Defining Regulators of Human Hematopoietic Stem Cells
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DEFINING REGULATORS OF HUMAN HEMATOPOIETIC STEM CELLS Christine Karlsson Division of Molecular Medicine and Gene Therapy Department of Laboratory Medicine Faculty of Medicine DOCTORAL DISSERTATION With the approval of the Lund University Faculty of Medicine, this thesis will be defended on June 15, 2013 at 09.00 in the Segerfalk lecture hall, BMC A10, Sölvegatan 17, Lund, Sweden. Faculty opponent Professor Hal E. Broxmeyer, PhD Indiana School of Medicine Indiana, USA Organization Document name LUND UNIVERSITY DOCTORAL DISSERTATION Date of issue Division of Molecular Medicine and Gene Therapy June 15, 2013 Institution of Laboratory Medicine, Lund Author(s) Sponsoring organization Christine Karlsson Title and subtitle DEFINING REGULATORS OF HUMAN HEMATOPOIETIC STEM CELLS Abstract Bone marrow transplantation (BMT) is a conceptual and elegant example of stem cell therapy, rendered possible by the dual capacity of hematopoietic stem cells (HSCs) to self-renew and differentiate. These defining features ultimately ensure the production of all blood cell lineages and simultaneous maintenance of the stem cell pool, thereby maintaining lifelong homeostasis. Banked umbilical cord blood (CB) is an abundant and readily available stem cell resource. However, the relatively low numbers of hematopoietic stem- and progenitor cells (HSPCs) present in a typical single CB unit and the associated delay in engraftment restrict its routine applicability to primarily children. Despite intense global efforts to find strategies that would enable the ex vivo amplification of transplantable stem cells, the culture of HSCs outside their natural environment has proven difficult. It is therefore important to understand how HSC self-renewal, proliferation, and differentiation are integrated, which molecules participate in their regulation and how these could be modified for clinical benefit. With the aim to discover new growth factors supporting stem cells in culture, we have assessed 276 extrinsic signaling molecules for their effect on CB-derived HSPCs. We identified the immunoregulatory chemokine (C-C motif) ligand 28 (CCL28) as a novel growth- and survival factor for primitive hematopoietic cells. CCL28 strongly supported the proliferation and clonogenic potential of hematopoietic progenitors from different ontogenetic origins, and significantly enhanced the ability of cultured putative HSCs to long-term reconstitute immunodeficient mice. Thus, CCL28 represents one of the few cytokines that can maintain the primitive properties and functional integrity of cultured human HSPCs. Furthermore, we identified myostatin propeptide, a naturally occurring inhibitor of the transforming growth factor-ß (TGF-ß) family member myostatin, as a novel promoter of HSPC proliferation during ex vivo culture. Based on the limited self-renewal capacity of HSCs in vitro, we have developed a forward RNA interference (RNAi)-based screening method that allows the discovery of novel genes implicated in stem- and progenitor cell proliferation. Using pooled lentiviral short hairpin RNA (shRNA) libraries transduced into CB cells, we have identified short hairpins designed against exostoses 1 (shExt1), phospholipase C zeta 1 (shPLCZ1) and serine threonine kinase 38 (shSTK38) as new fate determinants for HSPC differentiation, proliferation, and self-renewal, respectively. However, first-generation RNAi screening in primary cells yielded a considerable amount of target gene-unrelated, yet shRNA-specific events (so-called ‘off-target effects’), exemplified by shSTK38. Tracking of library-transduced HSPCs by next-generation sequencing significantly improved the resolution and feasibility of the screening approach and identified inhibition of MAPK14/p38α as means to promote expansion of undifferentiated cells, as shown by both RNAi and pharmacological modification of p38 using small molecule inhibitors. Taken together, in this thesis we employed different screening approaches to identify novel regulators of primitive human hematopoietic cells. We conclude that systematic growth factor screenings as well as forward RNAi-based technologies are powerful tools to detect and subsequently define novel mediators of HSPC fate decisions. Key words Hematopoietic stem cell, cord blood, expansion, high-throughput screening, RNA interference, extrinsic signaling molecules Classification system and/or index terms (if any) Supplementary bibliographical information Language English ISSN and key title ISBN 1652-8220 978-91-87449-41-3 Recipient´s notes Number of pages Price 99 Security classification Disribtion by (name and address) Christine Karlsson, BMC A12, 22184 Lund, Sweden. [email protected] I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sources permission to publish and disseminate the abstract of the above-mentioned dissertation. Signature Date May 13, 2013 2 DEFINING REGULATORS OF HUMAN HEMATOPOIETIC STEM CELLS Christine Karlsson Division of Molecular Medicine and Gene Therapy Faculty of Medicine Lund University 2013 Copyright © Christine Karlsson LUND UNIVERSITY Faculty of Medicine, Doctoral Dissertation Series 2013:71 ISBN 978-91-87449-41-3 ISSN 1652-8220 Printed by Media-Tryck, Lund University Lund 2013 To my family TABLE OF CONTENTS TABLE OF CONTENTS 7 ABBREVIATIONS 9 LIST OF PUBLICATIONS 11 PREFACE 13 HEMATOPOIESIS 15 HEMATOPOIETIC STEM CELLS 16 METHODS TO STUDY HEMATOPOIETIC CELLS 18 HSC FATE OPTIONS IN THE HSC NICHE 21 REGULATION OF HSCS 24 HSC ONTOGENY 26 STEM CELL-BASED THERAPY 28 CORD BLOOD – FROM BENCH TO BEDSIDE 31 HISTORY OF CORD BLOOD TRANSPLANTATION AND CLINICAL RELEVANCE 31 STRATEGIES TO IMPROVE CORD BLOOD TRANSPLANTATION 34 EFFORTS TO ENHANCE STARTING CELL NUMBERS 34 EX VIVO EXPANSION OF HEMATOPOIETIC STEM- AND PROGENITOR CELLS 35 EFFORTS TO ENHANCE HOMING AND ENGRAFTMENT 41 SCREENING STRATEGIES TO IDENTIFY HSC REGULATORS 44 GENERAL ASPECTS 44 RNAI-BASED FORWARD GENETIC SCREENS 45 PRESENT INVESTIGATION 47 FOCUS AND AIM 47 SUMMARY OF RESULTS 48 CONCLUSIONS FROM PRESENT STUDIES 51 GENERAL DISCUSSION 52 MODELING HUMAN HEMATOPOIESIS 52 HETEROGENEITY AND LIMITATIONS WITH CURRENT HSPC ASSAYS 52 STEM CELL EXPANSION 53 IDENTIFICATION OF NOVEL HEMATOPOIETIC CYTOKINES 53 INTRINSIC CONTROL 55 PROSPECTS TO HSC EXPANSION 56 HIGH-THROUGHPUT SCREENING IN HSPCS 56 CHALLENGES IN TARGET VALIDATION 57 VALIDATION OF THE SCREENING STRATEGY 57 VALIDATION OF THE TARGET IDENTITY 58 FUTURE DIRECTIONS 60 OPTIMIZATION OF CULTURE CONDITIONS FOR HUMAN HSPCS 60 THE ROLE OF CCL28 IN HEMATOPOIESIS 61 7 SAMMANFATTNING PÅ SVENSKA 63 ZUSAMMENFASSUNG AUF DEUTSCH 65 ACKNOWLEDGEMENTS 67 REFERENCES 70 APPENDICES (ARTICLES I-IV) 99 8 ABBREVIATIONS AGM aorta-gonad-mesonephros AML acute myeloid leukemia Angpt angiopoietin Angptl angiopoietin-like BFU-E burst forming unit erythroid BM bone marrow BMP bone morphogenetic protein BMT bone marrow transplantation BrdU bromodeoxyuridine CB cord blood CBT cord blood transplantation CCL28 chemokine (C-C motif) ligand 28 CD cluster of differentiation CDC cell division cycle CDKI cyclin-dependent kinase inhibitor CFC colony forming cell CFU-E colony forming unit erythroid CFU-GM colony forming unit granulocyte/macrophage CFU-S colony forming unit spleen CLP common lymphoid progenitor CMP common myeloid progenitor CRU competitive repopulation unit CXCR4 C-X-C chemokine receptor type 4 DNA deoxyribonucleic acid E embryonic day ECM extracellular matrix EPO erythropoietin ESC embryonic stem cell EXT1 exostoses 1 FACS fluorescence activated cell sorting FA Fanconi Anemia FDR false discovery rate FGF fibroblast growth factor FL fetal liver FLT3 fms-related tyrosine kinase 3 FNBP1L forming binding protein 1-like G-CSF granulocyte colony-stimulating factor GDF growth and differentiation factor GFP green fluorescent protein GMP granulocyte/macrophage progenitor HCT hematopoietic cell transplantation HLA human leukocyte antigen HPRT hypoxanthine guanine phosphoribosyl transferase HPC hematopoietic progenitor cell HSC hematopoietic stem cell HSPC hematopoietic stem- and progenitor cell IDDb insertional dominance database IGF insulin growth factor IGFBP2 IGF-binding protein 2 IL interleukin KO knockout 9 Lin lineage LSK Lin- Sca-1+ c-kit+ LTC-IC long-term culture initiating cell LT-HSC long-term HSC LTR long terminal repeats MAPK mitogen-activated protein kinase MEP megakaryocyte/erythrocyte progenitor MHC major histocompatibility complex miRNA microRNA MP myostatin propeptide MPP multipotent progenitor MSTN myostatin NOD non-obese diabetic NSG NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ OTE off-target effect PB peripheral blood PLCZ1 phospholipase C zeta1 RISC RNA-induced silencing complex RNA ribonucleic acid RNAi RNA interference ROS reactive oxygen species RTCGD retrovirus-tagged cancer gene database SAM significance analysis of microarrays Sca-1 stem cell antigen 1 SCF stem cell factor SCID severe combined immunodeficiency SD standard deviation SDF-1 stromal derived factor-1, also called CXCL12 SEM standard error of the mean shRNA short hairpin RNA SIN self-inactivating siRNA short interfering RNA SRC SCID-repopulating cell ST-HSC short-term HSC STK38 serine/threonine kinase 38 TGF-β transforming growth factor β Tie2 tyrosine kinase receptor 2 TPO thrombopoietin 10 LIST OF PUBLICATIONS This thesis is based on the articles listed below, which are referred to in the text by their roman numerals (I-IV). I Karlsson C, Baudet A, Miharada N, Soneji S, Gupta R, Magnusson M, Enver T, Karlsson G, and Larsson