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INCOMPLETE REJUVENATION OF AGED HSCS IN YOUNG BONE MARROW NICHE
S72
Poster Presentations / Experimental Hematology 2019;76 (Suppl):S52−S96
3070 - THE SINUSOIDAL ENDOTHELIUM FUNCTIONS AS
A HEMATOPOIETIC NICHE IN THE ZEBRAFISH KIDNEY Mao Kondo1, Shiori Yamamori1, Jingjing Kobayashi-Sun1 , Makoto Taniguchi2 , Kaori Kanemaru3, David Traver4, Isao Kobayashi1 1 Kanazawa University, Kanazawa, Japan; 2 Kanazawa Medical University, Uchinada, Japan; 3Tokyo University of Science, Noda, Japan; 4University of California at San Diego, San Diego, United States
Hematopoietic stem cells (HSCs) are maintained in the specific microenvironment, termed the HSC niche. Some essential cellular components of HSC niches have been identified in the murine bone marrow, such as sinusoidal endothelial cells, perivascular cells, and mesenchymal stem cells. The zebrafish is an excellent model for the study of HSCs due to its many unique advantages, including valuable tools and experimental methods (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.). Moreover, the major hematopoietic organ in zebrafish is the kidney, so-called the “kidney marrow”, providing a parallel view of the HSC niche over evolution. Little is known, however, regarding which cell types plays a role in HSC niches in the kidney. This is due in part to the lack of robust methods to purify HSCs from the zebrafish kidney. Here, we show that zebrafish HSCs are highly enriched in the double-positive fraction of gata2a:GFP and runx1: mCherry (gata2a+ runx1+), and that gata2a+ runx1+ cells are closely associated with the sinusoidal endothelium in the adult kidney. An in vivo competitive repopulation assay showed that the frequency of HSCs was at least 550 times higher in gata2a+ runx1+ cells than total hematopoietic cells in the zebrafish kidney. Histological analyses revealed that gata2a+ runx1+ cells were mainly observed in the dorsal lateral area of the kidney where sinusoidal capillaries are abundantly observed. Loss of Jam1a, which is expressed in both sinusoidal endothelial cells and hematopoietic cells, led to a remarkable reduction in the sinusoidal area of the kidney and defect in supporting the hematopoietic repopulation. Our data thus suggest that the sinusoidal endothelium is an evolutionarily conserved component of HSC niches in vertebrates.
3071 - INCOMPLETE REJUVENATION OF AGED HSCS IN
YOUNG BONE MARROW NICHE Wakako Kuribayashi1,2, Atsushi Iwama3, Motohiko Oshima3 1 Division of Stem Cell and Molecular Medicine Center for Stem Cell Biology and Regenerative Medicine The Institute of Medical Science, The University of Tokyo, Shinagawa-ku, Japan; 2Chiba Univercity, Shinagawa-ku, Japan; 3Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Hematopoietic stem cell (HSCs) give rise to all kinds of blood cells throughout life. As they age, HSCs exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation. HSC niche, which is essential for the maintenance of HSCs, also undergo considerable changes with aging. However, detailed molecular mechanisms that regulate HSC aging have not been fully understood. Although it is technically difficult to directly evaluate the relationship between aging of HSCs and BM niche, we assumed that engrafting aged HSCs into young niche would rejuvenate them. In this study, we transplanted 20-month-old aged HSCs into 10-week-old young mice without irradiation following the report by Shimoto et al. that the number of empty HSC niches available for engraftment is larger than the number of niches occupied by host HSCs (Blood, 2017). Two months after transplantation, we collected aged HSCs engrafted in young mice (Aged/Y), then performed secondary transplantation and RNA sequence analysis. Secondary transplantation revealed no rejuvenation of aged HSCs in terms of repopulating capacity and non-biased differentiation. However, the gene expression profiles of Aged/Y HSCs were reprogrammed to a large extent similar to those of young HSCs. We detected many genes, the aberrant expression of which in aged HSCs was restored close to the levels of young HSCs. These genes were related to KEGG pathways such as phosphorylation, chemokine signaling pathway, and proteolysis. We are currently engaged in the epigenome profiling of Aged/Y HSCs engrafted in the young niche. These results indicate that a significant portion of changes in transcriptional profiles of HSCs with aging is dependent on aging of niche. Rejuvenation of niche can largely restore the age-related transcriptional profiles of HSCs but cannot restore the functional defects of aged HSCs.
3072 - RETINOIC ACID RECEPTOR GAMMA ACTIVITY IN
ENDOTHELIAL CELLS REGULATES B LYMPHOPOIESIS, ERYTHROPOIESIS, PLATELET PRODUCTION, VASCULATURE STRUCTURE AND ADIPOCYTE SIZE IN A SEX-DEPENDENT MANNER Diannita Kwang1,2 , Kelli Schleibs3 , Lenny straszkowski3, org Heierhorst4, Gavin Tjin4, Louise Purton4 Ashleigh King3, J€ 1 St Vincent’s Institute of Medical Research, Melbourne, Australia; 2 Department of Medicine at St. Vincent’s Hospital, The University of Melbourne, Melbourne, Australia; 3 St. Vincent’s Institute, Melbourne, Australia; 4St Vincent’s Institute, Melbourne, Australia
We have previously shown that mice null for the vitamin A receptor, retinoic acid receptor gamma (Rarg-/- mice) developed a myeloproliferative-like syndrome, accompanied by bone marrow (BM) B lymphopenia and reduced BM erythrocytes, and we have shown that all of these phenotypes were microenvironment-induced. Here, we deleted Rarg in tamoxifen-inducible Cdh5(PAC)CreERT2 (VE-Cadherin)-expressing endothelial cells (ECs) in 3-week old mice and investigated their haematopoiesis and BM niche parameters at 12 weeks of age. All mice were crossed to Rosa26mTmG reporter mice which confirmed that Rarg deletion was restricted to the ECs. Male and female EC:Rarg-/-mTmG mice had significant reductions of B lymphocytes in their peripheral blood (PB), accompanied by significant reductions in BM mature recirculating B cells compared to sex-matched EC: Rarg+/+mTmG mice. No significant phenotypes were observed in the maturing B lymphocyte populations in the BM or spleen of female mice, whereas male mice had reduced numbers of pre-pro-B and increased numbers of pre-B lymphocytes in their BM. Female EC: Rarg-/-mTmG mice also developed PB thrombocytopenia and macrocytic anaemia, accompanied by increased numbers of erythrocytes in the spleen. Male EC:Rarg-/-mTmG mice developed thrombocytosis and had significantly reduced numbers of mature BM erythrocytes. The BM sinusoidal vessels in female, but not male, EC:Rarg-/-mTmG mice were significantly dilated, and these female mice had smaller adipocytes in their BM. Collectively, our data show that RARg activity in ECs is essential for mature cell B lymphopoiesis, platelet production and erythropoiesis in male and female mice. Furthermore, RARg activity in ECs is essential for the regulation of sinusoidal vasculature structure and adipocyte production in female mice. The mechanisms underlying these phenotypes are currently under investigation.
3073 - NEW DIRECT TARGETS OF PSTAT3 AND PSTAT5
IN HUMAN ERYTHROID AND MEGAKARYOCYTIC CELLS Charlene Lam, Kevin Gillinder, Graham Magor, Helen Mitchell, Andrew Perkins Monash University, Melbourne, Australia
Myeloproliferative neoplasms (MPNs) are characterised by excess production of mature blood cells accompanied by an increased risk of thrombosis and progression to marrow fibrosis and AML. Most are driven by a mutation (V617F) in the pseudo-kinase domain of JAK2, which leads to unrestrained cell proliferation. To find direct targets of JAK2-STAT signalling in MPNs, we undertook ChIP-Seq for pSTAT5 and pSTAT3 in cell lines, HEL and SET2. HEL cells have 13-16 copies of JAK2-V617F as determined by FISH and SNP arrays 1. They have high levels of pSTAT5 and pSTAT3 by phosphoflow and Western blotting. SET-2 cells were derived from a patient with JAK2-V617F+ ET; they also have high levels of pSTATs. We found »690 pSTAT5-occupied sites and >10,000 pSTAT3-occupied sites in HEL cells. The majority reside in distal enhancers. We found new enhancers in wellknown target genes such as BCL2L1, which encodes the pro-survival protein, BCL-X. The human enhancer is in a similar relative position to the recently described mouse EPO-dependent enhancer 2. We found new direct target genes that encode for proteins with interesting predicted functions. SET-2 cells have similar pSTAT5 and pSTAT3-bound sites suggesting commonality of functions. We show pSTAT3 and pSTAT5 bind as dimers in vivo to typical GAS elements, but with slightly different site preferences. This has implications for differential target gene regulation. We undertook expression profiling using SLAM-seq following treatment with ruxolitinib and validated novel target genes by qRT-PCR. In short, we have discovered hundreds of direct JAK-STAT target genes involved in cell survival, proliferation, down regulation of cytokine signalling, and novel functions. The genes provide new insights into the biology of MPNs, and a source of potential new biomarkers and drug targets. We have validated some in primary MPN samples.
Poster Presentations / Experimental Hematology 2019;76 (Suppl):S52−S96
3070 - THE SINUSOIDAL ENDOTHELIUM FUNCTIONS AS
A HEMATOPOIETIC NICHE IN THE ZEBRAFISH KIDNEY Mao Kondo1, Shiori Yamamori1, Jingjing Kobayashi-Sun1 , Makoto Taniguchi2 , Kaori Kanemaru3, David Traver4, Isao Kobayashi1 1 Kanazawa University, Kanazawa, Japan; 2 Kanazawa Medical University, Uchinada, Japan; 3Tokyo University of Science, Noda, Japan; 4University of California at San Diego, San Diego, United States
Hematopoietic stem cells (HSCs) are maintained in the specific microenvironment, termed the HSC niche. Some essential cellular components of HSC niches have been identified in the murine bone marrow, such as sinusoidal endothelial cells, perivascular cells, and mesenchymal stem cells. The zebrafish is an excellent model for the study of HSCs due to its many unique advantages, including valuable tools and experimental methods (e.g. transgenic/mutant animals, transplantation assays, cell culture assays, etc.). Moreover, the major hematopoietic organ in zebrafish is the kidney, so-called the “kidney marrow”, providing a parallel view of the HSC niche over evolution. Little is known, however, regarding which cell types plays a role in HSC niches in the kidney. This is due in part to the lack of robust methods to purify HSCs from the zebrafish kidney. Here, we show that zebrafish HSCs are highly enriched in the double-positive fraction of gata2a:GFP and runx1: mCherry (gata2a+ runx1+), and that gata2a+ runx1+ cells are closely associated with the sinusoidal endothelium in the adult kidney. An in vivo competitive repopulation assay showed that the frequency of HSCs was at least 550 times higher in gata2a+ runx1+ cells than total hematopoietic cells in the zebrafish kidney. Histological analyses revealed that gata2a+ runx1+ cells were mainly observed in the dorsal lateral area of the kidney where sinusoidal capillaries are abundantly observed. Loss of Jam1a, which is expressed in both sinusoidal endothelial cells and hematopoietic cells, led to a remarkable reduction in the sinusoidal area of the kidney and defect in supporting the hematopoietic repopulation. Our data thus suggest that the sinusoidal endothelium is an evolutionarily conserved component of HSC niches in vertebrates.
3071 - INCOMPLETE REJUVENATION OF AGED HSCS IN
YOUNG BONE MARROW NICHE Wakako Kuribayashi1,2, Atsushi Iwama3, Motohiko Oshima3 1 Division of Stem Cell and Molecular Medicine Center for Stem Cell Biology and Regenerative Medicine The Institute of Medical Science, The University of Tokyo, Shinagawa-ku, Japan; 2Chiba Univercity, Shinagawa-ku, Japan; 3Division of Stem Cell and Molecular Medicine, Center for Stem Cell Biology and Regenerative Medicine The Institute of Medical Science, The University of Tokyo, Tokyo, Japan Hematopoietic stem cell (HSCs) give rise to all kinds of blood cells throughout life. As they age, HSCs exhibit functional alterations, such as reduced regenerative capacity and myeloid-biased differentiation. HSC niche, which is essential for the maintenance of HSCs, also undergo considerable changes with aging. However, detailed molecular mechanisms that regulate HSC aging have not been fully understood. Although it is technically difficult to directly evaluate the relationship between aging of HSCs and BM niche, we assumed that engrafting aged HSCs into young niche would rejuvenate them. In this study, we transplanted 20-month-old aged HSCs into 10-week-old young mice without irradiation following the report by Shimoto et al. that the number of empty HSC niches available for engraftment is larger than the number of niches occupied by host HSCs (Blood, 2017). Two months after transplantation, we collected aged HSCs engrafted in young mice (Aged/Y), then performed secondary transplantation and RNA sequence analysis. Secondary transplantation revealed no rejuvenation of aged HSCs in terms of repopulating capacity and non-biased differentiation. However, the gene expression profiles of Aged/Y HSCs were reprogrammed to a large extent similar to those of young HSCs. We detected many genes, the aberrant expression of which in aged HSCs was restored close to the levels of young HSCs. These genes were related to KEGG pathways such as phosphorylation, chemokine signaling pathway, and proteolysis. We are currently engaged in the epigenome profiling of Aged/Y HSCs engrafted in the young niche. These results indicate that a significant portion of changes in transcriptional profiles of HSCs with aging is dependent on aging of niche. Rejuvenation of niche can largely restore the age-related transcriptional profiles of HSCs but cannot restore the functional defects of aged HSCs.
3072 - RETINOIC ACID RECEPTOR GAMMA ACTIVITY IN
ENDOTHELIAL CELLS REGULATES B LYMPHOPOIESIS, ERYTHROPOIESIS, PLATELET PRODUCTION, VASCULATURE STRUCTURE AND ADIPOCYTE SIZE IN A SEX-DEPENDENT MANNER Diannita Kwang1,2 , Kelli Schleibs3 , Lenny straszkowski3, org Heierhorst4, Gavin Tjin4, Louise Purton4 Ashleigh King3, J€ 1 St Vincent’s Institute of Medical Research, Melbourne, Australia; 2 Department of Medicine at St. Vincent’s Hospital, The University of Melbourne, Melbourne, Australia; 3 St. Vincent’s Institute, Melbourne, Australia; 4St Vincent’s Institute, Melbourne, Australia
We have previously shown that mice null for the vitamin A receptor, retinoic acid receptor gamma (Rarg-/- mice) developed a myeloproliferative-like syndrome, accompanied by bone marrow (BM) B lymphopenia and reduced BM erythrocytes, and we have shown that all of these phenotypes were microenvironment-induced. Here, we deleted Rarg in tamoxifen-inducible Cdh5(PAC)CreERT2 (VE-Cadherin)-expressing endothelial cells (ECs) in 3-week old mice and investigated their haematopoiesis and BM niche parameters at 12 weeks of age. All mice were crossed to Rosa26mTmG reporter mice which confirmed that Rarg deletion was restricted to the ECs. Male and female EC:Rarg-/-mTmG mice had significant reductions of B lymphocytes in their peripheral blood (PB), accompanied by significant reductions in BM mature recirculating B cells compared to sex-matched EC: Rarg+/+mTmG mice. No significant phenotypes were observed in the maturing B lymphocyte populations in the BM or spleen of female mice, whereas male mice had reduced numbers of pre-pro-B and increased numbers of pre-B lymphocytes in their BM. Female EC: Rarg-/-mTmG mice also developed PB thrombocytopenia and macrocytic anaemia, accompanied by increased numbers of erythrocytes in the spleen. Male EC:Rarg-/-mTmG mice developed thrombocytosis and had significantly reduced numbers of mature BM erythrocytes. The BM sinusoidal vessels in female, but not male, EC:Rarg-/-mTmG mice were significantly dilated, and these female mice had smaller adipocytes in their BM. Collectively, our data show that RARg activity in ECs is essential for mature cell B lymphopoiesis, platelet production and erythropoiesis in male and female mice. Furthermore, RARg activity in ECs is essential for the regulation of sinusoidal vasculature structure and adipocyte production in female mice. The mechanisms underlying these phenotypes are currently under investigation.
3073 - NEW DIRECT TARGETS OF PSTAT3 AND PSTAT5
IN HUMAN ERYTHROID AND MEGAKARYOCYTIC CELLS Charlene Lam, Kevin Gillinder, Graham Magor, Helen Mitchell, Andrew Perkins Monash University, Melbourne, Australia
Myeloproliferative neoplasms (MPNs) are characterised by excess production of mature blood cells accompanied by an increased risk of thrombosis and progression to marrow fibrosis and AML. Most are driven by a mutation (V617F) in the pseudo-kinase domain of JAK2, which leads to unrestrained cell proliferation. To find direct targets of JAK2-STAT signalling in MPNs, we undertook ChIP-Seq for pSTAT5 and pSTAT3 in cell lines, HEL and SET2. HEL cells have 13-16 copies of JAK2-V617F as determined by FISH and SNP arrays 1. They have high levels of pSTAT5 and pSTAT3 by phosphoflow and Western blotting. SET-2 cells were derived from a patient with JAK2-V617F+ ET; they also have high levels of pSTATs. We found »690 pSTAT5-occupied sites and >10,000 pSTAT3-occupied sites in HEL cells. The majority reside in distal enhancers. We found new enhancers in wellknown target genes such as BCL2L1, which encodes the pro-survival protein, BCL-X. The human enhancer is in a similar relative position to the recently described mouse EPO-dependent enhancer 2. We found new direct target genes that encode for proteins with interesting predicted functions. SET-2 cells have similar pSTAT5 and pSTAT3-bound sites suggesting commonality of functions. We show pSTAT3 and pSTAT5 bind as dimers in vivo to typical GAS elements, but with slightly different site preferences. This has implications for differential target gene regulation. We undertook expression profiling using SLAM-seq following treatment with ruxolitinib and validated novel target genes by qRT-PCR. In short, we have discovered hundreds of direct JAK-STAT target genes involved in cell survival, proliferation, down regulation of cytokine signalling, and novel functions. The genes provide new insights into the biology of MPNs, and a source of potential new biomarkers and drug targets. We have validated some in primary MPN samples.