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Understanding Megakaryopoiesis and Thrombopoiesis Using Human Stem Cells Models

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Understanding Megakaryopoiesis and Thrombopoiesis Using Human Stem Cells Models University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2017 Understanding Megakaryopoiesis And Thrombopoiesis Using Human Stem Cells Models Xiu Li Sim University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Developmental Biology Commons Recommended Citation Sim, Xiu Li, "Understanding Megakaryopoiesis And Thrombopoiesis Using Human Stem Cells Models" (2017). Publicly Accessible Penn Dissertations. 2586. https://repository.upenn.edu/edissertations/2586 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/2586 For more information, please contact [email protected]. Understanding Megakaryopoiesis And Thrombopoiesis Using Human Stem Cells Models Abstract Human stem cell models (CD34+ hematopoietic progenitors, embryonic stem cells and induced pluripotent stem cells (iPSCs)) are powerful tools for the study of megakaryopoiesis and thrombopoiesis, particularly in situations where mouse models are unavailable or do not accurately recapitulate human physiology or development. In the first part of this thesis, we identified and characterized novel megakaryocyte (MK) maturation stages in MK cultures derived from human stem cells. An immature, low granular (LG) MK pool (defined yb side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and GPIbα (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently-labeled coagulation factor V (FV) from the medium into alpha-granules and releases functional FV+CD42b+ platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ platelets have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs. In the second part of this thesis, we used patient-specific iPSCs ot model Thrombocytopenia Absent Radius (TAR) syndrome, a rare congenital disorder characterized by low platelet counts and bilateral absence of the radius. We generated several iPSC lines from patients and controls and confirmed that the patient lines had decreased expression of RBM8A, the candidate disease gene for TAR syndrome. We differentiated patient and control iPSCs to hematopoietic progenitor cells (HPCs) and MKs and showed that HPCs derived from TAR iPSCs exhibited decreased MK colony-forming potential but differentiated normally into MKs in liquid culture. When we restored RBM8A expression in TAR iPSCs using a doxycycline inducible system, we saw no effect on megakaryopoiesis. We then knocked out RBM8A in TAR iPSCs and found that a complete deficiency in RBM8A expression was lethal for iPSCs, HPCs, MKs and erythrocytes. These studies suggest that we were unable to determine a specific oler for RBM8A in primitive (embryonic) megakaryopoiesis and perhaps future studies to direct iPSCs towards the definitive (adult) MK lineage may better elucidate RBM8A’s role in TAR syndrome. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Paul Gadue Keywords Factor V, Megakaryocytes, Platelets, Stem Cells, Transfusion Subject Categories Developmental Biology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/2586 UNDERSTANDING MEGAKARYOPOIESIS AND THROMBOPOIESIS USING HUMAN STEM CELLS MODELS Xiu Li Sim A DISSERTATION In Cell and Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2017 Supervisor of Dissertation: --------------------------------------- Paul Gadue, Associate Professor of Pathology and Laboratory Medicine Graduate Group Chair Person: --------------------------------------- Daniel S. Kessler, Professor of Cell and Developmental Biology Dissertation Committee Mortimer Poncz, Professor of Pediatrics Wei Tong, Associate Professor of Pediatrics Nancy A. Speck, Professor of Cell and Developmental Biology Peter S. Klein, Professor of Medicine UNDERSTANDING MEGAKARYOPOIESIS AND THROMBOPOIESIS USING HUMAN STEM CELLS MODELS COPYRIGHT 2017 Xiu Li Sim ACKNOWLEDGMENT This work would not have been possible without the help and support of my official advisor, Paul and my unofficial but equally important advisor, Debbie. I would like to thank them for the scientific discussions, and more importantly, the tremendous amount of moral support they have given me over the years. I am also grateful to my thesis committee chair, Morty, who only noticed my existence in the last 2 years of my PhD, but was instrumental in pushing me to finish this work. I am very fortunate to be surrounded by fantastic colleagues, who became my friends and occupy a special place in my heart. Thank you all for the drama and gossip that spiced up the otherwise monotonous daily grind. Last but not least, I would like to thank my family, in particular, my parents and my mother-in-law, who took care of us in numerous ways, my daughter, Emily, who brought so much joy and laughter to our family and of course, my husband, Lu, whose love and encouragement gave me the courage and confidence to reach the finish line. iii ABSTRACT UNDERSTANDING MEGAKARYOPOIESIS AND THROMBOPOIESIS USING HUMAN STEM CELLS MODELS Xiu Li Sim Paul Gadue Human stem cell models (CD34+ hematopoietic progenitors, embryonic stem cells and induced pluripotent stem cells (iPSCs)) are powerful tools for the study of megakaryopoiesis and thrombopoiesis, particularly in situations where mouse models are unavailable or do not accurately recapitulate human physiology or development. In the first part of this thesis, we identified and characterized novel megakaryocyte (MK) maturation stages in MK cultures derived from human stem cells. An immature, low granular (LG) MK pool (defined by side scatter on flow cytometry) gives rise to a mature high granular (HG) pool, which then becomes damaged by apoptosis and GPIbα (CD42b) shedding. We define an undamaged HG/CD42b+ MK subpopulation, which endocytoses fluorescently-labeled coagulation factor V (FV) from the medium into alpha- granules and releases functional FV+CD42b+ platelet-like particles in vitro and when infused into immunodeficient mice. Importantly, these FV+ platelets have the same size distribution as infused human donor platelets and are preferentially incorporated into clots after laser injury. Using drugs to protect HG MKs from apoptosis and CD42b shedding, we also demonstrate that apoptosis precedes CD42b shedding and that apoptosis inhibition enriches the FV+ HG/CD42b+ MKs, leading to increased platelet yield in vivo, but not in vitro. These studies identify a transition between distinct MK populations in vitro, including one that is primed for platelet release. Technologies to optimize and select these platelet-ready MKs may be important to efficiently generate functional platelets from in vitro-grown MKs. In the second part of this thesis, we used iv patient-specific iPSCs to model Thrombocytopenia Absent Radius (TAR) syndrome, a rare congenital disorder characterized by low platelet counts and bilateral absence of the radius. We generated several iPSC lines from patients and controls and confirmed that the patient lines had decreased expression of RBM8A, the candidate disease gene for TAR syndrome. We differentiated patient and control iPSCs to hematopoietic progenitor cells (HPCs) and MKs and showed that HPCs derived from TAR iPSCs exhibited decreased MK colony-forming potential but differentiated normally into MKs in liquid culture. When we restored RBM8A expression in TAR iPSCs using a doxycycline inducible system, we saw no effect on megakaryopoiesis. We then knocked out RBM8A in TAR iPSCs and found that a complete deficiency in RBM8A expression was lethal for iPSCs, HPCs, MKs and erythrocytes. These studies suggest that we were unable to determine a specific role for RBM8A in primitive (embryonic) megakaryopoiesis and perhaps future studies to direct iPSCs towards the definitive (adult) MK lineage may better elucidate RBM8A’s role in TAR syndrome. v TABLE OF CONTENTS ACKNOWLEDGMENT -----------------------------------------------------------------------------------iii ABSTRACT --------------------------------------------------------------------------------------------------iv TABLE OF CONTENTS ----------------------------------------------------------------------------------vi LIST OF TABLES -------------------------------------------------------------------------------------------x LIST OF ILLUSTRATIONS ------------------------------------------------------------------------------xi LIST OF ABBREVIATIONS ---------------------------------------------------------------------------xiii CHAPTER 1: Introduction and Overview ----------------------------------------------------------1 1.1 Characteristics and Functions of Megakaryocytes (MKs) and Platelets
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