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Characteristics of stromal stem cells in the foetal liver
S90
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3156 - TARGETING INTRACELLULAR TRAFFICKING OF FLT3-ITD AND KIT D816V BY CHLORPROMAZINE SHOWS PROMISING PRECLINICAL ACTIVITY AGAINST ACUTE MYELOID LEUKEMIA CELLS Shinya Rai1, Hirokazu Tanaka1, Toshio Watanabe2, Yuzuru Kanakura3, and Itaru Matsumura1 1 Kinki University Faculty of Medicine, Osaka, Japan; 2Nara Women’s University, Nara, Japan; 3Osaka University Graduate School of Medicine, Osaka University Graduate School of Medicine, Japan
3158 - MUSASHI-2 POSTRANSCRIPTIONALLY ATTENUATES ARYL HYDROCARBON RECEPTOR SIGNALING TO EXPAND HUMAN HEMATOPOIETIC STEM CELLS Stefan Rentas1, Nicholas Holzapfel1, Muluken Belew1, Veronique Voisin2, Gabriel Pratt3, Gary Bader2, Gene Yeo3, and Kristin Hope1 1 Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario, Canada; 2The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada; 3 University of California, San Diego, La Jolla, California, USA
FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V (vKIT) are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Recent studies have demonstrated these oncogenic receptor tyrosine kinases (RTKs) are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules. So, we speculated that manipulation of their trafficking between intracellular compartments may be a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of an inhibitor of membrane trafficking, chlorpromazine (CPZ). First, we examined the effect of CPZ on the intracellular trafficking of RTK mutants. Although both FLT3-ITD and v-KIT were mainly located close to endoplasmic reticulum (ER) and Golgi, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi by the treatment of CPZ, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment.Next, we evaluated the effect of CPZ using primary AML cells. As a result, CPZ inhibited the growth and survival of AML cells with mutant RTK. As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) AML cells. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. In mice transplanted with AML cells with FLT3-ITD or v-KIT, administration of CPZ significantly reduced their engraftment. Furthermore, we found that CPZ is cytotoxic to CD34+38- leukemic stem cell fractions isolated from FLT3-ITD(+) AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from cord blood. These results suggest that CPZ would be a promising therapeutic drug to eradicate AML cells with an established safety profile.
A greater understanding of the molecular pathways that underpin the unique human hematopoietic stem and progenitor cell (HSPC) self-renewal program will improve strategies to expand these critical cell types for regenerative therapies. The post-transcriptional mechanisms guiding HSPC fate during ex vivo expansion have not been closely investigated. Using shRNA-mediated knockdown, we show that the RNAbinding protein (RBP) Musashi-2 (MSI2) is required for human HSPC self-renewal and long-term repopulation. Conversely, when overexpressed, MSI2 induces multiple pro-self-renewal phenotypes, including significant ex vivo expansion of short- and long-term repopulating cells through direct attenuation of aryl hydrocarbon receptor (AHR) signaling. Using Crosslinking Immunoprecipitation Sequencing (CLIP-seq) to identify global MSI2-RNA interactions, we determined that MSI2 post-transcriptionally downregulates canonical AHR pathway components in cord blood HSPCs. Our study provides new mechanistic insight into RBP-controlled RNA networks that underlie the self-renewal process and provides evidence that manipulating such networks can offer a novel means to enhance the regenerative potential of human HSPCs expanded ex vivo.
3157 - CHARACTERISTICS OF STROMAL STEM CELLS IN THE FOETAL LIVER Qiao Qiao, Vashe Chandrakanthan, and John Pimanda University of New South Wales, Sydney, New South Wales, Australia
3159 - RNAI SCREEN IDENTIFIES CYTOHESIN1 AS A MEDIATOR OF ADHESION IN HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS Justyna M. Rak1, Katie Foster2, Katarzyna Potrzebowska1, Mehrnaz Safaee Talkhoncheh1, Karolina Komorowska1, Therese T€orngren1, Anders Kvist1, Lena Svensson1, Dominique Bonnet2, and Jonas Larsson1 1 Lund University, Lund, Sweden; 2Cancer Research UK, London, United Kingdom
Murine haematopoiesis progresses step-wise through a number of defined stages that includes the yolk-sac, aorta-gonad-mesonephros (AGM), foetal liver (FL), placenta and finally the bone marrow. The number of haematopoietic stem cells (HSCs) increases dramatically in the FL during mid-gestation. The cellular and signaling processes that govern HSC expansion and maturation are not fully understood. Mesenchymal stem cells (MSC) are known to provide a niche for HSC maintenance in the adult bone marrow. Therefore, we hypothesised that FL MSCs may also play a role in the maturation and expansion of HSCs during embryonic development. Due to the lack of bona fide markers for FL MSCs, we first estimated the number of hepatic MSCs between E11.5 and E18.5 using colony forming unit- fibroblast (CFU-F) assays. FL CFU-Fs demonstrate serial clonogenicity, long term self- renewal and in vitro and in vivo multipotency and their number progressively increases during FL development. HSC expansion in the FL occurs during E11.5-E16.5. To evaluate the contribution of FL CFU-Fs to HSC expansion, we focused our attention on the E13.5 FL. MSCs in the E13.5 FL are derived solely from MesP1+ paraxial mesoderm and are present within the PDGFRa+ cell fraction. PDGFRa+ cells reside near the peritoneal surface of the hepatic lobes, in the peri sinusoidal space of disse and in perivascular regions. Lineage tracing studies coupled with confocal microscopy revealed that PDGFRa+ cells contribute to both mesothelial and endothelial cells during FL development and thus to the structural integrity and vasculature of the developing liver. The contribution of this cell population to HSC expansion is currently under investigation.
To understand mechanisms of homing and retention of hematopoietic stem and progenitor cells (HSPCs) to their niche in the bone marrow (BM), we developed a functional screen for regulators of human HSPC adhesion. We used the firm adherence between cord blood CD34+ cells and primary BM derived mesenchymal stroma cells (MSCs) as a surrogate niche model in vitro to screen for modifiers (shRNAs) that disrupt adhesion. Among the novel candidate genes previously not implicated in HSCP adhesion, we focused on cytohesin1 (CYTH1). Validation assays showed that knockdown (KD) of CYTH1 from four independent shRNAs significantly increased the fraction of non-adherent cells in the stroma adhesion assay. CYTH1 KD similarly reduced adherence of CD34+ cells to the glycoproteins fibronectin (FN) and ICAM1. Interference reflection microscopy showed, in higher detail, a change in the adhesive properties of CD34+ cells with CYTH1 KD: a reduced attachment area and a higher dynamic attachment area compared to control cells when cells were seeded on FN and ICAM1. Preliminary results from flow cytometry and total internal reflection fluorescence microscopy indicate that lower expression of both total and active integrins is involved in the reduced adhesive capacity of CD34+ cells with CYTH1 KD. In vivo, shRNA-mediated KD of CYTH1 significantly decreased both short and longterm engraftment of CD34+ cells in NSG mice, indicating a role for CYTH1 in mediating homing and engraftment of HSPCs. Homing assays using intravital microscopy show increased mobility within the BM space of CD34+CD38- cells with CYTH1 KD, coupled with a tendency to localize further away from the bone surface and endothelium. Moreover, cells with CYTH1 KD showed a less spheric shape compared to control cells, consistent with a more polarized and motile phenotype. To sum up, our findings suggest that CYTH1 is essential to regulate adhesion and motility of HSPCs and thereby their ability to anchor in the niche.
Poster Presentations/Experimental Hematology 43 (2015) S51–S106
3156 - TARGETING INTRACELLULAR TRAFFICKING OF FLT3-ITD AND KIT D816V BY CHLORPROMAZINE SHOWS PROMISING PRECLINICAL ACTIVITY AGAINST ACUTE MYELOID LEUKEMIA CELLS Shinya Rai1, Hirokazu Tanaka1, Toshio Watanabe2, Yuzuru Kanakura3, and Itaru Matsumura1 1 Kinki University Faculty of Medicine, Osaka, Japan; 2Nara Women’s University, Nara, Japan; 3Osaka University Graduate School of Medicine, Osaka University Graduate School of Medicine, Japan
3158 - MUSASHI-2 POSTRANSCRIPTIONALLY ATTENUATES ARYL HYDROCARBON RECEPTOR SIGNALING TO EXPAND HUMAN HEMATOPOIETIC STEM CELLS Stefan Rentas1, Nicholas Holzapfel1, Muluken Belew1, Veronique Voisin2, Gabriel Pratt3, Gary Bader2, Gene Yeo3, and Kristin Hope1 1 Stem Cell and Cancer Research Institute, McMaster University, Hamilton, Ontario, Canada; 2The Donnelly Centre, University of Toronto, Toronto, Ontario, Canada; 3 University of California, San Diego, La Jolla, California, USA
FLT3 internal tandem duplications (ITD) and a KIT point mutation at D816V (vKIT) are frequently found in acute myeloid leukemia (AML) and associated with poor prognosis. Recent studies have demonstrated these oncogenic receptor tyrosine kinases (RTKs) are mislocalized in the cytoplasm, where they transmit aberrant leukemogenic signals to downstream molecules. So, we speculated that manipulation of their trafficking between intracellular compartments may be a new therapeutic approach for AML with mutant RTKs. In this study, we evaluated the in vitro and in vivo anti-leukemic effects of an inhibitor of membrane trafficking, chlorpromazine (CPZ). First, we examined the effect of CPZ on the intracellular trafficking of RTK mutants. Although both FLT3-ITD and v-KIT were mainly located close to endoplasmic reticulum (ER) and Golgi, both RTK mutants were predominantly localized in the compartment apart from ER and Golgi by the treatment of CPZ, indicating that the intracellular trafficking system that retains mutated RTKs at ER or Golgi is disrupted by CPZ treatment.Next, we evaluated the effect of CPZ using primary AML cells. As a result, CPZ inhibited the growth and survival of AML cells with mutant RTK. As for this mechanism, we found that CPZ significantly suppressed the activities of mutant RTKs and their downstream molecules such as Stat5 in all tested FLT3-ITD(+) AML cells. Next, we evaluated the in vivo anti-leukemic effect of CPZ with a xenograft model. In mice transplanted with AML cells with FLT3-ITD or v-KIT, administration of CPZ significantly reduced their engraftment. Furthermore, we found that CPZ is cytotoxic to CD34+38- leukemic stem cell fractions isolated from FLT3-ITD(+) AML patients, while displaying minimal toxicity to normal hematopoietic stem/progenitor cells from cord blood. These results suggest that CPZ would be a promising therapeutic drug to eradicate AML cells with an established safety profile.
A greater understanding of the molecular pathways that underpin the unique human hematopoietic stem and progenitor cell (HSPC) self-renewal program will improve strategies to expand these critical cell types for regenerative therapies. The post-transcriptional mechanisms guiding HSPC fate during ex vivo expansion have not been closely investigated. Using shRNA-mediated knockdown, we show that the RNAbinding protein (RBP) Musashi-2 (MSI2) is required for human HSPC self-renewal and long-term repopulation. Conversely, when overexpressed, MSI2 induces multiple pro-self-renewal phenotypes, including significant ex vivo expansion of short- and long-term repopulating cells through direct attenuation of aryl hydrocarbon receptor (AHR) signaling. Using Crosslinking Immunoprecipitation Sequencing (CLIP-seq) to identify global MSI2-RNA interactions, we determined that MSI2 post-transcriptionally downregulates canonical AHR pathway components in cord blood HSPCs. Our study provides new mechanistic insight into RBP-controlled RNA networks that underlie the self-renewal process and provides evidence that manipulating such networks can offer a novel means to enhance the regenerative potential of human HSPCs expanded ex vivo.
3157 - CHARACTERISTICS OF STROMAL STEM CELLS IN THE FOETAL LIVER Qiao Qiao, Vashe Chandrakanthan, and John Pimanda University of New South Wales, Sydney, New South Wales, Australia
3159 - RNAI SCREEN IDENTIFIES CYTOHESIN1 AS A MEDIATOR OF ADHESION IN HUMAN HEMATOPOIETIC STEM/PROGENITOR CELLS Justyna M. Rak1, Katie Foster2, Katarzyna Potrzebowska1, Mehrnaz Safaee Talkhoncheh1, Karolina Komorowska1, Therese T€orngren1, Anders Kvist1, Lena Svensson1, Dominique Bonnet2, and Jonas Larsson1 1 Lund University, Lund, Sweden; 2Cancer Research UK, London, United Kingdom
Murine haematopoiesis progresses step-wise through a number of defined stages that includes the yolk-sac, aorta-gonad-mesonephros (AGM), foetal liver (FL), placenta and finally the bone marrow. The number of haematopoietic stem cells (HSCs) increases dramatically in the FL during mid-gestation. The cellular and signaling processes that govern HSC expansion and maturation are not fully understood. Mesenchymal stem cells (MSC) are known to provide a niche for HSC maintenance in the adult bone marrow. Therefore, we hypothesised that FL MSCs may also play a role in the maturation and expansion of HSCs during embryonic development. Due to the lack of bona fide markers for FL MSCs, we first estimated the number of hepatic MSCs between E11.5 and E18.5 using colony forming unit- fibroblast (CFU-F) assays. FL CFU-Fs demonstrate serial clonogenicity, long term self- renewal and in vitro and in vivo multipotency and their number progressively increases during FL development. HSC expansion in the FL occurs during E11.5-E16.5. To evaluate the contribution of FL CFU-Fs to HSC expansion, we focused our attention on the E13.5 FL. MSCs in the E13.5 FL are derived solely from MesP1+ paraxial mesoderm and are present within the PDGFRa+ cell fraction. PDGFRa+ cells reside near the peritoneal surface of the hepatic lobes, in the peri sinusoidal space of disse and in perivascular regions. Lineage tracing studies coupled with confocal microscopy revealed that PDGFRa+ cells contribute to both mesothelial and endothelial cells during FL development and thus to the structural integrity and vasculature of the developing liver. The contribution of this cell population to HSC expansion is currently under investigation.
To understand mechanisms of homing and retention of hematopoietic stem and progenitor cells (HSPCs) to their niche in the bone marrow (BM), we developed a functional screen for regulators of human HSPC adhesion. We used the firm adherence between cord blood CD34+ cells and primary BM derived mesenchymal stroma cells (MSCs) as a surrogate niche model in vitro to screen for modifiers (shRNAs) that disrupt adhesion. Among the novel candidate genes previously not implicated in HSCP adhesion, we focused on cytohesin1 (CYTH1). Validation assays showed that knockdown (KD) of CYTH1 from four independent shRNAs significantly increased the fraction of non-adherent cells in the stroma adhesion assay. CYTH1 KD similarly reduced adherence of CD34+ cells to the glycoproteins fibronectin (FN) and ICAM1. Interference reflection microscopy showed, in higher detail, a change in the adhesive properties of CD34+ cells with CYTH1 KD: a reduced attachment area and a higher dynamic attachment area compared to control cells when cells were seeded on FN and ICAM1. Preliminary results from flow cytometry and total internal reflection fluorescence microscopy indicate that lower expression of both total and active integrins is involved in the reduced adhesive capacity of CD34+ cells with CYTH1 KD. In vivo, shRNA-mediated KD of CYTH1 significantly decreased both short and longterm engraftment of CD34+ cells in NSG mice, indicating a role for CYTH1 in mediating homing and engraftment of HSPCs. Homing assays using intravital microscopy show increased mobility within the BM space of CD34+CD38- cells with CYTH1 KD, coupled with a tendency to localize further away from the bone surface and endothelium. Moreover, cells with CYTH1 KD showed a less spheric shape compared to control cells, consistent with a more polarized and motile phenotype. To sum up, our findings suggest that CYTH1 is essential to regulate adhesion and motility of HSPCs and thereby their ability to anchor in the niche.