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In vitro human embryonic stem cell hematopoiesis mimics MYB independent yolk sac hematopoiesis
Poster Presentations/ Experimental Hematology 42 (2014) S23–S68
P1160 - LINEAGE REGULATORS AND SIGNALING TRANSCRIPTION FACTORS DURING ERYTHROPOIESIS Avik Choudhuri3, Brian Abraham2, William Mallard3, Barbara Hummel1, Sonja Boatman3, Teresa Bowman3, Anthony DiBiase3, John Rinn3, Richard Young2, Leonard Zon3, and Eirini Trompouki1 1 Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; 2Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA; 3Harvard Stem Cell Institute, Boston, Massachusetts, USA During hematopoietic differentiation there is a rapid turnover of cell stages. Our goal is to delineate how signal-responsive transcription factors get integrated in lineage choices. Thus, we followed CD34+ hematopoietic stem and progenitor cells (HSPCs) during erythropoiesis. Our previous work demonstrated that the signal-responsive factor SMAD1 co-localizes with the lineage regulators GATA2 and GATA1 at lineage specific genes in HSPCs. To discover how SMAD1 is integrated in lineage choices we performed ChIP-seq and expression analysis for GATA2, GATA1, SMAD1 and H3K27ac in HSPCs during erythroid differentiation. Expression analysis indicated that there is one major global gene expression transition during days 3-4 of differentiation. This transition is further verified by the binding events during differentiation. Indeed at day4 GATA2 is down-regulated and GATA1 replaces GATA2 in some genomic regions. These data indicate the existence of a single time point that HSPCs commit to an erythroid fate wherein dramatic changes in transcription factor binding and expression occur. Before this transition, more than 70% of GATA2 bound genes remain unchanged and after the transition, more than 70% of the GATA1 bound genes remain stably bound. In contrast, the SMAD1 binding profile differed greatly at each time point with less than 30% similarity between adjacent days along the differentiation time-course. Genomic loci bound by both GATA 1 or 2 and SMAD1 showed better correlation with H3K27ac and gene expression than genomic loci that GATA binds alone. Our results demonstrate that, although lineage regulators like GATA1 and GATA2 sculpt the transcriptional landscape that will determine cell fate decisions, fine tuning of these decisions is aided by signaling transcription factors.
P1161 - FEMORAL MICROARCHITECTURE AND ENDOSTEAL NICHE ALTERATIONS IN PROTEIN MALNUTRITION: POSSIBLE EFFECTS ON HEMATOPOIESIS? Maristela Tsujita1,2, Manuela Rodrigues1, Marılia Teixeira1, Araceli Hastreiter1, Graziela Silva1, Cecılia Gouveia1, Ricardo Fock1, and Primavera Borelli1 1 University of S~ao Paulo, S~ao Paulo, Brazil; 2Paulista University, S~ao Paulo, Brazil Hematopoiesis is a dynamic process that leads to blood cells production. At the centre of this process sits the hematopoietic stem cells (HSC), which preferentially reside along the trabecular metaphysis of long bones. Evidences suggest that specific microenvironments could support HSC self-renewal and differentiation. The endosteal niche hosts HSC in quiescence or self-renewing state. Protein malnutrition (PM) is associated with pancytopenia, bone marrow (BM) hypoplasia and changes in bone microenvironment. In this way, we hypothesized that PM may change the endosteal niche with a consequent impairment in the number and function of longterm HSC (LT-HSC). To test this hypothesis, C57Bl/6J mice were randomly assigned in control and malnourished groups which received normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5 weeks period. We characterized femoral bone tissue changes in vivo by measuring bone mineral density (BMD) and ex vivo, using computed microtomography. Compared to controls, malnourished animals (MA) presented a decrease in the BMD of different regions of the femur and a significant decrease in the trabecular bone volume (BV/TV), and trabecular number (Tb.N) of the distal metaphysis of the femur, followed by an increase in the trabecular separation (Tb.Sp) and a prevalence of rod-like trabeculae. Furthermore, osteoblasts and LT-HSC were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry. We found a smaller amount of LTHSC in the BM of MA. Osteoblasts from MA showed decreased mRNA expression of type I collagen and osteocalcin, however no difference was found on CXCL-12, angiopoetin, Jagged-2 or N-cadherin mRNA expression. Therefore, we may conclude that PM compromises trabecular bone structure and reduces the number of LT-HSC cells in the BM.
S63
P1162 - AGING OF HEMATOPOIETIC STEM CELLS IS ONLY MARGINALLY AFFECTED BY DIET Seka Lazare, Bertien Dethmers, Gerald de Haan, and Ronald van Os Laboratory of Ageing Biology and Stem Cells, European Research Institute on the Biology of Aging (ERIBA), University Medical Center Groningen, Groningen, Netherlands Aging of hematopoietic stem cells (HSC) is characterized by an increased number or phenotypically defined stem cells, reduced functional activity at the single cell level, reduced homing capacity, and a myeloid biased output. It has been previously shown that bone marrow from mice aged on a caloric restricted diet have a competitive advantage over mice aged on a normal diet and even over young bone marrow (Chen et al, Exp Hematol, 2000). However, little is known on aging on a high fat diet and on how phenotypically defined stem cells function under these conditions. Therefore, we aged mice on a normal, high fat and caloric restricted diet and tested for stem cell activity in vitro and in vivo. In this study, the stem cell frequency (LSK CD48-CD150+ CD34-EPCR+) increased during aging in all three groups but with a slight delay in caloric restricted mice and a faster increase in mice on a high fat diet. However, the in vitro activity of individual HSC as measured by outgrowth as cobblestone areas on a stromal cell line, was not affected except for a faster decline in mice on a fat diet. No evidence for enhanced myeloid skewing was observed when myeloid and lymphoid progenitor cell frequencies were measured by flow cytometry. Finally, transplantation of purified bone marrow stem cells showed a severe competitive disadvantage of all old stem cells compared to young cells. In conclusion, hematopoiesis in old mice is predominantly affected by age and less by caloric intake.
P1163 - IN VITRO HUMAN EMBRYONIC STEM CELL HEMATOPOIESIS MIMICS MYB INDEPENDENT YOLK SAC HEMATOPOIESIS Stijn Vanhee1, Katrien De Mulder1, Yasmine Van Caeneghem1, Nadine Van Roy2,1, Bj€orn Menten2,1, Imke Velghe1, Jan Philippe3,1, Tom Taghon1, Georges Leclercq1, Tessa Kerre1,3, and Bart Vandekerckhove1 1 Ghent University, Ghent, Belgium; 2Center for Medical Genetics, Belgium, Belgium; 3Ghent University Hospital, Ghent, Belgium The in vitro formation of HSC from hESC has been attempted by many scientists because of its clinical applicability. Here we investigated whether human in vitro hematopoiesis generates cMYB dependent HSC (precursors) besides HSC independent yolk sac (YS) type precursors. We generated cMYBeGFP reporter hESC using random integration BAC transgenesis and could show that GFP expression paralleled cMYB expression. Upon hematopoietic differentiation, eGFP weakly positive cells became first apparent from d11 onwards, forming a clear population by d14. These cells were CD34+CD43+CD45-/lo compatible with an early hematopoietic precursor. Using time lapse confocal microscopy, hemogenic endothelium sorted from d11 embryoid bodies of MYB reporter ESC started rounding up and formed CD34+CD43+ cells without expression of eGFP. Extensive phenotypic analysis of these early GFP+ cells evidenced expression of CD33, suggesting commitment towards the myeloid lineage. These cells were found expandable using conditions containing IL3. In agreement with these data, only myeloid cells and no erythroid, megakaryocytic or lymphoid cells could be derived in differentiation experiments. We next studied cMYBeGFP expression during directed myeloid differentiation of hematopoietic precursor cells. Strong GFP expression was found upon granulocytic commitment, while monocytes were found eGFP negative. Tissue macrophages were reported to develop from cMYB HSC independent YS progenitors. Therefore, we studied generation of these cells. Using time-lapse confocal microscopic analyses, CD14+ monocytes were shown to derive from eGFP- precursors. This was confirmed using flow cytometry. Using qPCR, we found markers for MYB-independent tissue macrophages to be expressed by these cells. In summary, no evidence was found for the in vitro generation of cMYBeGFP+ HSC; during hESC-initiated hematopoiesis. Instead, lineage progenitors form directly from hemogenic endothelium. Moreover, monocytic cells generated in vitro show similar characteristics to immature tissue macrophages.
P1160 - LINEAGE REGULATORS AND SIGNALING TRANSCRIPTION FACTORS DURING ERYTHROPOIESIS Avik Choudhuri3, Brian Abraham2, William Mallard3, Barbara Hummel1, Sonja Boatman3, Teresa Bowman3, Anthony DiBiase3, John Rinn3, Richard Young2, Leonard Zon3, and Eirini Trompouki1 1 Cellular and Molecular Immunology, Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany; 2Whitehead Institute for Biomedical Research, Cambridge, Massachusetts, USA; 3Harvard Stem Cell Institute, Boston, Massachusetts, USA During hematopoietic differentiation there is a rapid turnover of cell stages. Our goal is to delineate how signal-responsive transcription factors get integrated in lineage choices. Thus, we followed CD34+ hematopoietic stem and progenitor cells (HSPCs) during erythropoiesis. Our previous work demonstrated that the signal-responsive factor SMAD1 co-localizes with the lineage regulators GATA2 and GATA1 at lineage specific genes in HSPCs. To discover how SMAD1 is integrated in lineage choices we performed ChIP-seq and expression analysis for GATA2, GATA1, SMAD1 and H3K27ac in HSPCs during erythroid differentiation. Expression analysis indicated that there is one major global gene expression transition during days 3-4 of differentiation. This transition is further verified by the binding events during differentiation. Indeed at day4 GATA2 is down-regulated and GATA1 replaces GATA2 in some genomic regions. These data indicate the existence of a single time point that HSPCs commit to an erythroid fate wherein dramatic changes in transcription factor binding and expression occur. Before this transition, more than 70% of GATA2 bound genes remain unchanged and after the transition, more than 70% of the GATA1 bound genes remain stably bound. In contrast, the SMAD1 binding profile differed greatly at each time point with less than 30% similarity between adjacent days along the differentiation time-course. Genomic loci bound by both GATA 1 or 2 and SMAD1 showed better correlation with H3K27ac and gene expression than genomic loci that GATA binds alone. Our results demonstrate that, although lineage regulators like GATA1 and GATA2 sculpt the transcriptional landscape that will determine cell fate decisions, fine tuning of these decisions is aided by signaling transcription factors.
P1161 - FEMORAL MICROARCHITECTURE AND ENDOSTEAL NICHE ALTERATIONS IN PROTEIN MALNUTRITION: POSSIBLE EFFECTS ON HEMATOPOIESIS? Maristela Tsujita1,2, Manuela Rodrigues1, Marılia Teixeira1, Araceli Hastreiter1, Graziela Silva1, Cecılia Gouveia1, Ricardo Fock1, and Primavera Borelli1 1 University of S~ao Paulo, S~ao Paulo, Brazil; 2Paulista University, S~ao Paulo, Brazil Hematopoiesis is a dynamic process that leads to blood cells production. At the centre of this process sits the hematopoietic stem cells (HSC), which preferentially reside along the trabecular metaphysis of long bones. Evidences suggest that specific microenvironments could support HSC self-renewal and differentiation. The endosteal niche hosts HSC in quiescence or self-renewing state. Protein malnutrition (PM) is associated with pancytopenia, bone marrow (BM) hypoplasia and changes in bone microenvironment. In this way, we hypothesized that PM may change the endosteal niche with a consequent impairment in the number and function of longterm HSC (LT-HSC). To test this hypothesis, C57Bl/6J mice were randomly assigned in control and malnourished groups which received normoproteic and hypoproteic diets (12% and 2% protein, respectively) over a 5 weeks period. We characterized femoral bone tissue changes in vivo by measuring bone mineral density (BMD) and ex vivo, using computed microtomography. Compared to controls, malnourished animals (MA) presented a decrease in the BMD of different regions of the femur and a significant decrease in the trabecular bone volume (BV/TV), and trabecular number (Tb.N) of the distal metaphysis of the femur, followed by an increase in the trabecular separation (Tb.Sp) and a prevalence of rod-like trabeculae. Furthermore, osteoblasts and LT-HSC were isolated from the femoral region by immunomagnetic depletion and immunophenotyped by flow cytometry. We found a smaller amount of LTHSC in the BM of MA. Osteoblasts from MA showed decreased mRNA expression of type I collagen and osteocalcin, however no difference was found on CXCL-12, angiopoetin, Jagged-2 or N-cadherin mRNA expression. Therefore, we may conclude that PM compromises trabecular bone structure and reduces the number of LT-HSC cells in the BM.
S63
P1162 - AGING OF HEMATOPOIETIC STEM CELLS IS ONLY MARGINALLY AFFECTED BY DIET Seka Lazare, Bertien Dethmers, Gerald de Haan, and Ronald van Os Laboratory of Ageing Biology and Stem Cells, European Research Institute on the Biology of Aging (ERIBA), University Medical Center Groningen, Groningen, Netherlands Aging of hematopoietic stem cells (HSC) is characterized by an increased number or phenotypically defined stem cells, reduced functional activity at the single cell level, reduced homing capacity, and a myeloid biased output. It has been previously shown that bone marrow from mice aged on a caloric restricted diet have a competitive advantage over mice aged on a normal diet and even over young bone marrow (Chen et al, Exp Hematol, 2000). However, little is known on aging on a high fat diet and on how phenotypically defined stem cells function under these conditions. Therefore, we aged mice on a normal, high fat and caloric restricted diet and tested for stem cell activity in vitro and in vivo. In this study, the stem cell frequency (LSK CD48-CD150+ CD34-EPCR+) increased during aging in all three groups but with a slight delay in caloric restricted mice and a faster increase in mice on a high fat diet. However, the in vitro activity of individual HSC as measured by outgrowth as cobblestone areas on a stromal cell line, was not affected except for a faster decline in mice on a fat diet. No evidence for enhanced myeloid skewing was observed when myeloid and lymphoid progenitor cell frequencies were measured by flow cytometry. Finally, transplantation of purified bone marrow stem cells showed a severe competitive disadvantage of all old stem cells compared to young cells. In conclusion, hematopoiesis in old mice is predominantly affected by age and less by caloric intake.
P1163 - IN VITRO HUMAN EMBRYONIC STEM CELL HEMATOPOIESIS MIMICS MYB INDEPENDENT YOLK SAC HEMATOPOIESIS Stijn Vanhee1, Katrien De Mulder1, Yasmine Van Caeneghem1, Nadine Van Roy2,1, Bj€orn Menten2,1, Imke Velghe1, Jan Philippe3,1, Tom Taghon1, Georges Leclercq1, Tessa Kerre1,3, and Bart Vandekerckhove1 1 Ghent University, Ghent, Belgium; 2Center for Medical Genetics, Belgium, Belgium; 3Ghent University Hospital, Ghent, Belgium The in vitro formation of HSC from hESC has been attempted by many scientists because of its clinical applicability. Here we investigated whether human in vitro hematopoiesis generates cMYB dependent HSC (precursors) besides HSC independent yolk sac (YS) type precursors. We generated cMYBeGFP reporter hESC using random integration BAC transgenesis and could show that GFP expression paralleled cMYB expression. Upon hematopoietic differentiation, eGFP weakly positive cells became first apparent from d11 onwards, forming a clear population by d14. These cells were CD34+CD43+CD45-/lo compatible with an early hematopoietic precursor. Using time lapse confocal microscopy, hemogenic endothelium sorted from d11 embryoid bodies of MYB reporter ESC started rounding up and formed CD34+CD43+ cells without expression of eGFP. Extensive phenotypic analysis of these early GFP+ cells evidenced expression of CD33, suggesting commitment towards the myeloid lineage. These cells were found expandable using conditions containing IL3. In agreement with these data, only myeloid cells and no erythroid, megakaryocytic or lymphoid cells could be derived in differentiation experiments. We next studied cMYBeGFP expression during directed myeloid differentiation of hematopoietic precursor cells. Strong GFP expression was found upon granulocytic commitment, while monocytes were found eGFP negative. Tissue macrophages were reported to develop from cMYB HSC independent YS progenitors. Therefore, we studied generation of these cells. Using time-lapse confocal microscopic analyses, CD14+ monocytes were shown to derive from eGFP- precursors. This was confirmed using flow cytometry. Using qPCR, we found markers for MYB-independent tissue macrophages to be expressed by these cells. In summary, no evidence was found for the in vitro generation of cMYBeGFP+ HSC; during hESC-initiated hematopoiesis. Instead, lineage progenitors form directly from hemogenic endothelium. Moreover, monocytic cells generated in vitro show similar characteristics to immature tissue macrophages.