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Controlled generation of hematopoietic progenitor cells from pluripotent stem cells using microenvironmental cues
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Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1021 - DECLINED PRESENTATION NOVEL FUNCTIONAL ROLES FOR TEN-ELEVEN-TRANSLOCATION 2 (TET2) IN NORMAL AND LEUKEMIC GROWTH OF MAST CELLS Raghuveer Mali1, Valeria Visconte2, Tiu Ramon2, Helmut Hanenberg1, Mingjiang Xu1, and Reuben Kapur1 1 Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA; 2Translational Hematology and Oncology Research, Cleveland Clinic, Cleveland, Ohio, USA Mast cells play a vital role in innate and adaptive immunity. KIT receptor signaling is essential for mast cell growth and survival. Gain-of-function mutations in KIT receptor have been identified in humans in 90% of systemic mastocytosis (SM) patients. KITD816V mutation found in SM is imatinib-resistant and current antiKIT therapies for KITD816V+ SM are ineffective. Our recent clinical studies have identified the presence of Tet2 mutations in 23% of mastocytosis patients and are associated with poor overall prognosis. However, the physiologic role(s) of Tet2 in normal mast cell development and cooperation between Tet2 and KITD816V mutations in SM are not well studied. In this study, we have determined the functional role of Tet2 in normal mast cell development and molecular mechanisms of cooperation between Tet2 and KITD816V mutations in SM. We found significantly increased number of immature mast cells in the peritoneal cavity of Tet2-/- mice compared to WT mice in vivo. Bone marrow-derived mast cells (BMMCs) from Tet2-/- mice show significant reduction in 5-hydroxymethyl cytosine levels compared to WT BMMCs. Tet2-/- BMMCs show significant reduction in mast cell differentiation, reduced expression of mast cell-specific genes MCP-5, MCP-6 and CPA, and altered expression of key transcription factors Mitf, Gata-2 and PU.1 compared to WT BMMCs. In addition, Tet2-/- BMMCs show enhanced cytokine-induced growth and survival. Furthermore, Tet2-/- murine or knocked down human cells bearing KIT mutation show significantly enhanced growth compared to cells bearing KIT mutation alone. Hyperproliferation of Tet2-/- BMMCs in the presence and absence of KIT mutation is associated with reduced expression of Pten and enhanced activation of the PI3Kinase/AKT pathway. Treatment with demethylating agent 5azacytidine or PI3Kinase inhibitor GDC-0941 (p110a/d-specific), but not TGX221 (p110b-specific) or IC87114 (p110d-specific), significantly reduced the hyperproliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from mastocytosis patients bearing KITD816V mutation. Thus, combinatorial therapy involving both demethylating agent and PI3Kinase inhibitor is likely to be ideal for treating SM patients with Tet2 and KIT mutations.
P1022 - OPTIMIZATION OF X-LINKED CHRONIC GRANULOMATOUS DISEASE MODELIZATION BY USING PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLS Julie Brault1,2, Erwan Goutagny1,2, Tomo Saric3, Karl-Heinz Krause7, Marie-Jose Stasia1,2, K. Shao3, Mk Gupta3, D. Grunwald4, Jp Brion5, and D. Plantaz6 1 Centre Diagnostic et Recherche sur la granulomatose septique (CDiReC), Institut de Biologie et Pathologie, Grenoble, France; 2TheREx, TIMC/Imag, UMR CNRS 5525, Universite Joseph Fourier, Grenoble, France; 3Institute for Neurophysiology, Cologne, Germany; 4iRTSV/BCI, CEA, Grenoble, France; 5CEA, Grenoble, France; 6 P^ole de Medecine Aigue et Communautaire, P^ole Couple Enfant, Grenoble, France; 7 Dept. of Pathology and Immunology, P^ole Couple Enfant, Grenoble, Switzerland Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells with embryonic stem cell (ESC)-like characteristics generated by the introduction of combinations of specific transcription factors. Patient-specific iPSCs can be used to recapitulate disease-specific phenotypes for the screening of new therapies. Chronic granulomatous disease (CGD), a rare inherited immunodeficiency, is characterized by recurrent and severe infections in childhood. The most frequent form is the X-linked CGD (X-CGD) due to mutations in CYBB leading to the absence of Nox2 of the phagocytic NADPH oxidase complex, responsible for the production of microbicidal reactive oxygen species. Our objective was to optimize the generation of phagocytes reproducing the X-CGD physiopathology in order to test a new therapeutic approach based on protein therapy. X-CGD and control iPSC lines were reprogrammed from human fibroblasts with OSKM retroviral vectors. iPSC lines expressing pluripotency markers were able to form embryoid bodies (EBs) with the three embryonic germ layers. Hematopoietic differentiation induced after coculture of iPSCs or EBs with OP9 mouse bone marrow stromal cell line, was characterized by a fast decrease of pluripotency markers with a progressive appearance of CD34+ progenitors. Terminal differentiation into mature neutrophils or macrophages (5.10^5 cells) was obtained in 10-14 days by using specific hematopoietic cytokines. No significant difference between both coculture types (iPSC or EB) was noticed. Freezing and thawing of the CD34+ progenitors did not modify the progenitor ability. X-CGD neutrophils and macrophages derived from X-CGD iPSCs showed no Nox2 expression and NADPH oxidase activity unlike control cells. In conclusion, we succeeded to generate a cellular model of X-CGD ready to test new therapeutic approaches. P1023 - CONTROLLED GENERATION OF HEMATOPOIETIC PROGENITOR CELLS FROM PLURIPOTENT STEM CELLS USING MICROENVIRONMENTAL CUES Muhammad Rahman1, Patrick Brauer2, Juan Zu~niga-Pfl€ucker2, and Peter Zandstra1 1 Institute of Biomaterials & Biomedical Engineering, University of Toronto, Downtown Toronto, Ontario, Canada; 2Sunnybrook Research Institute, Toronto, Ontario, Canada Human pluripotent stem cells (hPSCs) have great potential for regenerative medicine due to their ability to develop into any of the somatic cells including blood cells. However, in vitro protocols for generating hPSC-derived hematopoietic progenitor cells (HPCs) utilize xenogenic products, display low yields and typically generate primitive blood phenotypes. Current evidence suggests a hemogenic endothelial (HE) population giving rise to definitive blood cells. Obtaining this population from hPSCs has been difficult since definitive hematopoiesis during embryogenesis is a dynamic and tightly controlled process. We hypothesize that hPSC-derived endothelial cells can be obtained using defined and controlled conditions and can be micropatterned into artificial niches to mimic blood forming HE. The AggreWellTM technology was used to generate variable aggregate sizes to culture PSCs towards HPCs in serum-free defined media. Expression of CD45 peaked by day 20 (35%) and colony forming cells from PSCderived cells were highest on day 13 at 200 colonies/105 seeded cells. HE expression was assessed by tracking CD34+VECAD+CD43- which was highest on day 8. Thereafter, day 8 cells were sorted into CD34+VECAD+ and were positive for von willebrand factor and uptake of low density lipoprotein confirming its endothelial nature. To assess if these cells can give rise to definitive-like blood cells, day 8 CD34+CD43- cells (29% expression pre-sort) were magnetically sorted and placed on non-irradiated OP9-DL1 cells for co-culture. By day 21 of co-culture, cells were 11% positive for CD5+CD7+ indicative of pro-T lymphoid cells thereby implicating their definitive potential. We have previously shown that precise control of colony size allows for the manipulation of hESC fate. Building on this, day 8 HE cells are currently being patterned as 200 mm diameter spots with a 500 mm pitch (distance b/w spots). These cells will be supplemented with factors involved in embryonic definitive hematopoiesis to enhance blood induction compared to our co-culture experiments. All in all, these studies demonstrate the use of tools that allow us to recapitulate aspects of the embryonic microenvironment to guide PSCs towards hematopoietic cells in an efficient and scalable fashion.
Poster Presentations/ Experimental Hematology 41 (2013) S23–S75
P1021 - DECLINED PRESENTATION NOVEL FUNCTIONAL ROLES FOR TEN-ELEVEN-TRANSLOCATION 2 (TET2) IN NORMAL AND LEUKEMIC GROWTH OF MAST CELLS Raghuveer Mali1, Valeria Visconte2, Tiu Ramon2, Helmut Hanenberg1, Mingjiang Xu1, and Reuben Kapur1 1 Department of Pediatrics, Indiana University School of Medicine, Indianapolis, Indiana, USA; 2Translational Hematology and Oncology Research, Cleveland Clinic, Cleveland, Ohio, USA Mast cells play a vital role in innate and adaptive immunity. KIT receptor signaling is essential for mast cell growth and survival. Gain-of-function mutations in KIT receptor have been identified in humans in 90% of systemic mastocytosis (SM) patients. KITD816V mutation found in SM is imatinib-resistant and current antiKIT therapies for KITD816V+ SM are ineffective. Our recent clinical studies have identified the presence of Tet2 mutations in 23% of mastocytosis patients and are associated with poor overall prognosis. However, the physiologic role(s) of Tet2 in normal mast cell development and cooperation between Tet2 and KITD816V mutations in SM are not well studied. In this study, we have determined the functional role of Tet2 in normal mast cell development and molecular mechanisms of cooperation between Tet2 and KITD816V mutations in SM. We found significantly increased number of immature mast cells in the peritoneal cavity of Tet2-/- mice compared to WT mice in vivo. Bone marrow-derived mast cells (BMMCs) from Tet2-/- mice show significant reduction in 5-hydroxymethyl cytosine levels compared to WT BMMCs. Tet2-/- BMMCs show significant reduction in mast cell differentiation, reduced expression of mast cell-specific genes MCP-5, MCP-6 and CPA, and altered expression of key transcription factors Mitf, Gata-2 and PU.1 compared to WT BMMCs. In addition, Tet2-/- BMMCs show enhanced cytokine-induced growth and survival. Furthermore, Tet2-/- murine or knocked down human cells bearing KIT mutation show significantly enhanced growth compared to cells bearing KIT mutation alone. Hyperproliferation of Tet2-/- BMMCs in the presence and absence of KIT mutation is associated with reduced expression of Pten and enhanced activation of the PI3Kinase/AKT pathway. Treatment with demethylating agent 5azacytidine or PI3Kinase inhibitor GDC-0941 (p110a/d-specific), but not TGX221 (p110b-specific) or IC87114 (p110d-specific), significantly reduced the hyperproliferation of Tet2-/- BMMCs and cell lines as well as primary BM blasts derived from mastocytosis patients bearing KITD816V mutation. Thus, combinatorial therapy involving both demethylating agent and PI3Kinase inhibitor is likely to be ideal for treating SM patients with Tet2 and KIT mutations.
P1022 - OPTIMIZATION OF X-LINKED CHRONIC GRANULOMATOUS DISEASE MODELIZATION BY USING PATIENT-SPECIFIC INDUCED PLURIPOTENT STEM CELLS Julie Brault1,2, Erwan Goutagny1,2, Tomo Saric3, Karl-Heinz Krause7, Marie-Jose Stasia1,2, K. Shao3, Mk Gupta3, D. Grunwald4, Jp Brion5, and D. Plantaz6 1 Centre Diagnostic et Recherche sur la granulomatose septique (CDiReC), Institut de Biologie et Pathologie, Grenoble, France; 2TheREx, TIMC/Imag, UMR CNRS 5525, Universite Joseph Fourier, Grenoble, France; 3Institute for Neurophysiology, Cologne, Germany; 4iRTSV/BCI, CEA, Grenoble, France; 5CEA, Grenoble, France; 6 P^ole de Medecine Aigue et Communautaire, P^ole Couple Enfant, Grenoble, France; 7 Dept. of Pathology and Immunology, P^ole Couple Enfant, Grenoble, Switzerland Induced pluripotent stem cells (iPSCs) are reprogrammed somatic cells with embryonic stem cell (ESC)-like characteristics generated by the introduction of combinations of specific transcription factors. Patient-specific iPSCs can be used to recapitulate disease-specific phenotypes for the screening of new therapies. Chronic granulomatous disease (CGD), a rare inherited immunodeficiency, is characterized by recurrent and severe infections in childhood. The most frequent form is the X-linked CGD (X-CGD) due to mutations in CYBB leading to the absence of Nox2 of the phagocytic NADPH oxidase complex, responsible for the production of microbicidal reactive oxygen species. Our objective was to optimize the generation of phagocytes reproducing the X-CGD physiopathology in order to test a new therapeutic approach based on protein therapy. X-CGD and control iPSC lines were reprogrammed from human fibroblasts with OSKM retroviral vectors. iPSC lines expressing pluripotency markers were able to form embryoid bodies (EBs) with the three embryonic germ layers. Hematopoietic differentiation induced after coculture of iPSCs or EBs with OP9 mouse bone marrow stromal cell line, was characterized by a fast decrease of pluripotency markers with a progressive appearance of CD34+ progenitors. Terminal differentiation into mature neutrophils or macrophages (5.10^5 cells) was obtained in 10-14 days by using specific hematopoietic cytokines. No significant difference between both coculture types (iPSC or EB) was noticed. Freezing and thawing of the CD34+ progenitors did not modify the progenitor ability. X-CGD neutrophils and macrophages derived from X-CGD iPSCs showed no Nox2 expression and NADPH oxidase activity unlike control cells. In conclusion, we succeeded to generate a cellular model of X-CGD ready to test new therapeutic approaches. P1023 - CONTROLLED GENERATION OF HEMATOPOIETIC PROGENITOR CELLS FROM PLURIPOTENT STEM CELLS USING MICROENVIRONMENTAL CUES Muhammad Rahman1, Patrick Brauer2, Juan Zu~niga-Pfl€ucker2, and Peter Zandstra1 1 Institute of Biomaterials & Biomedical Engineering, University of Toronto, Downtown Toronto, Ontario, Canada; 2Sunnybrook Research Institute, Toronto, Ontario, Canada Human pluripotent stem cells (hPSCs) have great potential for regenerative medicine due to their ability to develop into any of the somatic cells including blood cells. However, in vitro protocols for generating hPSC-derived hematopoietic progenitor cells (HPCs) utilize xenogenic products, display low yields and typically generate primitive blood phenotypes. Current evidence suggests a hemogenic endothelial (HE) population giving rise to definitive blood cells. Obtaining this population from hPSCs has been difficult since definitive hematopoiesis during embryogenesis is a dynamic and tightly controlled process. We hypothesize that hPSC-derived endothelial cells can be obtained using defined and controlled conditions and can be micropatterned into artificial niches to mimic blood forming HE. The AggreWellTM technology was used to generate variable aggregate sizes to culture PSCs towards HPCs in serum-free defined media. Expression of CD45 peaked by day 20 (35%) and colony forming cells from PSCderived cells were highest on day 13 at 200 colonies/105 seeded cells. HE expression was assessed by tracking CD34+VECAD+CD43- which was highest on day 8. Thereafter, day 8 cells were sorted into CD34+VECAD+ and were positive for von willebrand factor and uptake of low density lipoprotein confirming its endothelial nature. To assess if these cells can give rise to definitive-like blood cells, day 8 CD34+CD43- cells (29% expression pre-sort) were magnetically sorted and placed on non-irradiated OP9-DL1 cells for co-culture. By day 21 of co-culture, cells were 11% positive for CD5+CD7+ indicative of pro-T lymphoid cells thereby implicating their definitive potential. We have previously shown that precise control of colony size allows for the manipulation of hESC fate. Building on this, day 8 HE cells are currently being patterned as 200 mm diameter spots with a 500 mm pitch (distance b/w spots). These cells will be supplemented with factors involved in embryonic definitive hematopoiesis to enhance blood induction compared to our co-culture experiments. All in all, these studies demonstrate the use of tools that allow us to recapitulate aspects of the embryonic microenvironment to guide PSCs towards hematopoietic cells in an efficient and scalable fashion.