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Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells Strategies to Overcome the Hurdles ROGER E. RÖNN DIVISION OF MOLECULAR MEDICINE AND GENE THERAPY | LUND UNIVERSITY 2015 Lund University, Faculty of Medicine Doctoral Dissertation Series 2015:142 ISBN 978-91-7619-222-1 ISSN 1652-8220 PrintedbyMedia-Tryck,LundUniversity2015 9789176192221 ROGERE.RÖNN  TowardsGenerationofHumanHematopoieticStemCellsfromPluripotentStemCells 142 It has been more than 6 years since this journey began. I have spent this time studying how blood forms during early life for the purpose of increasing our understanding of this process, with the hope that this knowledge one day can contribute to therapeutic applications. These 6 years have been a great chapter in my life, and I hope that you enjoy reading this book as much as I have enjoyed writing it. Roger Emanuel Rönn, Lund, November 2015 It has been more than 6 years since this journey began. I have spent this time studying how blood forms and grows during early life, and the purpose has been to increase what we understand about this process, with the hope that this knowledge one day can be used for something positive. These 6 years have been a great chapter in my life, and I hope that you enjoy reading this book as much as I have enjoyed writing it. /Roger
1 Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells Strategies to Overcome the Hurdles Roger E. Rönn DOCTORAL DISSERTATION With the approval of the Faculty of Medicine, Lund University, Sweden, this thesis will be defended in the Belfrage lecture hall, BMC D15, Klinikgatan 32, Lund on December 17th 2015 at 14:00. Supervisor Niels-Bjarne Woods, Ph.D. Faculty opponent Professor George Q. Daley, M.D., Ph.D. Children’s Hospital Boston, U.S.A.
2 Organization LUND UNIVERSITY Division of Molecular Medicine and Gene Therapy Document name: Doctoral dissertation Institution of Laboratory Medicine, Lund Date of disputation: December 17th , 2015 Author: Roger E. Rönn Sponsoring organization Title and subtitle Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells. Strategies to Overcome the Hurdles. Abstract There is a pressing need for compatible Hematopoietic Stem Cells (HSCs), in high quantities and of good clinical quality, to ensure optimal treatment for patients with various blood malignancies. Pluripotent Stem Cells (PSCs) have the potential to be a source for generating HSCs. The indefinite expansion capacity of PSCs, combined with the possibility of creating these cells from patient-specific material could, in principle, allow for the generation of patient-derived HSCs. Generation of HSCs from PSCs could dramatically improve the treatment of patients in the clinic, including the possible correction of genetically carried hematological diseases. Furthermore, successful generation of HSCs would provide a tool to better study the nature of hematological malignancies, and could be employed for the purpose of drug screening With the ultimate goal of facilitating in vitro generation of functional HSCs from human PSCs, the studies included in this thesis explore potential key factors and mechanisms involved in the process of hematopoietic development. In paper I we describe that control over the amount of retinoic acid during in vitro differentiation of human PSCs increase blood generation, allowing for more defined and efficient production of relevant cells. In paper II we provide a molecular identification of Endothelial to Hematopoietic Transition (EHT), the conversion through which HSCs emerge from endothelial cells. This provides a foundation for further characterization of the cells undergoing this transition, and could potentially enable control over this process for the purpose of more efficient generation of hematopoietic cells from hemogenic endothelium. In paper III we explore how cAMP intrinsically regulates the generation of HSC-like cells, and we demonstrate the importance of cAMP signaling in the emergence of HSCs. Modulating cAMP provides an additional approach to increase the generation, and potentially the function, of HSC-like cells from human PSCs. In paper IV, we demonstrate that reactive oxygen species is increased due to in vitro culture, causing functional impairment of hematopoietic progenitors and HSC-like cells generated from human PSCs. This argues that we must revise our standardized culture methods, and that a more physiological oxygen concentration is likely to be a critical component for handling and generating HSCs outside of the body. In summary, the papers included in this thesis focuses on understanding the development of human HSCs, with the ultimate goal of developing methods that would enable the generation of functional HSCs in the laboratory, potentially translating into improved treatments against a multitude of hematological malignancies. Key words: hematopoietic stem cells, pluripotent stem cells, in vitro differentiation, embryonic development Classification system and/or index terms (if any) Supplementary bibliographical information Language: English ISSN and key title: 1652-8220 ISBN: 978-91-7619- 222-1 Recipient’s notes Number of pages Price Security classification I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sourcespermission to publish and disseminate the abstract of the above-mentioned dissertation. Signature Date
3 Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells Strategies to Overcome the Hurdles Roger E. Rönn
4 Cover artwork by Roger Rönn Copyright Roger Rönn Lund University, Faculty of Medicine, Institution of Laboratory Medicine, Division of Molecular Medicine and Gene Therapy Lund University, Faculty of Medicine Doctoral Dissertation Series 2015:142 ISBN 978-91-7619-222-1 ISSN 1652-8220 Printed in Sweden by Media-Tryck, Lund University Lund 2015 EndelavFörpacknings-och Tidningsinsamlingen(FTI)
5 Till min familj och mina vänner
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7 Content Populärvetenskaplig sammanfattning.......................................................................9 Original papers .......................................................................................................12 Papers not included in this thesis............................................................................13 Abbreviations .........................................................................................................14 Preface....................................................................................................................16 Introduction ............................................................................................................17 The Hematopoietic System 17 Hematopoietic Stem Cells 19 Characterization of the Human HSC...................................................19 The Bone Marrow Niche.....................................................................20 Regulation of HSC Self-Renewal........................................................21 HSC Sensitivity ...................................................................................22 Embryonic Development 24 Pluripotent Stem Cells.........................................................................24 Developmental Specification...............................................................26 Retinoic Acid During Embryonic Development .................................27 Hematopoiesis During Embryonic Development................................28 In Vitro Differentiation of hPSCs Towards HSCs 32 The Potential of in Vitro HSC Generation from hPSCs ......................32 Methodology .......................................................................................33 Previous Key Discoveries....................................................................34 Aims of the thesis...................................................................................................37 Summary of results.................................................................................................39 Paper I: 39 Paper II: 40 Paper III: 41 Paper IV: 42 Conclusions ............................................................................................................45 General discussion and future perspectives............................................................47 Acknowledgements ................................................................................................51 References ..............................................................................................................55
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9 Populärvetenskaplig sammanfattning Idag har patienter med blodcancer (leukemi) eller genetiskt betingade blodsjukdomar (sickelcellanemi etc.) begränsad tillgång till behandlingar som effektivt och riskfritt botar sjukdomstillståndet. Detta arbete inkluderar forskningsresultat ämnade att bidra till utveckling av konceptuellt nya behandlingsmetoder för olika typer av blodsjukdomar. Ett fungerande blodsystem är nödvändigt för varje individs överlevnad. De röda blodkropparna (erytrocyterna) förser kroppen med syre från lungorna, och de vita blodkropparna (leukocyterna) utgör vårt immunförsvar mot infektionssjukdomar. Blodceller slits snabbt ut och för att ersätta dessa behöver kroppen varje dag tillverka cirka 1 biljon nya blodceller. Ansvarig för denna kontinuerliga nyproduktion av blodceller är en sällsynt typ av blodceller som kallas för Hematopoetiska stamceller (HSCs), även kallade blodstamceller. Blodstamceller återfinns i benmärgen där de stundtals genomgår celldelning. Vid celldelning kan en dottercell påbörja en mognadsprocess där, efter många nya celldelningar, ett stort antal av färdiga blodceller resulterar. Blodstamceller är därför centrala för att våra kroppar ska kunna förses med nya blodceller genom livet. Blodstamceller är dock väldigt känsliga och kan fallera om de utsätts för skadliga faktorer som olika kemiska substanser, eller radioaktiv strålning. Vid behandling av blodcancer används idag ofta sådana faktorer för att ta död på cancercellerna. Dessa behandlingar medför ofta att även kroppens friska blodstamceller förstörs, vilket kan resultera i blodsystemets kollaps och patientens avlidande om detta inte åtgärdas. Därför genomgår patienter som genomgått cancerdödande behandling ofta en transplantation där blodstamceller från en donator införs i patientens kropp. Om transplantationen lyckats återetablerar donatorns blodstamceller en kontinuerlig nyproduktion av blodceller vilket resulterar i patientens överlevnad. Men donatorbaserad transplantation av blodstamceller kan medföra komplikationer. Mängden blodstamceller som kan inhämtas från en villig donator kan vara begränsad vilket resulterar i en oönskat lång återhämtning för patienten. En donator måste även vara immunkompatibel med patienten (att deras immunförsvar har liknande karaktär) för att minimera risken att det nya immunförsvaret reagerar mot patientens egna vävnader. Ett sådant tillstånd där transplantatet angriper värden, på engelska Graft Versus Host Diesease (GVHD), är livsfarligt för patienten. Därför krävs en hög kompatibilitet mellan patient och
10 donator vilket ytterligare begränsar antalet möjliga donatorer. Det finns därför ett stort behov av patientkompatibla blodstamceller för att kunna möjliggöra optimal och effektiv behandling mot olika blodsjukdomar. En möjlighet att förse detta behov är odling och produktion av blodstamceller i laboratoriet. I början av den embryonala utvecklingen (strax efter äggets befruktning) finns en typ av stamceller som kan ge upphov till alla olika typer av celler och vävnader i den vuxna kroppen. Dessa stamceller kallas därför pluripotenta och kan, om de stimuleras på rätt sätt, odlas i laboratorium för att utvecklas till olika vuxna celltyper inklusive blod. Pluripotenta stamceller behöver inte nödvändigtvis erhållas från embryon. År 2007 lyckades ett japanskt laboratorium, lett av professor Shinya Yamanaka, med att framställa mänskliga pluripotenta stamceller från vuxna hudceller. Denna bedrift belönades med Nobelpriset i medicin år 2012, och innebar nya möjligheter att framställa pluripotenta stamceller från enskilda individer som till exempel patienter. Om det vore möjligt att framställa funktionsdugliga blodstamceller från pluripotenta stamceller skulle behandlingen av blodsjukdomar kunna förbättras avsevärt eftersom det teoretiskt sett vore möjligt att framställa obegränsade mängder av nya patientkompatibla blodstamceller. Denna avhandling innehåller 4 olika forskningsstudier där det gemensamma målet är att utveckla en metod för att lyckas med att framställa mänskliga blodstamceller från pluripotenta stamceller. I den första studien demonstrerar vi att Retinoinsyra, en metabolit av Vitamin A, har en negativ påverkan under utvecklandet av pluripotenta stamceller till blod. Genom att använda en kemisk substans vid namn (DEAB) för att blockera cellernas egen produktion av Retinoinsyra kan vi flerfaldigt förbättra framställningen av celler vilka liknar blodstamceller. Ökad förståelse hur regleringen av utvecklingsprocessen går till är viktigt för att specifikt och effektivt kunna framställa de celler som är av intresse. I den andra studien undersöker vi i detalj hur de första blodstamcell-lika cellerna formas. Under utvecklingen uppstår de första blodstamcellerna från endotelceller som täcker blodkärlens insida. Denna process, där en icke-blodcell genomgår ett identitetsbyte till blod, kallas för Endotelisk till Hematopoetisk Övergång, på engelska Endothelial to Hematopoietic Transition (EHT), och detaljerad vetskap om hur denna process fungerar saknas. Genom att studera övergången från endotel till blodcell beskriver vi ett specifikt övergångstillstånd som sker centralt i denna process. Våra resultat låter oss för första gången identifiera och särskilja de celler som deltar i denna övergång, vilket kan komma väl tillhanda om vi bättre vill efterlikna den embryonala blodutvecklingen för att kunna framställa blodstamceller i laboratoriet.
11 I den tredje studien visar vi att utvecklingen av blodstamcell-lika celler är i kritiskt behov av Cykliskt adenosinmonofosfat (cAMP) vilket fungerar som en sekundär budbärare åt flera olika signalsubstanser. Genom att manipulera cellernas mängd av cAMP demonstrerar vi flerfaldigt ökad framställning av blodstamcell-lika celler. Våra resultat påvisar även att cAMP förmedlar sina positiva effekter genom ej hittills uppskattade signaleringsleder inom cellen. Dessa resultat ökar vår förståelse över vilka faktorer som krävs för effektiv framställning av blod. I den fjärde studien demonstrerar vi att majoriteten av alla blodstamcell-lika celler har förhöjda nivåer av reaktiva syregrupper, på engelska Reactive Oxygen Species (ROS), vilket orsakar funktionsoduglighet. Vi påvisar att denna förhöjning av reaktiva syregrupper orsakas av flera olika processer vilka grundar sig i de metoder för cellodling som idag är generell praxis. Genom att använda samt kombinera fyra olika metoder för att minska mängden av reaktiva syregrupper kan vi för första gången effektivt framställa blodstamcell-lika celler med reducerade mängder av ROS. Dessa celler uppvisar kraftigt förbättrad kapacitet för tillväxt. Våra resultat argumenterar för att reaktiva syregrupper vilka resulterar från våra cellodlingsmetoder utgör ett allvarligt hinder, samt att en reducering sannolikt är en nödvändighet, för lyckad framställning av funktionsdugliga blodstamceller i laboratoriet. Sammantaget presenteras i denna avhandling forskning inriktad på att förstå utvecklingsprocessen av blod, med syfte att möjliggöra framställning av blodstamceller från pluripotenta stamceller. Våra resultat ger ökad insikt i hur denna komplicerade process fungerar och det är vår förhoppning att dessa framsteg kan bidra till en framtida metod vilken möjliggör effektiv framställning av funktionella blodstamceller. Detta eftersom en sådan metod drastiskt skulle kunna förbättra behandlingen av patienter som lider av diverse blodsjukdomar.
12 Original papers Paper I, Retinoic acid regulates hematopoietic development from human pluripotent stem cells. Roger E. Rönn, Carolina Guibentif, Roksana Moraghebi, Patricia Chaves, Shobhit Saxena, Bradley Garcia, Niels-Bjarne Woods. Stem Cell Reports (2015) 4, 269-281. Paper II, Endothelial to hematopoietic transition state identified in differentiated human pluripotent stem cells. Carolina Guibentif, Roger Rönn, Charlotta Böiers, Shobhit Saxena, Stefan Lang, Shamit Soneji, Tariq Enver, Göran Karlsson, Niels-Bjarne Woods. Submitted Manuscript (2015). Paper III, Cyclic AMP induction enhances human hematopoietic cell emergence through the cAMP-Epac signaling axis, creates redox state balance, and increases CXCR4 expression. Shobhit Saxena, Roger E. Rönn, Carolina Guibentif, Roksana Moraghebi, Niels- Bjarne Woods. Submitted Manuscript (2015). Paper IV, Reactive oxygen species impair the function of hematopoietic stem / progenitor cells generated from human pluripotent stem cells. Roger E. Rönn, Carolina Guibentif, Shobhit Saxena, Niels-Bjarne Woods. Submitted Manuscript (2015).
13 Papers not included in this thesis Localization of SMAP2 to the TGN and its function in the regulation of TGN protein transport. Tomo Funaki, Shunsuke Kon, Roger E. Rönn, Yuji Henmi, Yuka Kobayashi, Toshio Watanabe, Keiko Nakayama, Kenji Tanabe, and Masanobu Satake. Cell Structure and Function (2011) 36, 83-95.
14 Abbreviations AGM Aorta Gonad Mesonephros AP Alkaline Phosphatase ATP Adenosine-5-Triphosphate BM Bone Marrow cAMP Cyclic Adenosine Mono Phosphate CAR Cell CXCL12-Abundant Reticular Cell CB Cord Blood CFU Colony Forming Unit DEAB Diethylaminobenzaldehyde DS Down Syndrome EB Embryoid Body EHT Endothelial to Hematopoietic Transition EPAC Exchange Proteins Activated by cAMP ES Cell Embryonic Stem Cell FACS Fluorescence Activated Cell Sorting GD Gaucher Disease GFP Green Fluorescent Protein GPX Glutathione Peroxidase GSR Glutathione S-reductase GVHD Graft Versus Host Disease HE Hemogenic Endothelium HLA Human Leukocyte Antigen HR Homologous Recombination HSC Hematopoietic Stem Cell IBMX Isobutyl-1-Methylxanthine ICM Inner Cell Mass iPS Cell Induced Pluripotent Stem Cell LDA Limited Dilution Assay LPM Lateral Plate Mesoderm MAPK Mitogen-Activated Protein Kinase MPO Myeloperoxidase MPP Multipotent Progenitor MSC Mesenchymal Stem Cell NK Cell Natural Killer Cell
15 NHEJ Non-Homologous End Joining NOD Nonobese Diabetic OTM Olive Tail Moment OXPHOS Oxidative Phosphorylation PCA Principal Component Analysis PD Parkinson Disease PGE2 Prostaglandin E2 PKA Protein Kinase A PSC Pluripotent Stem Cell RA Retinoic Acid RALDH Retinaldehyde Dehydrogenase RAR Retinoic Acid Receptor RARE Retinoic Acid Response Element RXR Retinoid X Receptor ROS Reactive Oxygen Species SBDS Shwachman-Bodian-Diamond Syndrome SCID Severe Combined Immunodeficiency SOD Superoxide Dismutase WAS Wiscott-Aldrich Syndrome YS Yolk Sac 4-ABAH 4-Aminobenzoic Acid
16 Preface It has been more than 6 years since this journey began. I have spent this time studying how blood forms during early life for the purpose of increasing our understanding of this process, with the hope that this knowledge one day can contribute to therapeutic applications. Today, there are limitations to how we can treat patients suffering from leukemia and other blood disorders. One key requirement for successful treatment is transplantation of hematopoietic stem cells capable of re-establishing the hematopoietic system. Today hematopoietic stem cells often need to be donated from other individuals. The reliance on donated cells for transplantation leads to limitations to the amount of suitable hematopoietic stem cells both in terms of quality and in quantity. Pluripotent stem cells are cells that have the capacity to form all types of cells in the body. If we could understand how hematopoietic stem cells form during early life, and then recreate the same conditions in the laboratory for the purpose of generating hematopoietic stem cells from pluripotent stem cells, we may have the means to provide patients with new effective treatments potentially curing and correcting many types of blood disease. The first part of this thesis gives an introduction to the hematopoietic system, the hematopoietic stem cell, embryonic development, and previous efforts to generate hematopoietic stem cells. The second part summarizes the present studies, and is followed by a general discussion where I elaborate on what I believe to be a key hurdle to overcome if we want to successfully generate hematopoietic stem cells in the laboratory. These 6 years have been a great chapter in my life, and I hope that you enjoy reading this book as much as I have enjoyed writing it. Roger Emanuel Rönn, Lund, November 2015
17 Introduction The Hematopoietic System Hematopoiesis (“creation of blood” in Greek) is the process of blood formation that sustains an individual’s requirement of circulating blood cells. Viewed as a liquid organ, the hematopoietic system provides key functions needed for the survival of the individual. There are several types of blood cells in the hematopoietic system, each having specialized functions. The red blood cells (erythrocytes) are required for the transport of oxygen and carbon dioxide between the lungs and the rest of the body and are the most abundant blood cell type (20-30 trillion in an adult human). The ability of blood to clot and stop bleeding is provided by platelets, small fragments of another hematopoietic cell type known as megakaryocytes. The hematopoietic system also includes cell types that are part of the innate and adaptive immune systems, protecting the organism from foreign pathogens. The innate immune system includes macrophages, granulocytes, and the dendritic cells. These cells facilitate immediate responses, including the engulfment of foreign material (phagocytosis), pathogen neutralization by release of toxic substances, modulation of the inflammatory response, and communication of information to the adaptive immune system. The adaptive immune system features the lymphocytes, providing memory and long-term protection against encountered pathogens. Lymphocytes include the B cells required for the production of antibodies, and T cells as well as Natural Killer (NK) cells that provides directed killing of infected cells. Together, these various cells of the hematopoietic system provide functions critical for the survival of the individual, but in order to replace aged or broken blood cells the body needs to create approximately 1 trillion new blood cells every day (Ogawa, 1993). While the various types of blood cells are specialized for their specific functions, they are all related to each other and are derived from a common precursor in a hierarchical manner (Figure 1). At the apex of the hematopoietic hierarchy are the Hematopoietic Stem Cells (HSCs). Formed during embryonic development and maintained throughout adult life, HSCs are multipotent progenitors that form all mature blood cells while simultaneously having the capacity to self-renew, forming cellular progeny that retain the same HSC characteristics. Fulfilling these criteria, the HSC have the capacity to provide long-term reconstitution of the
18 hematopoietic system. While other cell types in the hematopoietic hierarchy have limitations as to how many times they can divide, the HSCs, due to their ability to self-renew, provide a continuous source of new blood cells and are responsible for the homeostasis of the entire hematopoietic system. In absence of such self- renewing HSCs the continuous production of new blood cells cannot occur, resulting in the collapse of the hematopoietic system and compromised survival. Figure 1. The Hematopoietic System. All mature blood cells of the hematopoietic system are derived, in a hierarchical manner, from HSCs as their common precursors. HSC: Hematopoietic Stem Cell, MPP: Multipotent Progenitor, CMP: Common Myeloid Progenitor, MEP: Megakaryocyte/Erythroid Progenitor, GMP: Granulocyte/Macrophage Progenitor, MLP: immature Lymphoid Progenitor, B/NK: B cell / Natural Killer cell progenitor, proB: B cell precursor, ETP: Early T cell Precursor, NK: Natural Killer cell. Human HSCs are enriched by the following markers: Lin- CD34+ CD38- CD90+ CD49f+ . Illustration was inspired by figure 2 in Doulatov et al., 2012. HSC MPP CMP MLP Dendritic cells NK cells B cells T cellsMacrophages Granulocytes Erythrocytes Megakaryocytes ETP B/NK proBGMPMEP Lin- CD34+ CD38- CD90+ CD49f+
19 Hematopoietic Stem Cells The idea that a population of self-renewing HSC is responsible for the continuous regeneration of the hematopoietic system was initially proposed by Till and McCulloch in 1961 after observing that limited numbers of transplanted bone marrow cells formed colonies of hematopoietic cells in the spleens of recipient mice. Since then, due to the central role of HSCs in hematopoiesis, there have been numerous scientific studies aimed at characterizing and increasing the understanding of this cell type Characterization of the Human HSC Immunodeficient mice used as xenograft models have been invaluable tools for studying human hematopoiesis. Immunodeficiency allows for transplanted human cells to avoid rejection and this was first taken advantage of with the development of the Severe Combined Immune-Deficient (SCID) mouse model (Bosma et al., 1983; Fulop and Phillips 1990). These mice lack both B and T cells, and allows for mainly long-term engraftment of human lymphoid cells. Backcrossing the SCID mutation onto nonobese diabetic (NOD) mice (harboring further defects in innate immunity), with additional deletion of the IL-2R common γ chain gene, resulted in the NSG mouse model that features a complete loss of B, T, and NK cells. The NSG model allows for robust human lymphoid-myeloid engraftment and is commonly used to study human engraftment from HSCs (Doulatov et al., 2012). By analyzing the presence or absence of specific proteins on the cellular surface, various cell types can be identified and characterized. Development of fluorochrome-conjugated monoclonal antibodies capable of binding such cell surface markers, combined with Fluorescence Activated Cell Sorting (FACS) technology have enabled the separation and analysis of the various cell types in the hematopoietic hierarchy. To identify human HSC, the currently most stringent combination of surface markers is the following; negativity for specific markers of the T, B, NK, and myeloid-erythroid lineages (Lin- ), positivity for marker CD34 (CD34+ ), CD38- , CD90+ (Majeti et al., 2007), and CD49f+ (Notta et al., 2011) (Figure 1). Transplantation with reduced numbers of cells purified with these markers, using the Limited Dilution Assay (LDA), have demonstrated that only 1 in 10 of these cells are able to repopulate recipients (Notta et al., 2011) indicating the rarity of true HSCs and the challenge of identifying them reliantly.
20 The Bone Marrow Niche During homeostasis, the anatomical location of the HSCs in the adult is in the bone marrow (BM), which is the flexible “spongy” tissue inside the larger bones of the body. The BM is a highly complex environment that includes many different cell types, organized together within the vascularized space between the inner walls of endosteal bone. The BM as a whole provides an intricate environment (niche) that regulates and protects the HSC pool throughout an individual’s life (Scadden, 2006). Cells found in the bone marrow niche include bone-synthesizing osteoblasts, bone-resorbing osteoclasts, energy storing adipocytes, Mesenchymal Stem Cells (MSCs), CXCL12-Abundant Reticular cells (CAR cells) and macrophages, with HSCs comprising less than 0.01% of all cells in the BM (Walasek et al., 2012) (Figure 2). By secreting various signaling molecules, the relative position of specific niche cells to the HSC, combined with location dependent effects upon the HSC in the architecture of the BM (for instance the proximity to blood vessels providing oxygen), together provide a highly complex physiological regulatory system required for HSC maintenance, and thereby for hematopoiesis itself (Scadden, 2006; Walasek et al., 2012). Figure 2. The Bone Marrow Niche. This illustration provides a simplified model of the bone marrow niche. General structural features include blood vessels and the inner surface of endosteal bone. Cell types included in this illustration are: CAR cells (CXCL12- Abundant Reticular cells), macrophages, osteoblasts, osteoclasts, HSCs (Hematopoietic Stem Cells), MSCs (Mesenchymal Stem Cells), and adipocytes. Gradients of oxygen and calcium are also illustrated. Calcium Oxygen Blood Vessel CAR Cell Macrophage Adipocyte MSC HSC Osteoclast Osteoblast Endosteal Bone
21 Regulation of HSC Self-Renewal Since self-renewal of the HSC is required for maintained and continuous regeneration of the hematopoietic system, self-renewal is considered to be a core- defining feature of functional HSCs. The central role of self-renewing HSCs in hematopoiesis, together with the fact that HSCs rapidly lose their self-renewal capacity upon in vitro culture (limiting their potential expansion for therapeutic purposes), have resulted in efforts to better understand how self-renewal is regulated. It has been postulated that HSCs, when faced with changing circumstances, have a number of different fate options available (Wagers et al., 2002). These fate options include the division of an HSC into daughter cells whereupon either one or both cells retain self-renewal and HSC identity (asymmetric and symmetric division), in the latter case expanding the HSC-pool without giving rise to maturing (differentiating) blood cells. An HSC might also divide into daughter cells where both cells differentiate, resulting in loss of self- renewing cells and the reduction of the HSC pool. Additional fate options could be considered including the cell entering a state of inactive hibernation (quiescence), migration of the HSC into other environments, induction of premature senescence causing functional elimination of the HSC without the aspect of apoptosis (Ito et al., 2006; Ito and Suda, 2014), or termination of the cell by controlled cell death (apoptosis) (Figure 3). Thus, control over these fate options to favor symmetric division of self-renewing HSCs could potentially enable HSC expansion. Factors that regulate the behavior of HSCs can either be extrinsic or intrinsic. Extrinsic regulation includes environmental cues that affect the HSC. This includes factors that are secreted from other cells in the niche, or physical circumstances such as the distance to the vasculature and the available amount of oxygen (Ludin et al., 2014). Intrinsic regulation covers the actions of internal mediators affecting HSC behavior and identity. This includes genetic and epigenetic modulators regulating the transcriptional state and the division characteristics of the cell (Oguro et al., 2006). Other intrinsic regulators are factors associated with the apoptotic response (Domen et al., 1998), or involved in DNA repair mechanisms (Biechonski and Milyavsky, 2013). In addition, the metabolic state of the HSC is known to affect self-renewal capacity. Glycolysis-based production of adenosine-5-triphosphate (ATP) is associated with increased survival and engraftment capacity, while ATP production through oxidative phosphorylation (OXPHOS) is associated with loss of self-renewal and differentiation (Takubo et al., 2013; Ito and Suda, 2014). Intrinsic regulation also includes the availability of intracellular molecules involved in signal transduction. One such intracellular mediator is Cyclic Adenosine Mono Phosphate (cAMP) transmitting the effects of neurotransmitters, growth factors, and hormones such as Prostaglandin E2 (PGE2), binding to G- protein coupled receptors on surface of the HSC (Cutler et al., 2013). This allows the cell to respond to such extrinsic factors.
22 The individual importance of the factors mediating extrinsic and intrinsic regulation suggests that HSC self-renewal is a delicate state, easily lost upon imbalance and dysregulation. Figure 3. Fate Options of the HSC. The HSC can, depending on circumstance, undertake a range of different fate options. These fate options include; symmetric or asymmetric self-renewal division (expanding or maintaining the HSC pool), dividing into cells that both differentiate into maturing blood cells (reduction of the HSC pool), entering a state of inactive hibernation (quiescence), moving into other environments (migration), losing the capacity to divide (premature senescence), or to terminate by controlled cell death (apoptosis). HSC Sensitivity Various cell types have different tolerances to stressful insults such as harmful chemicals or ionizing radiation. The HSC has been demonstrated to be more sensitive to genotoxic insult as compared to the downstream effector cells in the hematopoietic hierarchy (Milyavsky et al., 2010). The central role of self-renewing HSCs to continuously resupply the hematopoietic system, combined with the high stress sensitivity of the HSC, makes the hematopoietic system one of the most radiosensitive organs of the body. Thus, full body radioactive exposure often results in lethal outcomes due to the collapse of the hematopoietic system (bone marrow failure). In steady state most HSCs are dividing infrequently, have minimal metabolic activity, and are suspended in a state of inactive hibernation called quiescence. If the body is in urgent need of more blood cells, quiescent Zzz... Differentiation Asymmetric Self-renewal Symmetric Self-renewal Quiescence Migration Premature Senescence Apoptosis
23 HSCs are capable of activating to facilitate rapid restoration of hematopoietic homeostasis. Since actively dividing cells are more prone to suffer from endogenous stress/damage as a result of increased cellular respiration and DNA replication it is believed that quiescence, by minimizing such stress, is an essential protective mechanism intended to ensure long-term performance of the otherwise stress sensitive HSC (Orford and Scadden, 2008). However, quiescence does not directly protect HSCs from external sources of stress. If faced with DNA damage, non-cycling human HSCs have limitations on how to carry out DNA repair, as more accurate DNA repair by Homologous Recombination (HR) (requiring cell cycling) cannot be utilized. This forces the quiescent HSC to rely on more error- prone Non-Homologous End Joining (NHEJ), more likely to result in genomic instability and loss of cellular function (Mohrin et al., 2010). The reduced metabolic activity associated with quiescence is believed to protect HSCs from the damaging effects of Reactive Oxygen Species (ROS) (Ito and Suda, 2014). ROS are highly reactive oxygen containing molecules, typically generated as a by- product of OXPHOS-based metabolism, that can cause oxidative damage to cellular components including DNA, proteins and lipids (Finkel, 2005). It has been suggested that accumulation of such oxidative damage contributes to the phenotypes associated with both aging and the development of cancer, as argued by the free radical theory of aging (Dröge, 2002; Harman, 1955). HSCs are highly sensitive to increased levels of ROS, with ROS-mediated oxidative damage impairing the self-renewal and long-term engraftment capacity of these cells (Ito et al., 2006; Jang and Sharkis, 2007; Yahata et al., 2011) (Figure 4). It is possible that ROS may also influence the performance of hematopoietic progenitors generated in a laboratory setting but this remains be elucidated.
24 Figure 4. HSC sensitivity to Reactive Oxygen Species. Human HSCs are highly sensitive to increased amounts of Reactive Oxygen Species (ROS), and their performance is inversely correlated with their ROS level. Quiescent HSCs, and self-renewing HSCs are both characterized by low ROS levels. Further increase of ROS results in HSC exhaustion through; differentiation, premature senescence, and apoptosis. Illustration was inspired from figures in Ludin et al., 2014, and Zhou et al., 2014. Embryonic Development Embryonic development is the process whereby the cellular progeny of a fertilized egg progressively differentiates towards specific cell fates, while at the same time becoming organized into specific patterns and structures, required for the formation of tissues, organs, and the higher body structure of a new organism. Thus, by studying the mechanisms of embryonic development, it becomes possible to define the conditions that facilitate the formation of specific cell types. Pluripotent Stem Cells Pluripotent Stem Cells (PSCs) are self-renewing cells that have the potential to differentiate into any cell type of the adult body. Upon fertilization (conception), the first cell of the new organism (the zygote) and its immediate cellular progeny are totipotent, having the potential to form all the cell types of the developing embryo and also extra-embryonic structures such as the placenta. During these early stages of development, specialization begins and the first pluripotent stem cells, now restricted to develop only into tissues of the embryo itself, form in the Zzz... Q uiescence Prem atureSenescence Self-renew al D ifferentiation A poptosis HSC Performance ReactiveOxygen Species
25 inner cell mass (ICM) of the blastocyst. Pluripotent stem cells that originate from the ICM of a blastocyst are known as Embryonic Stem cells (ES cells). ES cells were first derived from murine embryos in 1981 by two independent groups (Evans and Kaufman, 1981; Martin, 1981) together providing methods to cultivate and expand these cells in vitro. Later, human ES cells were successfully isolated from the inner cell mass of human blastocysts (Thomson et al., 1998). Due to the propagation potential of human ES cells, and due to their capacity to differentiate into adult cell types, they have a potential application in therapies that feature transplantation, regeneration, or tissue engineering. But, use of human ES cells has been a subject of ethical debate since the extraction and derivation of ES cells result in the destruction of early embryos. However, pluripotent stem cells can also be generated artificially by inducing pluripotency in adult somatic cell types. Such induced Pluripotent Stem cells (iPS cells) were first reported in 2006 when combinatorial overexpression of 4 transcription factors (c-Myc, Klf4, Oct4, and Sox2) induced pluripotency in murine fibroblasts (Takahashi and Yamanaka, 2006), followed later with the generation of human iPS cells (Takahashi et al., 2007). Similar to ES cells, iPS cells are pluripotent, capable of self-renewal, and can differentiate into all the cell types of the adult body (Figure 5). Commonly used markers for hPSCs includes staining positive for Alkaline Phosphatase (AP), Tra-1-81, Tra-1-60, OCT4, NANOG, SSEA3, and SSEA4 (Park et al., 2008). Functional evaluation of pluripotency in cells is possible by testing the cells capacity to form teratomas upon transplantation into immunodeficient mice (Lensch et al., 2007). If the transplanted cells form cystic masses featuring derivates of all 3 embryonic germ layers then the transplanted cells are considered to be pluripotent.
26 Figure 5. The Potential of Pluripotent Stem Cells. Pluripotent Stem Cells (PSCs) can either be isolated as Embryonic Stem cells (ES cells) from the Inner Cell Mass (ICM) of blastocysts, or generated as induced Pluripotent Stem cells (iPS cells) from somatic cell types through reprogramming by overexpressing specific transcription factors. PSCs can theoretically self-renew indefinitely, and have the capacity to differentiate into all cell types of the adult body. This includes derivates of the ectoderm (such as nerve cells), the mesoderm (such as vascular endothelial cells), and the endoderm (such as intestinal epithelial cells). Developmental Specification During embryonic development the blastocyst (also called blastula), containing the pluripotent stem cells of the inner cell mass, begins the process of gastrulation. During gastrulation the blastocyst invaginates and reorganizes itself into a three- layered structure (gastrula), with the pluripotent stem cells becoming committed into the 3 germ layers. Each germ layer gives rise to specific tissues and organs during the developmental process, with the ectoderm (outer layer) forming tissues including the epidermis (skin) and the nervous system, the mesoderm (middle layer) forming muscle based tissues and the hematopoietic system, and the endoderm (inner layer) forming the interior lining of the digestive system and the respiratory system. As the process of specification continues, epigenetic mechanisms (including methylation and chromatin condensation) gradually restrict access to parts of the genome that contains genetic information no longer required for further specification towards downstream developmental derivates. While such gradual specification restricts the number of possible cell fates available to the cell, the “choice” among available cell fates is instructed by spatial (positional) Somatic Cell Induced Pluripotent Stem Cells (iPS Cells) Embryonic Stem Cells (ES Cells) Pluripotent Stem Cell c-Myc Klf4 Oct4 Sox2 ICM Isolation Reprograming Blastocyst Ectoderm Mesoderm Endoderm
27 information. To illustrate, if 3 identical precursors are positioned with differing distance from a tissue that secretes a gradient of a differentiation-inducing factor (morphogen), these cells will be “instructed” by their relative position, since the exposure level of the morphogen determines cell fate commitment (Figure 6). One important morphogen that instructs towards hematopoietic development is the cytokine BMP4. BMP4 takes part in the sub-specification of early mesoderm during embryonic development as demonstrated by studies in birds (Pourquié et al., 1996; Tonegawa et al., 1997), and it is crucial for the successful establishment of both vasculature and hematopoiesis. Similar findings were also reported in mice where targeted mutagenesis of the Bmp4 gene compromised both blood and endothelial cell development (Winnier et al., 1995). Thus, positional information (communicated by morphogen exposure), combined with genetic/epigenetic information (temporal cell identity), determines cellular response and lineage commitment, together enabling the formation of distinct cell types organized in characteristic patterns required for tissue formation and higher body structure (Wolpert, 1969). Figure 6. Positional Information by Morphogen Exposure Instruct Cell Fate Commitment During Embryonic Development. This illustration provides a simplified model for how morphogen gradients serve to instruct commitment of precursor populations into different developmental lineages. The positional information, established by the exposure level of the morphogen (due to the distance to the morphogen source), instruct the precursors to differentiate towards specific cell fates. Cell fates that depend on high morphogen exposure are morphogen instructive, while cell fates that depend on low exposure are morphogen permissive. Retinoic Acid During Embryonic Development A morphogen (“form-maker” in Greek) denotes diffusible biochemical molecules that instruct cell fate by concentration. Several cytokines are known to act as morphogens during the developmental process, including members of the FGF, Cell Fate A (Morphogen Instructive) Cell Fate C (Morphogen Permissive) Cell Fate B Morphogen Gradient
28 WNT, Hedgehog, and TGF-β families (Kardel and Eaves, 2012). Retinoic Acid (RA), a derivate of Vitamin A, is a central morphogen required for successful embryonic development in all higher animal species ranging from fish to humans (Ross et al., 2000). RA is synthesized in cells that express the enzyme retinaldehyde dehydrogenase 1, 2, or 3 (RALDH1, RALDH2, RALDH3). Due to its diffusive nature, gradients of RA form across regions of the developing embryo, providing positional information and contributing to both patterning and cell differentiation along the embryonic anterior/posterior axis (Ross et al., 2000). When available, RA enters the nucleus where it binds to members of the Retinoic Acid Receptor (RAR) family of nuclear receptors that, in turn, form heterodimeric complexes with members of the Retinoid-X-Receptor (RXR) family. These then localize to specific Retinoic Acid Response Elements (RAREs) in promoter regions of the genome to drive transcription of RA target genes (Duester, 2008; Kumar and Duester, 2011). The use of animal models to study the effects of RA deficiency has proved essential for determining the functions of RA during embryonic development, with RA required for the development of several organs and tissues including the eye, hindbrain, spinal cord, heart, lung, pancreas and the skeleton (Duester, 2008). Studies where additional RA was added during the developmental process have also demonstrated that RA is a potent teratogen (“monster-maker” in Greek), due to the severe malformations that result from increased RA during embryogenesis (Ross et al., 2000). Because of its teratogenic nature, pregnant women must avoid exposure to external sources of RA due to the high risk of abnormal embryonic development and birth defects. Since RA instructs cell fate through exposure, there are cell fates that depend on either high or low RA for successful commitment. Accordingly, cell fates that depend on low RA exposure are RA-permissive, while cell fates that require high RA exposure are RA-instructive. For instance, the initial commitment towards ectoderm is increased, at the expense of the other germ layers, if additional RA is added (Bain et al., 1996). Thus, ectodermal commitment is RA-instructive. During the later developmental stage where sub-specification of Lateral Plate Mesoderm (LPM) occurs, RA has been demonstrated to direct specification towards cardiac tissue (anterior LPM) at the expense of hematopoietic development as observed in Xenopus (Deimling and Drysdale, 2009), zebrafish (de Jong et al., 2010), and mouse (Szatmari et al., 2010). These findings indicate that specification towards the hematopoietic lineage at this stage in development is RA-permissive. However, it remains to be elucidated if this also applies to the human setting. Hematopoiesis During Embryonic Development During embryonic development hematopoiesis occurs at several distinct time- points and anatomical locations (Dzierzak and Speck, 2008; Kardel and Eaves,
29 2012; Clements and Traver, 2013). These separate hematopoietic events are broadly classified as belonging to the primitive, or the definitive, wave of hematopoiesis. The primitive wave features the formation of the first hematopoietic cells during the development of the embryo. In the mouse this process begins at embryonic day 7 (E7) when myeloid-erythroid progenitors are formed in extra-embryonic structures including the Yolk Sac (YS) and the allantois (which will later contribute to the formation of the umbilical cord) (Moore and Metcalf, 1970). These cells have limited self-renewal but serve to provide the embryo with the first erythroid cells (expressing embryonic globins). The definitive wave of hematopoiesis initiates around E10.5 in the mouse, with self-renewing HSCs, capable of long-term lymphoid-myeloid engraftment, emerging at the ventral side of the dorsal aorta located in the anatomical region of Aorta Gonad Mesonephros (AGM) (Müller et al., 1994; Medvinsky and Dzierzak, 1996). Other anatomical sites have also been identified as harboring definitive hematopoietic cells, including the arteries connecting the YS (Gordon-Keylock et al., 2013), the placenta (Ottersbach and Dzierzak, 2005; Rhodes et al., 2008), as well as the head (Li et al., 2012). Similar waves of primitive and definitive hematopoiesis are present during human embryonic development, with myeloid- erythroid progenitors first appearing in the YS (Huyhn et al., 1995) and self- renewing HSCs with lymphoid-myeloid capacity later detected in the AGM region (Tavian et al., 2001). After the emergence of definitive HSCs, these cells travel through the circulatory system to colonize the fetal liver. The fetal liver provides a temporary environment where the HSCs expand in number, while also giving rise to large quantities of mature hematopoietic cells required to support the continued development of the growing organism (Mikkola and Orkin, 2006). Following the expansion phase in the fetal liver the HSCs migrate into the newly formed bone marrow, where they are housed and functionally maintained during the remaining lifespan of the adult (Kardel and Eaves, 2012). Differentiation of murine ES cells towards the hematopoietic lineage demonstrated that hematopoietic and endothelial cells develop from a shared precursor (Choi et al., 1998), supporting an earlier hypothesis that a mesodermal precursor, referred to as the hemangioblast, gives rise to both blood cells and vascular cells during embryonic development. Hemangioblast cells express Flk1 (Ema et al., 2003; Fehling et al., 2003), and ES-derived Flk1+ cells have been demonstrated to form blood through a transient endothelial state, prior to up-regulating hematopoietic markers and down-regulating endothelial markers (Mikkola et al., 2003). This transition of endothelial cells to hematopoietic cells was later observed in mouse embryos for both primitive wave hematopoiesis in the YS, as well as for definitive hematopoiesis in the AGM region (Lancrin et al., 2009). While embryonic development features separate hematopoietic events at multiple anatomical locations, the central features are that hematopoietic cells and vascular
30 endothelium develop from a shared mesodermal precursor, and that the emergence of hematopoietic cells occurs by transition through a hemogenic endothelial (HE) state (Costa et al., 2012). Indeed, hematopoietic progenitor cells have been directly confirmed to bud from endothelial precursors lining the luminal space on the ventral side of the dorsal aorta in murine embryos (Boisset et al., 2010), and has been additionally verified by lineage tracing experiments (Zovein et al., 2008). Live-cell imaging has also confirmed the emergence of blood from murine HE generated in vitro (Eilken et al., 2009). Studies aimed at characterizing this process of Endothelial to Hematopoietic Transition (EHT) have revealed the involvement of several intrinsic and extrinsic factors (Costa et al., 2012). The transcription factor Scl has been demonstrated to be critical for the establishment of HE from Flk1+ hemangioblast cells (D’Souza et al., 2005). Transition of HE into definitive hematopoietic progenitors is then critically dependent on the activity of transcription factor Runx1 (Yokomizo et al., 2001), with runx1 expression at the dorsal aorta overlapping with the emergence of hematopoietic cells as demonstrated by live imaging of zebrafish embryos (Lam et al., 2010). Runx1 then regulate EHT by acting through the two transcription factors Gfi1 and Gfi1b (Lancrin et al., 2012). Runx1 itself is regulated by both internal and external factors. For instance, Scl has been demonstrated to directly regulate the expression of Runx1 (Nottingham et al., 2007; Landry et al., 2008), and Bmp4 signaling, mediated by Smad1, is also capable of regulating Runx1 expression (Pimanda et al., 2007). Notch activity has also been demonstrated to induce runx1 expression and HSC expansion during embryonic development in zebrafish (Burns et al., 2005), and it has recently been reported that hPSC-derived HE depend on NOTCH signaling for EHT (Ditadi et al., 2015). Furthermore, Runx1 expression is positively regulated by biomechanical forces, mediated by nitric oxide based signaling mechanisms, due to fluid shear stress from the circulation (Adamo et al., 2009). Catecholamines, a class of neurotransmitters, have also been demonstrated to play a role in the emergence of HSCs at the murine AGM. Knockout of the enzyme tyrosine hydroxylase, responsible for synthesizing catecholamines, led to reductions in repopulating HSCs at the murine AGM, which could then be rescued by catecholamine supplementation (Fitch et al., 2012). Catecholamines are known to bind to β-adrenergic receptors on the cell surface, with downstream signaling mediated through cAMP pathways, thereby suggesting a possible role for cAMP in EHT.
31 Figure 7. Establishment of Hemogenic Endothelium, and the Emergence of Hematopoietic Cells through Endothelial to Hematopoietic Transition. The upper part of the figure illustrates the emergence of budding hematopoietic progenitors from Hemogenic Endothelium (HE) at the ventral side of the dorsal aorta. The lower part of the figure provides a simplified schematic over key transcription factors and external mediators involved in the establishment of HE, and the emergence of blood from HE through Endothelial to Hematopoietic Transition (EHT). The illustration was inspired by figure 3 and 4 in Costa et al., 2012. Working in the murine setting has allowed for in vivo observations to directly complement findings based on differentiating murine pluripotent stem cells towards blood in the laboratory. Since the process of EHT cannot be directly studied in human embryos due to ethical reasons, in vitro differentiation of hPSCs provides an alternative approach to study this process in the human setting. Differentiation of hPSCs demonstrated that a hemangioblast population, comparable to the murine counterpart, exists in the human setting (Kennedy et al., 2007). More recently, with the aim of identifying and characterizing human HE, several groups have successfully differentiated hPSCs into HE that express RUNX1 and VE-Cadherin, capable of giving rise to definitive hematopoietic progenitors with lymphoid-myeloid potential (Choi et al., 2012; Kennedy et al., 2012). Recently, the surface marker phenotype CD34+ CD73- CXCR4- has been reported to separate human HE from committed vascular endothelium (Ditadi et al., 2015). While these reports provide evidence that HE can be generated from hPSCs, full consensus on appropriate markers remains to be reached, and the developmental relationship between cells undergoing EHT remains to be elucidated. Scl Runx1 Gfi1 Gfi1b AorticLumen Mesodermal Precursor (Hemagioblast) Hemogenic Endothelium Hematopoietic Stem Cell Bmp4 Smad1 Shear Stress EHT Budding Hematopoietic Progenitors Hemogenic Endothelium Aortic Endothelium
32 In Vitro Differentiation of hPSCs Towards HSCs The Potential of in Vitro HSC Generation from hPSCs During treatment of patients suffering from hematological malignancies myeloablative conditioning, including radiotherapy and/or chemotherapy, is employed to facilitate eradication of malignant cells. During such treatment the potentially healthy HSCs in the patient are also destroyed. Therefore, re- introduction of functional HSCs after myeloablative treatment, often by transplantation of donor-derived cells, allows for the re-establishment of hematopoiesis and thereby the survival of the patient. However, donor-derived transplantation does not come without potential complications. Human Leukocyte Antigen (HLA) compatibility between donor and patient is required to reduce the risk of Graft Versus Host Disease (GVHD), where the establishing immune system of the transplant recognize the cells of the patient as foreign, commencing an immunological response. GVHD is highly associated with mortality in transplanted patients (Copelan, 2006) and due to the need for high HLA compatibility the number of available and compatible donors can be limited. Another potential limitation is due to the variable quality of donated HSCs. Aged HSCs have been demonstrated to have a reduced function as compared to young HSCs (Morrison et al., 1996; Sahin and DePinho, 2010). In addition, the number of available HSCs in a transplant may be too few to allow for successful and efficient repopulation of the patient (Brunstein et al., 2010; Doulatov et al., 2012). Thus, methods to grow and expand available self-renewing HSCs in vitro have long been pursued. However, while it is possible to expand cells from HSCs that carry the correct cell surface markers, self-renewal of HSCs cultured in vitro is rapidly lost, severely limiting the efficiency of such approaches (Walasek et al., 2012). It has been proposed that the loss of HSC self-renewal in vitro is due to the absence of the conditions associated with the bone marrow niche. To summarize, there is a pressing need for HLA-compatible HSCs, in high quantities and of good clinical quality, to ensure optimal treatment of myeloablated patients. Since the discovery of pluripotent stem cells, the potential use of these cells as a source for generating HSCs have presented an attractive goal for the scientific community. The indefinite expansion capacity of pluripotent stem cells, combined with the possibility of creating pluripotent stem cells from individual patients (patient-specific iPS cells) could, in principle, allow for the generation of patient- derived HSCs. The HLA compatibility of patient specific iPS-derived HSCs, together with the potential possibility of providing cells with high quality and in sufficient numbers, could dramatically improve the treatment of patients in the clinic (Figure 8). This also includes the possible correction of genetically carried
33 hematological diseases if applying additional methods of gene therapy. Furthermore, successful generation of HSCs could provide an improvement to better study the nature of hematological malignancies, and also for the purpose of drug screening (Vo and Daley, 2015). Figure 8. The Potential of Hematopoietic Stem Cells Generated from Pluripotent Stem Cells. Successful generation of functional human HSCs, of good clinical quality and in large quantity, could improve the treatment of patients with hematological malignancies. Theoretically, small amounts of somatic cells collected from the individual patient, could be reprogrammed into patient-specific iPS cells. If needed these cells could then be corrected for inherited or acquired mutations by methods of gene therapy. The patient-specific iPS cells could then be expanded in the laboratory before being instructed to differentiate into large quantities of functional HSCs. The generated HSCs would then be transplanted back into the patient after myeloablative conditioning, thereby re- establishing the hematopoietic system of the patient while circumventing the issue of HLA compatibility. Such patient- specific replacement therapy could provide new and safer treatments against a multitude of hematological malignancies. Methodology Many studies have reported protocols for generating hematopoietic cells from hPSCs. These protocols typically feature differentiation of hPSCs by Embryoid Body (EB) formation, alternatively differentiating the hPSCs as a monolayer. Additional approaches include culturing the cells on supportive stroma or extracellular matrix, with media either containing serum (undefined) or having a defined composition, supplemented with specific morphogens and growth factors to encourage the commitment of hPSCs towards the hematopoietic lineage (Kaufman et al., 2001; Chadwick et al., 2003; Vodyanik et al., 2005; 2006; Ledran et al., 2008; Choi et al., 2012; Kennedy et al., 2012; Ditadi et al., 2015). The cultures are then analyzed for HSC-like cells by methods including; cell surface marker analysis using flow cytometry, gene expression analysis, lymphoid- myeloid differentiation potential, and long-term engraftment capacity by transplantation into immunodeficient recipient mice. Interestingly, these approaches to differentiate pluripotent stem cells towards the hematopoietic lineage in vitro show considerable parallelism to embryonic blood development + Somatic Cells Pluripotent Stem Cells Hematopoietic Stem Cells Patient Drug Screening Disease Study
34 (Kardel and Eaves, 2012). This includes the initial mesodermal commitment, followed by an early wave of primitive hematopoiesis, and later the onset of definitive hematopoiesis as indicated by the emergence of hematopoietic progenitors with lymphoid-myeloid potential (Choi et al., 2012; Kennedy et al., 2012; Timmermans et al., 2009). Recently, bioinformatics have provided a new method to study hematopoietic development. Using computational methods of analysis to interpret biological data, bioinformatics can be used to identify candidate genes of interest for the purpose of understanding the genetic foundation of a biological process. Combined with high throughput analysis at the single cell level, bioinformatics could provide a powerful approach to elucidate the details of EHT but so far this has not been reported for the murine, or for the human setting. There have been several important discoveries indicating that obtaining functional HSCs from hPSCs should be possible (see next section). However, generation of hPSC-derived HSCs that are functionally comparable to true HSCs remains a challenge. It is generally assumed that either a lack of a HSC-specific gene signature, or a lack of appropriate developmental cues required for correct developmental specification, could explain the difficulty in generating self- renewing HSCs from hPSCs in vitro (Mikkola and Orkin, 2006; Murry and Keller, 2008; Vo and Daley, 2015). Previous Key Discoveries Human ES cells were first successfully differentiated (in vitro) into hematopoietic colony forming cells in 2001 (Kaufman et al., 2001). Later in 2002, murine ES cells, by overexpression of the transcription factor HoxB4, were differentiated into HSC-like cells that could self-renew and had lymphoid-myeloid engraftment potential (Kyba et al., 2002). The same method was used in conjunction with corrective gene therapy to cure immunodeficiency in Rag2- /- mice, demonstrating by proof-of-concept the therapeutic potential of pluripotent stem cells (Rideout et al., 2002). The concept of iPS cells was introduced in 2006 when murine iPS cells, generated from fibroblasts, were reported (Takahashi and Yamanaka, 2006). One year later, the same authors reproduced their findings in the human setting, generating the first human iPS cells (Takahashi et al., 2007). In 2007, it was also demonstrated that murine iPS cells could be used for autologous correction of sickle cell anemia in mice (Hanna et al., 2007). By 2008, human iPS cells were generated from patients with a variety of genetic diseases including adenosine deaminase deficiency-related severe combined immunodeficiency, Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher
35 disease (GD), Parkinson disease (PD), Down syndrome (DS), and more (Park et al., 2008), providing new means to study these diseases, and also a platform for the possible employment of corrective gene therapy. 5 years later in 2013, short-term myeloid-erythroid engraftment with cells generated from hPSCs was demonstrated by means of overexpressing 5 transcription factors (HOXA9, ERG, RORA, SOX4, and MYB) (Doulatov et al., 2013). The same year, long-term engrafting HSCs were extracted from human iPS-derived teratomas in mice (Amabile et al., 2013; Suzuki et al., 2013) providing evidence that functional HSCs can be generated from hPSCs in vivo. And in 2015, it was reported that co-culture of differentiating non-human primate iPSCs together with human endothelial cells (expressing DLL4 and JAG1) enabled generation of cells capable of long-term lymphoid-myeloid engraftment and serial transplantation (Gori et al., 2015). The same paper also differentiated human ES cells in a similar manner and demonstrated long-term lymphoid-myeloid engraftment in mice. However, serial transplantation was not reported raising the possibility that the generated hPSC-derived cells were long-lasting Multipotent Progenitors (MPPs). Together, colleagues in the scientific community have made significant progress that brings us closer to the final goal of improving therapies against hematological malignancies. Unfortunately the generation of hPSC-derived HSCs with engraftment capacity on par with bona fide HSCs has not yet been achieved.
36
37 Aims of the thesis With the ultimate goal of facilitating in vitro generation of functional Hematopoietic Stem Cells (HSCs) from human Pluripotent Stem Cells (hPSCs), the studies included in this thesis explore potential key factors and mechanisms involved in the process of hematopoietic development. More specifically, the aims were:  To evaluate the effects of Retinoic Acid (RA) on the development of blood and on generated hematopoietic progenitors (Paper I).  To molecularly dissect the process of Endothelial to Hematopoietic Transition (EHT) in the human setting (Paper II).  To explore the functions of cyclic Adenosine Mono Phosphate (cAMP) on the emergence of hematopoietic progenitors (Paper III).  To investigate the impact of Reactive Oxygen Species (ROS) on the functionality of generated hematopoietic progenitors (Paper IV).
38 New links must be forged as old ones rust. - Jane Howard
39 Summary of results Paper I: Retinoic Acid Regulates Hematopoietic Development from Human Pluripotent Stem Cells In Paper I, we dissected the role of Retinoic Acid (RA) signaling in human hematopoietic development by using in vitro differentiation of hPSCs towards the hematopoietic lineage. We report that RA decreases blood generation during this process. RA signaling inhibition, before the onset of hematopoiesis, establishes an increase in the final output of cells with an HSC-like surface marker phenotype, confirming the role of RA signaling in the early stages of human blood development. qRT-PCR analysis for genes marking specific developmental cell types identified the stages during development where decreased RA signaling increased commitment towards the hematopoietic lineage. This included the initial commitment of pluripotent stem cells to mesoderm, and also by increased specification toward hemangiogenic mesoderm while reducing cardiac mesoderm differentiation. These results suggest that increased levels of RA antagonize blood development, and that reduced RA facilitate increased generation efficiency of hematopoietic progenitors from hPSCs. Furthermore, we show that RA accelerated the differentiation of generated hematopoietic progenitors, and that reduction of RA signaling preserved the generated progenitor fraction from differentiation into more mature cell types. Taken together, we have provided evidence identifying multiple developmental stages that depend on restricted RA signaling for efficient differentiation towards blood, and we have shown that RA signaling inhibition can prevent loss of blood progenitors generated in vitro. Our findings show the utility of differentiating pluripotent stem cells in vitro to study human hematopoietic development, and to determine the effects and functions of RA in this process.
40 Graphical Abstract for Paper I. Paper II: Endothelial to Hematopoietic Transition State Identified in Differentiated Human Pluripotent Stem Cells In paper II, using iPS in vitro differentiation, we proposed a model for Endothelial to Hematopoietic Transition (EHT) in the human setting. Single-cell transcriptional analysis of index-sorted cells has allowed us to molecularly identify an EHT transcriptional state within a novel EHT differentiation hierarchy, where a subset of cells (termed 5b subset) express high levels of numerous key HSC regulators while also displaying a clear endothelial program. Together, Principal Component Analysis (PCA) and unsupervised hierarchical clustering analysis placed these cells at the interface between the endothelial and the hematopoietic populations. Also, index-sorting allowed for a molecular characterization of the currently best functionally described cell types in the human setting, i.e. Hemogenic Endothelium (HE) and HSC-like cells, within our EHT hierarchical model, placing the 5b subset developmentally between these functional cell types. Finally, correlation of the transcriptional profile of our identified successive groups with existing genome-wide transcriptional analysis data of bulk populations of interest from the mouse AGM, further supports our EHT hierarchical model and inferred cell program identities. Human Pluripotent Stem Cells High RA High RA Low RA Low RA Blood Progenitor Cells Differentiated Blood Cells
41 In summary, with single-cell analysis for 90 genes, relevant for both endothelial and hematopoietic regulation, we show for the first time, at a single-cell level and in the human setting, the existence of an EHT program at the interface between endothelial identity and hematopoietic commitment. Graphical Abstract for Paper II. Paper III: Cyclic AMP Induction Enhances Human Hematopoietic Cell Emergence Through the cAMP-Epac Signaling Axis, Creates Redox State Balance, and Increases CXCR4 Expression. In paper III, we explored the functions of cAMP on the emergence of HSC-like cells generated from hPSCs. Our findings demonstrate that cAMP, by signaling through the Epac axis, regulates the in vitro specification of hPSC-derived HSC- like cells. In addition to up-regulating the frequency of HSC-like cells in the differentiation cultures, cAMP induction also mitigated oxidative stress, created redox-state balance, and enhanced CXCR4 expression in hPSC-derived hematopoietic cells. Furthermore, we demonstrated that by inhibiting the downstream effectors of cAMP signaling, PKA and Epac, only Epac signaling is required for hematopoietic emergence during hPSC differentiation towards the hematopoietic lineage. Epac regulates endothelial cell-cell adhesion, and the involvement of endothelial cells for Endothelial to Hematopoietic Transition Hemogenic Endothelium HSC-like EHT HOXB4 MEN1 SPI1 RUNX1 LIN28B MEIS1 FLI1 ETV6 VAV1 LYL1 GATA2 CBFA2T3 TAL1 NFE2 BMI1 5 6 5b 74 Endothelial Genes WAS-GFP HSC Genes
42 (EHT) during the emergence of hematopoietic cells suggests a novel link between endothelial cell junctions and EHT. Epac inhibition resulted in impairment of Hemogenic Endothelium (HE) specification, reduced blood emergence, and decreased the numbers of HSC-like cells from hPSCs, thus demonstrating the critical role of Epac signaling in the specification of human HE and the generation of hPSC-derived HSC-like cells. Collectively, our findings show that cAMP regulates the generation of human HSC-like cells during in vitro hematopoiesis. By demonstrating the role of the cAMP-Epac axis in blood and HSC emergence, our study provides new insights in understanding the previously unknown role of the cAMP-signaling component in human hematopoietic development. Graphical Abstract for Paper III. Paper IV: Reactive Oxygen Species Impair the Function of Hematopoietic Stem / Progenitor Cells Generated from Human Pluripotent Stem Cells In Paper IV, we investigated the impact of ROS on hematopoietic progenitors generated in vitro from hPSCs. We report that the vast majority of all hPSC- derived hematopoietic progenitors display an increased ROS level, as compared to cord blood non-cultured progenitors. High ROS levels were associated with reduced colony forming capacity and increased DNA damage. Of hPSC-derived HSC-like cells, only cells with reduced amounts of ROS could proliferate upon sub-culture, demonstrating the detrimental impact of elevated ROS on these cells. Furthermore, we report 4 separate factors capable of reducing ROS in our cultures, Hemogenic Endothelium Human Pluripotent Stem Cells hPSC-Derived HSC-Like Cells ROS Redox Balance CXCR4 Cyclic AMP Epac
43 demonstrating that ROS build-up in vitro results not only from cellular metabolism but also from multiple processes including innate immunity, the cellular stress response, and the atmospheric oxygen concentration. Combining these factors greatly reduced the level of ROS and increased the generation of ROSlo hPSC-derived HSC-like cells capable of robust proliferation. Taken together, we have provided evidence that increased ROS, associated with multiple processes during in vitro culture, leads to functional decline in the majority of hematopoietic progenitors generated from hPSCs. Methods to normalize ROS towards physiological levels enabled a > 20-fold increased production of HSC-like cells with a reduced ROS phenotype and robust proliferative capacity, indicating that oxidative stress is occurring during standard in vitro culture and that it is functionally detrimental to these cells. Thus, normalization of ROS toward physiological levels will be a key requirement for future in vitro generation of engrafting HSCs. Graphical Abstract for Paper IV. ROS ReactiveOxygen Species hPSC-Derived HSC-LikeCells Functionality Antioxidants Innate Immunity Blocking Stress Response Inhibition 4% Oxygen Condition + + + + ROS Functionality
44
45 Conclusions  Decreased retinoic acid signaling increases the generation of hematopoietic progenitors from human pluripotent stem cells in vitro (paper I).  Retinoic acid signaling inhibition increases the early developmental commitment toward the hematopoietic lineage (paper I).  Retinoic acid signaling inhibition prevents differentiation and loss of more immature hematopoietic progenitors generated in vitro (paper I).  Single-cell transcriptional analysis of index-sorted cells provides molecular identification of an EHT transcriptional state in the human setting (paper II).  A proposed model for EHT argues for a linear progression from endothelial to hematopoietic commitment that features an intermediate stage (paper II).  cAMP, mediated through the Epac signaling axis, regulates the generation of HSC-like cells from human pluripotent stem cells in vitro (paper III).  Increased cAMP signaling is associated with reduced oxidative stress, redox-state balance, and enhanced CXCR4 expression in generated hematopoietic cells (paper III).  The majority of hPSC-derived hematopoietic progenitors have increased ROS levels, originating from multiple processes (paper IV).  ROShi hematopoietic progenitors have reduced colony forming capacity and increased DNA damage (paper IV).  A ROSlo phenotype is a prerequisite for proliferative capacity in hPSC- derived HSC-like cells (paper IV).  Combinatorial ROS reduction increases the generation of hPSC-derived HSC-like cells with proliferative capacity (paper IV).
46 It’s not what you look at that matters, it’s what you see. - Henry David Thoreau
47 General discussion and future perspectives In vitro generation of functionally relevant HSCs from hPSCs has the potential to revolutionize the treatment of hematological diseases and malignancies. In this thesis, by using in vitro differentiation of hPSCs towards the hematopoietic lineage, we elucidate several novel aspects of this process. By controlling the amount of retinoic acid we increase in vitro specification of hPSCs toward blood, allowing for a more defined and efficient development of relevant cells. Our molecular identification of EHT, the conversion through which HSCs emerge from endothelial cells provides a foundation for further characterization of cells undergoing this process, and could potentially enable control over this transition for the purpose of more efficient generation of hematopoietic cells from hemogenic endothelium. Increased understanding on how cAMP intrinsically regulates the generation of HSC-like cells, together with the ability to modulate it, provides an additional approach to increase the generation, and potentially the function, of HSC-like cells from hPSCs. Finally, the finding that reactive oxygen species is increased due to the in vitro culture conditions, causing functional impairment of hPSC-derived hematopoietic stem and progenitor cells, argues that we must revise our standardized culture methods and that a more physiological oxygen concentration is likely to be a critical component for handling and generating HSCs outside of the body. Unfortunately, while our work provides new insights into the process of hematopoietic development and HSC emergence, we have not been able to generate true HSCs capable of robustly engrafting into recipient mice. As mentioned previously, the scientific community has long pursued the successful generation of functionally relevant HSCs from hPSCs and over the years there has been significant progress bringing us closer to the final goal of improving therapies against hematological malignancies. However, the generation of hPSC- derived HSCs with engraftment capacity on par with bona fide HSCs has not been achieved. Theories as yet to why the generation of functional HSCs has remained such a challenge include a possible lack of specific developmental cues required for
48 correct specification of hPSCs towards the HSC, or alternatively that a gene regulatory signature of HSC self-renewal is missing (Mikkola and Orkin, 2006; Murry and Keller, 2008; Vo and Daley, 2015). I would argue that both theories mentioned above are based on assumptions that something is “lacking” for successful HSC generation,and that such approaches forget to consider the possibility that HSC function (and thereby generation) instead could be antagonized by external factors. In other words, assuming that a problem is complex and therefore looking for a complex solution, is risky if more simplistic possibilities have not been thoroughly evaluated. Illustration created by Hanna Rönn (2015). Based on findings included in this thesis, I would therefore like to propose an alternative theory where ROS, primarily resulting as an external artifact from our standardized in vitro culture conditions, cause functional impairment of cells that otherwise could have performed more satisfactory. Thus, correction of ROS would technically be a correction of a methodological oversight, rather than an improvement on developmental commitment or directly establishing an HSC gene signature. I would like to provide a range of arguments why this Model of Functional Impairment by External Oxidative Insult may be relevant for the generation of functional HSCs from hPSCs in vitro.
49 Firstly, generation of robustly engrafting HSCs from hPSCs is possible by in vivo teratoma formation (Amabile et al., 2013; Suzuki et al., 2013). Not only is this proof-of-principle that functional HSCs can be generated from hPSCs, but also that undirected and fully stochastic differentiation, giving rise to only a small percentage of blood, is sufficient for successful HSC generation. This indicates that strategies to further improve the developmental commitment towards the hematopoietic lineage are unlikely to confer HSC functionality. Furthermore, the fact that stochastic differentiation of hPSCs in vitro does not yield the same results suggests that the in vivo aspect is critical. Secondly, the fact that HSC function is rapidly impaired upon exposure to in vitro conditions not only questions the rationale of generating functional HSCs from hPSCs in similar settings, but also suggests that this impairment is directly linked to the very in vitro culture conditions that we employ. Thus, we need to consider the “standardized” way we often culture cells in vitro. Ever since Wilhelm Roux in 1885 demonstrated that embryonic chick cells could be kept alive in saline solution outside the body, cell culture has continued to evolve. One example is the work of Hayflick and Moorhead from 1961, demonstrating that human fibroblasts cultured in vitro can only undergo a finite number of divisions (Hayflick and Moorhead, 1961). Since then, in vitro culture has been refined, allowing us to study a multitude of different cell types in the laboratory. One central aspect of typical in vitro culture is the handling of cells with an atmospheric oxygen concentration at 21%. Arguably, an atmospheric oxygen concentration is much higher than what is encountered physiologically by cells inside the body, but since most cell types retain functionality during culture with this setting the level of oxygen has not been of great concern. However, if we agree that there could be cell types that are more critically dependent on more physiological conditions, we can no longer continue to assume that our traditional in vitro culture methods are appropriate for culturing such cells. An increased oxygen concentration will result in increased spontaneous formation of ROS (Baez and Shiloach, 2014). Adult HSCs are highly sensitive to ROS and multiple studies have demonstrated that increased ROS is incompatible with long-term engraftment capacity of both murine and human HSCs (Ito et al., 2006; Jang and Sharkis, 2007; Yahata et al., 2011). Finally, it should be taken into consideration that there may be species-specific differences between human and murine cells in terms of stress resistance. It has indeed been suggested that human and murine HSCs have differences in their DNA repair and damage response mechanisms (Biechonski and Milyavsky, 2013). Mice are also more resistant to radioactive exposure in comparison to humans, further indicating a difference between the species in terms of stress tolerance (Green et al., 2007; Donnelly et al., 2010). If we accept the likely possibility of a real difference in stress resistance between human and murine cells, and we also
50 agree that in vitro culture facilitates a stressful environment for HSCs, then it provides a possible explanation as to why strategies that enabled the generation of functional HSC-like cells in the murine setting (Kyba et al., 2002) have not been successfully reproduced in the human setting, despite more than a decade of scientific effort. In paper IV we were unable to reduce the amount of ROS in our generated HSC- like cells to a level equal to that of non-cultured cord blood blood progenitors, and we did not observe robust engraftment when we transplanted these cells. Thus, it would be very interesting to evaluate the engraftment capacity of hPSC-derived HSC-like cells if methods to fully normalize the ROS level were to be developed. However, while our work does provide clear evidence that ROS is detrimental to the functionality of HSCs, we cannot exclude the possibility that additional aspects may be required to facilitate successful generation of functional HSCs. In other words there may be additional hurdles to overcome, along with sufficient ROS reduction, before functional HSCs can be generated. But in the end we still must overcome every single hurdle to reach our goal. Previously, generation of self-renewing HSC-like cells from pluripotent stem cells, capable of long-term lymphoid-myeloid engraftment, was demonstrated in the murine setting by overexpression of the transcription factor HoxB4 (Kyba et al., 2002). While artificial overexpression of genes does not technically mimic the developmental process towards the emergence of blood, it does provide an alternative route for generating functional HSCs. If successful generation of human HSCs were to be achieved by gene expression strategies, it would be interesting to see how the functions of these cells might correlate with their level of ROS. The results included in this thesis provide new insights into the process of blood development and the emergence of HSCs, bringing us closer to establishing methods for safe and efficient HSC generation suitable for clinical applications.
51 Acknowledgements Thinking about all the time I have spent at work the last 6 years and how it now might come to an end fills me with both excitement and some sadness. I remember that when I started my primary motivation was just simple curiosity, and perhaps a desire to do something that would have some lasting relevance. There are so many people that have been with me during these years and without them I would not have been able to come this far. These people have enriched my days, supported me through both the good and the bad times, and have made the time as a PhD- student one of the best times in my life. Therefore I want to acknowledge the following people: First, I am very grateful to Niels-Bjarne Woods, my Supervisor, for his never- ending support, his scientific enthusiasm, and for encouraging me to always be creative. Because of his supervision I feel confident, as I will continue my journey and my scientific career. To Stefan Karlsson, my co-supervisor, for establishing the warm and highly scientific molmed environment that we all share at A12. Without that friendly, enthusiastic, and scientifically objective environment I would not have enjoyed my PhD as much as I did. To Jonas Larsson, Head of Department, for continuing the caretaking of our great department, and for always is having the best in mind for all of us who work here. To Anna Rignell-Hydbom, Deputy Director of the Medical Faculty, and Anders Malmström, Director of the Research School, for being great sources of support for us students, watching over us during our journeys. To Teia and Zhi for all their support in the FACS core. To Qianren and Emanuela for doing an awesome job running the iPS core. To the staff in the animal facility for always taking care of our mice. To Beata and Xiaojie for running the vector core. To Karin Olsson, for all your help and for answering my endless amount of questions during the first years. To Göran Karlsson for taking care of me and my fellow students during the time in the preparatory program, and Christine Karlsson for your organization of the department and your drive to get things done.
52 To the young PIs; Mattias Magnusson, Sofie Singbrant, Kenichi Miharada, and Johan Flygare for your scientific discussions, and for sharing your experience and advice to prepare me additionally for my future in science. To the members of the Woods-group: To Roksana for sharing this journey with me from the very start. Your kindness and hard work will take you forward towards your goal. To Carolina for being a great friend and a great co-worker. Your energy, creativity, and scientific drive will take you far. To Shobhit for sharing your friendship, your experience, and for reminding me about the bigger questions. I always enjoy our coffee-break discussions. All of my office mates: Abdul, Alexander, Sandra, Hooi Min, Matilda, Maria, and Roksana for all the good times and great Youtube videos ;-) (https://www.youtube.com/watch?v=HwSAkRKcc4g). To Pekka for being a great friend and a great inspiration. I just might come and crash on your couch in Cambridge a few more times. To Roman for your friendship, generosity, Arnold quotes, and your insane passion for both laborative and medical science. House FTW! To Praveen, Valli, Shub, and Alex for great basketball sessions. And to Sarah, Sara, Alexandra, Simon, Karolina, David, Terese, Mehrnaz, Svetlana, Justyna, Visnja, Natsumi, Ilana, Carmen, Ineke, Charlotta, Kavitha, Kristian, Lina, and all current and previous co-workers at BMC A12 and B12. To Andreas for great discussions, hangouts, and for being a good friend. To Ajoy, for great science and beer sessions (next pint is on me). To Maggie for training me in human stem cell culture (and Long Island Iced Tea drinking) during my time at the SALK. Special thanks to Bjarne, Carolina, Shobhit, and Alex for giving me valuable feedback on parts of this thesis.
53 To Professor Masanobu Satake and my dear friend and former mentor Shunsuke Kon (now an Assistant Professor at Hokkaido University) for taking care of me during my year at Tohoku University in Japan (2007-2008), giving me my first true lab experience. I am not very good at keeping in contact regularly but I will definitely keep coming for visits when I travel to Japan in the future. Because of you, I was able to set my aim on becoming a scientist. Till alla mina vänner utanför jobbet: Hemifrån; Rodney, Jacob, Joke. Från min tid i Skåneland; Emil, Alina, Henke, Hillan, PG, Olof, Stellan, Grodan, och Kungen. Jag vet att jag inte alltid gör mycket ljud av mig men jag värdesätter er vänskap oerhört och hoppas att ni fortsätter att ha tålamod med mig även i framtiden. Till min underbara familj. Till Mamma Irené och Pappa Lennart. Till mina tre lillasystrar Hanna (tack för den fina bilden till diskussionen ☺), Sofia, och Victoria. Till Farmor Gunilla, och även till min Farfar Gunnar som alltid stöttat mig i mina akademiska studier men som tyvärr inte finns med oss i livet nu när jag blir klar. Samt till mina bortgångna morföräldrar, Mormor Karin och Morfar Bertil. Jag älskar er alla! Och till sist till min trolovade Meike, för att du alltid finns där för mig och hjälper mig vara mer än jag någonsin kunde vara på egen hand. Utan dig och vår dotter Mia saknar min värld mening. Jag älskar er!
54
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57 Eilken, H.M., Nishikawa, S., and Schroeder, T. (2009). Continuous single-cell imaging of blood generation from haemogenic endothelium. Nature 457, 896–900. Ema, M., Faloon, P., Zhang, W.J., Hirashima, M., Reid, T., Stanford, W.L., Orkin, S., Choi, K., and Rossant, J. (2003). Combinatorial effects of Flk1 and Tal1 on vascular and hematopoietic development in the mouse. Genes & Development 17, 380–393. Evans, M.J., and Kaufman, M.H. (1981). Establishment in culture of pluripotential cells from mouse embryos. Nature 292, 154–156. Fehling, H.J., Lacaud, G., Kubo, A., Kennedy, M., Robertson, S., Keller, G., and Kouskoff, V. (2003). Tracking mesoderm induction and its specification to the hemangioblast during embryonic stem cell differentiation. Development 130, 4217– 4227. Finkel, T. (2005). Radical medicine: treating ageing to cure disease. Nat Rev Mol Cell Biol 6, 971–976. Fitch, S.R., Kimber, G.M., Wilson, N.K, Parker, A., Mirshekar-Syahkal, B., Göttgens, B., Medvinsky, A., Dzierzak, E., and Ottersbach, K. (2012). Signaling from the Sympathetic Nervous System Regulates Hematopoietic Stem Cell Emergence during Embryogenesis. Cell Stem Cell 11, 554–566. Fulop, G.M., and Phillips, R.A. (1990). The scid mutation in mice causes a general defect in DNA repair. Nature 347, 479–482. Gordon-Keylock, S., Sobiesiak, M., Rybtsov, S., Moore, K., and Medvinsky, A. (2013). Mouse extraembryonic arterial vessels harbor precursors capable of maturing into definitive HSCs. Blood 122, 2338–2345. Gori, J.L., Butler, J.M., Chan, Y.-Y., Chandrasekaran, D., Poulos, M.G., Ginsberg, M., Nolan, D.J., Elemento, O., Wood, B.L., Adair, J.E., et al. (2015). Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. J. Clin. Invest. 125, 1243–1254. Green, E.L., et al. (2007). BIOLOGY OF THE LABORATORY MOUSE, By the staff of the Jackson laboratory, Second revised edition, Chapter 22. Hanna, J., Wernig, M., Markoulaki, S., Sun, C.W., Meissner, A., Cassady, J.P., Beard, C., Brambrink, T., Wu, L.C., Townes, T.M., et al. (2007). Treatment of Sickle Cell Anemia Mouse Model with iPS Cells Generated from Autologous Skin. Science 318, 1920–1923. Harman, D. (1955). Aging: a theory based on free radical and radiation chemistry. Journal of Gerontology 298–300. Hayflick, L., and Moorhead, P.S. (1961). The serial cultivation of human diploid cell strains. Exp. Cell Res. 25, 585–621. Huyhn, A., Dommergues, M., Izac, B., Croisille, L., Katz, A., Vainchenker, W., and Coulombel, L. (1995). Characterization of hematopoietic progenitors from human yolk sacs and embryos. Blood 86, 4474–4485. Ito, K., and Suda, T. (2014). Metabolic requirements for the maintenance of self-renewing stem cells. Nat Rev Mol Cell Biol 15, 243–256.
PhD Thesis Without Papers - Roger Rönn e-spik
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PhD Thesis Without Papers - Roger Rönn e-spik

  • 1. Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells Strategies to Overcome the Hurdles ROGER E. RÖNN DIVISION OF MOLECULAR MEDICINE AND GENE THERAPY | LUND UNIVERSITY 2015 Lund University, Faculty of Medicine Doctoral Dissertation Series 2015:142 ISBN 978-91-7619-222-1 ISSN 1652-8220 PrintedbyMedia-Tryck,LundUniversity2015 9789176192221 ROGERE.RÖNN  TowardsGenerationofHumanHematopoieticStemCellsfromPluripotentStemCells 142 It has been more than 6 years since this journey began. I have spent this time studying how blood forms during early life for the purpose of increasing our understanding of this process, with the hope that this knowledge one day can contribute to therapeutic applications. These 6 years have been a great chapter in my life, and I hope that you enjoy reading this book as much as I have enjoyed writing it. Roger Emanuel Rönn, Lund, November 2015 It has been more than 6 years since this journey began. I have spent this time studying how blood forms and grows during early life, and the purpose has been to increase what we understand about this process, with the hope that this knowledge one day can be used for something positive. These 6 years have been a great chapter in my life, and I hope that you enjoy reading this book as much as I have enjoyed writing it. /Roger
  • 2.
  • 3. 1 Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells Strategies to Overcome the Hurdles Roger E. Rönn DOCTORAL DISSERTATION With the approval of the Faculty of Medicine, Lund University, Sweden, this thesis will be defended in the Belfrage lecture hall, BMC D15, Klinikgatan 32, Lund on December 17th 2015 at 14:00. Supervisor Niels-Bjarne Woods, Ph.D. Faculty opponent Professor George Q. Daley, M.D., Ph.D. Children’s Hospital Boston, U.S.A.
  • 4. 2 Organization LUND UNIVERSITY Division of Molecular Medicine and Gene Therapy Document name: Doctoral dissertation Institution of Laboratory Medicine, Lund Date of disputation: December 17th , 2015 Author: Roger E. Rönn Sponsoring organization Title and subtitle Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells. Strategies to Overcome the Hurdles. Abstract There is a pressing need for compatible Hematopoietic Stem Cells (HSCs), in high quantities and of good clinical quality, to ensure optimal treatment for patients with various blood malignancies. Pluripotent Stem Cells (PSCs) have the potential to be a source for generating HSCs. The indefinite expansion capacity of PSCs, combined with the possibility of creating these cells from patient-specific material could, in principle, allow for the generation of patient-derived HSCs. Generation of HSCs from PSCs could dramatically improve the treatment of patients in the clinic, including the possible correction of genetically carried hematological diseases. Furthermore, successful generation of HSCs would provide a tool to better study the nature of hematological malignancies, and could be employed for the purpose of drug screening With the ultimate goal of facilitating in vitro generation of functional HSCs from human PSCs, the studies included in this thesis explore potential key factors and mechanisms involved in the process of hematopoietic development. In paper I we describe that control over the amount of retinoic acid during in vitro differentiation of human PSCs increase blood generation, allowing for more defined and efficient production of relevant cells. In paper II we provide a molecular identification of Endothelial to Hematopoietic Transition (EHT), the conversion through which HSCs emerge from endothelial cells. This provides a foundation for further characterization of the cells undergoing this transition, and could potentially enable control over this process for the purpose of more efficient generation of hematopoietic cells from hemogenic endothelium. In paper III we explore how cAMP intrinsically regulates the generation of HSC-like cells, and we demonstrate the importance of cAMP signaling in the emergence of HSCs. Modulating cAMP provides an additional approach to increase the generation, and potentially the function, of HSC-like cells from human PSCs. In paper IV, we demonstrate that reactive oxygen species is increased due to in vitro culture, causing functional impairment of hematopoietic progenitors and HSC-like cells generated from human PSCs. This argues that we must revise our standardized culture methods, and that a more physiological oxygen concentration is likely to be a critical component for handling and generating HSCs outside of the body. In summary, the papers included in this thesis focuses on understanding the development of human HSCs, with the ultimate goal of developing methods that would enable the generation of functional HSCs in the laboratory, potentially translating into improved treatments against a multitude of hematological malignancies. Key words: hematopoietic stem cells, pluripotent stem cells, in vitro differentiation, embryonic development Classification system and/or index terms (if any) Supplementary bibliographical information Language: English ISSN and key title: 1652-8220 ISBN: 978-91-7619- 222-1 Recipient’s notes Number of pages Price Security classification I, the undersigned, being the copyright owner of the abstract of the above-mentioned dissertation, hereby grant to all reference sourcespermission to publish and disseminate the abstract of the above-mentioned dissertation. Signature Date
  • 5. 3 Towards Generation of Human Hematopoietic Stem Cells from Pluripotent Stem Cells Strategies to Overcome the Hurdles Roger E. Rönn
  • 6. 4 Cover artwork by Roger Rönn Copyright Roger Rönn Lund University, Faculty of Medicine, Institution of Laboratory Medicine, Division of Molecular Medicine and Gene Therapy Lund University, Faculty of Medicine Doctoral Dissertation Series 2015:142 ISBN 978-91-7619-222-1 ISSN 1652-8220 Printed in Sweden by Media-Tryck, Lund University Lund 2015 EndelavFörpacknings-och Tidningsinsamlingen(FTI)
  • 7. 5 Till min familj och mina vänner
  • 8. 6
  • 9. 7 Content Populärvetenskaplig sammanfattning.......................................................................9 Original papers .......................................................................................................12 Papers not included in this thesis............................................................................13 Abbreviations .........................................................................................................14 Preface....................................................................................................................16 Introduction ............................................................................................................17 The Hematopoietic System 17 Hematopoietic Stem Cells 19 Characterization of the Human HSC...................................................19 The Bone Marrow Niche.....................................................................20 Regulation of HSC Self-Renewal........................................................21 HSC Sensitivity ...................................................................................22 Embryonic Development 24 Pluripotent Stem Cells.........................................................................24 Developmental Specification...............................................................26 Retinoic Acid During Embryonic Development .................................27 Hematopoiesis During Embryonic Development................................28 In Vitro Differentiation of hPSCs Towards HSCs 32 The Potential of in Vitro HSC Generation from hPSCs ......................32 Methodology .......................................................................................33 Previous Key Discoveries....................................................................34 Aims of the thesis...................................................................................................37 Summary of results.................................................................................................39 Paper I: 39 Paper II: 40 Paper III: 41 Paper IV: 42 Conclusions ............................................................................................................45 General discussion and future perspectives............................................................47 Acknowledgements ................................................................................................51 References ..............................................................................................................55
  • 10. 8
  • 11. 9 Populärvetenskaplig sammanfattning Idag har patienter med blodcancer (leukemi) eller genetiskt betingade blodsjukdomar (sickelcellanemi etc.) begränsad tillgång till behandlingar som effektivt och riskfritt botar sjukdomstillståndet. Detta arbete inkluderar forskningsresultat ämnade att bidra till utveckling av konceptuellt nya behandlingsmetoder för olika typer av blodsjukdomar. Ett fungerande blodsystem är nödvändigt för varje individs överlevnad. De röda blodkropparna (erytrocyterna) förser kroppen med syre från lungorna, och de vita blodkropparna (leukocyterna) utgör vårt immunförsvar mot infektionssjukdomar. Blodceller slits snabbt ut och för att ersätta dessa behöver kroppen varje dag tillverka cirka 1 biljon nya blodceller. Ansvarig för denna kontinuerliga nyproduktion av blodceller är en sällsynt typ av blodceller som kallas för Hematopoetiska stamceller (HSCs), även kallade blodstamceller. Blodstamceller återfinns i benmärgen där de stundtals genomgår celldelning. Vid celldelning kan en dottercell påbörja en mognadsprocess där, efter många nya celldelningar, ett stort antal av färdiga blodceller resulterar. Blodstamceller är därför centrala för att våra kroppar ska kunna förses med nya blodceller genom livet. Blodstamceller är dock väldigt känsliga och kan fallera om de utsätts för skadliga faktorer som olika kemiska substanser, eller radioaktiv strålning. Vid behandling av blodcancer används idag ofta sådana faktorer för att ta död på cancercellerna. Dessa behandlingar medför ofta att även kroppens friska blodstamceller förstörs, vilket kan resultera i blodsystemets kollaps och patientens avlidande om detta inte åtgärdas. Därför genomgår patienter som genomgått cancerdödande behandling ofta en transplantation där blodstamceller från en donator införs i patientens kropp. Om transplantationen lyckats återetablerar donatorns blodstamceller en kontinuerlig nyproduktion av blodceller vilket resulterar i patientens överlevnad. Men donatorbaserad transplantation av blodstamceller kan medföra komplikationer. Mängden blodstamceller som kan inhämtas från en villig donator kan vara begränsad vilket resulterar i en oönskat lång återhämtning för patienten. En donator måste även vara immunkompatibel med patienten (att deras immunförsvar har liknande karaktär) för att minimera risken att det nya immunförsvaret reagerar mot patientens egna vävnader. Ett sådant tillstånd där transplantatet angriper värden, på engelska Graft Versus Host Diesease (GVHD), är livsfarligt för patienten. Därför krävs en hög kompatibilitet mellan patient och
  • 12. 10 donator vilket ytterligare begränsar antalet möjliga donatorer. Det finns därför ett stort behov av patientkompatibla blodstamceller för att kunna möjliggöra optimal och effektiv behandling mot olika blodsjukdomar. En möjlighet att förse detta behov är odling och produktion av blodstamceller i laboratoriet. I början av den embryonala utvecklingen (strax efter äggets befruktning) finns en typ av stamceller som kan ge upphov till alla olika typer av celler och vävnader i den vuxna kroppen. Dessa stamceller kallas därför pluripotenta och kan, om de stimuleras på rätt sätt, odlas i laboratorium för att utvecklas till olika vuxna celltyper inklusive blod. Pluripotenta stamceller behöver inte nödvändigtvis erhållas från embryon. År 2007 lyckades ett japanskt laboratorium, lett av professor Shinya Yamanaka, med att framställa mänskliga pluripotenta stamceller från vuxna hudceller. Denna bedrift belönades med Nobelpriset i medicin år 2012, och innebar nya möjligheter att framställa pluripotenta stamceller från enskilda individer som till exempel patienter. Om det vore möjligt att framställa funktionsdugliga blodstamceller från pluripotenta stamceller skulle behandlingen av blodsjukdomar kunna förbättras avsevärt eftersom det teoretiskt sett vore möjligt att framställa obegränsade mängder av nya patientkompatibla blodstamceller. Denna avhandling innehåller 4 olika forskningsstudier där det gemensamma målet är att utveckla en metod för att lyckas med att framställa mänskliga blodstamceller från pluripotenta stamceller. I den första studien demonstrerar vi att Retinoinsyra, en metabolit av Vitamin A, har en negativ påverkan under utvecklandet av pluripotenta stamceller till blod. Genom att använda en kemisk substans vid namn (DEAB) för att blockera cellernas egen produktion av Retinoinsyra kan vi flerfaldigt förbättra framställningen av celler vilka liknar blodstamceller. Ökad förståelse hur regleringen av utvecklingsprocessen går till är viktigt för att specifikt och effektivt kunna framställa de celler som är av intresse. I den andra studien undersöker vi i detalj hur de första blodstamcell-lika cellerna formas. Under utvecklingen uppstår de första blodstamcellerna från endotelceller som täcker blodkärlens insida. Denna process, där en icke-blodcell genomgår ett identitetsbyte till blod, kallas för Endotelisk till Hematopoetisk Övergång, på engelska Endothelial to Hematopoietic Transition (EHT), och detaljerad vetskap om hur denna process fungerar saknas. Genom att studera övergången från endotel till blodcell beskriver vi ett specifikt övergångstillstånd som sker centralt i denna process. Våra resultat låter oss för första gången identifiera och särskilja de celler som deltar i denna övergång, vilket kan komma väl tillhanda om vi bättre vill efterlikna den embryonala blodutvecklingen för att kunna framställa blodstamceller i laboratoriet.
  • 13. 11 I den tredje studien visar vi att utvecklingen av blodstamcell-lika celler är i kritiskt behov av Cykliskt adenosinmonofosfat (cAMP) vilket fungerar som en sekundär budbärare åt flera olika signalsubstanser. Genom att manipulera cellernas mängd av cAMP demonstrerar vi flerfaldigt ökad framställning av blodstamcell-lika celler. Våra resultat påvisar även att cAMP förmedlar sina positiva effekter genom ej hittills uppskattade signaleringsleder inom cellen. Dessa resultat ökar vår förståelse över vilka faktorer som krävs för effektiv framställning av blod. I den fjärde studien demonstrerar vi att majoriteten av alla blodstamcell-lika celler har förhöjda nivåer av reaktiva syregrupper, på engelska Reactive Oxygen Species (ROS), vilket orsakar funktionsoduglighet. Vi påvisar att denna förhöjning av reaktiva syregrupper orsakas av flera olika processer vilka grundar sig i de metoder för cellodling som idag är generell praxis. Genom att använda samt kombinera fyra olika metoder för att minska mängden av reaktiva syregrupper kan vi för första gången effektivt framställa blodstamcell-lika celler med reducerade mängder av ROS. Dessa celler uppvisar kraftigt förbättrad kapacitet för tillväxt. Våra resultat argumenterar för att reaktiva syregrupper vilka resulterar från våra cellodlingsmetoder utgör ett allvarligt hinder, samt att en reducering sannolikt är en nödvändighet, för lyckad framställning av funktionsdugliga blodstamceller i laboratoriet. Sammantaget presenteras i denna avhandling forskning inriktad på att förstå utvecklingsprocessen av blod, med syfte att möjliggöra framställning av blodstamceller från pluripotenta stamceller. Våra resultat ger ökad insikt i hur denna komplicerade process fungerar och det är vår förhoppning att dessa framsteg kan bidra till en framtida metod vilken möjliggör effektiv framställning av funktionella blodstamceller. Detta eftersom en sådan metod drastiskt skulle kunna förbättra behandlingen av patienter som lider av diverse blodsjukdomar.
  • 14. 12 Original papers Paper I, Retinoic acid regulates hematopoietic development from human pluripotent stem cells. Roger E. Rönn, Carolina Guibentif, Roksana Moraghebi, Patricia Chaves, Shobhit Saxena, Bradley Garcia, Niels-Bjarne Woods. Stem Cell Reports (2015) 4, 269-281. Paper II, Endothelial to hematopoietic transition state identified in differentiated human pluripotent stem cells. Carolina Guibentif, Roger Rönn, Charlotta Böiers, Shobhit Saxena, Stefan Lang, Shamit Soneji, Tariq Enver, Göran Karlsson, Niels-Bjarne Woods. Submitted Manuscript (2015). Paper III, Cyclic AMP induction enhances human hematopoietic cell emergence through the cAMP-Epac signaling axis, creates redox state balance, and increases CXCR4 expression. Shobhit Saxena, Roger E. Rönn, Carolina Guibentif, Roksana Moraghebi, Niels- Bjarne Woods. Submitted Manuscript (2015). Paper IV, Reactive oxygen species impair the function of hematopoietic stem / progenitor cells generated from human pluripotent stem cells. Roger E. Rönn, Carolina Guibentif, Shobhit Saxena, Niels-Bjarne Woods. Submitted Manuscript (2015).
  • 15. 13 Papers not included in this thesis Localization of SMAP2 to the TGN and its function in the regulation of TGN protein transport. Tomo Funaki, Shunsuke Kon, Roger E. Rönn, Yuji Henmi, Yuka Kobayashi, Toshio Watanabe, Keiko Nakayama, Kenji Tanabe, and Masanobu Satake. Cell Structure and Function (2011) 36, 83-95.
  • 16. 14 Abbreviations AGM Aorta Gonad Mesonephros AP Alkaline Phosphatase ATP Adenosine-5-Triphosphate BM Bone Marrow cAMP Cyclic Adenosine Mono Phosphate CAR Cell CXCL12-Abundant Reticular Cell CB Cord Blood CFU Colony Forming Unit DEAB Diethylaminobenzaldehyde DS Down Syndrome EB Embryoid Body EHT Endothelial to Hematopoietic Transition EPAC Exchange Proteins Activated by cAMP ES Cell Embryonic Stem Cell FACS Fluorescence Activated Cell Sorting GD Gaucher Disease GFP Green Fluorescent Protein GPX Glutathione Peroxidase GSR Glutathione S-reductase GVHD Graft Versus Host Disease HE Hemogenic Endothelium HLA Human Leukocyte Antigen HR Homologous Recombination HSC Hematopoietic Stem Cell IBMX Isobutyl-1-Methylxanthine ICM Inner Cell Mass iPS Cell Induced Pluripotent Stem Cell LDA Limited Dilution Assay LPM Lateral Plate Mesoderm MAPK Mitogen-Activated Protein Kinase MPO Myeloperoxidase MPP Multipotent Progenitor MSC Mesenchymal Stem Cell NK Cell Natural Killer Cell
  • 17. 15 NHEJ Non-Homologous End Joining NOD Nonobese Diabetic OTM Olive Tail Moment OXPHOS Oxidative Phosphorylation PCA Principal Component Analysis PD Parkinson Disease PGE2 Prostaglandin E2 PKA Protein Kinase A PSC Pluripotent Stem Cell RA Retinoic Acid RALDH Retinaldehyde Dehydrogenase RAR Retinoic Acid Receptor RARE Retinoic Acid Response Element RXR Retinoid X Receptor ROS Reactive Oxygen Species SBDS Shwachman-Bodian-Diamond Syndrome SCID Severe Combined Immunodeficiency SOD Superoxide Dismutase WAS Wiscott-Aldrich Syndrome YS Yolk Sac 4-ABAH 4-Aminobenzoic Acid
  • 18. 16 Preface It has been more than 6 years since this journey began. I have spent this time studying how blood forms during early life for the purpose of increasing our understanding of this process, with the hope that this knowledge one day can contribute to therapeutic applications. Today, there are limitations to how we can treat patients suffering from leukemia and other blood disorders. One key requirement for successful treatment is transplantation of hematopoietic stem cells capable of re-establishing the hematopoietic system. Today hematopoietic stem cells often need to be donated from other individuals. The reliance on donated cells for transplantation leads to limitations to the amount of suitable hematopoietic stem cells both in terms of quality and in quantity. Pluripotent stem cells are cells that have the capacity to form all types of cells in the body. If we could understand how hematopoietic stem cells form during early life, and then recreate the same conditions in the laboratory for the purpose of generating hematopoietic stem cells from pluripotent stem cells, we may have the means to provide patients with new effective treatments potentially curing and correcting many types of blood disease. The first part of this thesis gives an introduction to the hematopoietic system, the hematopoietic stem cell, embryonic development, and previous efforts to generate hematopoietic stem cells. The second part summarizes the present studies, and is followed by a general discussion where I elaborate on what I believe to be a key hurdle to overcome if we want to successfully generate hematopoietic stem cells in the laboratory. These 6 years have been a great chapter in my life, and I hope that you enjoy reading this book as much as I have enjoyed writing it. Roger Emanuel Rönn, Lund, November 2015
  • 19. 17 Introduction The Hematopoietic System Hematopoiesis (“creation of blood” in Greek) is the process of blood formation that sustains an individual’s requirement of circulating blood cells. Viewed as a liquid organ, the hematopoietic system provides key functions needed for the survival of the individual. There are several types of blood cells in the hematopoietic system, each having specialized functions. The red blood cells (erythrocytes) are required for the transport of oxygen and carbon dioxide between the lungs and the rest of the body and are the most abundant blood cell type (20-30 trillion in an adult human). The ability of blood to clot and stop bleeding is provided by platelets, small fragments of another hematopoietic cell type known as megakaryocytes. The hematopoietic system also includes cell types that are part of the innate and adaptive immune systems, protecting the organism from foreign pathogens. The innate immune system includes macrophages, granulocytes, and the dendritic cells. These cells facilitate immediate responses, including the engulfment of foreign material (phagocytosis), pathogen neutralization by release of toxic substances, modulation of the inflammatory response, and communication of information to the adaptive immune system. The adaptive immune system features the lymphocytes, providing memory and long-term protection against encountered pathogens. Lymphocytes include the B cells required for the production of antibodies, and T cells as well as Natural Killer (NK) cells that provides directed killing of infected cells. Together, these various cells of the hematopoietic system provide functions critical for the survival of the individual, but in order to replace aged or broken blood cells the body needs to create approximately 1 trillion new blood cells every day (Ogawa, 1993). While the various types of blood cells are specialized for their specific functions, they are all related to each other and are derived from a common precursor in a hierarchical manner (Figure 1). At the apex of the hematopoietic hierarchy are the Hematopoietic Stem Cells (HSCs). Formed during embryonic development and maintained throughout adult life, HSCs are multipotent progenitors that form all mature blood cells while simultaneously having the capacity to self-renew, forming cellular progeny that retain the same HSC characteristics. Fulfilling these criteria, the HSC have the capacity to provide long-term reconstitution of the
  • 20. 18 hematopoietic system. While other cell types in the hematopoietic hierarchy have limitations as to how many times they can divide, the HSCs, due to their ability to self-renew, provide a continuous source of new blood cells and are responsible for the homeostasis of the entire hematopoietic system. In absence of such self- renewing HSCs the continuous production of new blood cells cannot occur, resulting in the collapse of the hematopoietic system and compromised survival. Figure 1. The Hematopoietic System. All mature blood cells of the hematopoietic system are derived, in a hierarchical manner, from HSCs as their common precursors. HSC: Hematopoietic Stem Cell, MPP: Multipotent Progenitor, CMP: Common Myeloid Progenitor, MEP: Megakaryocyte/Erythroid Progenitor, GMP: Granulocyte/Macrophage Progenitor, MLP: immature Lymphoid Progenitor, B/NK: B cell / Natural Killer cell progenitor, proB: B cell precursor, ETP: Early T cell Precursor, NK: Natural Killer cell. Human HSCs are enriched by the following markers: Lin- CD34+ CD38- CD90+ CD49f+ . Illustration was inspired by figure 2 in Doulatov et al., 2012. HSC MPP CMP MLP Dendritic cells NK cells B cells T cellsMacrophages Granulocytes Erythrocytes Megakaryocytes ETP B/NK proBGMPMEP Lin- CD34+ CD38- CD90+ CD49f+
  • 21. 19 Hematopoietic Stem Cells The idea that a population of self-renewing HSC is responsible for the continuous regeneration of the hematopoietic system was initially proposed by Till and McCulloch in 1961 after observing that limited numbers of transplanted bone marrow cells formed colonies of hematopoietic cells in the spleens of recipient mice. Since then, due to the central role of HSCs in hematopoiesis, there have been numerous scientific studies aimed at characterizing and increasing the understanding of this cell type Characterization of the Human HSC Immunodeficient mice used as xenograft models have been invaluable tools for studying human hematopoiesis. Immunodeficiency allows for transplanted human cells to avoid rejection and this was first taken advantage of with the development of the Severe Combined Immune-Deficient (SCID) mouse model (Bosma et al., 1983; Fulop and Phillips 1990). These mice lack both B and T cells, and allows for mainly long-term engraftment of human lymphoid cells. Backcrossing the SCID mutation onto nonobese diabetic (NOD) mice (harboring further defects in innate immunity), with additional deletion of the IL-2R common γ chain gene, resulted in the NSG mouse model that features a complete loss of B, T, and NK cells. The NSG model allows for robust human lymphoid-myeloid engraftment and is commonly used to study human engraftment from HSCs (Doulatov et al., 2012). By analyzing the presence or absence of specific proteins on the cellular surface, various cell types can be identified and characterized. Development of fluorochrome-conjugated monoclonal antibodies capable of binding such cell surface markers, combined with Fluorescence Activated Cell Sorting (FACS) technology have enabled the separation and analysis of the various cell types in the hematopoietic hierarchy. To identify human HSC, the currently most stringent combination of surface markers is the following; negativity for specific markers of the T, B, NK, and myeloid-erythroid lineages (Lin- ), positivity for marker CD34 (CD34+ ), CD38- , CD90+ (Majeti et al., 2007), and CD49f+ (Notta et al., 2011) (Figure 1). Transplantation with reduced numbers of cells purified with these markers, using the Limited Dilution Assay (LDA), have demonstrated that only 1 in 10 of these cells are able to repopulate recipients (Notta et al., 2011) indicating the rarity of true HSCs and the challenge of identifying them reliantly.
  • 22. 20 The Bone Marrow Niche During homeostasis, the anatomical location of the HSCs in the adult is in the bone marrow (BM), which is the flexible “spongy” tissue inside the larger bones of the body. The BM is a highly complex environment that includes many different cell types, organized together within the vascularized space between the inner walls of endosteal bone. The BM as a whole provides an intricate environment (niche) that regulates and protects the HSC pool throughout an individual’s life (Scadden, 2006). Cells found in the bone marrow niche include bone-synthesizing osteoblasts, bone-resorbing osteoclasts, energy storing adipocytes, Mesenchymal Stem Cells (MSCs), CXCL12-Abundant Reticular cells (CAR cells) and macrophages, with HSCs comprising less than 0.01% of all cells in the BM (Walasek et al., 2012) (Figure 2). By secreting various signaling molecules, the relative position of specific niche cells to the HSC, combined with location dependent effects upon the HSC in the architecture of the BM (for instance the proximity to blood vessels providing oxygen), together provide a highly complex physiological regulatory system required for HSC maintenance, and thereby for hematopoiesis itself (Scadden, 2006; Walasek et al., 2012). Figure 2. The Bone Marrow Niche. This illustration provides a simplified model of the bone marrow niche. General structural features include blood vessels and the inner surface of endosteal bone. Cell types included in this illustration are: CAR cells (CXCL12- Abundant Reticular cells), macrophages, osteoblasts, osteoclasts, HSCs (Hematopoietic Stem Cells), MSCs (Mesenchymal Stem Cells), and adipocytes. Gradients of oxygen and calcium are also illustrated. Calcium Oxygen Blood Vessel CAR Cell Macrophage Adipocyte MSC HSC Osteoclast Osteoblast Endosteal Bone
  • 23. 21 Regulation of HSC Self-Renewal Since self-renewal of the HSC is required for maintained and continuous regeneration of the hematopoietic system, self-renewal is considered to be a core- defining feature of functional HSCs. The central role of self-renewing HSCs in hematopoiesis, together with the fact that HSCs rapidly lose their self-renewal capacity upon in vitro culture (limiting their potential expansion for therapeutic purposes), have resulted in efforts to better understand how self-renewal is regulated. It has been postulated that HSCs, when faced with changing circumstances, have a number of different fate options available (Wagers et al., 2002). These fate options include the division of an HSC into daughter cells whereupon either one or both cells retain self-renewal and HSC identity (asymmetric and symmetric division), in the latter case expanding the HSC-pool without giving rise to maturing (differentiating) blood cells. An HSC might also divide into daughter cells where both cells differentiate, resulting in loss of self- renewing cells and the reduction of the HSC pool. Additional fate options could be considered including the cell entering a state of inactive hibernation (quiescence), migration of the HSC into other environments, induction of premature senescence causing functional elimination of the HSC without the aspect of apoptosis (Ito et al., 2006; Ito and Suda, 2014), or termination of the cell by controlled cell death (apoptosis) (Figure 3). Thus, control over these fate options to favor symmetric division of self-renewing HSCs could potentially enable HSC expansion. Factors that regulate the behavior of HSCs can either be extrinsic or intrinsic. Extrinsic regulation includes environmental cues that affect the HSC. This includes factors that are secreted from other cells in the niche, or physical circumstances such as the distance to the vasculature and the available amount of oxygen (Ludin et al., 2014). Intrinsic regulation covers the actions of internal mediators affecting HSC behavior and identity. This includes genetic and epigenetic modulators regulating the transcriptional state and the division characteristics of the cell (Oguro et al., 2006). Other intrinsic regulators are factors associated with the apoptotic response (Domen et al., 1998), or involved in DNA repair mechanisms (Biechonski and Milyavsky, 2013). In addition, the metabolic state of the HSC is known to affect self-renewal capacity. Glycolysis-based production of adenosine-5-triphosphate (ATP) is associated with increased survival and engraftment capacity, while ATP production through oxidative phosphorylation (OXPHOS) is associated with loss of self-renewal and differentiation (Takubo et al., 2013; Ito and Suda, 2014). Intrinsic regulation also includes the availability of intracellular molecules involved in signal transduction. One such intracellular mediator is Cyclic Adenosine Mono Phosphate (cAMP) transmitting the effects of neurotransmitters, growth factors, and hormones such as Prostaglandin E2 (PGE2), binding to G- protein coupled receptors on surface of the HSC (Cutler et al., 2013). This allows the cell to respond to such extrinsic factors.
  • 24. 22 The individual importance of the factors mediating extrinsic and intrinsic regulation suggests that HSC self-renewal is a delicate state, easily lost upon imbalance and dysregulation. Figure 3. Fate Options of the HSC. The HSC can, depending on circumstance, undertake a range of different fate options. These fate options include; symmetric or asymmetric self-renewal division (expanding or maintaining the HSC pool), dividing into cells that both differentiate into maturing blood cells (reduction of the HSC pool), entering a state of inactive hibernation (quiescence), moving into other environments (migration), losing the capacity to divide (premature senescence), or to terminate by controlled cell death (apoptosis). HSC Sensitivity Various cell types have different tolerances to stressful insults such as harmful chemicals or ionizing radiation. The HSC has been demonstrated to be more sensitive to genotoxic insult as compared to the downstream effector cells in the hematopoietic hierarchy (Milyavsky et al., 2010). The central role of self-renewing HSCs to continuously resupply the hematopoietic system, combined with the high stress sensitivity of the HSC, makes the hematopoietic system one of the most radiosensitive organs of the body. Thus, full body radioactive exposure often results in lethal outcomes due to the collapse of the hematopoietic system (bone marrow failure). In steady state most HSCs are dividing infrequently, have minimal metabolic activity, and are suspended in a state of inactive hibernation called quiescence. If the body is in urgent need of more blood cells, quiescent Zzz... Differentiation Asymmetric Self-renewal Symmetric Self-renewal Quiescence Migration Premature Senescence Apoptosis
  • 25. 23 HSCs are capable of activating to facilitate rapid restoration of hematopoietic homeostasis. Since actively dividing cells are more prone to suffer from endogenous stress/damage as a result of increased cellular respiration and DNA replication it is believed that quiescence, by minimizing such stress, is an essential protective mechanism intended to ensure long-term performance of the otherwise stress sensitive HSC (Orford and Scadden, 2008). However, quiescence does not directly protect HSCs from external sources of stress. If faced with DNA damage, non-cycling human HSCs have limitations on how to carry out DNA repair, as more accurate DNA repair by Homologous Recombination (HR) (requiring cell cycling) cannot be utilized. This forces the quiescent HSC to rely on more error- prone Non-Homologous End Joining (NHEJ), more likely to result in genomic instability and loss of cellular function (Mohrin et al., 2010). The reduced metabolic activity associated with quiescence is believed to protect HSCs from the damaging effects of Reactive Oxygen Species (ROS) (Ito and Suda, 2014). ROS are highly reactive oxygen containing molecules, typically generated as a by- product of OXPHOS-based metabolism, that can cause oxidative damage to cellular components including DNA, proteins and lipids (Finkel, 2005). It has been suggested that accumulation of such oxidative damage contributes to the phenotypes associated with both aging and the development of cancer, as argued by the free radical theory of aging (Dröge, 2002; Harman, 1955). HSCs are highly sensitive to increased levels of ROS, with ROS-mediated oxidative damage impairing the self-renewal and long-term engraftment capacity of these cells (Ito et al., 2006; Jang and Sharkis, 2007; Yahata et al., 2011) (Figure 4). It is possible that ROS may also influence the performance of hematopoietic progenitors generated in a laboratory setting but this remains be elucidated.
  • 26. 24 Figure 4. HSC sensitivity to Reactive Oxygen Species. Human HSCs are highly sensitive to increased amounts of Reactive Oxygen Species (ROS), and their performance is inversely correlated with their ROS level. Quiescent HSCs, and self-renewing HSCs are both characterized by low ROS levels. Further increase of ROS results in HSC exhaustion through; differentiation, premature senescence, and apoptosis. Illustration was inspired from figures in Ludin et al., 2014, and Zhou et al., 2014. Embryonic Development Embryonic development is the process whereby the cellular progeny of a fertilized egg progressively differentiates towards specific cell fates, while at the same time becoming organized into specific patterns and structures, required for the formation of tissues, organs, and the higher body structure of a new organism. Thus, by studying the mechanisms of embryonic development, it becomes possible to define the conditions that facilitate the formation of specific cell types. Pluripotent Stem Cells Pluripotent Stem Cells (PSCs) are self-renewing cells that have the potential to differentiate into any cell type of the adult body. Upon fertilization (conception), the first cell of the new organism (the zygote) and its immediate cellular progeny are totipotent, having the potential to form all the cell types of the developing embryo and also extra-embryonic structures such as the placenta. During these early stages of development, specialization begins and the first pluripotent stem cells, now restricted to develop only into tissues of the embryo itself, form in the Zzz... Q uiescence Prem atureSenescence Self-renew al D ifferentiation A poptosis HSC Performance ReactiveOxygen Species
  • 27. 25 inner cell mass (ICM) of the blastocyst. Pluripotent stem cells that originate from the ICM of a blastocyst are known as Embryonic Stem cells (ES cells). ES cells were first derived from murine embryos in 1981 by two independent groups (Evans and Kaufman, 1981; Martin, 1981) together providing methods to cultivate and expand these cells in vitro. Later, human ES cells were successfully isolated from the inner cell mass of human blastocysts (Thomson et al., 1998). Due to the propagation potential of human ES cells, and due to their capacity to differentiate into adult cell types, they have a potential application in therapies that feature transplantation, regeneration, or tissue engineering. But, use of human ES cells has been a subject of ethical debate since the extraction and derivation of ES cells result in the destruction of early embryos. However, pluripotent stem cells can also be generated artificially by inducing pluripotency in adult somatic cell types. Such induced Pluripotent Stem cells (iPS cells) were first reported in 2006 when combinatorial overexpression of 4 transcription factors (c-Myc, Klf4, Oct4, and Sox2) induced pluripotency in murine fibroblasts (Takahashi and Yamanaka, 2006), followed later with the generation of human iPS cells (Takahashi et al., 2007). Similar to ES cells, iPS cells are pluripotent, capable of self-renewal, and can differentiate into all the cell types of the adult body (Figure 5). Commonly used markers for hPSCs includes staining positive for Alkaline Phosphatase (AP), Tra-1-81, Tra-1-60, OCT4, NANOG, SSEA3, and SSEA4 (Park et al., 2008). Functional evaluation of pluripotency in cells is possible by testing the cells capacity to form teratomas upon transplantation into immunodeficient mice (Lensch et al., 2007). If the transplanted cells form cystic masses featuring derivates of all 3 embryonic germ layers then the transplanted cells are considered to be pluripotent.
  • 28. 26 Figure 5. The Potential of Pluripotent Stem Cells. Pluripotent Stem Cells (PSCs) can either be isolated as Embryonic Stem cells (ES cells) from the Inner Cell Mass (ICM) of blastocysts, or generated as induced Pluripotent Stem cells (iPS cells) from somatic cell types through reprogramming by overexpressing specific transcription factors. PSCs can theoretically self-renew indefinitely, and have the capacity to differentiate into all cell types of the adult body. This includes derivates of the ectoderm (such as nerve cells), the mesoderm (such as vascular endothelial cells), and the endoderm (such as intestinal epithelial cells). Developmental Specification During embryonic development the blastocyst (also called blastula), containing the pluripotent stem cells of the inner cell mass, begins the process of gastrulation. During gastrulation the blastocyst invaginates and reorganizes itself into a three- layered structure (gastrula), with the pluripotent stem cells becoming committed into the 3 germ layers. Each germ layer gives rise to specific tissues and organs during the developmental process, with the ectoderm (outer layer) forming tissues including the epidermis (skin) and the nervous system, the mesoderm (middle layer) forming muscle based tissues and the hematopoietic system, and the endoderm (inner layer) forming the interior lining of the digestive system and the respiratory system. As the process of specification continues, epigenetic mechanisms (including methylation and chromatin condensation) gradually restrict access to parts of the genome that contains genetic information no longer required for further specification towards downstream developmental derivates. While such gradual specification restricts the number of possible cell fates available to the cell, the “choice” among available cell fates is instructed by spatial (positional) Somatic Cell Induced Pluripotent Stem Cells (iPS Cells) Embryonic Stem Cells (ES Cells) Pluripotent Stem Cell c-Myc Klf4 Oct4 Sox2 ICM Isolation Reprograming Blastocyst Ectoderm Mesoderm Endoderm
  • 29. 27 information. To illustrate, if 3 identical precursors are positioned with differing distance from a tissue that secretes a gradient of a differentiation-inducing factor (morphogen), these cells will be “instructed” by their relative position, since the exposure level of the morphogen determines cell fate commitment (Figure 6). One important morphogen that instructs towards hematopoietic development is the cytokine BMP4. BMP4 takes part in the sub-specification of early mesoderm during embryonic development as demonstrated by studies in birds (Pourquié et al., 1996; Tonegawa et al., 1997), and it is crucial for the successful establishment of both vasculature and hematopoiesis. Similar findings were also reported in mice where targeted mutagenesis of the Bmp4 gene compromised both blood and endothelial cell development (Winnier et al., 1995). Thus, positional information (communicated by morphogen exposure), combined with genetic/epigenetic information (temporal cell identity), determines cellular response and lineage commitment, together enabling the formation of distinct cell types organized in characteristic patterns required for tissue formation and higher body structure (Wolpert, 1969). Figure 6. Positional Information by Morphogen Exposure Instruct Cell Fate Commitment During Embryonic Development. This illustration provides a simplified model for how morphogen gradients serve to instruct commitment of precursor populations into different developmental lineages. The positional information, established by the exposure level of the morphogen (due to the distance to the morphogen source), instruct the precursors to differentiate towards specific cell fates. Cell fates that depend on high morphogen exposure are morphogen instructive, while cell fates that depend on low exposure are morphogen permissive. Retinoic Acid During Embryonic Development A morphogen (“form-maker” in Greek) denotes diffusible biochemical molecules that instruct cell fate by concentration. Several cytokines are known to act as morphogens during the developmental process, including members of the FGF, Cell Fate A (Morphogen Instructive) Cell Fate C (Morphogen Permissive) Cell Fate B Morphogen Gradient
  • 30. 28 WNT, Hedgehog, and TGF-β families (Kardel and Eaves, 2012). Retinoic Acid (RA), a derivate of Vitamin A, is a central morphogen required for successful embryonic development in all higher animal species ranging from fish to humans (Ross et al., 2000). RA is synthesized in cells that express the enzyme retinaldehyde dehydrogenase 1, 2, or 3 (RALDH1, RALDH2, RALDH3). Due to its diffusive nature, gradients of RA form across regions of the developing embryo, providing positional information and contributing to both patterning and cell differentiation along the embryonic anterior/posterior axis (Ross et al., 2000). When available, RA enters the nucleus where it binds to members of the Retinoic Acid Receptor (RAR) family of nuclear receptors that, in turn, form heterodimeric complexes with members of the Retinoid-X-Receptor (RXR) family. These then localize to specific Retinoic Acid Response Elements (RAREs) in promoter regions of the genome to drive transcription of RA target genes (Duester, 2008; Kumar and Duester, 2011). The use of animal models to study the effects of RA deficiency has proved essential for determining the functions of RA during embryonic development, with RA required for the development of several organs and tissues including the eye, hindbrain, spinal cord, heart, lung, pancreas and the skeleton (Duester, 2008). Studies where additional RA was added during the developmental process have also demonstrated that RA is a potent teratogen (“monster-maker” in Greek), due to the severe malformations that result from increased RA during embryogenesis (Ross et al., 2000). Because of its teratogenic nature, pregnant women must avoid exposure to external sources of RA due to the high risk of abnormal embryonic development and birth defects. Since RA instructs cell fate through exposure, there are cell fates that depend on either high or low RA for successful commitment. Accordingly, cell fates that depend on low RA exposure are RA-permissive, while cell fates that require high RA exposure are RA-instructive. For instance, the initial commitment towards ectoderm is increased, at the expense of the other germ layers, if additional RA is added (Bain et al., 1996). Thus, ectodermal commitment is RA-instructive. During the later developmental stage where sub-specification of Lateral Plate Mesoderm (LPM) occurs, RA has been demonstrated to direct specification towards cardiac tissue (anterior LPM) at the expense of hematopoietic development as observed in Xenopus (Deimling and Drysdale, 2009), zebrafish (de Jong et al., 2010), and mouse (Szatmari et al., 2010). These findings indicate that specification towards the hematopoietic lineage at this stage in development is RA-permissive. However, it remains to be elucidated if this also applies to the human setting. Hematopoiesis During Embryonic Development During embryonic development hematopoiesis occurs at several distinct time- points and anatomical locations (Dzierzak and Speck, 2008; Kardel and Eaves,
  • 31. 29 2012; Clements and Traver, 2013). These separate hematopoietic events are broadly classified as belonging to the primitive, or the definitive, wave of hematopoiesis. The primitive wave features the formation of the first hematopoietic cells during the development of the embryo. In the mouse this process begins at embryonic day 7 (E7) when myeloid-erythroid progenitors are formed in extra-embryonic structures including the Yolk Sac (YS) and the allantois (which will later contribute to the formation of the umbilical cord) (Moore and Metcalf, 1970). These cells have limited self-renewal but serve to provide the embryo with the first erythroid cells (expressing embryonic globins). The definitive wave of hematopoiesis initiates around E10.5 in the mouse, with self-renewing HSCs, capable of long-term lymphoid-myeloid engraftment, emerging at the ventral side of the dorsal aorta located in the anatomical region of Aorta Gonad Mesonephros (AGM) (Müller et al., 1994; Medvinsky and Dzierzak, 1996). Other anatomical sites have also been identified as harboring definitive hematopoietic cells, including the arteries connecting the YS (Gordon-Keylock et al., 2013), the placenta (Ottersbach and Dzierzak, 2005; Rhodes et al., 2008), as well as the head (Li et al., 2012). Similar waves of primitive and definitive hematopoiesis are present during human embryonic development, with myeloid- erythroid progenitors first appearing in the YS (Huyhn et al., 1995) and self- renewing HSCs with lymphoid-myeloid capacity later detected in the AGM region (Tavian et al., 2001). After the emergence of definitive HSCs, these cells travel through the circulatory system to colonize the fetal liver. The fetal liver provides a temporary environment where the HSCs expand in number, while also giving rise to large quantities of mature hematopoietic cells required to support the continued development of the growing organism (Mikkola and Orkin, 2006). Following the expansion phase in the fetal liver the HSCs migrate into the newly formed bone marrow, where they are housed and functionally maintained during the remaining lifespan of the adult (Kardel and Eaves, 2012). Differentiation of murine ES cells towards the hematopoietic lineage demonstrated that hematopoietic and endothelial cells develop from a shared precursor (Choi et al., 1998), supporting an earlier hypothesis that a mesodermal precursor, referred to as the hemangioblast, gives rise to both blood cells and vascular cells during embryonic development. Hemangioblast cells express Flk1 (Ema et al., 2003; Fehling et al., 2003), and ES-derived Flk1+ cells have been demonstrated to form blood through a transient endothelial state, prior to up-regulating hematopoietic markers and down-regulating endothelial markers (Mikkola et al., 2003). This transition of endothelial cells to hematopoietic cells was later observed in mouse embryos for both primitive wave hematopoiesis in the YS, as well as for definitive hematopoiesis in the AGM region (Lancrin et al., 2009). While embryonic development features separate hematopoietic events at multiple anatomical locations, the central features are that hematopoietic cells and vascular
  • 32. 30 endothelium develop from a shared mesodermal precursor, and that the emergence of hematopoietic cells occurs by transition through a hemogenic endothelial (HE) state (Costa et al., 2012). Indeed, hematopoietic progenitor cells have been directly confirmed to bud from endothelial precursors lining the luminal space on the ventral side of the dorsal aorta in murine embryos (Boisset et al., 2010), and has been additionally verified by lineage tracing experiments (Zovein et al., 2008). Live-cell imaging has also confirmed the emergence of blood from murine HE generated in vitro (Eilken et al., 2009). Studies aimed at characterizing this process of Endothelial to Hematopoietic Transition (EHT) have revealed the involvement of several intrinsic and extrinsic factors (Costa et al., 2012). The transcription factor Scl has been demonstrated to be critical for the establishment of HE from Flk1+ hemangioblast cells (D’Souza et al., 2005). Transition of HE into definitive hematopoietic progenitors is then critically dependent on the activity of transcription factor Runx1 (Yokomizo et al., 2001), with runx1 expression at the dorsal aorta overlapping with the emergence of hematopoietic cells as demonstrated by live imaging of zebrafish embryos (Lam et al., 2010). Runx1 then regulate EHT by acting through the two transcription factors Gfi1 and Gfi1b (Lancrin et al., 2012). Runx1 itself is regulated by both internal and external factors. For instance, Scl has been demonstrated to directly regulate the expression of Runx1 (Nottingham et al., 2007; Landry et al., 2008), and Bmp4 signaling, mediated by Smad1, is also capable of regulating Runx1 expression (Pimanda et al., 2007). Notch activity has also been demonstrated to induce runx1 expression and HSC expansion during embryonic development in zebrafish (Burns et al., 2005), and it has recently been reported that hPSC-derived HE depend on NOTCH signaling for EHT (Ditadi et al., 2015). Furthermore, Runx1 expression is positively regulated by biomechanical forces, mediated by nitric oxide based signaling mechanisms, due to fluid shear stress from the circulation (Adamo et al., 2009). Catecholamines, a class of neurotransmitters, have also been demonstrated to play a role in the emergence of HSCs at the murine AGM. Knockout of the enzyme tyrosine hydroxylase, responsible for synthesizing catecholamines, led to reductions in repopulating HSCs at the murine AGM, which could then be rescued by catecholamine supplementation (Fitch et al., 2012). Catecholamines are known to bind to β-adrenergic receptors on the cell surface, with downstream signaling mediated through cAMP pathways, thereby suggesting a possible role for cAMP in EHT.
  • 33. 31 Figure 7. Establishment of Hemogenic Endothelium, and the Emergence of Hematopoietic Cells through Endothelial to Hematopoietic Transition. The upper part of the figure illustrates the emergence of budding hematopoietic progenitors from Hemogenic Endothelium (HE) at the ventral side of the dorsal aorta. The lower part of the figure provides a simplified schematic over key transcription factors and external mediators involved in the establishment of HE, and the emergence of blood from HE through Endothelial to Hematopoietic Transition (EHT). The illustration was inspired by figure 3 and 4 in Costa et al., 2012. Working in the murine setting has allowed for in vivo observations to directly complement findings based on differentiating murine pluripotent stem cells towards blood in the laboratory. Since the process of EHT cannot be directly studied in human embryos due to ethical reasons, in vitro differentiation of hPSCs provides an alternative approach to study this process in the human setting. Differentiation of hPSCs demonstrated that a hemangioblast population, comparable to the murine counterpart, exists in the human setting (Kennedy et al., 2007). More recently, with the aim of identifying and characterizing human HE, several groups have successfully differentiated hPSCs into HE that express RUNX1 and VE-Cadherin, capable of giving rise to definitive hematopoietic progenitors with lymphoid-myeloid potential (Choi et al., 2012; Kennedy et al., 2012). Recently, the surface marker phenotype CD34+ CD73- CXCR4- has been reported to separate human HE from committed vascular endothelium (Ditadi et al., 2015). While these reports provide evidence that HE can be generated from hPSCs, full consensus on appropriate markers remains to be reached, and the developmental relationship between cells undergoing EHT remains to be elucidated. Scl Runx1 Gfi1 Gfi1b AorticLumen Mesodermal Precursor (Hemagioblast) Hemogenic Endothelium Hematopoietic Stem Cell Bmp4 Smad1 Shear Stress EHT Budding Hematopoietic Progenitors Hemogenic Endothelium Aortic Endothelium
  • 34. 32 In Vitro Differentiation of hPSCs Towards HSCs The Potential of in Vitro HSC Generation from hPSCs During treatment of patients suffering from hematological malignancies myeloablative conditioning, including radiotherapy and/or chemotherapy, is employed to facilitate eradication of malignant cells. During such treatment the potentially healthy HSCs in the patient are also destroyed. Therefore, re- introduction of functional HSCs after myeloablative treatment, often by transplantation of donor-derived cells, allows for the re-establishment of hematopoiesis and thereby the survival of the patient. However, donor-derived transplantation does not come without potential complications. Human Leukocyte Antigen (HLA) compatibility between donor and patient is required to reduce the risk of Graft Versus Host Disease (GVHD), where the establishing immune system of the transplant recognize the cells of the patient as foreign, commencing an immunological response. GVHD is highly associated with mortality in transplanted patients (Copelan, 2006) and due to the need for high HLA compatibility the number of available and compatible donors can be limited. Another potential limitation is due to the variable quality of donated HSCs. Aged HSCs have been demonstrated to have a reduced function as compared to young HSCs (Morrison et al., 1996; Sahin and DePinho, 2010). In addition, the number of available HSCs in a transplant may be too few to allow for successful and efficient repopulation of the patient (Brunstein et al., 2010; Doulatov et al., 2012). Thus, methods to grow and expand available self-renewing HSCs in vitro have long been pursued. However, while it is possible to expand cells from HSCs that carry the correct cell surface markers, self-renewal of HSCs cultured in vitro is rapidly lost, severely limiting the efficiency of such approaches (Walasek et al., 2012). It has been proposed that the loss of HSC self-renewal in vitro is due to the absence of the conditions associated with the bone marrow niche. To summarize, there is a pressing need for HLA-compatible HSCs, in high quantities and of good clinical quality, to ensure optimal treatment of myeloablated patients. Since the discovery of pluripotent stem cells, the potential use of these cells as a source for generating HSCs have presented an attractive goal for the scientific community. The indefinite expansion capacity of pluripotent stem cells, combined with the possibility of creating pluripotent stem cells from individual patients (patient-specific iPS cells) could, in principle, allow for the generation of patient- derived HSCs. The HLA compatibility of patient specific iPS-derived HSCs, together with the potential possibility of providing cells with high quality and in sufficient numbers, could dramatically improve the treatment of patients in the clinic (Figure 8). This also includes the possible correction of genetically carried
  • 35. 33 hematological diseases if applying additional methods of gene therapy. Furthermore, successful generation of HSCs could provide an improvement to better study the nature of hematological malignancies, and also for the purpose of drug screening (Vo and Daley, 2015). Figure 8. The Potential of Hematopoietic Stem Cells Generated from Pluripotent Stem Cells. Successful generation of functional human HSCs, of good clinical quality and in large quantity, could improve the treatment of patients with hematological malignancies. Theoretically, small amounts of somatic cells collected from the individual patient, could be reprogrammed into patient-specific iPS cells. If needed these cells could then be corrected for inherited or acquired mutations by methods of gene therapy. The patient-specific iPS cells could then be expanded in the laboratory before being instructed to differentiate into large quantities of functional HSCs. The generated HSCs would then be transplanted back into the patient after myeloablative conditioning, thereby re- establishing the hematopoietic system of the patient while circumventing the issue of HLA compatibility. Such patient- specific replacement therapy could provide new and safer treatments against a multitude of hematological malignancies. Methodology Many studies have reported protocols for generating hematopoietic cells from hPSCs. These protocols typically feature differentiation of hPSCs by Embryoid Body (EB) formation, alternatively differentiating the hPSCs as a monolayer. Additional approaches include culturing the cells on supportive stroma or extracellular matrix, with media either containing serum (undefined) or having a defined composition, supplemented with specific morphogens and growth factors to encourage the commitment of hPSCs towards the hematopoietic lineage (Kaufman et al., 2001; Chadwick et al., 2003; Vodyanik et al., 2005; 2006; Ledran et al., 2008; Choi et al., 2012; Kennedy et al., 2012; Ditadi et al., 2015). The cultures are then analyzed for HSC-like cells by methods including; cell surface marker analysis using flow cytometry, gene expression analysis, lymphoid- myeloid differentiation potential, and long-term engraftment capacity by transplantation into immunodeficient recipient mice. Interestingly, these approaches to differentiate pluripotent stem cells towards the hematopoietic lineage in vitro show considerable parallelism to embryonic blood development + Somatic Cells Pluripotent Stem Cells Hematopoietic Stem Cells Patient Drug Screening Disease Study
  • 36. 34 (Kardel and Eaves, 2012). This includes the initial mesodermal commitment, followed by an early wave of primitive hematopoiesis, and later the onset of definitive hematopoiesis as indicated by the emergence of hematopoietic progenitors with lymphoid-myeloid potential (Choi et al., 2012; Kennedy et al., 2012; Timmermans et al., 2009). Recently, bioinformatics have provided a new method to study hematopoietic development. Using computational methods of analysis to interpret biological data, bioinformatics can be used to identify candidate genes of interest for the purpose of understanding the genetic foundation of a biological process. Combined with high throughput analysis at the single cell level, bioinformatics could provide a powerful approach to elucidate the details of EHT but so far this has not been reported for the murine, or for the human setting. There have been several important discoveries indicating that obtaining functional HSCs from hPSCs should be possible (see next section). However, generation of hPSC-derived HSCs that are functionally comparable to true HSCs remains a challenge. It is generally assumed that either a lack of a HSC-specific gene signature, or a lack of appropriate developmental cues required for correct developmental specification, could explain the difficulty in generating self- renewing HSCs from hPSCs in vitro (Mikkola and Orkin, 2006; Murry and Keller, 2008; Vo and Daley, 2015). Previous Key Discoveries Human ES cells were first successfully differentiated (in vitro) into hematopoietic colony forming cells in 2001 (Kaufman et al., 2001). Later in 2002, murine ES cells, by overexpression of the transcription factor HoxB4, were differentiated into HSC-like cells that could self-renew and had lymphoid-myeloid engraftment potential (Kyba et al., 2002). The same method was used in conjunction with corrective gene therapy to cure immunodeficiency in Rag2- /- mice, demonstrating by proof-of-concept the therapeutic potential of pluripotent stem cells (Rideout et al., 2002). The concept of iPS cells was introduced in 2006 when murine iPS cells, generated from fibroblasts, were reported (Takahashi and Yamanaka, 2006). One year later, the same authors reproduced their findings in the human setting, generating the first human iPS cells (Takahashi et al., 2007). In 2007, it was also demonstrated that murine iPS cells could be used for autologous correction of sickle cell anemia in mice (Hanna et al., 2007). By 2008, human iPS cells were generated from patients with a variety of genetic diseases including adenosine deaminase deficiency-related severe combined immunodeficiency, Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher
  • 37. 35 disease (GD), Parkinson disease (PD), Down syndrome (DS), and more (Park et al., 2008), providing new means to study these diseases, and also a platform for the possible employment of corrective gene therapy. 5 years later in 2013, short-term myeloid-erythroid engraftment with cells generated from hPSCs was demonstrated by means of overexpressing 5 transcription factors (HOXA9, ERG, RORA, SOX4, and MYB) (Doulatov et al., 2013). The same year, long-term engrafting HSCs were extracted from human iPS-derived teratomas in mice (Amabile et al., 2013; Suzuki et al., 2013) providing evidence that functional HSCs can be generated from hPSCs in vivo. And in 2015, it was reported that co-culture of differentiating non-human primate iPSCs together with human endothelial cells (expressing DLL4 and JAG1) enabled generation of cells capable of long-term lymphoid-myeloid engraftment and serial transplantation (Gori et al., 2015). The same paper also differentiated human ES cells in a similar manner and demonstrated long-term lymphoid-myeloid engraftment in mice. However, serial transplantation was not reported raising the possibility that the generated hPSC-derived cells were long-lasting Multipotent Progenitors (MPPs). Together, colleagues in the scientific community have made significant progress that brings us closer to the final goal of improving therapies against hematological malignancies. Unfortunately the generation of hPSC-derived HSCs with engraftment capacity on par with bona fide HSCs has not yet been achieved.
  • 38. 36
  • 39. 37 Aims of the thesis With the ultimate goal of facilitating in vitro generation of functional Hematopoietic Stem Cells (HSCs) from human Pluripotent Stem Cells (hPSCs), the studies included in this thesis explore potential key factors and mechanisms involved in the process of hematopoietic development. More specifically, the aims were:  To evaluate the effects of Retinoic Acid (RA) on the development of blood and on generated hematopoietic progenitors (Paper I).  To molecularly dissect the process of Endothelial to Hematopoietic Transition (EHT) in the human setting (Paper II).  To explore the functions of cyclic Adenosine Mono Phosphate (cAMP) on the emergence of hematopoietic progenitors (Paper III).  To investigate the impact of Reactive Oxygen Species (ROS) on the functionality of generated hematopoietic progenitors (Paper IV).
  • 40. 38 New links must be forged as old ones rust. - Jane Howard
  • 41. 39 Summary of results Paper I: Retinoic Acid Regulates Hematopoietic Development from Human Pluripotent Stem Cells In Paper I, we dissected the role of Retinoic Acid (RA) signaling in human hematopoietic development by using in vitro differentiation of hPSCs towards the hematopoietic lineage. We report that RA decreases blood generation during this process. RA signaling inhibition, before the onset of hematopoiesis, establishes an increase in the final output of cells with an HSC-like surface marker phenotype, confirming the role of RA signaling in the early stages of human blood development. qRT-PCR analysis for genes marking specific developmental cell types identified the stages during development where decreased RA signaling increased commitment towards the hematopoietic lineage. This included the initial commitment of pluripotent stem cells to mesoderm, and also by increased specification toward hemangiogenic mesoderm while reducing cardiac mesoderm differentiation. These results suggest that increased levels of RA antagonize blood development, and that reduced RA facilitate increased generation efficiency of hematopoietic progenitors from hPSCs. Furthermore, we show that RA accelerated the differentiation of generated hematopoietic progenitors, and that reduction of RA signaling preserved the generated progenitor fraction from differentiation into more mature cell types. Taken together, we have provided evidence identifying multiple developmental stages that depend on restricted RA signaling for efficient differentiation towards blood, and we have shown that RA signaling inhibition can prevent loss of blood progenitors generated in vitro. Our findings show the utility of differentiating pluripotent stem cells in vitro to study human hematopoietic development, and to determine the effects and functions of RA in this process.
  • 42. 40 Graphical Abstract for Paper I. Paper II: Endothelial to Hematopoietic Transition State Identified in Differentiated Human Pluripotent Stem Cells In paper II, using iPS in vitro differentiation, we proposed a model for Endothelial to Hematopoietic Transition (EHT) in the human setting. Single-cell transcriptional analysis of index-sorted cells has allowed us to molecularly identify an EHT transcriptional state within a novel EHT differentiation hierarchy, where a subset of cells (termed 5b subset) express high levels of numerous key HSC regulators while also displaying a clear endothelial program. Together, Principal Component Analysis (PCA) and unsupervised hierarchical clustering analysis placed these cells at the interface between the endothelial and the hematopoietic populations. Also, index-sorting allowed for a molecular characterization of the currently best functionally described cell types in the human setting, i.e. Hemogenic Endothelium (HE) and HSC-like cells, within our EHT hierarchical model, placing the 5b subset developmentally between these functional cell types. Finally, correlation of the transcriptional profile of our identified successive groups with existing genome-wide transcriptional analysis data of bulk populations of interest from the mouse AGM, further supports our EHT hierarchical model and inferred cell program identities. Human Pluripotent Stem Cells High RA High RA Low RA Low RA Blood Progenitor Cells Differentiated Blood Cells
  • 43. 41 In summary, with single-cell analysis for 90 genes, relevant for both endothelial and hematopoietic regulation, we show for the first time, at a single-cell level and in the human setting, the existence of an EHT program at the interface between endothelial identity and hematopoietic commitment. Graphical Abstract for Paper II. Paper III: Cyclic AMP Induction Enhances Human Hematopoietic Cell Emergence Through the cAMP-Epac Signaling Axis, Creates Redox State Balance, and Increases CXCR4 Expression. In paper III, we explored the functions of cAMP on the emergence of HSC-like cells generated from hPSCs. Our findings demonstrate that cAMP, by signaling through the Epac axis, regulates the in vitro specification of hPSC-derived HSC- like cells. In addition to up-regulating the frequency of HSC-like cells in the differentiation cultures, cAMP induction also mitigated oxidative stress, created redox-state balance, and enhanced CXCR4 expression in hPSC-derived hematopoietic cells. Furthermore, we demonstrated that by inhibiting the downstream effectors of cAMP signaling, PKA and Epac, only Epac signaling is required for hematopoietic emergence during hPSC differentiation towards the hematopoietic lineage. Epac regulates endothelial cell-cell adhesion, and the involvement of endothelial cells for Endothelial to Hematopoietic Transition Hemogenic Endothelium HSC-like EHT HOXB4 MEN1 SPI1 RUNX1 LIN28B MEIS1 FLI1 ETV6 VAV1 LYL1 GATA2 CBFA2T3 TAL1 NFE2 BMI1 5 6 5b 74 Endothelial Genes WAS-GFP HSC Genes
  • 44. 42 (EHT) during the emergence of hematopoietic cells suggests a novel link between endothelial cell junctions and EHT. Epac inhibition resulted in impairment of Hemogenic Endothelium (HE) specification, reduced blood emergence, and decreased the numbers of HSC-like cells from hPSCs, thus demonstrating the critical role of Epac signaling in the specification of human HE and the generation of hPSC-derived HSC-like cells. Collectively, our findings show that cAMP regulates the generation of human HSC-like cells during in vitro hematopoiesis. By demonstrating the role of the cAMP-Epac axis in blood and HSC emergence, our study provides new insights in understanding the previously unknown role of the cAMP-signaling component in human hematopoietic development. Graphical Abstract for Paper III. Paper IV: Reactive Oxygen Species Impair the Function of Hematopoietic Stem / Progenitor Cells Generated from Human Pluripotent Stem Cells In Paper IV, we investigated the impact of ROS on hematopoietic progenitors generated in vitro from hPSCs. We report that the vast majority of all hPSC- derived hematopoietic progenitors display an increased ROS level, as compared to cord blood non-cultured progenitors. High ROS levels were associated with reduced colony forming capacity and increased DNA damage. Of hPSC-derived HSC-like cells, only cells with reduced amounts of ROS could proliferate upon sub-culture, demonstrating the detrimental impact of elevated ROS on these cells. Furthermore, we report 4 separate factors capable of reducing ROS in our cultures, Hemogenic Endothelium Human Pluripotent Stem Cells hPSC-Derived HSC-Like Cells ROS Redox Balance CXCR4 Cyclic AMP Epac
  • 45. 43 demonstrating that ROS build-up in vitro results not only from cellular metabolism but also from multiple processes including innate immunity, the cellular stress response, and the atmospheric oxygen concentration. Combining these factors greatly reduced the level of ROS and increased the generation of ROSlo hPSC-derived HSC-like cells capable of robust proliferation. Taken together, we have provided evidence that increased ROS, associated with multiple processes during in vitro culture, leads to functional decline in the majority of hematopoietic progenitors generated from hPSCs. Methods to normalize ROS towards physiological levels enabled a > 20-fold increased production of HSC-like cells with a reduced ROS phenotype and robust proliferative capacity, indicating that oxidative stress is occurring during standard in vitro culture and that it is functionally detrimental to these cells. Thus, normalization of ROS toward physiological levels will be a key requirement for future in vitro generation of engrafting HSCs. Graphical Abstract for Paper IV. ROS ReactiveOxygen Species hPSC-Derived HSC-LikeCells Functionality Antioxidants Innate Immunity Blocking Stress Response Inhibition 4% Oxygen Condition + + + + ROS Functionality
  • 46. 44
  • 47. 45 Conclusions  Decreased retinoic acid signaling increases the generation of hematopoietic progenitors from human pluripotent stem cells in vitro (paper I).  Retinoic acid signaling inhibition increases the early developmental commitment toward the hematopoietic lineage (paper I).  Retinoic acid signaling inhibition prevents differentiation and loss of more immature hematopoietic progenitors generated in vitro (paper I).  Single-cell transcriptional analysis of index-sorted cells provides molecular identification of an EHT transcriptional state in the human setting (paper II).  A proposed model for EHT argues for a linear progression from endothelial to hematopoietic commitment that features an intermediate stage (paper II).  cAMP, mediated through the Epac signaling axis, regulates the generation of HSC-like cells from human pluripotent stem cells in vitro (paper III).  Increased cAMP signaling is associated with reduced oxidative stress, redox-state balance, and enhanced CXCR4 expression in generated hematopoietic cells (paper III).  The majority of hPSC-derived hematopoietic progenitors have increased ROS levels, originating from multiple processes (paper IV).  ROShi hematopoietic progenitors have reduced colony forming capacity and increased DNA damage (paper IV).  A ROSlo phenotype is a prerequisite for proliferative capacity in hPSC- derived HSC-like cells (paper IV).  Combinatorial ROS reduction increases the generation of hPSC-derived HSC-like cells with proliferative capacity (paper IV).
  • 48. 46 It’s not what you look at that matters, it’s what you see. - Henry David Thoreau
  • 49. 47 General discussion and future perspectives In vitro generation of functionally relevant HSCs from hPSCs has the potential to revolutionize the treatment of hematological diseases and malignancies. In this thesis, by using in vitro differentiation of hPSCs towards the hematopoietic lineage, we elucidate several novel aspects of this process. By controlling the amount of retinoic acid we increase in vitro specification of hPSCs toward blood, allowing for a more defined and efficient development of relevant cells. Our molecular identification of EHT, the conversion through which HSCs emerge from endothelial cells provides a foundation for further characterization of cells undergoing this process, and could potentially enable control over this transition for the purpose of more efficient generation of hematopoietic cells from hemogenic endothelium. Increased understanding on how cAMP intrinsically regulates the generation of HSC-like cells, together with the ability to modulate it, provides an additional approach to increase the generation, and potentially the function, of HSC-like cells from hPSCs. Finally, the finding that reactive oxygen species is increased due to the in vitro culture conditions, causing functional impairment of hPSC-derived hematopoietic stem and progenitor cells, argues that we must revise our standardized culture methods and that a more physiological oxygen concentration is likely to be a critical component for handling and generating HSCs outside of the body. Unfortunately, while our work provides new insights into the process of hematopoietic development and HSC emergence, we have not been able to generate true HSCs capable of robustly engrafting into recipient mice. As mentioned previously, the scientific community has long pursued the successful generation of functionally relevant HSCs from hPSCs and over the years there has been significant progress bringing us closer to the final goal of improving therapies against hematological malignancies. However, the generation of hPSC- derived HSCs with engraftment capacity on par with bona fide HSCs has not been achieved. Theories as yet to why the generation of functional HSCs has remained such a challenge include a possible lack of specific developmental cues required for
  • 50. 48 correct specification of hPSCs towards the HSC, or alternatively that a gene regulatory signature of HSC self-renewal is missing (Mikkola and Orkin, 2006; Murry and Keller, 2008; Vo and Daley, 2015). I would argue that both theories mentioned above are based on assumptions that something is “lacking” for successful HSC generation,and that such approaches forget to consider the possibility that HSC function (and thereby generation) instead could be antagonized by external factors. In other words, assuming that a problem is complex and therefore looking for a complex solution, is risky if more simplistic possibilities have not been thoroughly evaluated. Illustration created by Hanna Rönn (2015). Based on findings included in this thesis, I would therefore like to propose an alternative theory where ROS, primarily resulting as an external artifact from our standardized in vitro culture conditions, cause functional impairment of cells that otherwise could have performed more satisfactory. Thus, correction of ROS would technically be a correction of a methodological oversight, rather than an improvement on developmental commitment or directly establishing an HSC gene signature. I would like to provide a range of arguments why this Model of Functional Impairment by External Oxidative Insult may be relevant for the generation of functional HSCs from hPSCs in vitro.
  • 51. 49 Firstly, generation of robustly engrafting HSCs from hPSCs is possible by in vivo teratoma formation (Amabile et al., 2013; Suzuki et al., 2013). Not only is this proof-of-principle that functional HSCs can be generated from hPSCs, but also that undirected and fully stochastic differentiation, giving rise to only a small percentage of blood, is sufficient for successful HSC generation. This indicates that strategies to further improve the developmental commitment towards the hematopoietic lineage are unlikely to confer HSC functionality. Furthermore, the fact that stochastic differentiation of hPSCs in vitro does not yield the same results suggests that the in vivo aspect is critical. Secondly, the fact that HSC function is rapidly impaired upon exposure to in vitro conditions not only questions the rationale of generating functional HSCs from hPSCs in similar settings, but also suggests that this impairment is directly linked to the very in vitro culture conditions that we employ. Thus, we need to consider the “standardized” way we often culture cells in vitro. Ever since Wilhelm Roux in 1885 demonstrated that embryonic chick cells could be kept alive in saline solution outside the body, cell culture has continued to evolve. One example is the work of Hayflick and Moorhead from 1961, demonstrating that human fibroblasts cultured in vitro can only undergo a finite number of divisions (Hayflick and Moorhead, 1961). Since then, in vitro culture has been refined, allowing us to study a multitude of different cell types in the laboratory. One central aspect of typical in vitro culture is the handling of cells with an atmospheric oxygen concentration at 21%. Arguably, an atmospheric oxygen concentration is much higher than what is encountered physiologically by cells inside the body, but since most cell types retain functionality during culture with this setting the level of oxygen has not been of great concern. However, if we agree that there could be cell types that are more critically dependent on more physiological conditions, we can no longer continue to assume that our traditional in vitro culture methods are appropriate for culturing such cells. An increased oxygen concentration will result in increased spontaneous formation of ROS (Baez and Shiloach, 2014). Adult HSCs are highly sensitive to ROS and multiple studies have demonstrated that increased ROS is incompatible with long-term engraftment capacity of both murine and human HSCs (Ito et al., 2006; Jang and Sharkis, 2007; Yahata et al., 2011). Finally, it should be taken into consideration that there may be species-specific differences between human and murine cells in terms of stress resistance. It has indeed been suggested that human and murine HSCs have differences in their DNA repair and damage response mechanisms (Biechonski and Milyavsky, 2013). Mice are also more resistant to radioactive exposure in comparison to humans, further indicating a difference between the species in terms of stress tolerance (Green et al., 2007; Donnelly et al., 2010). If we accept the likely possibility of a real difference in stress resistance between human and murine cells, and we also
  • 52. 50 agree that in vitro culture facilitates a stressful environment for HSCs, then it provides a possible explanation as to why strategies that enabled the generation of functional HSC-like cells in the murine setting (Kyba et al., 2002) have not been successfully reproduced in the human setting, despite more than a decade of scientific effort. In paper IV we were unable to reduce the amount of ROS in our generated HSC- like cells to a level equal to that of non-cultured cord blood blood progenitors, and we did not observe robust engraftment when we transplanted these cells. Thus, it would be very interesting to evaluate the engraftment capacity of hPSC-derived HSC-like cells if methods to fully normalize the ROS level were to be developed. However, while our work does provide clear evidence that ROS is detrimental to the functionality of HSCs, we cannot exclude the possibility that additional aspects may be required to facilitate successful generation of functional HSCs. In other words there may be additional hurdles to overcome, along with sufficient ROS reduction, before functional HSCs can be generated. But in the end we still must overcome every single hurdle to reach our goal. Previously, generation of self-renewing HSC-like cells from pluripotent stem cells, capable of long-term lymphoid-myeloid engraftment, was demonstrated in the murine setting by overexpression of the transcription factor HoxB4 (Kyba et al., 2002). While artificial overexpression of genes does not technically mimic the developmental process towards the emergence of blood, it does provide an alternative route for generating functional HSCs. If successful generation of human HSCs were to be achieved by gene expression strategies, it would be interesting to see how the functions of these cells might correlate with their level of ROS. The results included in this thesis provide new insights into the process of blood development and the emergence of HSCs, bringing us closer to establishing methods for safe and efficient HSC generation suitable for clinical applications.
  • 53. 51 Acknowledgements Thinking about all the time I have spent at work the last 6 years and how it now might come to an end fills me with both excitement and some sadness. I remember that when I started my primary motivation was just simple curiosity, and perhaps a desire to do something that would have some lasting relevance. There are so many people that have been with me during these years and without them I would not have been able to come this far. These people have enriched my days, supported me through both the good and the bad times, and have made the time as a PhD- student one of the best times in my life. Therefore I want to acknowledge the following people: First, I am very grateful to Niels-Bjarne Woods, my Supervisor, for his never- ending support, his scientific enthusiasm, and for encouraging me to always be creative. Because of his supervision I feel confident, as I will continue my journey and my scientific career. To Stefan Karlsson, my co-supervisor, for establishing the warm and highly scientific molmed environment that we all share at A12. Without that friendly, enthusiastic, and scientifically objective environment I would not have enjoyed my PhD as much as I did. To Jonas Larsson, Head of Department, for continuing the caretaking of our great department, and for always is having the best in mind for all of us who work here. To Anna Rignell-Hydbom, Deputy Director of the Medical Faculty, and Anders Malmström, Director of the Research School, for being great sources of support for us students, watching over us during our journeys. To Teia and Zhi for all their support in the FACS core. To Qianren and Emanuela for doing an awesome job running the iPS core. To the staff in the animal facility for always taking care of our mice. To Beata and Xiaojie for running the vector core. To Karin Olsson, for all your help and for answering my endless amount of questions during the first years. To Göran Karlsson for taking care of me and my fellow students during the time in the preparatory program, and Christine Karlsson for your organization of the department and your drive to get things done.
  • 54. 52 To the young PIs; Mattias Magnusson, Sofie Singbrant, Kenichi Miharada, and Johan Flygare for your scientific discussions, and for sharing your experience and advice to prepare me additionally for my future in science. To the members of the Woods-group: To Roksana for sharing this journey with me from the very start. Your kindness and hard work will take you forward towards your goal. To Carolina for being a great friend and a great co-worker. Your energy, creativity, and scientific drive will take you far. To Shobhit for sharing your friendship, your experience, and for reminding me about the bigger questions. I always enjoy our coffee-break discussions. All of my office mates: Abdul, Alexander, Sandra, Hooi Min, Matilda, Maria, and Roksana for all the good times and great Youtube videos ;-) (https://www.youtube.com/watch?v=HwSAkRKcc4g). To Pekka for being a great friend and a great inspiration. I just might come and crash on your couch in Cambridge a few more times. To Roman for your friendship, generosity, Arnold quotes, and your insane passion for both laborative and medical science. House FTW! To Praveen, Valli, Shub, and Alex for great basketball sessions. And to Sarah, Sara, Alexandra, Simon, Karolina, David, Terese, Mehrnaz, Svetlana, Justyna, Visnja, Natsumi, Ilana, Carmen, Ineke, Charlotta, Kavitha, Kristian, Lina, and all current and previous co-workers at BMC A12 and B12. To Andreas for great discussions, hangouts, and for being a good friend. To Ajoy, for great science and beer sessions (next pint is on me). To Maggie for training me in human stem cell culture (and Long Island Iced Tea drinking) during my time at the SALK. Special thanks to Bjarne, Carolina, Shobhit, and Alex for giving me valuable feedback on parts of this thesis.
  • 55. 53 To Professor Masanobu Satake and my dear friend and former mentor Shunsuke Kon (now an Assistant Professor at Hokkaido University) for taking care of me during my year at Tohoku University in Japan (2007-2008), giving me my first true lab experience. I am not very good at keeping in contact regularly but I will definitely keep coming for visits when I travel to Japan in the future. Because of you, I was able to set my aim on becoming a scientist. Till alla mina vänner utanför jobbet: Hemifrån; Rodney, Jacob, Joke. Från min tid i Skåneland; Emil, Alina, Henke, Hillan, PG, Olof, Stellan, Grodan, och Kungen. Jag vet att jag inte alltid gör mycket ljud av mig men jag värdesätter er vänskap oerhört och hoppas att ni fortsätter att ha tålamod med mig även i framtiden. Till min underbara familj. Till Mamma Irené och Pappa Lennart. Till mina tre lillasystrar Hanna (tack för den fina bilden till diskussionen ☺), Sofia, och Victoria. Till Farmor Gunilla, och även till min Farfar Gunnar som alltid stöttat mig i mina akademiska studier men som tyvärr inte finns med oss i livet nu när jag blir klar. Samt till mina bortgångna morföräldrar, Mormor Karin och Morfar Bertil. Jag älskar er alla! Och till sist till min trolovade Meike, för att du alltid finns där för mig och hjälper mig vara mer än jag någonsin kunde vara på egen hand. Utan dig och vår dotter Mia saknar min värld mening. Jag älskar er!
  • 56. 54
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