- Email: [email protected]
Global landscape of hematopoietic stem cells and multipotent progenitors
Oral Short Talk Presentations/ Experimental Hematology 41 (2013) S11–S21
O1008 - DIVERSE AND HERITABLE LINEAGE IMPRINTING OF EARLY HEMATOPOIETIC PROGENITORS Shalin Naik1,2, Leila Perie2, Erwin Zwart2, Carmen Gerlach2, Nienke van Rooij2, Rob de Boer3, and Ton Schumacher2 1 Immunology, WEHI, Melbourne, Victoria, Australia; 2Immunology, NKI, Amsterdam, NH, Netherlands; 3Theoretical Biology & Bioinformatics, Utrecht University, Utrecht, Netherlands Hematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is contentious. In one model, lymphoid and myeloid production split after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells (DCs). However, this model is largely based on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single cell complexity. Here we avoid these pitfalls using a new quantitative version of "cellular barcoding", to trace the in vivo fate of hundreds of LMPPs and HSCs at the single cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types they produce, separating into combinations of lymphoid-, myeloid-, and DC-biased producers. Conversely, while we also observe a known lineage bias of HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs demonstrates that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for DC-biased LMPPs, DCs may be considered a distinct lineage on the principle of separate ancestry. These data suggest a ‘graded commitment’ model of hematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously appreciated.
O1009 - GLOBAL LANDSCAPE OF HEMATOPOIETIC STEM CELLS AND MULTIPOTENT PROGENITORS Daniel Klimmeck1,2,3, Nina Cabezas-Wallscheid1,2, Jenny Hansson3, Lisa Dohrn1,2, Alejandro Reyes3, Wolfgang Huber3, Jeroen Krijgsveld3, and Andreas Trumpp1,2 1 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HISTEM), Heidelberg, Baden-W€urttemberg, Germany; 2Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, BadenW€urttemberg, Germany; 3Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Baden-W€urttemberg, Germany In the hematopoietic system, hematopoietic stem cells (HSC) harbor the highest selfrenewal activity and generate a series of multipotent progenitors (MPP) that differentiate into lineage-committed progenitors. To explore essential HSC features such as self-renewal and quiescence, we performed an extensive global analysis combining the latest generation of quantitative proteome and transcriptome (RNA-seq) analyses. Therefore, we ex vivo-isolated and FACS-sorted HSC (Lin-Sca1+cKit+ (LSK) CD34- Flt3- CD150+ CD48-), MPP1 (LSK CD34+ Flt3- CD150+ CD48-), MPP2 (LSK CD34+ Flt3- CD150+ CD48+), MPP3 (LSK CD34+ Flt3- CD150- CD48+) and MPP4 (LSK CD34+ Flt3+ CD150+ CD48+), as previously described in our lab (Wilson et al.). By employing stable isotope dimethyl labeling and high-resolution tandem mass spectrometry, more than 7,000 proteins were identified. Expression profiling highlights energy metabolism, immune response, cell cycle and DNA repair to be modulated along differentiation. To our knowledge, these data represent the first global protein signature of HSC defined at this level. Furthermore, using in-depth RNA-seq we achieved more than 11-fold coverage of the genome enabling for robust identification of over 22,000 genes. We describe specific expression clusters of cell adhesion molecules and TFs and present a comprehensive landscape of polyA-long non-coding RNAs (lncRNA) in HSC or MPP. Finally, to address differentiation potential of MPP2, 3 and 4 we complement our OMICs approach with functional transplant experiments. In summary, the gene and protein signatures for stemness and multipotency defined in this study represent a novel unique resource for the scientific community and will significantly extend the current understanding of HSC biology.
S13
O1010 - ESTABLISHMENT OF LYMPHO-MYELOID RESTRICTED PROGENITORS PRIOR TO THE EMERGENCE OF DEFINITIVE HEMATOPOIETIC STEM CELLS Charlotta B€oiers2,1, Michael Lutteropp3, Sidinh Luc3, Petter Woll3, Adam Mead3, Anne Hultquist2, Joana Carrelha3, Iain Macaulay3, Gemma Swiers4, Luca Melchiori3, Tiago Luis3, Shabnam Kharazi2, Tiphaine Bouriez-Jones3, Qiaolin Deng5, Annica Ponten6, Christina Jensen3, Ewa Sitnicka2, Rickard Sandberg5, Marella de Bruijn4, and Sten Eirik Jacobsen3,2,7 1 Div of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund, Sweden; 2Haematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund, Sweden; 3Haematopoietic Stem Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom; 4MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom; 5 Department of Cell and Molecular Biology, Karolinska Institutet and Ludwig Institute for Cancer Research, Stockholm, Sweden; 6Cardiovascular group, Lund Stem Cell Center, Lund, Sweden; 7Wallenberg Institute for Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the evolutionary older innate immune-system are formed early in development, while lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we prospectively identified a lymphomyeloid restricted embryonic progenitor as early as E11.5 that expresses IL7Ra and that sustains combined lymphoid and GM transcriptional lineage priming and lineage potentials at the single cell level but no MkE potential. Using Rag1-GFP knock-in mice, we tracked the establishment of this lympho-myeloid restricted progenitor back to as early as E9.5, preceding both hematopoietic colonization of the FL and the establishment of definitive HSCs. Moreover, through in vivo fate mapping we confirmed the inability of Rag1 expressing early embryonic progenitors to contribute to the MkE lineage, while unequivocally and robustly contributing to the myeloid innate as well as lymphoid adaptive immune systems of the mammalian embryo. These findings identify the developmentally earliest immune-restricted progenitor and establish the lymphomyeloid restriction step as a physiologically important lineage commitment step in embryonic mammalian hematopoiesis, preceding the emergence of definitive HSCs. O1011 - RHO GTPASE ACTIVATING PROTEIN P190-B REGULATES HEMATOPOIETIC STEM CELL SELF-RENEWAL DECISIONS Ashwini Hinge1, Bruce Aronow2, and Marie Filippi1 1 Division of Experimental Hematolofy and cancer biology, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, USA; 2Division of biomedical informatics, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, USA Hematopoietic stem cells (HSC) can self-renew or commit to differentiate into all blood cells. The signaling network that regulates these choices is poorly understood. We previously showed that p190-B loss, a negative regulator of Rho activity, enhanced long-term engraftment without altering HSC proliferation or survival or lineage development (Xu, Blood 2009), suggesting an effect on HSC fate decisions. Here, we compared single HSC divisions ex vivo by analyzing multilineage potential of the paired-daughter cells. Non-transplanted (NT) HSCs generated two multipotent daughter cells (self-renewing divisions). However, after secondary transplantation, wild-type (WT) HSCs generated one multipotent and one committed daughter cells (differentiating divisions). p190-B-/- HSCs maintained self-renewal divisions, indicating p190-B loss mediates higher probability of ‘stemness’ inheritance through divisions to prevent HSC depletion following transplantation. Unexpectedly, p190B loss does this by decreasing autocrine TGF-b. Active TGF-b protein levels, TGF-b target genes increased in WT but not in p190-B-/- HSCs upon transplantation relative to NT HSC. TGF-b signaling inhibitors restored self-renewal divisions of transplanted WT HSC as seen in vitro and in engraftment in vivo. TGF-b inhibitor treatment of transplanted mice significantly increased HSC frequency - without changing their proliferation - that yielded higher chimera in tertiary transplant compared to DMSO treated mice. Conversely, recombinant TGF-b1 changed nontransplanted HSC choice to differentiating divisions in vitro. Finally, p38MAPK activity mediated TGF-b effect on HSC fate decisions. Hence, HSC decisions to self-renew or differentiate are specified by a p190-B/TGF-b signaling pathway during HSC regeneration. This study uncovers a novel role for autocrine TGF-b in HSC fate decisions uncoupled from its role on HSC quiescence that may have important implications for regenerative medicine.
O1008 - DIVERSE AND HERITABLE LINEAGE IMPRINTING OF EARLY HEMATOPOIETIC PROGENITORS Shalin Naik1,2, Leila Perie2, Erwin Zwart2, Carmen Gerlach2, Nienke van Rooij2, Rob de Boer3, and Ton Schumacher2 1 Immunology, WEHI, Melbourne, Victoria, Australia; 2Immunology, NKI, Amsterdam, NH, Netherlands; 3Theoretical Biology & Bioinformatics, Utrecht University, Utrecht, Netherlands Hematopoietic stem cells (HSCs) and their subsequent progenitors produce blood cells, but the precise nature and kinetics of this production is contentious. In one model, lymphoid and myeloid production split after the lymphoid-primed multipotent progenitor (LMPP), with both branches subsequently producing dendritic cells (DCs). However, this model is largely based on in vitro clonal assays and population-based tracking in vivo, which could miss in vivo single cell complexity. Here we avoid these pitfalls using a new quantitative version of "cellular barcoding", to trace the in vivo fate of hundreds of LMPPs and HSCs at the single cell level. These data demonstrate that LMPPs are highly heterogeneous in the cell types they produce, separating into combinations of lymphoid-, myeloid-, and DC-biased producers. Conversely, while we also observe a known lineage bias of HSCs, most cellular output is derived from a small number of HSCs that each generates all cell types. Crucially, in vivo analysis of the output of sibling cells derived from single LMPPs demonstrates that they often share a similar fate, suggesting that the fate of these progenitors was imprinted. Furthermore, as this imprinting is also observed for DC-biased LMPPs, DCs may be considered a distinct lineage on the principle of separate ancestry. These data suggest a ‘graded commitment’ model of hematopoiesis, in which heritable and diverse lineage imprinting occurs earlier than previously appreciated.
O1009 - GLOBAL LANDSCAPE OF HEMATOPOIETIC STEM CELLS AND MULTIPOTENT PROGENITORS Daniel Klimmeck1,2,3, Nina Cabezas-Wallscheid1,2, Jenny Hansson3, Lisa Dohrn1,2, Alejandro Reyes3, Wolfgang Huber3, Jeroen Krijgsveld3, and Andreas Trumpp1,2 1 Heidelberg Institute for Stem Cell Technology and Experimental Medicine (HISTEM), Heidelberg, Baden-W€urttemberg, Germany; 2Division of Stem Cells and Cancer, Deutsches Krebsforschungszentrum (DKFZ), Heidelberg, BadenW€urttemberg, Germany; 3Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Heidelberg, Baden-W€urttemberg, Germany In the hematopoietic system, hematopoietic stem cells (HSC) harbor the highest selfrenewal activity and generate a series of multipotent progenitors (MPP) that differentiate into lineage-committed progenitors. To explore essential HSC features such as self-renewal and quiescence, we performed an extensive global analysis combining the latest generation of quantitative proteome and transcriptome (RNA-seq) analyses. Therefore, we ex vivo-isolated and FACS-sorted HSC (Lin-Sca1+cKit+ (LSK) CD34- Flt3- CD150+ CD48-), MPP1 (LSK CD34+ Flt3- CD150+ CD48-), MPP2 (LSK CD34+ Flt3- CD150+ CD48+), MPP3 (LSK CD34+ Flt3- CD150- CD48+) and MPP4 (LSK CD34+ Flt3+ CD150+ CD48+), as previously described in our lab (Wilson et al.). By employing stable isotope dimethyl labeling and high-resolution tandem mass spectrometry, more than 7,000 proteins were identified. Expression profiling highlights energy metabolism, immune response, cell cycle and DNA repair to be modulated along differentiation. To our knowledge, these data represent the first global protein signature of HSC defined at this level. Furthermore, using in-depth RNA-seq we achieved more than 11-fold coverage of the genome enabling for robust identification of over 22,000 genes. We describe specific expression clusters of cell adhesion molecules and TFs and present a comprehensive landscape of polyA-long non-coding RNAs (lncRNA) in HSC or MPP. Finally, to address differentiation potential of MPP2, 3 and 4 we complement our OMICs approach with functional transplant experiments. In summary, the gene and protein signatures for stemness and multipotency defined in this study represent a novel unique resource for the scientific community and will significantly extend the current understanding of HSC biology.
S13
O1010 - ESTABLISHMENT OF LYMPHO-MYELOID RESTRICTED PROGENITORS PRIOR TO THE EMERGENCE OF DEFINITIVE HEMATOPOIETIC STEM CELLS Charlotta B€oiers2,1, Michael Lutteropp3, Sidinh Luc3, Petter Woll3, Adam Mead3, Anne Hultquist2, Joana Carrelha3, Iain Macaulay3, Gemma Swiers4, Luca Melchiori3, Tiago Luis3, Shabnam Kharazi2, Tiphaine Bouriez-Jones3, Qiaolin Deng5, Annica Ponten6, Christina Jensen3, Ewa Sitnicka2, Rickard Sandberg5, Marella de Bruijn4, and Sten Eirik Jacobsen3,2,7 1 Div of Molecular Medicine and Gene Therapy, Lund Strategic Center for Stem Cell Biology, Lund, Sweden; 2Haematopoietic Stem Cell Laboratory, Lund Stem Cell Center, Lund, Sweden; 3Haematopoietic Stem Cell Laboratory, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom; 4MRC Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, Oxford, United Kingdom; 5 Department of Cell and Molecular Biology, Karolinska Institutet and Ludwig Institute for Cancer Research, Stockholm, Sweden; 6Cardiovascular group, Lund Stem Cell Center, Lund, Sweden; 7Wallenberg Institute for Regenerative Medicine, Karolinska Institutet, Stockholm, Sweden In jawed vertebrates, development of an adaptive immune-system is essential for protection of the born organism against otherwise life-threatening pathogens. Myeloid cells of the evolutionary older innate immune-system are formed early in development, while lymphopoiesis has been suggested to initiate much later, following emergence of definitive hematopoietic stem cells (HSCs). Herein, we prospectively identified a lymphomyeloid restricted embryonic progenitor as early as E11.5 that expresses IL7Ra and that sustains combined lymphoid and GM transcriptional lineage priming and lineage potentials at the single cell level but no MkE potential. Using Rag1-GFP knock-in mice, we tracked the establishment of this lympho-myeloid restricted progenitor back to as early as E9.5, preceding both hematopoietic colonization of the FL and the establishment of definitive HSCs. Moreover, through in vivo fate mapping we confirmed the inability of Rag1 expressing early embryonic progenitors to contribute to the MkE lineage, while unequivocally and robustly contributing to the myeloid innate as well as lymphoid adaptive immune systems of the mammalian embryo. These findings identify the developmentally earliest immune-restricted progenitor and establish the lymphomyeloid restriction step as a physiologically important lineage commitment step in embryonic mammalian hematopoiesis, preceding the emergence of definitive HSCs. O1011 - RHO GTPASE ACTIVATING PROTEIN P190-B REGULATES HEMATOPOIETIC STEM CELL SELF-RENEWAL DECISIONS Ashwini Hinge1, Bruce Aronow2, and Marie Filippi1 1 Division of Experimental Hematolofy and cancer biology, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, USA; 2Division of biomedical informatics, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, USA Hematopoietic stem cells (HSC) can self-renew or commit to differentiate into all blood cells. The signaling network that regulates these choices is poorly understood. We previously showed that p190-B loss, a negative regulator of Rho activity, enhanced long-term engraftment without altering HSC proliferation or survival or lineage development (Xu, Blood 2009), suggesting an effect on HSC fate decisions. Here, we compared single HSC divisions ex vivo by analyzing multilineage potential of the paired-daughter cells. Non-transplanted (NT) HSCs generated two multipotent daughter cells (self-renewing divisions). However, after secondary transplantation, wild-type (WT) HSCs generated one multipotent and one committed daughter cells (differentiating divisions). p190-B-/- HSCs maintained self-renewal divisions, indicating p190-B loss mediates higher probability of ‘stemness’ inheritance through divisions to prevent HSC depletion following transplantation. Unexpectedly, p190B loss does this by decreasing autocrine TGF-b. Active TGF-b protein levels, TGF-b target genes increased in WT but not in p190-B-/- HSCs upon transplantation relative to NT HSC. TGF-b signaling inhibitors restored self-renewal divisions of transplanted WT HSC as seen in vitro and in engraftment in vivo. TGF-b inhibitor treatment of transplanted mice significantly increased HSC frequency - without changing their proliferation - that yielded higher chimera in tertiary transplant compared to DMSO treated mice. Conversely, recombinant TGF-b1 changed nontransplanted HSC choice to differentiating divisions in vitro. Finally, p38MAPK activity mediated TGF-b effect on HSC fate decisions. Hence, HSC decisions to self-renew or differentiate are specified by a p190-B/TGF-b signaling pathway during HSC regeneration. This study uncovers a novel role for autocrine TGF-b in HSC fate decisions uncoupled from its role on HSC quiescence that may have important implications for regenerative medicine.