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Regulation of Hematopoietic Progenitor Formation in a Shwachman Diamond Syndrome Induced Pluripotent Stem Cell Disease Model

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Regulation of Hematopoietic Progenitor Formation in a Shwachman Diamond Syndrome Induced Pluripotent Stem Cell Disease Model Regulation of Hematopoietic Progenitor Formation in a Shwachman Diamond Syndrome Induced Pluripotent Stem Cell Disease Model by Alice Maria Luca A thesis submitted in conformity with the requirements for the degree of Masters of Science Institute of Medical Science University of Toronto © Copyright by Alice Maria Luca 2015 Regulation of Hematopoietic Progenitor Formation in a Shwachman-Diamond Syndrome Induced Pluripotent Stem Cell Disease Model Alice Maria Luca Master of Science Institute of Medical Sciences University of Toronto 2015 Abstract Shwachman-Diamond syndrome (SDS) is an inherited bone marrow failure disease, with 90% of SDS patients carrying a mutation in the SBDS gene. Due to limited efficacy or toxicity of current treatments available, and the otherwise reduced life expectancy of SDS patients, novel therapeutic strategies are needed. Since the main morbidity and mortality are related to the blood dyscrasia, studying hematopoiesis will help characterize the hematological phenotype. We hypothesized that the definitive wave of hematopoiesis is markedly impaired. We generated SDS iPSCs that recapitulated the human SDS disease, specifically, the reduced blood cell formation. The SDS iPSCs showed a defect in definitive hematopoiesis, with a marked reduction in the hemogenic endothelium population. We did not observe a defect in primitive hematopoiesis. Our study sheds light on the onset and progression of the SDS hematopoietic phenotype, and provides a platform for the development of novel, potential therapeutic targets to improve patient care. ii Acknowledgments My graduate journey has been a wonderful learning experience, and this work would not have been possible without the guidance, support and encouragement I have received from a number of influential people. I would like to take a moment to extend my deepest gratitude to all that were involved in this process. First of all, I would like to thank my supervisor, Dr.Yigal Dror, without whom this project would not have been possible. His guidance, critical-thinking and support were instrumental to my journey. I am grateful for all the time and attention that he always put into ensuring that I was successful every step of the way. Thank you for always pushing me to think critically, and for your unwavering support, even when I doubted myself. I would also like to thank Mathura Sabanayagam, for the ribosome profile experiments, and her willingness to spend her time performing them in a 4°C room. A special thank you to Bozana Zlateska for her instrumental role in the generation of the patient-specific iPSCs. Thank you to Hongbing and other lab members for always providing valuable input, and for your continuous encouragement. To Santhosh Dhanraj, thank you for your kind words and friendship. I am thankful for all the valuable discussion, constructive-criticism and encouragement that Dr.Gordon Keller has provided. I am also very grateful for all the time and patience that Marion Kennedy offered me, as well as her expertise, guidance and support. To iii Dr.Gordon Keller, Marion Kennedy, and the rest of the Keller lab, thank you for always keeping me laughing, and always making me feel welcomed. I would also like to thank my committee members, Dr.Michael Glogauer and Walter Kahr for all of their critical insight to my work. Last but not least, I would like to thank my personal cheerleaders, my parents, my sister, Delia, NV and Ryan for their constant words of encouragement every step of the way. iv Contributions I would like to thank Dr. Yigal Dror for his instrumental role in designing the research, critically interpreting the data and editing the thesis. Mathura Sabanayagam performed the ribosome profile experiments and analyzed the acquired data. Members of the Sickkids-University Health Network Flow Cytometry Facility provided their assistance for the cell sorting experiments. Members of The Centre for Applied Genomics (TCAG) at Sickkids Hospital provided their expertise in sequencing the SBDS mutations. The Centre for Commercialization of Regenerative Medicine (CCRM) generated the Sendai- derived iPSCs, performed reverse transcription quantitative real time polymerase chain reaction and immunofluorescence staining of stem cell pluripotency markers. Gordon Keller helped in designing the research, as well as critically interpreting the data. Marion Kennedy helped design the research, critically interpreted the data, and also trained me in induced pluripotent stem cells culturing and hematopoietic development. The rest of the members of the Dror and Keller labs provided their input regarding the design of the project. Dr. Michael Glogauer and Dr. Walter Kahr helped in designing the research. I performed the DNA isolation, polymerase chain reaction amplification, hematopoietic differentiation, clonogenic assay, flow cytometry and microscopy experiments, and analyzed the data. I was also responsible for designing the research and writing the thesis. This work would not have been possible without funding from the Institute of Medical Sciences, the Butterfly Guild and Nicola’s Triathlon for Kids. v Table of Contents Abstract…………………………………………………………………………………. ii Acknowledgments……………………………………………………………………… iii Contributions…………………………………………………………………………… v Table of Contents………………………………………………………………………. vi List of Tables and Figures……………………………………………………………… ix List of Abbreviations…………………………………………………………………… x Chapter I General Introduction 1.1 Shwachman Diamond Syndrome 1 1.1.1 Hematological abnormalities 2 1.1.2 Non-hematological abnormalities 6 1.1.3 Treatment 8 1.1.4 Characterization of Shwachman-Bodian Diamond Syndrome gene 9 1.1.5 Current disease models 14 1.2 Hematopoietic development and hematopoietic differentiation models 16 1.2.1 Primitive hematopoiesis 21 1.2.2 Definitive hematopoiesis 22 1.2.3 Hematopoietic differentiation model using control PSCs 28 1.3 Disease-specific hiPSCs 32 1.4 Objective of Study 35 1.4.1 Rationale 35 1.4.2 Hypothesis 39 1.4.3 Specific aims 39 Chapter II Materials and Methods 2.1 Cell culture 2.1.1 Cell lines 40 2.1.1.1 Mouse embryonic fibroblasts 40 2.1.1.2 OP9-DL1 40 2.1.1.3 hiPSCs 41 2.1.2 Feeder depletion 42 2.1.3 Establishing embryoid bodies for hematopoietic differentiation 42 2.1.3.1 Primitive hematopoiesis 43 2.1.3.2 Definitive hematopoiesis 45 2.1.4 Preparation of single-cell suspension from embryoid bodies 48 2.1.5 Preparation of single-cell suspensions from OP9-DL1 co-cultures 48 2.1.6 Clonogenic assay 48 2.1.7 Morphological analysis by Wright-Giemsa staining 49 2.2 DNA isolation 49 2.3 PCR amplification and sequencing 50 2.4 Flow cytometry and cell sorting 50 2.4.1 Antibodies 50 2.4.2. Fluorescence-activated cell sorting and flow cytometry analysis 50 vi 2.5 Sucrose density gradient ultracentrifugation 51 2.6 Statistics 52 Chapter III Results 3.1 SDS and control iPCSs can be generated from SDS patient and control fibroblasts 53 3.1.1 Generation efficiency 53 3.1.2 SDS iPSCs carry the original patient mutations 55 3.1.3 Pluripotency markers 56 3.1.4 Germ layer differentiation 58 3.2 SDS iPSCs are characterized by an abnormal ribosome profile 60 3.2.1 SDS iPSCs show reduced levels of 80S ribosome subunit 60 3.3 SDS iPSCs can form definitive hematopoietic progenitors 62 3.4 SDS iPSCs can form terminally differentiated blood cells characteristic of 64 definitive hematopoiesis 3.4.1 Terminally differentiated blood cells have a morphology characteristic of macrophages, 64 erythrocytes and granulocytes ……------------------------------------…………………….. 3.5 SDS iPSCs have a reduced capacity to form hematopoietic progenitors characteristic of definitive hematopoiesis 65 3.6 SDS iPSCs have a developmental defect in definitive hematopoiesis 67 3.6.1 Mesoderm induction is not affected in SDS iPSCs 67 3.6.2 Induction of cells with hemogenic endothelium potential is reduced in SDS iPSCs 70 3.6.3 Early hematopoietic progenitor induction is reduced in SDS iPSCs 73 3.6.4 Myeloid cell induction is reduced in SDS iPSCs 76 3.6.5 Granulocyte/Monocyte induction is reduced in SDS iPSCs 78 3.7 SDS iPSCs manifest a delay in the development of primitive 80 hematopoiesis, without a quantitative defect 3.7.1 Mesoderm/Hemangioblast induction is intact in SDS iPSCs 80 3.7.2 Induction of cells with hemogenic endothelium potential is not impaired in SDS iPSCs 82 3.7.3 Early hematopoietic progenitor induction is not affected in SDS iPSCs 85 3.7.4 Mature blood cell induction is not impaired in SDS iPSCs 88 3.7.5 SDS iPSCs showcase a delay in their ability to form hematopoietic progenitors characteristic of primitive hematopoiesis 92 Chapter IV Discussion 4.1 Discussion of results 94 4.1.1 SDS iPSCs can be generated using integrative and non-integrative transgenes 96 4.1.2 Recapitulation of SDS using SDS iPSCs ………………………………... 98 4.1.3 Mesoderm development is intact when SDS iPSCs are induced to undergo definitive hematopoiesis 102 4.1.4 The SDS definitive hematopoietic defect was first noticed at the hemogenic 103 endothelium induction phase 4.1.5 During primitive hematopoiesis, clonogenic potential is delayed, but not impaired 106 4.2 Limitations of study 107 4.3 Significance 109 vii Chapter V Conclusion 112 Chapter VI 114 Future Directions References 116 viii List of Tables and Figures Table 1. Human induced pluripotent stem cells generation efficiency 54 Table 2. Human induced pluripotent stem cells germ layer differentiation 59 Figure 1. Human SBDS gene structure 13 Figure 2. Human SBDS protein structure 13 Figure 3. Sites of human hematopoietic development during fetal life and early infancy 20 Figure 4. Current model of human hematopoietic hierarchy 27 Figure 5. Model of primitive and definitive hematopoiesis using pluripotent stem cells 31 Figure 6. Primitive hematopoiesis differentiation scheme for human pluripotent stem cells 44 Figure 7. Definitive hematopoiesis differentiation scheme for human pluripotent stem cells 47 Figure 8. Sequencing of SBDS in SDS induced pluripotent stem cells 55 Figure 9.
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