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NEUROGENIC HETEROTOPIC OSSIFICATIONS ARE DERIVED FROM FIBROADIPOGENIC PROGENITORS IN THE SKELETAL MUSCLE NOT FROM SATELLITE CELLS
Poster Presentations / Experimental Hematology 2019;76 (Suppl):S52−S96
3136 - CHRONIC LYMPHOCYTIC LEUKAEMIA RELIES ON LIPID SCAVENGING AND SYNTHESIS AS AN ENERGY SOURCE Lauren Thurgood1, Lara Escane1, Christie Bader2, Karen Lower1, Doug Brooks2, Bryone Kuss1 1 Flinders University, Bedford Park, Australia; 2University of South Australia, Adelaide, Australia Dysregulation of cancer cell bioenergetics is one of the hallmarks of cancer. The Warburg effect is one such documented change. However, glucose metabolism is not universally increased in cancer cells. Uptake of 18F-FDG in chronic lymphocytic leukemia (CLL) fails as a marker of proliferation and whilst the underlying reason is poorly understood it suggests that CLL cells utilize energy sources other than glucose to proliferate. Using genetic, proteomic and lipidomic analyses, complemented with microscopy and nutrient uptake assays the preferred metabolic pathways of CLL cells have been identified. We measured the uptake of fluorescently labelled short, medium and long-chain fatty acids (LCFA) and the glucose analog 2-NBDG by flow cytometry and confocal microscopy. Three CLL lines (MEC1, MEC2, OSU-CLL) prefer LCFA, over short and medium chain and show a low uptake of 2-NBDG. We have also confirmed these findings in primary CLL samples. Using qPCR and western blot analysis we have identified varying levels of LCFA uptake receptors. We found an up-regulation of proteins involved in lipogenesis in quiescent peripheral CLL cells, and an increase in b-oxidation proteins in the proliferative compartment of the lymph node. Together with our morphological examination using electron and confocal microscopy we suggest that peripheral CLL cells scavenge lipids, which are stored in lipid droplets and protected from degradation by a high expression of PLIN proteins. These cells circulate back to the proliferation centres, the lipid droplets are degraded, likely by lipophagy which frees fatty acids for b-oxidation. Our results begin to unravel CLL bioenergetics and dysregulation of cellular metabolism that occurs in this disease. We are now investigating whether the manipulation of these pathways, particularly lipophagy, may represent a novel therapeutic approach in CLL.
3137 - IN SITU CHARACTERIZATION OF THE
HEMATOPOIETIC STEM AND PROGENITOR CELL MICROENVIRONMENT WITH FLUORESCENCE MULTIPLEXING AND SPECTRAL UNMIXING Gavin Tjin, Louise Purton St Vincent’s Institute of Medical Research, Melbourne, Australia
The hematopoietic stem cell (HSC) microenvironment (niche) is an integral part of hematopoietic regulation. Efforts to characterize these niche cells have been challenging due to technological limitations. Determining niche and HSC interactions in situ requires labelling with immunofluorescence (IF), but the degree of complexity increases exponentially with every additional marker (HSCs require at least five). To minimize the markers required, previous studies used reporter mice to label cells/lineages but reporter mice do not identify specific cells and are also not available in all labs. We overcome these limitations using IF multiplexing in situ to identify HSCs and niches on paraffin embedded mouse bone marrow. Following primary antibody labelling, a tyramide-conjugated fluorophore binds covalently to the tissue localized at the marker of interest. Heat treatment then strips the antibody complex, leaving only the covalently bound fluorophore. Stripping of the antibody-complex clears the tissue for a new pair of marker and fluorophore; the process is repeated until all markers are labelled. Imaging is done on the Perkin Elmer Vectra, and spectral unmixing is used to identify the unique spectral fingerprint of fluorophores on the tissue. These techniques allow labelling of at least six markers plus DAPI (with recent developments increasing this limit to nine). Therefore, we can visualize and quantify interactions between stem and progenitor cells within their niches in 2D, and potentially 3D. The versatility of multiplexing allows it to be adapted to identify other cells/markers of interest, including reporter mice and frozen samples, making it a valuable tool for researchers studying a wide range of stem cells in different tissues, including human biopsies.
S89
3138 - DACH1 DOWNREGULATION DELINEATE A
LYMPHOID PRIMED PROGENITOR (LPP) POPULATION WITHIN THE MPP4 CLUSTER Sara Tomei, Daniela Zalcenstein, Luyi Tian, Jaring Schreuder, Dawn Lin, Mark McKenzie, Andrew Jarratt, Adrienne Hilton, Jacob Jackson, Ben Shields, Ladina Di Rago, Matt McCormack, Doug Hilton, Matt Ritchie, Ashley Ng, Shalin Naik The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
Classical models of haematopoiesis dictate that a single hematopoietic stem cell (HSC) with self-renewal capacity can give rise to all the hematopoietic lineage through subsequent division and loss of potential. This loss of potential is captured through surface markers that classify different stem and progenitor stages including long-term (LT-) and short-term haematopoietic stem cells (ST-HSC) that give rise to 4 categories of multipotent progenitor (MPP) subpopulations 1-4. The MPP4 cluster Lin−c-kit+Sca1+Flt3+ is also known as the lymphoid multipotent primed progenitor (LMPPs) and lack of megakaryocyte and erythroid potential. We demonstrate through cellular barcoding and scRNA-seq that the MPP4 cluster is heterogenous for fate. In particular, Dach1 expression is heterogenous and co-expressed with myeloid and stemlike genes but inversely correlates with lymphoid genes. Through the generation of a Dach1-GFP reporter mouse we demonstrate that Dach1− MPP4s represents a novel progenitor subpopulation that definitively lacks myeloid potential but retains lymphoid potential, thus termed lymphoid-primed progenitors or LPPs. This work describes the earliest definitive branch point of lymphoid development in within haematopoiesis and shows the power of scRNA-seq in identifying progenitor subpopulations.
3139 - NEUROGENIC HETEROTOPIC OSSIFICATIONS ARE DERIVED FROM FIBROADIPOGENIC PROGENITORS IN THE SKELETAL MUSCLE NOT FROM SATELLITE CELLS Hsu-Wen Tseng1, Susan Millard1, Kylie Alexander1, Whitney Fleming1, Irina Kulina1, Bianca Nowlan1, Beulah Jose1, Marjorie Salga2, Allison Pettit1, Fran¸c ois Genet2, Jean-Pierre Levesque1 1 Mater Research Institute-The University of Queensland, Brisbane, Queensland, Australia, Brisbane, Australia; 2Service de Medecine Physique et de Readaptation, Raymond Poincare Hospital, Garches, France, END:ICAP U1179 INSERM, UFR des Sciences de la SanteSimone Veil, Universite Versailles Saint Quentin en Yvelines, Montigny le Bretonneux, France, Garches, France Neurological heterotopic ossifications (NHO) are frequent complications of traumatic brain and spinal cord injuries (SCI). NHO are abnormal ossifications within periarticular muscles resulting in joint ankylosis, vessel and nerve compression. To elucidate NHO pathogenesis, we have developed the first murine model of SCI-NHO, in which NHO is induced in cardiotoxin-injured muscle only following a SCI. However, what stem cell populations within muscle differentiate into osteoblasts and directly contribute to NHO is unclear. We hypothesized that injured muscles do not repair normally after SCI, and instead osteogenic differentiation of skeletal muscle-resident stem cells takes place. We focused on the two progenitor populations known to reside in muscles: satellite cells (SCs) and fibro- adipogenic progenitors (FAPs). SCs, which specifically express the transcription factor Pax7, differentiate into myoblasts, whereas FAPs, which express are of mesenchymal origin and express Prrx1+ instead, can differentiate into fibroblasts and adipocytes in vitro. We had shown that both populations can deposit calcium under osteogenic condition in vitro. We have now generated Pax7CreER x R26-ZsGreen (Pax7ZsG) and Prrx1Cre x R26-ZsGreen (Prrx1ZsG) to specifically trace the fate of SCs and FAPs respectively in our NHO model in vivo. We demonstrate that after SCI, Pax7+ SCs fail to proliferate and regenerate myofibers with instead persistent proliferation of Prrx1+ FAPs differentiating into osteocalcin+ osteoblasts in SCI-NHO at 21 days post-surgery. The above suggest that SCI causes muscle repair failure with instead proliferation and osteogenic differentiation of FAPs residing in the muscle. In addition, SCI significantly upregulated platelet-derived growth factor receptor (PDGFR) expression in FAPs indicating dysregulation of PDGFR signalling may be involved in the NHO development.
3136 - CHRONIC LYMPHOCYTIC LEUKAEMIA RELIES ON LIPID SCAVENGING AND SYNTHESIS AS AN ENERGY SOURCE Lauren Thurgood1, Lara Escane1, Christie Bader2, Karen Lower1, Doug Brooks2, Bryone Kuss1 1 Flinders University, Bedford Park, Australia; 2University of South Australia, Adelaide, Australia Dysregulation of cancer cell bioenergetics is one of the hallmarks of cancer. The Warburg effect is one such documented change. However, glucose metabolism is not universally increased in cancer cells. Uptake of 18F-FDG in chronic lymphocytic leukemia (CLL) fails as a marker of proliferation and whilst the underlying reason is poorly understood it suggests that CLL cells utilize energy sources other than glucose to proliferate. Using genetic, proteomic and lipidomic analyses, complemented with microscopy and nutrient uptake assays the preferred metabolic pathways of CLL cells have been identified. We measured the uptake of fluorescently labelled short, medium and long-chain fatty acids (LCFA) and the glucose analog 2-NBDG by flow cytometry and confocal microscopy. Three CLL lines (MEC1, MEC2, OSU-CLL) prefer LCFA, over short and medium chain and show a low uptake of 2-NBDG. We have also confirmed these findings in primary CLL samples. Using qPCR and western blot analysis we have identified varying levels of LCFA uptake receptors. We found an up-regulation of proteins involved in lipogenesis in quiescent peripheral CLL cells, and an increase in b-oxidation proteins in the proliferative compartment of the lymph node. Together with our morphological examination using electron and confocal microscopy we suggest that peripheral CLL cells scavenge lipids, which are stored in lipid droplets and protected from degradation by a high expression of PLIN proteins. These cells circulate back to the proliferation centres, the lipid droplets are degraded, likely by lipophagy which frees fatty acids for b-oxidation. Our results begin to unravel CLL bioenergetics and dysregulation of cellular metabolism that occurs in this disease. We are now investigating whether the manipulation of these pathways, particularly lipophagy, may represent a novel therapeutic approach in CLL.
3137 - IN SITU CHARACTERIZATION OF THE
HEMATOPOIETIC STEM AND PROGENITOR CELL MICROENVIRONMENT WITH FLUORESCENCE MULTIPLEXING AND SPECTRAL UNMIXING Gavin Tjin, Louise Purton St Vincent’s Institute of Medical Research, Melbourne, Australia
The hematopoietic stem cell (HSC) microenvironment (niche) is an integral part of hematopoietic regulation. Efforts to characterize these niche cells have been challenging due to technological limitations. Determining niche and HSC interactions in situ requires labelling with immunofluorescence (IF), but the degree of complexity increases exponentially with every additional marker (HSCs require at least five). To minimize the markers required, previous studies used reporter mice to label cells/lineages but reporter mice do not identify specific cells and are also not available in all labs. We overcome these limitations using IF multiplexing in situ to identify HSCs and niches on paraffin embedded mouse bone marrow. Following primary antibody labelling, a tyramide-conjugated fluorophore binds covalently to the tissue localized at the marker of interest. Heat treatment then strips the antibody complex, leaving only the covalently bound fluorophore. Stripping of the antibody-complex clears the tissue for a new pair of marker and fluorophore; the process is repeated until all markers are labelled. Imaging is done on the Perkin Elmer Vectra, and spectral unmixing is used to identify the unique spectral fingerprint of fluorophores on the tissue. These techniques allow labelling of at least six markers plus DAPI (with recent developments increasing this limit to nine). Therefore, we can visualize and quantify interactions between stem and progenitor cells within their niches in 2D, and potentially 3D. The versatility of multiplexing allows it to be adapted to identify other cells/markers of interest, including reporter mice and frozen samples, making it a valuable tool for researchers studying a wide range of stem cells in different tissues, including human biopsies.
S89
3138 - DACH1 DOWNREGULATION DELINEATE A
LYMPHOID PRIMED PROGENITOR (LPP) POPULATION WITHIN THE MPP4 CLUSTER Sara Tomei, Daniela Zalcenstein, Luyi Tian, Jaring Schreuder, Dawn Lin, Mark McKenzie, Andrew Jarratt, Adrienne Hilton, Jacob Jackson, Ben Shields, Ladina Di Rago, Matt McCormack, Doug Hilton, Matt Ritchie, Ashley Ng, Shalin Naik The Walter and Eliza Hall Institute of Medical Research, Parkville, Australia
Classical models of haematopoiesis dictate that a single hematopoietic stem cell (HSC) with self-renewal capacity can give rise to all the hematopoietic lineage through subsequent division and loss of potential. This loss of potential is captured through surface markers that classify different stem and progenitor stages including long-term (LT-) and short-term haematopoietic stem cells (ST-HSC) that give rise to 4 categories of multipotent progenitor (MPP) subpopulations 1-4. The MPP4 cluster Lin−c-kit+Sca1+Flt3+ is also known as the lymphoid multipotent primed progenitor (LMPPs) and lack of megakaryocyte and erythroid potential. We demonstrate through cellular barcoding and scRNA-seq that the MPP4 cluster is heterogenous for fate. In particular, Dach1 expression is heterogenous and co-expressed with myeloid and stemlike genes but inversely correlates with lymphoid genes. Through the generation of a Dach1-GFP reporter mouse we demonstrate that Dach1− MPP4s represents a novel progenitor subpopulation that definitively lacks myeloid potential but retains lymphoid potential, thus termed lymphoid-primed progenitors or LPPs. This work describes the earliest definitive branch point of lymphoid development in within haematopoiesis and shows the power of scRNA-seq in identifying progenitor subpopulations.
3139 - NEUROGENIC HETEROTOPIC OSSIFICATIONS ARE DERIVED FROM FIBROADIPOGENIC PROGENITORS IN THE SKELETAL MUSCLE NOT FROM SATELLITE CELLS Hsu-Wen Tseng1, Susan Millard1, Kylie Alexander1, Whitney Fleming1, Irina Kulina1, Bianca Nowlan1, Beulah Jose1, Marjorie Salga2, Allison Pettit1, Fran¸c ois Genet2, Jean-Pierre Levesque1 1 Mater Research Institute-The University of Queensland, Brisbane, Queensland, Australia, Brisbane, Australia; 2Service de Medecine Physique et de Readaptation, Raymond Poincare Hospital, Garches, France, END:ICAP U1179 INSERM, UFR des Sciences de la SanteSimone Veil, Universite Versailles Saint Quentin en Yvelines, Montigny le Bretonneux, France, Garches, France Neurological heterotopic ossifications (NHO) are frequent complications of traumatic brain and spinal cord injuries (SCI). NHO are abnormal ossifications within periarticular muscles resulting in joint ankylosis, vessel and nerve compression. To elucidate NHO pathogenesis, we have developed the first murine model of SCI-NHO, in which NHO is induced in cardiotoxin-injured muscle only following a SCI. However, what stem cell populations within muscle differentiate into osteoblasts and directly contribute to NHO is unclear. We hypothesized that injured muscles do not repair normally after SCI, and instead osteogenic differentiation of skeletal muscle-resident stem cells takes place. We focused on the two progenitor populations known to reside in muscles: satellite cells (SCs) and fibro- adipogenic progenitors (FAPs). SCs, which specifically express the transcription factor Pax7, differentiate into myoblasts, whereas FAPs, which express are of mesenchymal origin and express Prrx1+ instead, can differentiate into fibroblasts and adipocytes in vitro. We had shown that both populations can deposit calcium under osteogenic condition in vitro. We have now generated Pax7CreER x R26-ZsGreen (Pax7ZsG) and Prrx1Cre x R26-ZsGreen (Prrx1ZsG) to specifically trace the fate of SCs and FAPs respectively in our NHO model in vivo. We demonstrate that after SCI, Pax7+ SCs fail to proliferate and regenerate myofibers with instead persistent proliferation of Prrx1+ FAPs differentiating into osteocalcin+ osteoblasts in SCI-NHO at 21 days post-surgery. The above suggest that SCI causes muscle repair failure with instead proliferation and osteogenic differentiation of FAPs residing in the muscle. In addition, SCI significantly upregulated platelet-derived growth factor receptor (PDGFR) expression in FAPs indicating dysregulation of PDGFR signalling may be involved in the NHO development.