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A rare subpopulation of dormant, drug resistant stem cells in all MIMICS cells of minimal residual disease
S94
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
reciprocal, blunting of functional activity of both factors in vitro and in vivo. To investigate effects of DPP4 on EPHOSS, and vice versa, mBM was harvested (air/ hypoxia) with a DPP4 inhibitor (DPA), or from DPP4 K/O mice. This resulted in significant increases in the number of phenotypic LT-HSC (p5.017) in air, suggesting that DPP4 inhibition blunts EPHOSS mediated loss of phenotypic LT-HSC. Also, the percentage of DPP4+ cells was increased in primitive fractions of mBM or hCB, (LSK w15%, LSKCD150 40%, CD34+CD38- w10%, CD34+CD38CD45RA-CD90+CD49F+ w40% p5.007) and further enhanced 15- 20% when cells are isolated in hypoxia (p5.005). Unexpectedly, LT-HSC ROS levels (mitochondrial/ total) were not diminished in DPA or DPP4 K/O groups harvested in air despite the increase in phenotypic LT-HSC over air harvest alone, suggesting a non ROS/MPTP mechanism. In conclusion, DPP4 expression/activity serves heretofore unknown roles in the regulation/signaling of multiple protein types as well as cellular responses to EPHOSS and hematopoiesis.
3121 - A RARE SUBPOPULATION OF DORMANT, DRUG RESISTANT STEM CELLS IN ALL MIMICS CELLS OF MINIMAL RESIDUAL DISEASE 1 2 € € Erbey Ozdemir , Erbey Ozdemir , Sarah Ebinger2, Sebastian Tiedt2, Catarina Castro Alves2, Christoph Ziegenhain3, Wolfgang Enard3, Aloys Schepers2, and Irmela Jeremias2 1 Helmholtz Zentrum M€unchen, German Research Center for Environmental Health (HMGU); 2Helmholtz Zentrum M€unchen, Oberschleißheim, Germany; 3Ludwig-Maximilians University Munich, Munich, Germany
3120 - DISCOVERY OF A NEW PATH FOR RED BLOOD CELL GENERATION IN THE MOUSE EMBRYO € Irina Pinheiro1, Ozge Vargel2, Isabelle Bergiers2, Christina Nikolakopoulou2, Laura Sabou2, and Christophe Lancrin2 1 EMBL Monterotondo; 2EMBL Monterotondo, Rome, Italy
3122 - REJUVENATION OF AGED VASCULAR NICHES TO ENHANCE HEMATOPOIETIC FUNCTION Michael Poulos, Pradeep Ramalingam, Michael Gutkin, and Jason Butler Weill Cornell Medicine, New York City, USA
The Endothelial to Hematopoietic Transition (EHT) constitutes an essential step in the formation of hematopoietic progenitor and stem cells. However, it is still unclear whether there is a common hemogenic endothelium (HE) to all hematopoietic progenitor/stem cells, or a number of already specified endothelial cells that can give rise to only some, or even one specific blood lineage. We have identified for the first time, an endothelial population with erythroid gene expression (HE-Ery), using single cell transcriptomics and a miR144/451-eGFP mouse model that allows the tracking of the erythroid cell lineage. The HE-Ery population was found to be present in the Yolk Sac, emerging at a time-point that follows the onset of both primitive and Erythro-myeloid progenitor generating erythroid waves. Co-culture with OP9 stromal cells showed that this population could give rise to erythroid cells. Furthermore, we demonstrated that HE-Ery emergence is Runx1 independent. Together our results suggest the existence of a hemogenic endothelium specified towards the erythroid lineage, which could be responsible for a new erythroid wave during embryonic development.
Acute lymphoblastic leukemia (ALL) represents a tumor disease with dismal prognosis upon relapse and novel treatment options are intensively desired especially targeting drug resistant cells. As major challenge, the subpopulation of drug resistant cells is rare and difficult to study. In the individualized mouse model, primary patients’ ALL cells grow orthotopically in NSG mice as patient derived xenografts (PDX). Here, we developed the model further in order to be able to study the challenging rare subpopulation of drug resistant cells. ALL PDX cells were lentivirally transduced to generate what we called ‘‘genetically engineered PDX (GEPDX)’’ models in parallel to genetically engineered mouse models (GEMM). This model provides a novel platform for developing targeted therapies in ALL and was used here to express tags in PDX cells for purification of rare cell populations. In a first approach, we used dormancy as characteristic to identify drug resistant cells. GEPDX models were established where PDX cells express several transgenes allowing to enrich minor subpopulations from murine bone marrow. To identify resting PDX cells, PDX cells were labeled with the proliferation marker CFSE. Thereby, we could identify a rare subpopulation of ALL PDX cells which remained dormant over prolonged periods of time in mice; dormant cells revealed severe resistance against systemic drug treatment in mice. In a second approach, we performed prolonged chemotherapy of GEPDX models closely mimicking polychemotherapy of ALL patients. Remaining drug resistant cells at minimal residual disease (MRD) were isolated from murine bone marrow using their transgenes as anchors. Gene expression profiles of single RNA sequencing revealed that dormant and MRD cells shared major characteristics while both were clearly distinct from their cycling or drug sensitive counterparts. Taken together, complex in vivo modelling allowed direct studies on the enriched rare subpopulation of drug resistant patients ALL cells. Our approach will help characterizing these cells for developing future therapies to prevent ALL relapse.
The molecular and cellular pathways involved in the aging of hematopoietic stem cells (HSCs) are not fully elucidated. To date, most studies describing age-related alterations in the hematopoietic compartment have focused on cell-intrinsic alterations at the level of the HSC. These studies have shown that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential and self-renewing capabilities. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, making the system prone to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately taken into account the effects of the aging microenvironment. There is a large body of evidence demonstrating functional interactions between the HSC and its niche suggesting that local and systemic factors may regulate HSC function; however, the role of bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding this intimate relationship between the BM microenvironment and the HSC and its role in supporting HSC function during aging may prove to be beneficial in reversing or preventing the age-related functional decline observed in the hematopoietic system. Within the hematopoietic microenvironment, we have shown that Akt-activated endothelial cells (ECs) are indispensable to supporting HSC self-renewal and differentiation into lineage-committed progeny during steady-state and regenerative
Poster Presentations/ Experimental Hematology 44 (2016) S56–S110
reciprocal, blunting of functional activity of both factors in vitro and in vivo. To investigate effects of DPP4 on EPHOSS, and vice versa, mBM was harvested (air/ hypoxia) with a DPP4 inhibitor (DPA), or from DPP4 K/O mice. This resulted in significant increases in the number of phenotypic LT-HSC (p5.017) in air, suggesting that DPP4 inhibition blunts EPHOSS mediated loss of phenotypic LT-HSC. Also, the percentage of DPP4+ cells was increased in primitive fractions of mBM or hCB, (LSK w15%, LSKCD150 40%, CD34+CD38- w10%, CD34+CD38CD45RA-CD90+CD49F+ w40% p5.007) and further enhanced 15- 20% when cells are isolated in hypoxia (p5.005). Unexpectedly, LT-HSC ROS levels (mitochondrial/ total) were not diminished in DPA or DPP4 K/O groups harvested in air despite the increase in phenotypic LT-HSC over air harvest alone, suggesting a non ROS/MPTP mechanism. In conclusion, DPP4 expression/activity serves heretofore unknown roles in the regulation/signaling of multiple protein types as well as cellular responses to EPHOSS and hematopoiesis.
3121 - A RARE SUBPOPULATION OF DORMANT, DRUG RESISTANT STEM CELLS IN ALL MIMICS CELLS OF MINIMAL RESIDUAL DISEASE 1 2 € € Erbey Ozdemir , Erbey Ozdemir , Sarah Ebinger2, Sebastian Tiedt2, Catarina Castro Alves2, Christoph Ziegenhain3, Wolfgang Enard3, Aloys Schepers2, and Irmela Jeremias2 1 Helmholtz Zentrum M€unchen, German Research Center for Environmental Health (HMGU); 2Helmholtz Zentrum M€unchen, Oberschleißheim, Germany; 3Ludwig-Maximilians University Munich, Munich, Germany
3120 - DISCOVERY OF A NEW PATH FOR RED BLOOD CELL GENERATION IN THE MOUSE EMBRYO € Irina Pinheiro1, Ozge Vargel2, Isabelle Bergiers2, Christina Nikolakopoulou2, Laura Sabou2, and Christophe Lancrin2 1 EMBL Monterotondo; 2EMBL Monterotondo, Rome, Italy
3122 - REJUVENATION OF AGED VASCULAR NICHES TO ENHANCE HEMATOPOIETIC FUNCTION Michael Poulos, Pradeep Ramalingam, Michael Gutkin, and Jason Butler Weill Cornell Medicine, New York City, USA
The Endothelial to Hematopoietic Transition (EHT) constitutes an essential step in the formation of hematopoietic progenitor and stem cells. However, it is still unclear whether there is a common hemogenic endothelium (HE) to all hematopoietic progenitor/stem cells, or a number of already specified endothelial cells that can give rise to only some, or even one specific blood lineage. We have identified for the first time, an endothelial population with erythroid gene expression (HE-Ery), using single cell transcriptomics and a miR144/451-eGFP mouse model that allows the tracking of the erythroid cell lineage. The HE-Ery population was found to be present in the Yolk Sac, emerging at a time-point that follows the onset of both primitive and Erythro-myeloid progenitor generating erythroid waves. Co-culture with OP9 stromal cells showed that this population could give rise to erythroid cells. Furthermore, we demonstrated that HE-Ery emergence is Runx1 independent. Together our results suggest the existence of a hemogenic endothelium specified towards the erythroid lineage, which could be responsible for a new erythroid wave during embryonic development.
Acute lymphoblastic leukemia (ALL) represents a tumor disease with dismal prognosis upon relapse and novel treatment options are intensively desired especially targeting drug resistant cells. As major challenge, the subpopulation of drug resistant cells is rare and difficult to study. In the individualized mouse model, primary patients’ ALL cells grow orthotopically in NSG mice as patient derived xenografts (PDX). Here, we developed the model further in order to be able to study the challenging rare subpopulation of drug resistant cells. ALL PDX cells were lentivirally transduced to generate what we called ‘‘genetically engineered PDX (GEPDX)’’ models in parallel to genetically engineered mouse models (GEMM). This model provides a novel platform for developing targeted therapies in ALL and was used here to express tags in PDX cells for purification of rare cell populations. In a first approach, we used dormancy as characteristic to identify drug resistant cells. GEPDX models were established where PDX cells express several transgenes allowing to enrich minor subpopulations from murine bone marrow. To identify resting PDX cells, PDX cells were labeled with the proliferation marker CFSE. Thereby, we could identify a rare subpopulation of ALL PDX cells which remained dormant over prolonged periods of time in mice; dormant cells revealed severe resistance against systemic drug treatment in mice. In a second approach, we performed prolonged chemotherapy of GEPDX models closely mimicking polychemotherapy of ALL patients. Remaining drug resistant cells at minimal residual disease (MRD) were isolated from murine bone marrow using their transgenes as anchors. Gene expression profiles of single RNA sequencing revealed that dormant and MRD cells shared major characteristics while both were clearly distinct from their cycling or drug sensitive counterparts. Taken together, complex in vivo modelling allowed direct studies on the enriched rare subpopulation of drug resistant patients ALL cells. Our approach will help characterizing these cells for developing future therapies to prevent ALL relapse.
The molecular and cellular pathways involved in the aging of hematopoietic stem cells (HSCs) are not fully elucidated. To date, most studies describing age-related alterations in the hematopoietic compartment have focused on cell-intrinsic alterations at the level of the HSC. These studies have shown that the absolute number of immunophenotypically defined HSCs increases with age but that aged HSCs exhibit decreased long-term reconstitution potential and self-renewing capabilities. Furthermore, old HSCs exhibit a significant myeloid bias at the expense of lymphopoiesis, making the system prone to the development of myeloid neoplasms. While these studies show that cell-intrinsic changes contribute to the aging of the hematopoietic system, most have not adequately taken into account the effects of the aging microenvironment. There is a large body of evidence demonstrating functional interactions between the HSC and its niche suggesting that local and systemic factors may regulate HSC function; however, the role of bone marrow (BM) microenvironment in regulating HSC aging has not been fully elucidated. Understanding this intimate relationship between the BM microenvironment and the HSC and its role in supporting HSC function during aging may prove to be beneficial in reversing or preventing the age-related functional decline observed in the hematopoietic system. Within the hematopoietic microenvironment, we have shown that Akt-activated endothelial cells (ECs) are indispensable to supporting HSC self-renewal and differentiation into lineage-committed progeny during steady-state and regenerative