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Lineage specific metabolic activity in haematopoiesis
S62
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1148 - POLYMERIC SCAFFOLDS WITH DIFFERENT ALIGNMENT AND THEIR INTERACTIONS WITH BONE MARROW-DERIVED MESENCHYMAL STEM CELLS Sevil Kose1, Fatima Aerts Kaya1, Emir Denkbas2, Petek Korkusuz3, and Duygu Uckan1,4 1 Center for Stem Cell Research and Development/PEDI-STEM, Hacettepe University, Ankara, Turkey; 2Chemistry department, Biochemistry division, Hacettepe University, Ankara, Turkey; 3Department of Histology and Embryology, Hacettepe University, Ankara, Turkey; 4Pediatric Hematology, BMT unit, Hacettepe University Childrens Hospital, Ankara, Turkey Introduction: The development of bone tissue engineering is directly related to changes in materials technology. Both material characteristics and clinical requirements are critical when engineering a clinically relevant bone substitute. In this study, we aimed to investigate the interaction of mesenchymal stromal cells (MSCs) with randomly or aligned nanofiber PHB membranes prepared to mimic the physical structure of bone tissue and spinal axonal design, respectively. Materials and Methods: PHB membrane with its two different kinds of surface features as randomly arranged nanoscale fibers and nanoscale fibers oriented in one direction was used as a tissue scaffold. Human bone marrow MSCs were expanded on two different tissue scaffolds with different surface morphology and evaluated in terms of their orientation on tissue scaffolds by confocal and scanning electrone microscopes (SEM). Cells were also assessed for their adhesion characteristics and viability (MTT Assay, real time cell analysis, population doubling methods and Annexin V Assay); differentiation (adipogenic and osteogenic) and surface antigen properties. Results: The results of analysis showed that MSCs conserve their stem cell properties, adhesiveness and viability when they were expanded on both types of tissue scaffolds. While adhesiveness of MSCs that were expanded on oriented tissue scaffolds was more than knitted ones, proliferation rates was lower. Although, MSCs on oriented PHB tissue scaffolds showed spindle shape morphology and they were positioned in the direction of spindles, whereas MSCs on knitted nPHB tissue scaffolds distributed homogenously by conserving their poligonal morphology that mimicked bone marrow microenvironment. Conclusion: In the present study, it was found that MSCs cultivated on nanofiber PHB tissue scaffolds conserved their multpotentiality and attained morphology compatible with physical alignment of the scaffold; therefore they may provide effective tissue engineering approach by repairing cellular microenvironment in bone tissue engineering. P1149 - LACTIC ACID BACTERIA CAN REDUCE TLR-4 EXPRESSION ON INFLAMMATED GINGIVAL FIBROBLASTS AND MESENCHYMAL STEM CELLS IN VITRO Aysegul Mendi1, Sevil Kose2, Fatima Aerts Kaya2, Duygu Uckan2,3, Dervis Yilmaz1, and Levent Aral1 1 Department of Medical Microbiology, Gazi University Faculty of Dentistry, Ankara, Turkey; 2Center for Stem Cell Research and Development/PEDI-STEM, Hacettepe University, Ankara, Turkey; 3Pediatric Hematology/BMT Unit, Hacettepe University Children’s Hospital, Ankara, Turkey Introduction: Lactic acid bacteria (LAB) have been studied for their health-promoting and immunomodulatory effects. The main field of research has been in gastrointestinal tract. In the past few years LAB have also been investigated in the oral health perspective. We studied competitive adhesion of Lactobacillus strains with Porpyhromonas gingivalis on human gingival fibrolasts (GF) and bone marrow derived mesenchymal stem cells (MSCs). P. gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues and accounted for periodontitis. It has been shown that bacterial ligands stimulate Toll-like receptor (TLR) expression on a variety of cells. However it is unknown whether whole bacteria can induce TLR expression on GF and MSCs. . In this study, we evaluated the effect of LAB on cellular adhesion and TLR 4 and 2 expression induced by interferon (IFN)-g. Material and Methods: Adhesion assay was conducted (Lactobacillus rhamnosus ATCC 9595, L. brevis OML 28, P. gingivalis ATCC 33277) and competition of LAB and P. gingivalis on GF and MSCs was studied. To determine the suppressive effect of Lactobacillus strains on IFN-g- and P. gingivalis-based inflammation, TLR 4 and 2 expression by flow cytometer was investigated. Results: Adhesion of bacteria on GF and MSCs was significantly different. LAB prevented adhesion of P. gingivalis as shown by light and fluoresence microscopy. In addition,LAB also reduced TLR 4 expression on GF and MSCs, in in-vitro assays with IFN-g and P. gingivalis. Conclusion: Adhesion of probiotic bacteria to oral soft tissues promotes their beneficial effect to the host. Obtained data suggested that cells from different tissues may achieve specific functional characteristics according to the microenvironmental needs. Further data showed differential responses on TLR expression of GF vs MSCs suggesting participation of GFs on immune response at oral cavity by TLRs. On the other hand, lower TLR expression on bone marrow derived MSCs may partially be attributed to the immunprivileged state of the bone marrow niche.
P1150 - LINEAGE SPECIFIC METABOLIC ACTIVITY IN HAEMATOPOIESIS Claudia Billing1, Michael Walker2, Lydia Schnapka-Hille1, Uta Ceglarek3, Anthony Whetton2, Dietger Niederwieser1, and Michael Cross1 1 Haematology and Oncology, University of Leipzig, Leipzig, Germany; 2Wolfson Molecular Imaging Centre, University of Manchester, Manchester, UK; 3Institute of Laboratory Medicine, University of Leipzig, Leipzig, Germany Erythroid and neutrophil cells have widely different metabolic requirements and tend to be produced in distinct regions of the bone marrow associated with arterioles or sinusoids respectively. This is suggestive of metabolic compartmentalisation of haematopoietic marrow, and raises questions concerning the partitioning of metabolism in solid tissues, the interdependence of metabolic and signalling pathways in the control of normal and leukemic haematopoiesis and the achievement of high density in vitro culture conditions. A key issue is whether the early progenitor cells of each lineage employ distinct metabolic strategies to satisfy the biosynthetic demands of proliferation. To address this question we have purified mitochondria from large scale cultures of FDCP-Mix cells at various stages of erythroid or granulocyte/macrophage differentiation and subjected them to proteomic analysis using 8-plex iTRAQ liquid chromatography tandem mass spectrometry (LC-MS/MS). In a complementary approach, the uptake and release of key metabolites at various stages of differentiation was determined by mass spectrometry and biochemical assay of conditioned media. We find lineage specific changes in the balance of TCA cycle enzymes and of components of the electron transport chain, as well as in proteins controlling apoptosis and reactive oxygen species. Further changes in co-purified cytoplasmic enzymes suggest differential use of available substrates that is confirmed by lineage specific differences in metabolite uptake. Our results suggests that early neutrophil and early erythroid progenitors do indeed adopt different metabolic strategies to biosynthesis that optimise metabolite usage in bone marrow.
P1151 - DISTINCT LEUKEMIA-INITIATING SUBSETS ARE MAINTAINED BY IL-2/CD25 AXIS IN CHRONIC MYELOID LEUKEMIA NICHE Keiyo Takubo, Chiharu Kobayashi, and Toshio Suda Department of Cell Differentiation, the Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, Tokyo, Japan Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder originated from hematopoietic stem cells (HSCs) with the Philadelphia chromosome which produces BCR-ABL fusion protein. Targeting tyrosine kinase activity of BCR-ABL by tyrosine kinase inhibitors can deplete most of the differentiated CML cells but not CML leukemia initiating cells (LICs). Therefore it is mandatory to develop the eradication strategy of CML LICs. Here we show that CML LICs show a severely hypoxic phenotype then the normal HSCs in the niche. HIF-1alpha, a master regulator of cellular hypoxia response, plays a critical role for maintaining HSC in the hypoxic niche. HIF-1alpha-deficient p210BCR-ABL retrovirus transduced Lineage marker- Sca-1+ c-Kit+ (LSK) cells are defective in establishing LIC population in vivo. Under normoxic conditions, HIF-1alpha is recognized by an E3 ubiquitin ligase VHL. Although VHL deficient HSCs lose transplantation capacity, they show an accelerated formation of CML LIC after p210BCR-ABL retrovirus transduction. To elucidate the putative LIC markers in the hypoxic niche, we have screened various surface markers using p210BCR-ABL retrovirus-induced CML-like myeloproliferative disease model. Among them, we demonstrate that CML LICs are divided into CD25+FcεRIa- Lineage marker (Lin)- Sca-1+ c-Kit+ (F-LSK) cells and CD25-FLSK cells. We show that CD25+F-LSK cells have multipotent differentiation capacity with preference to cytokine-producing mast cell commitment. Although both CD25-F-LSK and CD25+F-LSK cells differentiate into one another, CD25+FLSK cells show higher LIC capacity than CD25-F-LSK cells. High expression of CD25 in CD34+CD38- fraction of human CML indicate that CD25+LICs constitute "LIC-derived niche" that can be a preferential therapeutic target for CML LICs.
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1148 - POLYMERIC SCAFFOLDS WITH DIFFERENT ALIGNMENT AND THEIR INTERACTIONS WITH BONE MARROW-DERIVED MESENCHYMAL STEM CELLS Sevil Kose1, Fatima Aerts Kaya1, Emir Denkbas2, Petek Korkusuz3, and Duygu Uckan1,4 1 Center for Stem Cell Research and Development/PEDI-STEM, Hacettepe University, Ankara, Turkey; 2Chemistry department, Biochemistry division, Hacettepe University, Ankara, Turkey; 3Department of Histology and Embryology, Hacettepe University, Ankara, Turkey; 4Pediatric Hematology, BMT unit, Hacettepe University Childrens Hospital, Ankara, Turkey Introduction: The development of bone tissue engineering is directly related to changes in materials technology. Both material characteristics and clinical requirements are critical when engineering a clinically relevant bone substitute. In this study, we aimed to investigate the interaction of mesenchymal stromal cells (MSCs) with randomly or aligned nanofiber PHB membranes prepared to mimic the physical structure of bone tissue and spinal axonal design, respectively. Materials and Methods: PHB membrane with its two different kinds of surface features as randomly arranged nanoscale fibers and nanoscale fibers oriented in one direction was used as a tissue scaffold. Human bone marrow MSCs were expanded on two different tissue scaffolds with different surface morphology and evaluated in terms of their orientation on tissue scaffolds by confocal and scanning electrone microscopes (SEM). Cells were also assessed for their adhesion characteristics and viability (MTT Assay, real time cell analysis, population doubling methods and Annexin V Assay); differentiation (adipogenic and osteogenic) and surface antigen properties. Results: The results of analysis showed that MSCs conserve their stem cell properties, adhesiveness and viability when they were expanded on both types of tissue scaffolds. While adhesiveness of MSCs that were expanded on oriented tissue scaffolds was more than knitted ones, proliferation rates was lower. Although, MSCs on oriented PHB tissue scaffolds showed spindle shape morphology and they were positioned in the direction of spindles, whereas MSCs on knitted nPHB tissue scaffolds distributed homogenously by conserving their poligonal morphology that mimicked bone marrow microenvironment. Conclusion: In the present study, it was found that MSCs cultivated on nanofiber PHB tissue scaffolds conserved their multpotentiality and attained morphology compatible with physical alignment of the scaffold; therefore they may provide effective tissue engineering approach by repairing cellular microenvironment in bone tissue engineering. P1149 - LACTIC ACID BACTERIA CAN REDUCE TLR-4 EXPRESSION ON INFLAMMATED GINGIVAL FIBROBLASTS AND MESENCHYMAL STEM CELLS IN VITRO Aysegul Mendi1, Sevil Kose2, Fatima Aerts Kaya2, Duygu Uckan2,3, Dervis Yilmaz1, and Levent Aral1 1 Department of Medical Microbiology, Gazi University Faculty of Dentistry, Ankara, Turkey; 2Center for Stem Cell Research and Development/PEDI-STEM, Hacettepe University, Ankara, Turkey; 3Pediatric Hematology/BMT Unit, Hacettepe University Children’s Hospital, Ankara, Turkey Introduction: Lactic acid bacteria (LAB) have been studied for their health-promoting and immunomodulatory effects. The main field of research has been in gastrointestinal tract. In the past few years LAB have also been investigated in the oral health perspective. We studied competitive adhesion of Lactobacillus strains with Porpyhromonas gingivalis on human gingival fibrolasts (GF) and bone marrow derived mesenchymal stem cells (MSCs). P. gingivalis, an important periodontal pathogen, is an effective colonizer of oral tissues and accounted for periodontitis. It has been shown that bacterial ligands stimulate Toll-like receptor (TLR) expression on a variety of cells. However it is unknown whether whole bacteria can induce TLR expression on GF and MSCs. . In this study, we evaluated the effect of LAB on cellular adhesion and TLR 4 and 2 expression induced by interferon (IFN)-g. Material and Methods: Adhesion assay was conducted (Lactobacillus rhamnosus ATCC 9595, L. brevis OML 28, P. gingivalis ATCC 33277) and competition of LAB and P. gingivalis on GF and MSCs was studied. To determine the suppressive effect of Lactobacillus strains on IFN-g- and P. gingivalis-based inflammation, TLR 4 and 2 expression by flow cytometer was investigated. Results: Adhesion of bacteria on GF and MSCs was significantly different. LAB prevented adhesion of P. gingivalis as shown by light and fluoresence microscopy. In addition,LAB also reduced TLR 4 expression on GF and MSCs, in in-vitro assays with IFN-g and P. gingivalis. Conclusion: Adhesion of probiotic bacteria to oral soft tissues promotes their beneficial effect to the host. Obtained data suggested that cells from different tissues may achieve specific functional characteristics according to the microenvironmental needs. Further data showed differential responses on TLR expression of GF vs MSCs suggesting participation of GFs on immune response at oral cavity by TLRs. On the other hand, lower TLR expression on bone marrow derived MSCs may partially be attributed to the immunprivileged state of the bone marrow niche.
P1150 - LINEAGE SPECIFIC METABOLIC ACTIVITY IN HAEMATOPOIESIS Claudia Billing1, Michael Walker2, Lydia Schnapka-Hille1, Uta Ceglarek3, Anthony Whetton2, Dietger Niederwieser1, and Michael Cross1 1 Haematology and Oncology, University of Leipzig, Leipzig, Germany; 2Wolfson Molecular Imaging Centre, University of Manchester, Manchester, UK; 3Institute of Laboratory Medicine, University of Leipzig, Leipzig, Germany Erythroid and neutrophil cells have widely different metabolic requirements and tend to be produced in distinct regions of the bone marrow associated with arterioles or sinusoids respectively. This is suggestive of metabolic compartmentalisation of haematopoietic marrow, and raises questions concerning the partitioning of metabolism in solid tissues, the interdependence of metabolic and signalling pathways in the control of normal and leukemic haematopoiesis and the achievement of high density in vitro culture conditions. A key issue is whether the early progenitor cells of each lineage employ distinct metabolic strategies to satisfy the biosynthetic demands of proliferation. To address this question we have purified mitochondria from large scale cultures of FDCP-Mix cells at various stages of erythroid or granulocyte/macrophage differentiation and subjected them to proteomic analysis using 8-plex iTRAQ liquid chromatography tandem mass spectrometry (LC-MS/MS). In a complementary approach, the uptake and release of key metabolites at various stages of differentiation was determined by mass spectrometry and biochemical assay of conditioned media. We find lineage specific changes in the balance of TCA cycle enzymes and of components of the electron transport chain, as well as in proteins controlling apoptosis and reactive oxygen species. Further changes in co-purified cytoplasmic enzymes suggest differential use of available substrates that is confirmed by lineage specific differences in metabolite uptake. Our results suggests that early neutrophil and early erythroid progenitors do indeed adopt different metabolic strategies to biosynthesis that optimise metabolite usage in bone marrow.
P1151 - DISTINCT LEUKEMIA-INITIATING SUBSETS ARE MAINTAINED BY IL-2/CD25 AXIS IN CHRONIC MYELOID LEUKEMIA NICHE Keiyo Takubo, Chiharu Kobayashi, and Toshio Suda Department of Cell Differentiation, the Sakaguchi Laboratory of Developmental Biology, Keio University School of Medicine, Tokyo, Japan Chronic myeloid leukemia (CML) is a clonal hematopoietic disorder originated from hematopoietic stem cells (HSCs) with the Philadelphia chromosome which produces BCR-ABL fusion protein. Targeting tyrosine kinase activity of BCR-ABL by tyrosine kinase inhibitors can deplete most of the differentiated CML cells but not CML leukemia initiating cells (LICs). Therefore it is mandatory to develop the eradication strategy of CML LICs. Here we show that CML LICs show a severely hypoxic phenotype then the normal HSCs in the niche. HIF-1alpha, a master regulator of cellular hypoxia response, plays a critical role for maintaining HSC in the hypoxic niche. HIF-1alpha-deficient p210BCR-ABL retrovirus transduced Lineage marker- Sca-1+ c-Kit+ (LSK) cells are defective in establishing LIC population in vivo. Under normoxic conditions, HIF-1alpha is recognized by an E3 ubiquitin ligase VHL. Although VHL deficient HSCs lose transplantation capacity, they show an accelerated formation of CML LIC after p210BCR-ABL retrovirus transduction. To elucidate the putative LIC markers in the hypoxic niche, we have screened various surface markers using p210BCR-ABL retrovirus-induced CML-like myeloproliferative disease model. Among them, we demonstrate that CML LICs are divided into CD25+FcεRIa- Lineage marker (Lin)- Sca-1+ c-Kit+ (F-LSK) cells and CD25-FLSK cells. We show that CD25+F-LSK cells have multipotent differentiation capacity with preference to cytokine-producing mast cell commitment. Although both CD25-F-LSK and CD25+F-LSK cells differentiate into one another, CD25+FLSK cells show higher LIC capacity than CD25-F-LSK cells. High expression of CD25 in CD34+CD38- fraction of human CML indicate that CD25+LICs constitute "LIC-derived niche" that can be a preferential therapeutic target for CML LICs.