in the Hoechst dye excluding side population (SP); yet, their Sca-1 (S) and c-Kit (K) expression levels are increased and severely reduced, respectively, with a concurrent increase in the proportion of SPSK cells positive for established indicators of HSC presence: CD150+/CD105+ and Tie2+. Virtually all HSCs quickly but transiently mobilize to replenish the bone marrow of myelo-ablated mice. Ten weeks after, whereas bone marrow cellularity has recovered and hematopoietic homeostasis is restored, major phenotypic modifications can be observed within the c-Kit+ Sca-1+ Lin−/low (KSL) stem/progenitor compartment: CD150+/Flk2− and Flk2+ KSL cell frequencies are increased and dramatically reduced, respectively. CD150+ KSL cells also show impaired reconstitution capacity, accrued γ-H2AX foci and increased tendency to apoptosis. This demonstrates that the KSL compartment is not properly restored 10 weeks after sublethal exposure, and that long-term IR-induced injury to the bone marrow proceeds, at least partially, through direct damage to the stem cell pool. Since thrombopoietin (TPO) has been shown to reduce haematopoietic injury when administered immediately after exposure to radiations, we asked whether TPO could restore the permanent IR-induced damage we observed in the HSC compartment. We first found in competitive transplant experiments that a single TPO administration rescued the impaired reconstitution capacity of HSC’s from animals exposed to sublethal IR. In addition, we observed that TPO injection right after irradiation considerably attenuates IR-induced long-term injury to the stem/progenitor compartment. Finally, the use of marrow cells from transgenic ubiquitous luciferase-expressing donors combined with bioluminescence imaging technology provided a valuable strategy that allowed visualizing HSC homing improvements of TPO-treated compared to untreated irradiated donors, and enabled the identification of a preferential cellular expansion sites which were inaccessible to investigation in most studies. Electronic microscopy analysis revealed that these sites show also differential activity of megakaryocytopoiesis with marked differences in the proplatelets reaching the vascular sinus. Altogether, our data provide novel insights in the cellular response of HSC to IR and the beneficial effects of TPO administration to these cells.

In Vivo Modulation of Mitochondrial Activity Determines HSC Engraftment and Post-Transplant Survival in Mice

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 213-213
Author(s):  
Nicola Vannini ◽  
Olaia M. Naveiras ◽  
Vasco Campos ◽  
Eija Pirinen ◽  
Riekelt Houtkooper ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
High Fat Diet ◽  
Mitochondrial Activity ◽  
High Fat ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Progenitor Compartment

Abstract Abstract 213 Cellular metabolism is emerging as a potential fate determinant in cancer and stem cell biology, constituting a crucial regulator of the hematopoietic stem cell (HSC) pool [1–4]. The extremely low oxygen tension in the HSC microenvironment of the adult bone marrow forces HSCs into a low metabolic profile that is thought to enable their maintenance by protecting them from reactive oxygen species (ROS). Although HSC quiescence has for long been associated with low mitochondrial activity, as testified by the low rhodamine stain that marks primitive HSCs, we hypothesized that mitochondrial activation could be an HSC fate determinant in its own right. We thus set to investigate the implications of pharmacologically modulating mitochondrial activity during bone marrow transplantation, and have found that forcing mitochondrial activation in the post-transplant period dramatically increases survival. Specifically, we examined the mitochondrial content and activation profile of each murine hematopoietic stem and progenitor compartment. Long-term-HSCs (LT-HSC, Lin-cKit+Sca1+ (LKS) CD150+CD34-), short-term-HSCs (ST-HSC, LKS+150+34+), multipotent progenitors (MPPs, LKS+150-) and committed progenitors (PROG, Lin-cKit+Sca1-) display distinct mitochondrial profiles, with both mitochondrial content and activity increasing with differentiation. Indeed, we found that overall function of the hematopoietic progenitor and stem cell compartment can be resolved by mitochondrial activity alone, as illustrated by the fact that low mitochondrial activity LKS cells (TMRM low) can provide efficient long-term engraftment, while high mitochondrial activity LKS cells (TMRM high) cannot engraft in lethally irradiated mice. Moreover, low mitochondrial activity can equally predict efficiency of engraftment within the LT-HSC and ST-HSC compartments, opening the field to a novel method of discriminating a population of transitioning ST-HSCs that retain long-term engraftment capacity. Based on previous experience that a high-fat bone marrow microenvironment depletes short-term hematopoietic progenitors while conserving their long-term counterparts [5], we set to measure HSC mitochondrial activation in high-fat diet fed mice, known to decrease metabolic rate on a per cell basis through excess insulin/IGF-1 production. Congruently, we found lower mitochondrial activation as assessed by flow cytometry and RT-PCR analysis as well as a depletion of the short-term progenitor compartment in high fat versus control chow diet fed mice. We then tested the effects of a mitochondrial activator known to counteract the negative effects of high fat diet. We first analyzed the in vitro effect on HSC cell cycle kinetics, where no significant change in proliferation or division time was found. However, HSCs responded to the mitochondrial activator by increasing asynchrony, a behavior that is thought to directly correlate with asymmetric division [6]. As opposed to high-fat diet fed mice, mice fed with the mitochondrial activator showed an increase in ST-HSCs, while all the other hematopoietic compartments were comparable to mice fed on control diet. Given the dependency on short-term progenitors to rapidly reconstitute hematopoiesis following bone marrow transplantation, we tested the effect of pharmacological mitochondrial activation on the recovery of mice transplanted with a limiting HSC dose. Survival 3 weeks post-transplant was 80% in the treated group compared to 0% in the control group, as predicted by faster recovery of platelet and neutrophil counts. In conclusion, we have found that mitochondrial activation regulates the long-term to short-term HSC transition, unraveling mitochondrial modulation as a valuable drug target for post-transplant therapy. Identification of molecular pathways accountable for the metabolically mediated fate switch is currently ongoing. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Multicolor Flowcytometry Analysis of Hematopoietic Stem and Progenitor Cells Subsets Among Basal and Mobilized Peripheral CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5117-5117
Author(s):  
Valentina Giai ◽  
Elona Saraci ◽  
Eleonora Marzanati ◽  
Christian Scharenberg ◽  
Monica De Stefanis ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Healthy Volunteers ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Progenitor Compartment

Abstract BACKGROUND: In the recent years, numerous studies based on multicolor flowcytometry have analyzed the different subpopulations of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) (Manz MG et al, PNAS 2002; Majeti R et al, Cell Stem Cell 2007): the common myeloid progenitors (CMPs: Lin-CD34+CD38+CD45RA-CD123+), the granulocyte-macrophage progenitors (GMPs: Lin-CD34+CD38+CD45RA+CD123+) and the megakaryocyte-erythroid progenitors (MEPs: Lin-CD34+CD38+CD45RA-CD123-) constitute the progenitor compartment, while the hematopoietic stem cells (HSCs: Lin-CD34+CD38- CD45RA-CD90+), the multipotent progenitors (MPPs: Lin-CD34+CD38- CD45RA-CD90-) and the lymphoid-myeloid multipotent progenitors (LMPPs: Lin-CD34+CD38- CD45RA+CD90-) represent the more immature HSPCs. In animal models, the progenitor compartment includes short-term repopulating cells, leading to the hematological recovery in the first 5 weeks after transplantation, whereas the stem cell compartment comprehends the long-term repopulation cells, responsible for the long-term hematological recovery. However, very little is known about the different subpopulations of HSPCs among peripheral blood (PB) CD34+ in basal state and after mobilization for harvest and transplantation. Our study was conducted to analyze PB CD34+ cells from healthy volunteers and from hematological patients during CD34+ cells mobilization. Our main aim was to understand if the proportions of different HSPCs among PB CD34+ cells were similar to those found in BM and whether the mobilizing regimens employed in chemo treated patients differently affected CD34+ cells subfractions in PB. METHODS: multicolor flowcytometry was used to analyze CD34+ cells from 4 BM samples and 9 PB samples from healthy volunteers and 32 PB samples from hematological patients prior CD34+ cells harvesting. RESULTS: Percentages of CD34+ cells subpopulations were different in basal PB compared to the BM: indeed, CMPs, GMPs and MEPs constituted respectively 27.6% ± 9.5, 23.8% ± 7.2 and 27.6% ± 16.2 of BM CD34+ cells and 47.8% ± 9.5, 10.3% ± 6.9 and 16.1% ± 7.6 of the total PB CD34+ cells. HSCs constituted 2.1% of BM and 1.5% of PB CD34+ cells. The differences between BM and circulating CMPs and GMPs were significant (p<0.005 and p<0.01). No differences in subpopulations proportions were shown comparing G-CSF mobilized and basal PB CD34+ cells. Interestingly, the 2 patients mobilized with AMD3100 (the inhibitory molecule for CXCR4) showed a higher percentage of GMPs (33.8% and 37.8% versus the average 16.3% ± 9.8 in G-CSF mobilized samples) and a lower fraction of CMPs (29.5% and 41.6% versus the average 58% ± 12 in G-CSF mobilized samples). In order to understand this result, we looked then at the CXCR4 mean fluorescence intensity among the progenitor subsets: GMPs showed significantly higher levels of this molecule compared to CMPs and MEPs. Regarding the mobilizing chemotherapy regimens, CMPs percentages were higher (61.1% versus 49.1%, p: 0.038) and GMPs’ were significantly lower (11.1% versus 27.6%, p<0.0001) in cyclophosphamide treated patients, compared to patients mobilized with other chemotherapy regimens. The percentage of HSCs did not significantly differ among bone marrow, unmobilized and mobilized PB CD34+ cells. Therefore, since an average collection of mobilized PB cells contains approximately one log more CD34+ cells than a BM harvest, a similarly higher amount of HSC are infused with mobilized CD34+ cell transplantation. A linear positive correlation between the number of mobilized CD34+ cells and the number of mobilized CMPs, GMPs, and MEPs was observed indicating that the proportions of different HSPCs did not significantly change among high- and low-mobilizers. There were no correlations between the number of mobilized subpopulations and leucocytes, hemoglobin and platelets levels. CONCLUSIONS: Our data displayed the heterogeneity of HSPC compartment between PB and BM. Many factors could contribute to this variegated scenario. These mechanisms comprehension can help us to choose the most suitable chemotherapy and cytokine administrations in order to improve clinical outcomes as infections complications, length of aplasia and transfusion requirements during an hematopoietic stem cell transplantation. Disclosures Palumbo: Bristol-Myers Squibb: Consultancy, Honoraria; Genmab A/S: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Janssen-Cilag: Consultancy, Honoraria; Millennium Pharmaceuticals, Inc.: Consultancy, Honoraria; Onyx Pharmaceuticals: Consultancy, Honoraria; Array BioPharma: Honoraria; Amgen: Consultancy, Honoraria; Sanofi: Honoraria. Boccadoro:Celgene: Honoraria; Janssen: Honoraria; Onyx: Honoraria.

Leukemia Stem Cells in Murine MLL−AF9 AML Are Downstream Myeloid Lineage Cells That Have Acquired Stem−Like Biological and Genetic Properties.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 771-771
Author(s):  
Tim C.P. Somervaille ◽  
Michael L. Cleary
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Leukemia Cell ◽  
Leukemia Stem Cells ◽  
Human Leukemia ◽  
Self Renewal ◽  
Myeloid Lineage ◽  
Hematopoietic Stem ◽  
Progenitor Compartment

Abstract An essential prerequisite for the development of more effective targeted therapies in AML is a characterization of the frequency and biological properties of leukemia stem cells (LSCs), which sustain the disease and mediate relapse. Previous studies have shown that hematopoietic stem cells, as well as progenitors, may be targeted by MLL oncoproteins to give rise to AML, however it was unclear whether these lineage negative cells also functioned as LSCs to sustain disease. To address this issue, we have identified and characterized LSCs in a somatic, genetic mouse model of leukemia that faithfully recapitulates many of the pathologic and clinical attributes of human leukemia initiated by the MLL−AF9 oncogene. In this model, CFCs in the bone marrow and spleen of leukemic mice were demonstrated to be LSCs, based on the observation that secondary transplantation of the progeny of individually isolated AML CFCs cultured in vitro for seven days or longer invariably resulted in transfer of short latency disease. These self−renewing cells were remarkably frequent, accounting for 25–30% of myeloid lineage cells at late−stage disease. Unexpectedly, they expressed mature myeloid lineage antigens (91.3 ± 3.8% Mac1+ Gr1+ immunophenotype, versus fewer than 0.2% lineage negative), placing them downstream of the known hematopoietic progenitor compartment. Furthermore, LSCs in this model generated a phenotypic, morphologic and functional leukemia cell hierarchy loosely defined by expression of c−kit. When compared with immortalized progenitors, LSCs exhibited a markedly enhanced ability to engraft secondary recipients, which was not due to differences in bone marrow homing, which was equivalently poor in the compared populations. Rather, LSCs exhibited an acquired ability to proliferate in response to stromal cell derived cytokines, an enhanced SDF1 induced chemotaxis, and increased proliferation in contact with OP9 stromal cells demonstrating that LSCs exhibit altered microenvironmental interactions by comparison with the oncogene immortalized CFCs that initiated the disease. Thus, the LSCs responsible for sustaining, expanding and regenerating MLL−AF9 AML are downstream myeloid lineage cells, outside of the normal stem and progenitor compartment. They have acquired an aberrant Hox−associated self−renewal program as well as other biologic features of hematopoietic stem cells. Our findings support a revision of the prevailing hypothesis that AML LSCs are always rare and solely located within the most immature bone marrow progenitor compartment. Furthermore, LSCs exhibit markedly different microenvironmental interactions, by comparison with cells simply immortalized by MLL−AF9, indicating that acquisition of sensitivity to stromal derived survival and proliferative signals is a critical feature of LSCs, in addition to their extensive self−renewal capabilities.

scholarly journals An Altered Bone Marrow Vascular Niche Impacts Normal Hematopoiesis and Tilts the Balance Between Myelopoiesis and Lymphopoiesis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 174-174
Author(s):  
Cindy L Hochstetler ◽  
Yuxin Feng ◽  
Yi Zheng
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Growth Factor ◽  
Mouse Model ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem ◽  
Vascular Niche ◽  
Progenitor Compartment

Abstract The bone marrow niche is an important milieu where hematopoietic stem and progenitor cells (HSPCs) are maintained to ensure their lifelong contribution to hematopoiesis. Recent evidence has highlighted the critical importance of the perivascular bone marrow (BM) niche as the key host and regulator of HSPCs. Bone marrow endothelial cells (BMECs) are major components of the vascular niche. While studies have shown that an alteration in a component of the niche can affect hematopoiesis and promote the development of myeloproliferative disorders/myelodysplastic syndromes, it remains unclear how altered BMECs can impact hematopoiesis. To this end, we have generated a Tamoxifen (TAM)-inducible Tie2-CreER/LSL-KRasG12D;tdTomato mouse model to introduce an oncogenic KRas mutation specifically in adult endothelial cells. The tdTomato reporter overlaps with the CD31 and vascular endothelial growth factor receptor 2 (VEGFR2) endothelial cell markers and shows no detectable leakage into the adult hematopoietic compartment. To evaluate changes in hematopoiesis, we performed complete blood counts at 12 weeks post TAM injection and found that the Tie2-CreER/LSL-KRasG12D mice (KRasG12D mice) had significantly more leukocytes (p=0.031) and neutrophils (p=0.002) than controls. Flow cytometry analysis confirmed that the KRasG12D mice had a significantly higher percentage of myeloid cells with concurrent decrease in lymphocyte percentage in the peripheral blood (p=0.016). At 16 weeks post TAM injection, a significant decrease in B cells could also be noted in the blood of KRasG12D mice (p=0.028). Compared to controls, the KRasG12D mice displayed splenomegaly (p=0.025) and their spleens had a higher percentage of myeloid cells (p=0.002). There was an increase in the common myeloid progenitor compartment in the spleen and a significant increase in the granulocyte macrophage progenitor compartment (p=0.014) of KRasG12D mice. These mice also had an increase in the short-term hematopoietic stem cell (ST-HSC) compartment both in the BM and spleen. Colony forming assays revealed that KRasG12D mice had a higher number of total colonies formed from BM (p=0.044), spleen (p=0.007) and blood cells (p=0.56). Genotyping PCR showed no KRasG12D activation in hematopoietic cells, confirming that the observed phenotypes were due to an effect in BMECs. To complement our native inducible mouse model, we transplanted BM cells from syngeneic BoyJ mice into lethally irradiated Tie2-CreER;KRasG12D or KRasWT recipients. The endothelial KRasG12D recipientsdied between 75-200 days post transplantation (p=0.0079) while the KRasWT recipients remained alive. The KRasG12D recipients also displayed splenomegaly (p=0.004). Competitive transplant studies with donor cells from KRasG12D or KRasWT mice with competitor cells from syngeneic mice (CD45.1) showed that BM cells from the KRasG12D mice (CD45.2) outcompeted cells from KRasWT mice with a significantly higher percentage of CD45.2 donor chimerism in all blood lineages examined. To uncover any molecular events underpinning these hematopoietic changes, we performed quantitative real-time polymerase chain reaction. Our preliminary experiments from total BM RNA of KRasG12D or KRasWT mice indicate that there is a significant increase in VEGFα and a decrease in transforming growth factor β in KRasG12D mice, accompanying the above noted increase in the ST-HSC population. Collectively, our data provide strong evidence that an abnormal vascular niche caused by oncogenic insults in BMECs can disrupt normal hematopoiesis and promote a myeloproliferative phenotype, thereby implicating abnormal BMECs as novel contributors to blood pathogenesis. Studies are underway to further assess the molecular contributions from the disrupted vascular niche and the resulting HSPCs. Uncovering the mechanism of how altered BMECs can remodel hematopoiesis holds the exciting promise of better therapeutic strategies. Disclosures No relevant conflicts of interest to declare.

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