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Hypoxia enhances the radio-resistance of mouse mesenchymal stromal cells
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
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P1137 - HYPOXIA ENHANCES THE RADIO-RESISTANCE OF MOUSE MESENCHYMAL STROMAL CELLS Tara Sugrue1,2, Rhodri Ceredig1, and Noel Lowndes2 1 Immunology, Regenerative Medicine Institute, National University of Ireland, Galway, Galway city, Ireland; 2Genome Stability Laboratory, Centre for Chromosome Biology, National University of Ireland, Galway, Galway city, Ireland
P1139 - DECLINED PRESENTATION ALTERATIONS IN HIERARCHY OF MULTIPOTENT MESENCHYMAL STROMAL CELLS POPULATION OCCURRING WITH CULTIVATION Natalia Petinati, Irina Shipounova, Natalia Sats, and Nina Drize lab Physiology of hematopoiesis, National Hematological Scientific Centre, Moscow, Russian Federation
Haematopoietic re-constitution by donor haematopoietic stem cells (HSCs) following total body irradiation is supported by host-derived radio-resistant mesenchymal stromal cells (MSCs). MSCs are also an integral part of tumours where they promote tumour cell growth and inhibit tumour-specific immune responses. The DNA Damage Response (DDR) represents a network of signalling pathways enabling cells to respond to genotoxic damage. We have previously shown that the execution of DDR pathways, including repair of DNA double strand breaks (DNA DSBs), promotes MSC survival post irradiation (IR) [1]. MSCs reside in a hypoxic (2-5% O2) microenvironment in the bone marrow. Hypoxia is known to enhance the radio-resistance of cancer cells by altering their response to IR-induced DNA damage. However, whether hypoxia affects the radio-resistance of MSCs is currently unknown. We have studied the DDR of g-irradiated mouse MSC lines, MS5 and ST2, cultured in normoxia (21% O2) and hypoxia (5% O2). Hypoxia increased MSC growth rate and enhanced long-term survival post irradiation. Cell cycle analysis by flow cytometry demonstrated that MSC recovery from IR (10 Gy) -induced cell cycle arrest was improved under hypoxic conditions. In MSCs, hypoxia accelerated the resolution of g-H2AX expression (a marker of DNA DSBs) and the disappearance of g-H2AX foci. Hypoxia also induced increased expression of DNA DSB repair proteins, including DNA-PKcs and DNA ligase IV in MSCs. Our results demonstrate, for the first time, that hypoxia enhances MSC radio-resistance in vitro most likely by up-regulating DNA DSB repair mechanisms, leading to alterations in the DDR to IR-induced DNA DSBs and thereby enhancing MSC survival. Our results have important implications for our understanding of the roles of MSCs in haematopoietic reconstitution and in the tumour microenvironment. 1. Sugrue T, Brown JAL, Lowndes NF & Ceredig Rh. (2013) Multiple facets of the DNA Damage Response contribute to the radio-resistance of mouse mesenchymal stromal cell lines. Stem Cells 31: 137-145.
Mesenchymal multipotent stromal cells (MMSC) are the fibroblast-like cells adherent to plastic and able to differentiate along main stromal lineages. The population of MMSC is heterogeneous. The aim of this study was to investigate the hierarchical alterations occurring in MMSC with cultivation. MMSC were isolated from the bone marrow of 50 donors and cultivated under standard conditions for 5 passages. The cloning efficiency and differentiation abilities were analyzed on 1-2nd and 5th passages. Relative expression levels (REL) of morphogenetic and differentiation marker genes were evaluated by real time PCR. Cloning efficiency of MMSC differed between the 2nd and 5th passages. It was associated with the REL of FGFR2 (R50.75, p50.02), which shows the dependence of MMSC proliferation capacity on the ability to respond to FGF2. A strong negative relationship between cloning efficiency and the REL of SPP1 was revealed at the 5th passage (R50.73, p50.03). Thus, proliferative potential reduced during maturation. So, MMSC populations changed with passages, and the portion of cells committed to differentiation became more pronounced. Most cells among MMSC did not proliferate. MMSC proliferative potential did not change significantly during cultivation, and more than half of all dividing cells were clones that were unable to perform more than 14 mitosis. When MMSC were exposed to osteogenic inducers at the 1st passage, no significant alterations in the REL of both SPP1 and BGLAP were revealed. Further MMSC cultivation up to the 5th passage made these cells more sensitive to differentiation stimuli. The increase in the REL of SPP1 was moderate, while the expression of BGLAP was up-regulated by 6-fold. Thus, cultivation increased the proportion of cells committed to differentiation. Prolonged MMSC cultivation had no effect on the response to adipogenic inducers. So, one could conclude that MMSC differentiation capacities did not change with cultivation, while in the population itself the proportion of cells committed to differentiation increased. Summarizing the data, the proportion of cells in different hierarchical subpopulations altered with MMSC cultivation. The aptitude to differentiation increased among the MMSC population.
P1138 - MOBILISING DOSES OF G-CSF STOP MEDULLARY ERYTHROPOIESIS BY DEPLETING CD169+ MACROPHAGES Rebecca Jacobsen1,2, Allison Pettit1, Valerie Barbier1, Bianca Nowlan1, Liza Raggatt1, Ingrid Winkler1, and Jean-Pierre Levesque1 1 Mater Research, Woolloongabba, Queensland, Australia; 2School of Medicine, University of Queensland, Brisbane, Queensland, Australia
P1140 - DECLINED PRESENTATION BONE MARROW DONOR’S AGE RELATED ALTERATIONS IN HUMAN STROMAL PRECURSOR CELLS Natalia Petinati, Irina Shipounova, Natalia Sats, and Nina Drize lab Physiology of hematopoiesis, National Hematological ScientificCentre, Moscow, Russian Federation
We have previously reported that G-CSF mobilises HSCs by suppressing a subset of HSC niche supportive macrophages. As macrophages are the central component of erythropoietic islands, we examined G-CSF effect on erythropoiesis. Mobilising doses of G-CSF caused a marked whitening of the bone marrow (BM), a 15 fold decrease in the number of phenotypic erythroblasts, a 2 fold decrease in polychromatic erythroblasts, a 1.4 fold decrease in orthochromatic erythoblasts and 5 fold decrease in reticulocytes. Conversely, pro-erythroblasts increased 4 fold. Concomitantly, numbers of CD169+ macrophages and erythropoietic island ER-HR3+ macrophages were decreased in mobilised BM. In contrast with the BM, G-CSF did not deplete macrophages in the spleen. As a result, splenic erythropoiesis was up-regulated to compensate for the loss of medullary erythropoiesis (5-8 fold increase in proerythroblast and erythroblast populations). This suggests that mobilising doses of G-CSF block the pro-erythroblast to erythroblast transition specifically in the BM (but not the spleen) by affecting central macrophages in erythropoietic island. Next, macrophages were depleted by injecting clodronate loaded liposomes. This caused a arrest in erythropoiesis and macrophage depletion in both the BM and the spleen. As subsets of macrophages depleted from the BM and spleen following G-CSF or clodronate liposome administration were CD169+, we selectively depleted CD169+ macrophages in CD169DTR/+ mice. Injection of diphtheria toxin caused a loss of CD169+ and ER-HR3+ macrophages in both BM and spleen and also a blockage in erythropoiesis at the proerythroblast-erythroblast transition similar to G-CSF and clodronate liposomes in wild-type mice. In conclusion, we propose that 1) CD169+ macrophages include nursing macrophages at the centre of erythropoietic islands and are essential for the transition of proerythoblasts to erythroblasts and 2) mobilising doses of G-CSF stop medullary erythropoiesis by depleting CD169+ macrophages in erythropoietic islands in the BM, but not the spleen.
Multipotent mesenchymal stromal cells (MMSC) are utilized in many therapeutic approaches. The aim of the study was to characterize stromal precursor cells (MMSC and colonyforming unit-fibroblasts - CFU-F), from the bone marrow of 50 healthy donors. The donors were divided in 2 groups (younger and older than 32.5) on the base of median age. The growth characteristics of MMSC and CFU-F, their proliferative potential, cloning efficiency and differentiation abilities were analyzed. MMSC were cultivated for 5 passages. For proliferative potential studies, MMSC from 2 and 5 passages were cloned from 1 cell. The differentiation capacity was estimated at passages 1 and 5 by standard stimulation. Relative expression levels (REL) of genes were investigated in MMSC by real-time quantitative PCR. Significant increase in CFU-F concentration was revealed in young donors (p!0.02). Cumulative MMSC production was significantly decreased in older donors (p! 0.01). REL of FGF2 and FGFR2 in MMSC decreased with donor age (R5-0.44, p50.001) and this was also true for the VEGF gene (R5-0.31, p50.03). The REL of the adipogenic differentiation marker PPARG increased, and the osteogenic one SPP1 decreased with age. The chondrogenic marker SOX9 did not depend on donor age. REL were analyzed in MMSC cultivated without differentiation inducers. REL of differentiation markers can reflect the decreasing with age capacity of mesenchymal cells for osteogenesis and osteoporosis in elderly persons. According cloning capacity MMSC were divided into 3 groups: MMSC with cloning efficiencies that increased from the 2 to the 5 passage, decreased and remained stable. The average donor age was 24.761.5 years in the 1 group, 34.664.7 years in the 2 group and 4666.8 years in the 3 group. This means that MMSC populations of young donors were enriched for cells with high proliferative potential. In the group of older donors, the MMSC proliferative potential notably decreased with cultivation, and in the eldest group of donors cloning capacity was decreased for all period of cultivation. The data are worth to take into account when choosing MMSC for gene therapy, as MMSC are now top candidates for many therapeutic approaches. Thus, donor age is very important for the stable function of MMSC.
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P1137 - HYPOXIA ENHANCES THE RADIO-RESISTANCE OF MOUSE MESENCHYMAL STROMAL CELLS Tara Sugrue1,2, Rhodri Ceredig1, and Noel Lowndes2 1 Immunology, Regenerative Medicine Institute, National University of Ireland, Galway, Galway city, Ireland; 2Genome Stability Laboratory, Centre for Chromosome Biology, National University of Ireland, Galway, Galway city, Ireland
P1139 - DECLINED PRESENTATION ALTERATIONS IN HIERARCHY OF MULTIPOTENT MESENCHYMAL STROMAL CELLS POPULATION OCCURRING WITH CULTIVATION Natalia Petinati, Irina Shipounova, Natalia Sats, and Nina Drize lab Physiology of hematopoiesis, National Hematological Scientific Centre, Moscow, Russian Federation
Haematopoietic re-constitution by donor haematopoietic stem cells (HSCs) following total body irradiation is supported by host-derived radio-resistant mesenchymal stromal cells (MSCs). MSCs are also an integral part of tumours where they promote tumour cell growth and inhibit tumour-specific immune responses. The DNA Damage Response (DDR) represents a network of signalling pathways enabling cells to respond to genotoxic damage. We have previously shown that the execution of DDR pathways, including repair of DNA double strand breaks (DNA DSBs), promotes MSC survival post irradiation (IR) [1]. MSCs reside in a hypoxic (2-5% O2) microenvironment in the bone marrow. Hypoxia is known to enhance the radio-resistance of cancer cells by altering their response to IR-induced DNA damage. However, whether hypoxia affects the radio-resistance of MSCs is currently unknown. We have studied the DDR of g-irradiated mouse MSC lines, MS5 and ST2, cultured in normoxia (21% O2) and hypoxia (5% O2). Hypoxia increased MSC growth rate and enhanced long-term survival post irradiation. Cell cycle analysis by flow cytometry demonstrated that MSC recovery from IR (10 Gy) -induced cell cycle arrest was improved under hypoxic conditions. In MSCs, hypoxia accelerated the resolution of g-H2AX expression (a marker of DNA DSBs) and the disappearance of g-H2AX foci. Hypoxia also induced increased expression of DNA DSB repair proteins, including DNA-PKcs and DNA ligase IV in MSCs. Our results demonstrate, for the first time, that hypoxia enhances MSC radio-resistance in vitro most likely by up-regulating DNA DSB repair mechanisms, leading to alterations in the DDR to IR-induced DNA DSBs and thereby enhancing MSC survival. Our results have important implications for our understanding of the roles of MSCs in haematopoietic reconstitution and in the tumour microenvironment. 1. Sugrue T, Brown JAL, Lowndes NF & Ceredig Rh. (2013) Multiple facets of the DNA Damage Response contribute to the radio-resistance of mouse mesenchymal stromal cell lines. Stem Cells 31: 137-145.
Mesenchymal multipotent stromal cells (MMSC) are the fibroblast-like cells adherent to plastic and able to differentiate along main stromal lineages. The population of MMSC is heterogeneous. The aim of this study was to investigate the hierarchical alterations occurring in MMSC with cultivation. MMSC were isolated from the bone marrow of 50 donors and cultivated under standard conditions for 5 passages. The cloning efficiency and differentiation abilities were analyzed on 1-2nd and 5th passages. Relative expression levels (REL) of morphogenetic and differentiation marker genes were evaluated by real time PCR. Cloning efficiency of MMSC differed between the 2nd and 5th passages. It was associated with the REL of FGFR2 (R50.75, p50.02), which shows the dependence of MMSC proliferation capacity on the ability to respond to FGF2. A strong negative relationship between cloning efficiency and the REL of SPP1 was revealed at the 5th passage (R50.73, p50.03). Thus, proliferative potential reduced during maturation. So, MMSC populations changed with passages, and the portion of cells committed to differentiation became more pronounced. Most cells among MMSC did not proliferate. MMSC proliferative potential did not change significantly during cultivation, and more than half of all dividing cells were clones that were unable to perform more than 14 mitosis. When MMSC were exposed to osteogenic inducers at the 1st passage, no significant alterations in the REL of both SPP1 and BGLAP were revealed. Further MMSC cultivation up to the 5th passage made these cells more sensitive to differentiation stimuli. The increase in the REL of SPP1 was moderate, while the expression of BGLAP was up-regulated by 6-fold. Thus, cultivation increased the proportion of cells committed to differentiation. Prolonged MMSC cultivation had no effect on the response to adipogenic inducers. So, one could conclude that MMSC differentiation capacities did not change with cultivation, while in the population itself the proportion of cells committed to differentiation increased. Summarizing the data, the proportion of cells in different hierarchical subpopulations altered with MMSC cultivation. The aptitude to differentiation increased among the MMSC population.
P1138 - MOBILISING DOSES OF G-CSF STOP MEDULLARY ERYTHROPOIESIS BY DEPLETING CD169+ MACROPHAGES Rebecca Jacobsen1,2, Allison Pettit1, Valerie Barbier1, Bianca Nowlan1, Liza Raggatt1, Ingrid Winkler1, and Jean-Pierre Levesque1 1 Mater Research, Woolloongabba, Queensland, Australia; 2School of Medicine, University of Queensland, Brisbane, Queensland, Australia
P1140 - DECLINED PRESENTATION BONE MARROW DONOR’S AGE RELATED ALTERATIONS IN HUMAN STROMAL PRECURSOR CELLS Natalia Petinati, Irina Shipounova, Natalia Sats, and Nina Drize lab Physiology of hematopoiesis, National Hematological ScientificCentre, Moscow, Russian Federation
We have previously reported that G-CSF mobilises HSCs by suppressing a subset of HSC niche supportive macrophages. As macrophages are the central component of erythropoietic islands, we examined G-CSF effect on erythropoiesis. Mobilising doses of G-CSF caused a marked whitening of the bone marrow (BM), a 15 fold decrease in the number of phenotypic erythroblasts, a 2 fold decrease in polychromatic erythroblasts, a 1.4 fold decrease in orthochromatic erythoblasts and 5 fold decrease in reticulocytes. Conversely, pro-erythroblasts increased 4 fold. Concomitantly, numbers of CD169+ macrophages and erythropoietic island ER-HR3+ macrophages were decreased in mobilised BM. In contrast with the BM, G-CSF did not deplete macrophages in the spleen. As a result, splenic erythropoiesis was up-regulated to compensate for the loss of medullary erythropoiesis (5-8 fold increase in proerythroblast and erythroblast populations). This suggests that mobilising doses of G-CSF block the pro-erythroblast to erythroblast transition specifically in the BM (but not the spleen) by affecting central macrophages in erythropoietic island. Next, macrophages were depleted by injecting clodronate loaded liposomes. This caused a arrest in erythropoiesis and macrophage depletion in both the BM and the spleen. As subsets of macrophages depleted from the BM and spleen following G-CSF or clodronate liposome administration were CD169+, we selectively depleted CD169+ macrophages in CD169DTR/+ mice. Injection of diphtheria toxin caused a loss of CD169+ and ER-HR3+ macrophages in both BM and spleen and also a blockage in erythropoiesis at the proerythroblast-erythroblast transition similar to G-CSF and clodronate liposomes in wild-type mice. In conclusion, we propose that 1) CD169+ macrophages include nursing macrophages at the centre of erythropoietic islands and are essential for the transition of proerythoblasts to erythroblasts and 2) mobilising doses of G-CSF stop medullary erythropoiesis by depleting CD169+ macrophages in erythropoietic islands in the BM, but not the spleen.
Multipotent mesenchymal stromal cells (MMSC) are utilized in many therapeutic approaches. The aim of the study was to characterize stromal precursor cells (MMSC and colonyforming unit-fibroblasts - CFU-F), from the bone marrow of 50 healthy donors. The donors were divided in 2 groups (younger and older than 32.5) on the base of median age. The growth characteristics of MMSC and CFU-F, their proliferative potential, cloning efficiency and differentiation abilities were analyzed. MMSC were cultivated for 5 passages. For proliferative potential studies, MMSC from 2 and 5 passages were cloned from 1 cell. The differentiation capacity was estimated at passages 1 and 5 by standard stimulation. Relative expression levels (REL) of genes were investigated in MMSC by real-time quantitative PCR. Significant increase in CFU-F concentration was revealed in young donors (p!0.02). Cumulative MMSC production was significantly decreased in older donors (p! 0.01). REL of FGF2 and FGFR2 in MMSC decreased with donor age (R5-0.44, p50.001) and this was also true for the VEGF gene (R5-0.31, p50.03). The REL of the adipogenic differentiation marker PPARG increased, and the osteogenic one SPP1 decreased with age. The chondrogenic marker SOX9 did not depend on donor age. REL were analyzed in MMSC cultivated without differentiation inducers. REL of differentiation markers can reflect the decreasing with age capacity of mesenchymal cells for osteogenesis and osteoporosis in elderly persons. According cloning capacity MMSC were divided into 3 groups: MMSC with cloning efficiencies that increased from the 2 to the 5 passage, decreased and remained stable. The average donor age was 24.761.5 years in the 1 group, 34.664.7 years in the 2 group and 4666.8 years in the 3 group. This means that MMSC populations of young donors were enriched for cells with high proliferative potential. In the group of older donors, the MMSC proliferative potential notably decreased with cultivation, and in the eldest group of donors cloning capacity was decreased for all period of cultivation. The data are worth to take into account when choosing MMSC for gene therapy, as MMSC are now top candidates for many therapeutic approaches. Thus, donor age is very important for the stable function of MMSC.