The Role of Third Complement Component (C3) in Homing of Hematopoietic Stem/Progenitor Cells into Bone Marrow

2007 ◽  
pp. 35-51 ◽  
Author(s):  
Ryan Reca ◽  
Marcin Wysoczynski ◽  
Jun Yan ◽  
John D. Lambris ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Complement Component ◽  
Hematopoietic Stem ◽  
Complement Component C3

Differential Role for VLA-5 in Mobilization of Heamotopoieic Stem Cells Toward Peripheral Blood and Homing to Bone Marrow and Spleen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2190-2190 ◽  
Author(s):  
Pieter K. Wierenga ◽  
Ellen Weersing ◽  
Bert Dontje ◽  
Gerald de Haan ◽  
Ronald P. van Os
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Hematopoietic Stem ◽  
Late Antigen ◽  
Cell Mobilization

Abstract Adhesion molecules have been implicated in the interactions of hematopoietic stem and progenitor cells with the bone marrow extracellular matrix and stromal cells. In this study we examined the role of very late antigen-5 (VLA-5) in the process of stem cell mobilization and homing after stem cell transplantation. In normal bone marrow (BM) from CBA/H mice 79±3 % of the cells in the lineage negative fraction express VLA-5. After mobilization with cyclophosphamide/G-CSF, the number of VLA-5 expressing cells in mobilized peripheral blood cells (MPB) decreases to 36±4%. The lineage negative fraction of MPB cells migrating in vitro towards SDF-1α (M-MPB) demonstrated a further decrease to 3±1% of VLA-5 expressing cells. These data are suggestive for a downregulation of VLA-5 on hematopoietic cells during mobilization. Next, MPB cells were labelled with PKH67-GL and transplanted in lethally irradiated recipients. Three hours after transplantation an increase in VLA-5 expressing cells was observed which remained stable until 24 hours post-transplant. When MPB cells were used the percentage PKH-67GL+ Lin− VLA-5+ cells increased from 36% to 88±4%. In the case of M-MPB cells the number increased from 3% to 33±5%. Although the increase might implicate an upregulation of VLA-5, we could not exclude selective homing of VLA-5+ cells as a possible explanation. Moreover, we determined the percentage of VLA-5 expressing cells immediately after transplantation in the peripheral blood of the recipients and were not able to observe any increase in VLA-5+ cells in the first three hours post-tranpslant. Finally, we separated the MPB cells in VLA-5+ and VLA-5− cells and plated these cells out in clonogenic assays for progenitor (CFU-GM) and stem cells (CAFC-day35). It could be demonstared that 98.8±0.5% of the progenitor cells and 99.4±0.7% of the stem cells were present in the VLA-5+ fraction. Hence, VLA-5 is not downregulated during the process of mobilization and the observed increase in VLA-5 expressing cells after transplantation is indeed caused by selective homing of VLA-5+ cells. To shed more light on the role of VLA-5 in the process of homing, BM and MPB cells were treated with an antibody to VLA-5. After VLA-5 blocking of MPB cells an inhibition of 59±7% in the homing of progenitor cells in bone marrow could be found, whereas homing of these subsets in the spleen of the recipients was only inhibited by 11±4%. For BM cells an inhibition of 60±12% in the bone marrow was observed. Homing of BM cells in the spleen was not affected at all after VLA-5 blocking. Based on these data we conclude that mobilization of hematopoietic progenitor/stem cells does not coincide with a downregulation of VLA-5. The observed increase in VLA-5 expressing cells after transplantation is caused by preferential homing of VLA-5+ cells. Homing of progenitor/stem cells to the bone marrow after transplantation apparantly requires adhesion interactions that can be inhibited by blocking VLA-5 expression. Homing to the spleen seems to be independent of VLA-5 expression. These data are indicative for different adhesive pathways in the process of homing to bone marrow or spleen.

scholarly journals p53-dependent induction of P2X7 on hematopoietic stem and progenitor cells regulates hematopoietic response to genotoxic stress

2021 ◽  
Vol 12 (10) ◽  
Author(s):  
Lin Tze Tung ◽  
HanChen Wang ◽  
Jad I. Belle ◽  
Jessica C. Petrov ◽  
David Langlais ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Transcriptional Response ◽  
Genotoxic Stress ◽  
Whole Body ◽  
Hematopoietic Stem ◽  
Hematopoietic System ◽  
Stem And Progenitor Cells ◽  
Hematopoietic Response

AbstractStem and progenitor cells are the main mediators of tissue renewal and repair, both under homeostatic conditions and in response to physiological stress and injury. Hematopoietic system is responsible for the regeneration of blood and immune cells and is maintained by bone marrow-resident hematopoietic stem and progenitor cells (HSPCs). Hematopoietic system is particularly susceptible to injury in response to genotoxic stress, resulting in the risk of bone marrow failure and secondary malignancies in cancer patients undergoing radiotherapy. Here we analyze the in vivo transcriptional response of HSPCs to genotoxic stress in a mouse whole-body irradiation model and, together with p53 ChIP-Seq and studies in p53-knockout (p53KO) mice, characterize the p53-dependent and p53-independent branches of this transcriptional response. Our work demonstrates the p53-independent induction of inflammatory transcriptional signatures in HSPCs in response to genotoxic stress and identifies multiple novel p53-target genes induced in HSPCs in response to whole-body irradiation. In particular, we establish the direct p53-mediated induction of P2X7 expression on HSCs and HSPCs in response to genotoxic stress. We further demonstrate the role of P2X7 in hematopoietic response to acute genotoxic stress, with P2X7 deficiency significantly extending mouse survival in irradiation-induced hematopoietic failure. We also demonstrate the role of P2X7 in the context of long-term HSC regenerative fitness following sublethal irradiation. Overall our studies provide important insights into the mechanisms of HSC response to genotoxic stress and further suggest P2X7 as a target for pharmacological modulation of HSC fitness and hematopoietic response to genotoxic injury.

Unexpected Evidence That Dimethylsulphoxide (DMSO) Upregulates Expression of CXCR4 on Hematopoietic Stem/Progenitor Cells (HSPC), Increases Their Responsiveness to an SDF-1 Gradient and Enhances Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1973-1973
Author(s):  
Marcin Majka ◽  
Danuta Jarocha ◽  
Marcin Wysoczynski ◽  
Duygu Sag ◽  
Ewa Zuba-Surma ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Short Exposure ◽  
Hematopoietic Stem ◽  
Akt Phosphorylation ◽  
Human Cd34 ◽  
New Strategy ◽  
First Time

Abstract Cryopreservation of bone marrow (BM), mobilized peripheral blood (mPB) and cord blood (CB) cells is a routine procedure to store hematopoietic stem/progenitor cells (HSPC) for transplantation. Dimethylsulphoxide (DMSO), the most commonly used cryoprotectant, is toxic to cells at higher concentrations (>10%); moreover, the freezing-thawing procedure itself is inevitably connected with the loss of HSPC. However, by chance we observed that short exposure of HSPC to DMSO enhances the responsiveness of these cells to an SDF-1 gradient and since SDF-1 is a major chemoattractant that navigates homing of HSPC to BM we became interested in elucidating this phenomenon. We found that short incubation (5–10 min) of human CB mononuclear cells (MNC) with DMSO at concentrations employed for cryopreservation (5–10%) significantly upregulates the expression of both CXCR4 (x 2–3) and CD34 (x 1.5) on CB MNC (as measured by FACS). Furthermore, DMSO significantly increased the chemotactic responsiveness (x 2–4) of CB MNC, BM MNC and selected CXCR4+ human hematopoietic cell lines (Jurkat, THP-1 cells) when the cells were exposed to 5–10% DMSO before chemotaxis assay. These responses to an SDF-1 gradient correlated with enhanced chemotaxis also of human CD34+, CD34+ CD38+, CD34+ CD38−, and CD34+ CXCR4+ clonogeneic progenitor cells, suggesting that DMSO directly enhances the responsiveness of human early progenitors (p<0.0001). At the molecular level, 5–10% DMSO strongly stimulated and prolonged SDF-1-dependent AKT phosphorylation. However, at the same time DMSO inhibited phosphorylation of MAPKp42/44. Similar observations were made for Sca-1+ BM-derived murine cells. In parallel experiments we found that murine Sca-1+ cells when preincubated with DMSO formed more 12 day-CFU-S colonies in spleens after transplantation into irradiated syngeneic recipients. Accordingly, x 2 more CFU-S were formed when Sca-1+ cells were exposed before transplantation to 5% DMSO and about x 4 more after exposure to 10% DMSO. Finally we employed a Ly5.1/Ly5.2 congeneic transplant model and showed that transplantation of Ly5.1 Sca-1+ cells exposed to 10% DMSO before transplantation resulted in higher chimerism in transplanted Ly5.2 mice as compared to untreated cells (control) (p<0.0001). In conclusion, we show for the first time an unexpected beneficial role of DMSO (5–10%) in regulation of homing of HSPC after transplantation and suggest that a short priming of HSPC with DMSO, even of non-cryopreserved cells, before transplantation may become a new strategy to enhance engraftment

Homeostatic Role of the Krüppel-Like Factor 4 (KLF4) in Proliferation and Differentiation of Primitive Hematopoietic Progenitor Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1497-1497 ◽  
Author(s):  
Chun Shik Park ◽  
Takeshi Yamada ◽  
H. Daniel Lacorazza
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Proliferative Potential ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Deficient Mice

Abstract Abstract 1497 Poster Board I-520 KLF4 is a tumor suppressor in the gastrointestinal tract known to induce cell cycle arrest in a cell context dependent manner. We recently reported that KLF4 maintains quiescence of T lymphocytes downstream of T-cell receptor signaling (Yamada et al., Nature Immunology, 2009). The role of KLF4 in reprogramming adult somatic cells into pluripotent stem cells along with Oct3/4, c-Myc and Sox2 suggests that KLF4 restricts proliferation of undifferentiated cells. In spite of a redundant role of KLF4 in fetal liver hematopoietic stem cells (HSC), its role in the maintenance of adult bone marrow HSCs has not been studied yet. To study the role of KLF4 in the hematopoietic system we used gain- and loss-of-function mouse models. Retroviral transfer of KLF4 into wild type bone marrow (BM) cells led to significant reduction of colony forming units (CFU) in methylcellulose cultures due to increased apoptosis and lower proliferation. Then, Mx1-Cre was used to induce deletion of Klf4-floxed mice by polyI:C administration. Analysis of peripheral blood cells up to 6-9 months post polyI:C administration showed significant reduction of monocytes, as previously reported, and expansion of CD8+CD44+ T cells due to their increased proliferative potential. BM cells from Klf4-deficient mice exhibited increased number of myeloid progenitor cells measured by flow cytometry (Lin-Sca-1-c-kit+FcRII/III+CD34+ cells), CFU and CFU-S8. Cytoablation with 5-fluorouracil (5-FU) showed lower nadir of peripheral white blood cells in Klf4-deficient mice compared to control mice. In spite of normal multilineage reconstitution in BM transplants experiments, competitive reconstitution with Klf4-deficient and normal BM cells resulted in reduced contribution of Klf4-deficient cells to peripheral blood, likely due to homing and proliferative differences. Collectively, our data shows that KLF4 has an important role in function of hematopoietic stem and progenitor cells. Disclosures: No relevant conflicts of interest to declare.

Distinct Effects of SCF and Hes1 On Normal Hematopoietic Stem and Progenitor Cells During Leukemic Cell Expansion in a T-ALL Mouse Model

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1216-1216
Author(s):  
Chen Tian ◽  
Zhipan Cao ◽  
Qiao Li ◽  
Jinhong Wang ◽  
Zhenyu Ju ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mouse Model ◽  
Progenitor Cells ◽  
Real Time ◽  
Hematopoietic Cells ◽  
Leukemic Cell ◽  
Leukemic Cells ◽  
Rt Pcr ◽  
Hematopoietic Stem

Abstract Abstract 1216 During leukemia development, emerging leukemic cells out-compete normal hematopoietic cells and become predominant in the body. How hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) respond to the growth of leukemic cell population is an important, yet less investigated area. Our previous study demonstrated differential effects of a leukemic environment on normal HSCs and HPCs in the Notch1-induced T-ALL mouse model (Hu X, et al. Blood 2009). We found that normal HSCs were better preserved in the leukemic bone marrow in part due to increased quiescence of the HSCs and in contrast, HPCs were exhausted during the expansion of leukemic cells. Our current work is aimed to further explore the molecular mechanisms concerning the distinct impacts of leukemic environment on normal HSCs and HPCs in the T-ALL mouse model. Given the previous report by others showing that increased secretion of stem cell factor (SCF) by myeloid leukemia cells played an important role in inducing normal HSCs/HPCs out of their niche and thus allowing leukemic cells to occupy the niche in the human-NOD/SCID xeno-graft model (Sipkins DA et al, Science 2008), we first examined the expression of SCF by ELISA, Western blot and real-time RT PCR in both normal hematopoietic and leukemic cell fractions in the Notch1-induced T-ALL mouse model as previously reported. We found that while expression of SCF in peripheral blood (PB) or bone marrow (BM) was increased in the leukemic mice, both mRNA and protein levels of SCF in normal hematopoietic cells were higher than that in leukemic cells, thereby suggesting that elevated SCF might be mainly secreted by non-leukemic cells in the leukemic hosts of our model. Further assessments on the role of SCF in leukemogenesis with the mice specifically deficient in SCF in different niche cell types are currently under investigation in our laboratory. In order to define potential mediators in HSCs in response to leukemic cell growth, a microarray study on normal HSCs isolated from T-ALL leukemic mice and the control mice was conducted. Gene expression profiling showed significantly differed expression of 169 genes (127 up and 42 down). Especially, real-time RT PCR confirmed an increase of Hes1, p21, Fbxw11, IL-18R1 and Itgb3, and a decrease of CXCR4 and Mmp2. Interestingly, the expression of Hes1 and its target gene, p21 were elevated in normal HSCs but not in HPCs, letting us to hypothesize that Hes1 might be in part mediate the different responses of HSCs and HPCs to the T-ALL leukemic environment. To test this hypothesis, we ectopically expressed Hes1 in normal hematopoietic cells and then examined their functions under the leukemic condition. BM cells from B6.SJL mice were transduced with either MSCV-Hes1-IRES-GFP or control MSCV-GFP vector. After transduction, Hes1-GFP+or control-GFP+cells were co-transplanted with the Notch1-induced T-ALL cells into lethally irradiated C57BL/6J recipients. The engrafted cells from the leukemic BM were analyzed and Hes1-GFP+or control-GFP+cells were sorted for functional assessments. Interestingly, although over-expression of Hes1 inhibited the growth of colony forming cell (CFC) in vitro, it could potentiate the long-term repopulating cells by maintaining more cells in the quiescent (G0) state in vivo. Taken together, our current study supports a role of Hes1 in mediating the distinct responses of normal HSCs and HPCs to the T-ALL leukemic environment. Disclosures: No relevant conflicts of interest to declare.

Dual Role Of MERIT40 In Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 797-797
Author(s):  
Krasimira Rozenova ◽  
Jing Jiang ◽  
Chao Wu ◽  
Junmin Wu ◽  
Bernadette Aressy ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Peripheral Blood ◽  
Bone Marrow Failure ◽  
Adaptor Protein ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The balance between self-renewal and differentiation of hematopoietic stem cells (HSCs) is maintained by cell intrinsic and extrinsic mechanisms, including tight regulation of signaling pathways such as Tpo-Mpl and SCF-ckit. Posttranslational modifications, such as phosphorylation and ubiquitination, regulate these pathways. While the role of protein phosphorylation is well established, the importance of ubiquitination in HSC self-renewal has not been well addressed. It is known that of the seven different lysines on ubiquitin, Lys48 polyubiquitination is a marker for protein degradation, and Lys63 polyubiquitination is associated with regulation of kinase activity, protein trafficking, and localization. In this study, we provide evidence that the adaptor protein MERIT40 has multiple roles in hematopoietic stem/progenitor cells (HSPCs). MERIT40 is a scaffolding protein shared by two distinct complexes with Lys63 deubiquitinase (DUB) activities: the nuclear RAP80 complex with a known role in DNA damage repair in breast/ovarian cancer cells, whereas the functions of the cytoplasmic BRISC remains less characterized. MERIT40 is important for integrity of both complexes, and its deficiency leads to their destabilization and a >90% reduction in deubiquitinase activity. By using MERIT40 knockout (M40-/-) mice, we found that lack of MERIT40 leads to a two-fold increase in phenotypic and functional HSCs determined by FACS and limiting dilution bone marrow transplantation (BMT), respectively. More importantly, M40-/- HSCs have increased regenerative capability demonstrated by increased chimerism in the peripheral blood after BMT of purified HSCs. The higher self-renewal potential of these HSCs provides a survival advantage to M40-/- mice and HSCs after repetitive administration of the cytotoxic agent 5-flurouracil (5FU). MERIT40 deficiency also preserves HSC stemness in culture as judged by an increase in peripheral blood chimerism in recipient mice transplanted with M40-/- Lin-Sca1+Kit+ (LSK) cells cultured in cytokines for nine days compared to recipient mice receiving cultured wildtype (WT) LSK cells. In contrast to the increased HSC homeostasis and superior stem cell activity due to MERIT40 deficiency, M40-/- mice are hypersensitive to DNA damaging agents caused by inter-cross linking (ICL), such as Mitomycin C (MMC) and acetaldehydes that are generated as side products of intracellular metabolism. MMC injection caused increased mortality in M40-/- mice compared to WT controls attributable to DNA damage-induced bone marrow failure. MMC-treated M40-/- mice showed marked reduction in LSK progenitor numbers accompanied by increased DNA damage, in comparison to WT mice. Consistent with the in vivo studies, M40-/- progenitor cells are hypersensitive to MMC and acetaldehyde treatment in a cell-autonomous manner in colony forming assays. ICL repair is known to require Fanconi Anemia (FA) proteins, an ICL repair network of which mutations in at least 15 different genes in humans cause bone marrow failure and cancer predisposition. Thus, M40-/- mice represent a novel mouse model to study ICL repair in HSPCs with potential relevance to bone marrow failure syndromes. Taken together, our data establishes a complex role of MERIT40 in HSPCs, warranting future investigation to decipher functional events downstream of two distinct deubiquitinating complexes associated with MERIT40 that may regulate distinct aspects of HSPC function. Furthermore, our findings reveal novel regulatory pathways involving a previously unappreciated role of K63-DUB in stem cell biology, DNA repair regulation and possibly bone marrow failure. DUBs are specialized proteases and have emerged as potential “druggable” targets for a variety of diseases. Hence, our work may provide insights into novel therapies for the treatment of bone marrow failure and associated malignancies that occur in dysregulated HSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Defective engraftment of C3aR−/− hematopoietic stem progenitor cells shows a novel role of the C3a–C3aR axis in bone marrow homing

Leukemia ◽  
2009 ◽  
Vol 23 (8) ◽  
pp. 1455-1461 ◽  
Author(s):  
M Wysoczynski ◽  
R Reca ◽  
H Lee ◽  
W Wu ◽  
J Ratajczak ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Hematopoietic Stem

scholarly journals Ing4 Regulates Stress Hematopoiesis and Represses Self-Renewal in Multipotent Progenitor Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 724-724
Author(s):  
Zanshe Thompson ◽  
Melanie Rodriguez ◽  
Seth Gabriel ◽  
Georgina Anderson ◽  
Vera Binder ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Progenitor Cells ◽  
Cell Transplant ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Multipotent Progenitor Cells ◽  
Equity Ownership

Hematopoiesis is tightly regulated by a network of transcription factors and complexes that are required for the maintenance and development of HSCs. In a screen for epigenetic regulators of hematopoiesis in zebrafish, we identified a requirement of the tumor suppressor protein, Ing4, in hematopoietic stem and progenitor cell (HSPC) specification. Though the Ing4 mechanism of action remains poorly characterized, it has been shown to promote stem-like cell characteristics in malignant cells and is a frequent target of inactivation in various cancer types. The tumor suppressive activity is, in part, due to the inhibitory role of Ing4 in the NF-kB signaling pathway. In zebrafish, loss of Ing4 results in loss of HSC specification and a significant increase in NF-kB target gene expression. Knockdown of NF-kB expression in Ing4 deficient zebrafish recovered HSC marker expression in the aorta suggesting that NF-kB inhibition could remediate the loss of Ing4 expression. Small molecule NF-kB pathway inhibitors with varying mechanisms were also observed to rescue of HSC marker staining in the zebrafish aorta. Ing4 deficient embryos incubated with a lower dose of inhibitor had a 31% recovery of marker staining and 82% of embryos incubated in the highest dose recovered HSC marker staining emphasizing a dose dependent rescue of HSC specification through NF-kB suppression. As in the zebrafish, we have identified a requirement for Ing4 in murine hematopoiesis. Ing4-/- bone marrow has aberrant hematopoiesis resulting in an increase in the number of short term-HSCs (ST-HSCs) (11.4% vs 31.7%) and a dramatic decrease in multipotent progenitor cells (MPPs) (47.9% vs 19.3%) along with a concurrent modest increase in the population of long-term HSCs (LT-HSCs) (2.4% vs 5.5%). Analysis of differentiation in Ing4 null bone marrow also reveals skewed hematopoiesis. We see a 14% increase in granulocytes in the null mouse marrow and observe similar skewing in CFU assays. Additionally, there were alterations in stress hematopoiesis following hematopoietic stem cell transplant. Sorted LT-HSCs fail to engraft, suggesting an evolutionarily conserved requirement for Ing4 in HSCs. Surprisingly, competitive transplantation assay with Ing4-defecient MPPs versus wild-type showed dramatic increase in peripheral blood multilineage chimerism up to 9 months post-transplantation (19% vs. 59%). This lends to the hypothesis that Ing4 deficient MPPs gain self-renewal capabilities. In further characterization of these cells, we found an increase in MPPs that express lower levels of CD34 (55.5% vs 67.7%). CD34 expression is a marker of HSCs. This CD34+/mid population also express CD229 (85% positive), which is barely detectable in wildtype marrow (less that 0.01%). CD229 is also an HSC marker. Based on these exciting findings, we hypothesize that we have identified a subset of CD34+/midCD229+ MPPs in Ing4 deficient mice that retain self-renewal characteristics. Our data suggest that Ing4 normally functions as a critical suppressor for genes required for self-renewal and developmental potency in MPPs. Overall, our findings suggest that Ing4 plays a crucial role in the regulation of hematopoiesis and provides key tools for further identification and characterization of Ing4 pathways and functions. Given the role of Ing4 in both normal hematopoiesis and cancer, this gene likely has a critical role in regulation of stem cell self-renewal and maintenance. Disclosures Zon: CAMP4: Equity Ownership; Fate Therapeutics: Equity Ownership; Scholar Rock: Equity Ownership.

scholarly journals NIPA As a Novel Regulator of Aging and Stress Response of the Primitive HSC Pool

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 1155-1155
Author(s):  
Stefanie Kreutmair ◽  
Rouzanna Istvanffy ◽  
Cathrin Klingeberg ◽  
Christine Dierks ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Damage ◽  
Stress Response ◽  
Progenitor Cells ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Hematopoietic Stem ◽  
Age Related

Abstract Accumulation of DNA damage in hematopoietic stem cells (HSCs) is associated with aging, bone marrow failure and development of hematological malignancies. Although HSCs numerically expand with age, their functional activity declines over time and the protection mechanism from DNA damage accumulation remains to be elucidated. NIPA (Nuclear Interaction Partner of ALK) is highly expressed in hematopoietic stem and progenitor cells, especially in the most primitive long-term repopulating HSCs (CD34-Flt3-Lin-Sca1+cKit+). Loss of NIPA leads to a significant exhaustion of primitive hematopoietic cells, where Lin-Sca1+cKit+ (LSK) cells were reduced to 40% of wildtype (wt) littermates (p<0.001). All LSK-subgroups, LT-HSCs (p<0.001), ST-HSCs (CD34+Flt3-LSK; p<0.01) and MPPs (CD34+Flt3+LSK; p<0.05) of NIPA deficient animals are affected and failed to age-related increase, whereas the lineage differentiation of Nipako/ko progenitor cells showed no gross differences. Myeloid depression by 5-FU treatment led to severely reduced HSC self renewal in Nipako/ko mice independent of age (p<0.001). Moreover, weekly 5-FU activation showed reduced survival of Nipako/ko vs. wt animals (11 vs. 14.5 days). To further examine the role of NIPA in HSC maintenance and exhaustion, we performed in vivo repopulationexperiments, where Nipa deletion causes bone marrow failure in case of competition, as Nipako/ko cells contributed to less than 10% of transplanted BM cells 6 month after transplantation (TX). According to this, colony formation assays and limiting dilution transplantation showed a dramatic reduction of competitive repopulation units (p<0.0001) in Nipako/ko animals. Serial LSK transplantation assays revealed loss of Nipa-deficient LSKs shortly after TX, whereas long-term repopulation capacity seemed to be maintained, suggesting a role of NIPA in critical stress response. To further investigate the stress response in Nipa-deficient HSCs, we irradiated LSKs with 3 Gy and stained for DNA-Damage foci by pH2ax. Remarkably, loss of NIPA led to significant higher numbers of pH2ax foci in irradiated HSCs (46% > 6 foci vs. 17% > 6 foci in wt cells) and highly increased the rates of apoptotic cells especially in the primitive CD34-LSK population. Taken together our results highlight the importance of the DNA damage response at HSC level for lifelong hematopoiesis and establish NIPA as a novel regulator of aging and stress response of the primitive HSC pool. Disclosures No relevant conflicts of interest to declare.

Involvement of Transcriptional Mediator Subunit TRAP220/MED1 in Action of Niche Cells to Support Hematopoietic Stem/Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3581-3581
Author(s):  
Kana Inoue ◽  
Akiko Sumitomo ◽  
Natsumi Hasegawa ◽  
Ayuko Kasai ◽  
Kenji Yonezawa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Dna Synthesis ◽  
Progenitor Cells ◽  
Target Genes ◽  
Bone Marrow Cells ◽  
Live Cells ◽  
Hematopoietic Stem ◽  
Marrow Cells

Abstract The mammalian TRAP/Mediator complex is a master transcriptional regulatory complex that integrates signals of diverse activators and recruits RNA polymerase II and other general factors to activate transcription. The TRAP220/MED1 subunit was originally identified as a ligand-dependent coactivator specific for nuclear receptors. We have previously shown through biochemical and mouse genetic studies that MED1 is essential for embryogenesis, cell growth/differentiation and homeostasis, and that it stimulates nuclear receptor-mediated myelomonopoiesis. MED1 also integrates other activators such as GATA-1 and C/EBPβ and appears to mediate erythropoiesis as well. The niche cells in the bone marrow plays a pivotal role in the maintenance of hematopoietic stem/progenitor cells (HSPCs). In this study, we employed mouse embryonic fibroblasts (MEFs) as a model to analyze the role of MED1 in the niche, since MEFs have a mesenchymal feature with the osteoblastic precursor lineage and are known to support HSPCs. To establish an experimental system, we crossed Med1 and p53 double knockouts to obtain Med1+/+/p53−/− and Med1−/−/p53−/− E10.0 embryos from a single female and prepared stable MEF lines. Then the Med1−/−/p53−/− MEFs were stably transfected with a MED1 expression vector (Rev-Med1−/− MEFs) or a control empty vector. When normal mouse bone marrow cells were cocultured with these MEFs treated with mitomycin C for a short period of 2 weeks, cell counts, live cells (MTT assay) and a DNA synthesis (BrdU incorporation) of marrow cells were measured. The number of live cells as well as DNA synthesis on Med1−/− MEFs was significantly decreased during this period, but those on Rev-Med1−/− MEFs recovered to the control levels. Thus the growth stress on MEFs appears to be attenuated on Med1−/− MEFs. When apoptosis of the marrow cells was measured, both the FITC-dUTP incorporation by TdT and annexin V/PI double positive cells were lower for Med1−/− MEFs, indicating that apoptosis was also attenuated. We next assessed the role of MED1 in MEFs to support long-term bone marrow culture. After bone marrow cells were cultured on mitomycin C-treated MEFs for 8 weeks in Myelocult M5300 (StemCell Technologies) or IMDM supplemented with BIT9500 (StemCell Technologies) and LDL, progenitor cells (adherent and nonadherent) were collected and cultured in complete methylcellulose (Methocult M3434; StemCell Technologies), and colonies were counted. The number of both myeloid and erythroid colonies were significantly attenuated (0 to 40% depending on experimental conditions) for cells on Med1−/− MEFs, but colonies for cells cultured on Rev-Med1−/− MEFs recovered to the control level. In order to exclude the possibility that lot differences among MEFs or p53 depletion might have affected the results, we next prepared primary Med1+/+ and Med1−/− MEFs by crossing Med1+/− mice and conducted the long-term culture experiments using these MEFs. The attenuated number of colonies for cells cocultured with Med1−/− MEFs (circa 10% of the control) was reproduced repeatedly, indicating that the observed role of MED1 in MEFs to support HSPCs is intrinsic. Since MED1 converges signals from a series of activators on specific promoters and activates transcription, one or some products of the downstream target genes in MEFs may be responsible for the observed activity to maintain HSPCs. In search for candidate MED1 target gene products among a series of known molecules that possess an activity on HSPCs, only the expression of osteopontin was found to be attenuated in Med1−/− MEFs and reverted in Rev-Med1−/− MEFs. Other factors including Angiopoietin-1 and Jagged-1 were comparable. This fact contrasts with the previous observation of osteopontin knockouts where the null niche cells that restricted the size of HSPC number overexpressed these factors. We next assessed the role of MED1 on the osteopontin promoter. We focused on vitamin D receptor (VDR) and Runx2 among the activators and tested MEFs by luciferase reporter assays. The basal level of transcription without any activators in Med1−/− MEFs was about half of the control. Moreover, both the activation by Runx2 and the liganddependent VDR function were significantly attenuated in Med1−/− MEFs. These results indicate that transcriptional coactivator MED1 in niche cells plays an important role in HSPCs support, and that osteopontin may be one of the downstream candidate target genes for MED1.

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