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

Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3143-3149 ◽  
Author(s):  
Anna Janowska-Wieczorek ◽  
Marcin Majka ◽  
Jacek Kijowski ◽  
Monika Baj-Krzyworzeka ◽  
Ryan Reca ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Clinical Implications ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Platelet Binding ◽  
Mobilized Peripheral Blood

Abstract Because human CD34+ and murine Sca-1+hematopoietic stem–progenitor cells (HSPCs) express platelet-binding sialomucin P-selectin (CD162) and integrin Mac-1 (CD11b–CD18) antigen, it was inferred that these cells might interact with platelets. As a result of this interaction, microparticles derived from platelets (PMPs) may transfer many platelet antigens (CD41, CD61, CD62, CXCR4, PAR-1) to the surfaces of HSPCs. To determine the biologic significance of the presence of PMPs on human CD34+ and murine Sca-1+ cells, their expressions on mobilized peripheral blood (mPB) and on nonmobilized PB- and bone marrow (BM)–derived CD34+ cells were compared. In addition, the effects of PMPs on the proliferation of CD34+ and Sca-1+ cells and on adhesion of HSPCs to endothelium and immobilized SDF-1 were studied. Finally, the hematopoietic reconstitution of lethally irradiated mice receiving transplanted BM mononuclear cells covered or not covered with PMPs was examined. It was found that PMPs are more numerous on mPB than on BM CD34+cells, do not affect the clonogenicity of human and murine HSPCs, and increase adhesion of these cells to endothelium and immobilized SDF-1. Moreover, murine BM cells covered with PMPs engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of HSPCs. This could explain why in a clinical setting human mPB HSPCs (densely covered with PMPs) engraft more rapidly than BM HSPCs (covered with fewer PMPs). These findings indicate a new role for PMPs in stem cell transplantation and may have clinical implications for the optimization of transplantations.

Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment

Blood ◽  
2001 ◽  
Vol 98 (10) ◽  
pp. 3143-3149
Author(s):  
Anna Janowska-Wieczorek ◽  
Marcin Majka ◽  
Jacek Kijowski ◽  
Monika Baj-Krzyworzeka ◽  
Ryan Reca ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Mononuclear Cells ◽  
Clinical Implications ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Hematopoietic Reconstitution ◽  
Cd34 Cells ◽  
Platelet Binding ◽  
Mobilized Peripheral Blood

Because human CD34+ and murine Sca-1+hematopoietic stem–progenitor cells (HSPCs) express platelet-binding sialomucin P-selectin (CD162) and integrin Mac-1 (CD11b–CD18) antigen, it was inferred that these cells might interact with platelets. As a result of this interaction, microparticles derived from platelets (PMPs) may transfer many platelet antigens (CD41, CD61, CD62, CXCR4, PAR-1) to the surfaces of HSPCs. To determine the biologic significance of the presence of PMPs on human CD34+ and murine Sca-1+ cells, their expressions on mobilized peripheral blood (mPB) and on nonmobilized PB- and bone marrow (BM)–derived CD34+ cells were compared. In addition, the effects of PMPs on the proliferation of CD34+ and Sca-1+ cells and on adhesion of HSPCs to endothelium and immobilized SDF-1 were studied. Finally, the hematopoietic reconstitution of lethally irradiated mice receiving transplanted BM mononuclear cells covered or not covered with PMPs was examined. It was found that PMPs are more numerous on mPB than on BM CD34+cells, do not affect the clonogenicity of human and murine HSPCs, and increase adhesion of these cells to endothelium and immobilized SDF-1. Moreover, murine BM cells covered with PMPs engrafted lethally irradiated mice significantly faster than those not covered, indicating that PMPs play an important role in the homing of HSPCs. This could explain why in a clinical setting human mPB HSPCs (densely covered with PMPs) engraft more rapidly than BM HSPCs (covered with fewer PMPs). These findings indicate a new role for PMPs in stem cell transplantation and may have clinical implications for the optimization of transplantations.

Defective Engraftment of HSPC from C3aR−/ − Mice Reveals an Underappreciated Role of C3a-C3aR Axis in Stem Cell Homing to Bone Marrow.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1259-1259
Author(s):  
Ryan Reca ◽  
Magda Kucia ◽  
Jarek Baran ◽  
Janina Ratajczak ◽  
Mariusz Z. Ratajczak
Keyword(s):  
Bone Marrow ◽  
Membrane Lipid ◽  
Small Gtpases ◽  
Scid Mice ◽  
Classical Pathway ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Stem Cell Homing ◽  
Chemotactic Gradient

Abstract Recently we demonstrated that complement (C) is activated in bone marrow (BM) during conditioning for transplantation and hematopoietic stem/progenitor cells (HSPC) express on their surface the C3a receptor (C3aR) and in the presence of the third C component - C3 cleavage fragments (C3a and desArgC3a) respond more robustly to a chemotactic gradient of stromal-derived factor (SDF)-1 (Reca et al., Blood2003, 101, 3784). The molecular explanation for this phenomenon is a C3a mediated increase in the incorporation of CXCR4 into membrane lipid rafts what enables CXCR4 to interact better with small GTPases from the Rho/Rac family (Wysoczynski et al. Blood2005, 105, 40–48). To elucidate this phenomenon better and to learn more on the role of the C3a-C3aR axis in homing/engraftment of HSPC we studied i) engraftment of murine HSPC derived from C3aR-deficient mice into wild type littermates and ii) human HSPC on which C3aR was blocked by C3aR antagonist SB290157 into NOD/SCID mice. We noticed that wt mice transplanted with C3aR−/ − HSPC engrafted significantly worse compared to normal littermates. Accordingly, transplantation of the same numbers of Sca-1+ cells from C3aR−/ − mice into wt littermates as compared to transplantation of wt cells resulted in i) delay by ~5–7 days in recovery of platelets and leukocytes, ii) decrease in day 12 CFU-S, and iii) decrease in the number of CFU-GM progenitors detectable in the BM cavities at day 16 after transplantation. Similarly in parallel experiments, human CD34+ cells exposed to nontoxic doses of C3aR antagonist SB29007 engrafted worse in NOD/SCID mice (p<0.0001). Next, we studied the different steps of homing of HSPC and noticed that sensitization of cells to an SDF-1 chemotactic gradient was compensated in C3aR−/ − mice probably by the activation of another putative receptor for C3a, however, the C3aR was indispensable for optimal adhesion of HSPC to endothelium and SDF-1-dependent MMP-9 secretion. In conclusion, activation of the C cascade in BM during conditioning for transplantation exposes a natural neoantigen which is recognized by immunoglobulins activating C by the classical pathway. As a consequence, C3 cleavage product, C3a, activates the C3aR on transplanted HSPC increasing the SDF-1 mediated homing of these cells.

3′ Distal Regulatory Elements Required for Human CD34 Expression in Transgenic Mice.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 125-125
Author(s):  
Elena Levantini ◽  
Yutaka Okuno ◽  
Pu Zhang ◽  
Steffen Koschmieder ◽  
Hanna S. Radomska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Progenitor Cells ◽  
Regulatory Element ◽  
Restriction Enzymes ◽  
Regulatory Elements ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Expression

Abstract CD34 is the best-defined human hematopoietic stem cell (HSC) marker, however the regulation of its gene expression is still largely unknown. Therefore, unraveling the elements that regulate human CD34 expression would be an invaluable tool for a broad range of studies, including the establishment of models of leukemia in mice, which require targeting of the transgene to stem and/or early progenitor cells. Moreover, identification of such regulatory elements will provide important insights into the transcriptional agenda of stem and progenitor cells and most importantly will prove useful for gene therapy protocols. Studies from our laboratory demonstrated that human CD34 transgenes are expressed in murine repopulating HSCs, which resembles the expression of the CD34 gene in human hematopoiesis, thus indicating the mouse model as an excellent way to study the expression of human CD34. Using P1 derived artificial chromosome (PAC) clones encompassing the human CD34 gene to generate transgenic mice, we showed that 90kb of upstream and 26kb of downstream flanking sequences were capable of regulating human CD34 expression in murine transgenic lines. Successive deletions of this larger construct were then performed to identify the important control regions. Deletion of the 5′ region from −90kb to −18kb did not result in any loss of activity. PAC54A19, a clone extending from −18kb to +26kb, expressed RNA in various tissues in a manner similar to that of larger fragments. In contrast, deletions creating a construct spanning from −10kb to +17kb led to complete loss of expression in transgenic animals, indicating that critical distal elements are located between −18kb to −10kb and/or +17kb to +26kb. In order to facilitate identification of important regulatory elements present in the upstream (−18kb to −10 kb) and/or downstream (+17kb to +26kb) regions of human CD34, we created further deletions of PAC54A19 using rare-cutting restriction enzymes, and studied the effects of the deletions on human CD34 expression in transgenic mice. Interestingly, we did not detect any human CD34 mRNA and protein expression in bone marrow and HSCs from transgenic mice carrying a construct spanning from −18kb to +17.4kb. In contrast, we observed expression of human CD34 transcripts in the bone marrow of transgenic mice containing a PAC spanning from −12.8kb to +26kb. Furthermore, HSCs from this latter group of mice presented the human CD34 antigen on their surface, as detected by FACS. Taken together, these data are highly suggestive that critical cis regulatory element(s) required to drive human CD34 in vivo expression are located in a 8.6kb fragment placed between +17.4kb and +26kb downstream of the human CD34 gene. Our current efforts focus on identifying the element(s) within the 8.6kb 3′ region that might be required to achieve human CD34 expression in HSCs.

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.

Enrichment of CD34+ Hematopoietic Stem and Progenitor Cells from Human Bone Marrow Using a P-Selectin-Coated Microtube.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1219-1219
Author(s):  
Srinivas D. Narasipura ◽  
Jane L. Liesveld ◽  
Joel C. Wojciechowski ◽  
Nichola Charles ◽  
Karen Rosell ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Progenitor Cells ◽  
Adhesion Molecule ◽  
Mononuclear Cells ◽  
Single Cells ◽  
Bone Marrow Mononuclear Cells ◽  
Cell Capture ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells ◽  
Coated Surfaces

Abstract Enrichment and purification of hematopoietic stem and progenitor cells (HSPCs) is important in transplantation therapies for hematological disorders and for basic stem cell research. Primitive CD34+ HSPCs have demonstrated stronger rolling adhesion than mature CD34- mononuclear cells on selectins (Blood2000; 95:478–486). We have exploited this differential rolling behavior to capture and purify HSPCs from bone marrow, by perfusing mononuclear cells through selectin-coated microtubes. Bone marrow mononuclear cells were perfused through the cell capture microtubes coated with adhesion molecules. These utilized a parallel plate flow chamber (Glycotech), and the P-selectin was adsorbed with laboratory tubing of appropriate lengths attached to the inlet, outlet, and vacuum ports of the gasket chamber. After perfusion, the device lumen was washed and captured cells were visualized and estimated by video microscopy. “Rolling” cells were defined as cells translating at less than 50% of the calculated hydrodynamic free stress velocity. Velocities of single cells were determined using a MATLAB program designed to measure the change in position of the cell centroid in a given time period. Adherent cells were eluted by high shear, calcium free buffer and air embolism. Immunofluorescence staining followed by flow cytometry was used to analyze CD34+ HSPCs. CD34+ HSPC purity of cells captured in adhesion molecule-coated devices was significantly higher than the fraction of CD34+ cells found in bone marrow- mononuclear cells (2.5 ± 0.8%). P-selectin coated surfaces yielded 16–20% CD34+ cell purity, while antibody coated surfaces yielded 12–18%. Although the CD34+ cell purities were comparable between selectin and antibody surfaces, the total number of CD34+ HSPCs captured was significantly higher in P-selectin devices (∼5.7–7.1 × 104) when compared to the antibody device (∼1.74–2.61 × 104). Furthermore, analysis for cells positive for CD133, a surface marker for more primitive HSPCs, depicted approximately 10–14 fold enrichment in P-selectin samples over control bone marrow mononuclear cells. The captured cells were viable and exhibited in vitro colony forming capabilities. Thus, P-selectin can be used in a compact flow device to capture and enrich HSPCs. This study supports the hypothesis that flow-based adhesion molecule-mediated capture may be a viable physiologic approach to the capture and purification of HSPCs.

Reversibility of CD34 expression on human hematopoietic stem cells that retain the capacity for secondary reconstitution

Blood ◽  
2003 ◽  
Vol 101 (1) ◽  
pp. 112-118 ◽  
Author(s):  
Mo A. Dao ◽  
Jesusa Arevalo ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cell Surface ◽  
Hematopoietic Stem Cells ◽  
Progenitor Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cd34 Expression

Abstract The cell surface protein CD34 is frequently used as a marker for positive selection of human hematopoietic stem/progenitor cells in research and in transplantation. However, populations of reconstituting human and murine stem cells that lack cell surface CD34 protein have been identified. In the current studies, we demonstrate that CD34 expression is reversible on human hematopoietic stem/progenitor cells. We identified and functionally characterized a population of human CD45+/CD34− cells that was recovered from the bone marrow of immunodeficient beige/nude/xid (bnx) mice 8 to 12 months after transplantation of highly purified human bone marrow–derived CD34+/CD38− stem/progenitor cells. The human CD45+ cells were devoid of CD34 protein and mRNA when isolated from the mice. However, significantly higher numbers of human colony-forming units and long-term culture-initiating cells per engrafted human CD45+ cell were recovered from the marrow of bnx mice than from the marrow of human stem cell–engrafted nonobese diabetic/severe combined immunodeficient mice, where 24% of the human graft maintained CD34 expression. In addition to their capacity for extensive in vitro generative capacity, the human CD45+/CD34− cells recovered from thebnx bone marrow were determined to have secondary reconstitution capacity and to produce CD34+ progeny following retransplantation. These studies demonstrate that the human CD34+ population can act as a reservoir for generation of CD34− cells. In the current studies we demonstrate that human CD34+/CD38− cells can generate CD45+/CD34− progeny in a long-term xenograft model and that those CD45+/CD34− cells can regenerate CD34+ progeny following secondary transplantation. Therefore, expression of CD34 can be reversible on reconstituting human hematopoietic stem cells.

Inducible expression of BCR/ABL using human CD34 regulatory elements results in a megakaryocytic myeloproliferative syndrome

Blood ◽  
2003 ◽  
Vol 102 (9) ◽  
pp. 3363-3370 ◽  
Author(s):  
Claudia S. Huettner ◽  
Steffen Koschmieder ◽  
Hiromi Iwasaki ◽  
Junko Iwasaki-Arai ◽  
Hanna S. Radomska ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Chronic Myeloid Leukemia ◽  
B Cell ◽  
Progenitor Cells ◽  
Myeloid Leukemia ◽  
Regulatory Elements ◽  
Cell Leukemia ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
B Cell Leukemia

AbstractThe BCR/ABL fusion protein is found in more than 90% of patients with chronic myeloid leukemia (CML) as well as in a subset of patients with acute B-cell leukemia. We have previously described a transgenic model for an inducible and reversible acute B-cell leukemia caused by p210 BCR/ABL. Here, we describe a new model of an inducible BCR/ABL disease by directing the expression of the oncogene to megakaryocytic progenitor cells within the murine bone marrow using the tetracycline-responsive expression system under the control of human CD34 regulatory elements. The predominant feature was the development of a chronic thrombocytosis. The condition progressed with the development of splenomegaly accompanied by lymphadenopathy in some mice. Affected animals demonstrated a dramatic increase in the number of megakaryocytes in the bone marrow and the spleen. Immunohistochemistry demonstrated that the reporter gene was expressed in hematopoietic stem cells (HSCs), common myeloid progenitor (CMP) cells, as well as in megakaryocytic/erythroid progenitor cells (MEPs). Although these mice did not display the increase in granulopoiesis commonly found in chronic myeloid leukemia (CML), the phenotype closely resembles a myeloproliferative disorder affecting the megakaryocytic lineage observed in some patients with the BCR/ABL P210 translocation.

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.

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.

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