Hematopoiesis in the adult organism is maintained by a complex, hierarchically organized cascade of differentiating cells that ultimately originate from adult hematopoietic stem cells (HSCs). HSCs have been extensively studied in perturbative settings (e.g transplantation assays), which are well suited for exploring cell potential but tell very little about cell behaviour in a physiological state. Novel \textit{in situ} techniques have made it possible to observe hematopoiesis in its natural environment, yet many questions about native HSC behaviour remain controversial. It is yet unclear whether and to what extent HSCs contribute to steady-state blood production, and whether they represent a reserve population that can be activated upon emergency. As, to date, common definitions of HSCs comprise cells of heterogeneous function, incoherent results are likely linked by another fundamental question: what is the identity of the HSC population residing on the apex of the hematopoietic hierarchy? To answer these questions is essential to gain a deeper understanding of fatal human diseases like leukemia or aplastic anemia. We describe an integrative approach combining non-invasive experimental methods with mathematical inference to uncover underlying pathways and differentiation patterns of steady state hematopoiesis. By this, we identify a population of Sca-1\textsuperscript{hi} CD201(EPCR)\textsuperscript{hi} HSCs as the hithertho elusive apex population, and show that they contribute marginally, but continuously, to native steady-state hematopoiesis. Further, we clarify the architecture of a shortcut route of thrombopoiesis, which emanates directly from HSCs and links them trough a previously undefined CD48\textsuperscript{-/low} megakaryocyte progenitor population directly to platelets. Finally, we provide an extensive analysis of the dynamic label propagation and proliferation behaviour of hematopoietic stem and progenitor cells in prototypical stress situations mimicking blood loss, infection and inflammation. We find that apex stem cells do not directly contribute to emergency hematopoiesis and are only activated upon severe myeloablation and chronic inflammation. Further, innate immune training did not influence HSC contribution in response to reoccurring stimulation. In sum, we demonstrate that primitive HSCs do neither represent a major contributor to steady-state blood production, nor a reservoir for emergency supply. We thus question current dogma of hematopoiesis, and argue that the primary function of HSCs in the adult organism remains yet to be discovered.:Contents
Abstract iv
Zusammenfassung vii
List of Figures xii
List of Tables xiii
List of Abbreviations xv
1 Introduction 1
1.1 Hematopoiesis and the hematopoietic system 1
1.1.1 Developmental origins 1
1.1.2 The hematopoietic hierarchy 3
1.2 Hematopoietic stem cells 5
1.2.1 Characteristics of hematopoietic stem cells 7
1.2.2 Identification of hematopoietic stem and progenitor cells 7
1.2.3 HSC heterogeneity 11
1.3 Novel genetic tools to analyse native hematopoiesis 12
1.3.1 Conditional gene targeting 12
1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15
1.4.1 Models of native Hematopoiesis 16
1.4.2 Models of HSC quiescence 23
1.4.3 Aged hematopoiesis 25
1.5 Hematopoietic stem cell behaviour in stress response 26
1.5.1 Inflammation and Infection 27
1.5.2 Myeloablation 30
1.5.3 Innate immune training 32
2 Aim of Thesis 35
3 Material and Methods 37
3.1 Materials 37
3.1.1 Antibodies 37
3.1.2 Buffer and Solutions 38
3.1.3 Chemicals and Reagents 39
3.1.4 Cultivation Media 40
3.1.5 Kit Systems 41
3.1.6 Software 41
3.2 Mice 42
3.2.1 Animal housing and husbandry 42
3.2.2 Mouse strains 42
3.3 Genotyping of mice 43
3.4 Treatments of mice 46
3.4.1 Reporter induction 46
3.4.2 Hematopoietic stress induction 46
3.5 Hemograms 48
3.6 Transplantation and in vivo repopulation assays 49
3.6.1 Preconditioning and transplantation of recipients 49
3.6.2 Competitive repopulation assay 50
3.6.3 Limiting dilution assay 50
3.6.4 Transplantation into sublethally irradiated hosts 51
3.7 Cell preparation for transplantation and flow cytometry 51
3.7.1 Bone marrow 51
3.7.2 Splenocytes 51
3.7.3 Peripheral blood 51
3.7.4 Peritoneal cells 52
3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52
3.8 Flow Cytometry 53
3.8.1 Cell staining 54
3.8.2 Cell counting 55
3.8.3 Cell identification and gating 55
3.8.4 Chimerism analysis 57
3.8.5 Cell sorting 58
3.9 Cytokine detection assay 58
3.10 in vitro single cell expansion assay 59
3.11 Sequencing 61
3.11.1 Single cell RNA sequencing 61
3.11.2 Bulk RNA sequencing 62
3.11.3 Single cell and bulk transcriptome analysis 62
3.12 Mathematical modelling 62
3.13 Data Normalization 63
3.14 Statistical Analysis 64
4 Results 67
4.1 Identification of an apex HSC population 67
4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68
4.1.2 ES HSCs reside at the top of the hierarchy 71
4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76
4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77
4.2.2 The myeloid lineage diverges within phenotypic HSCs 80
4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82
4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82
4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88
4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92
4.4 Ageing modulates the dynamics of fate mapping 95
4.5 HSCs in times of crisis: Contribution to stress recovery 99
4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100
4.5.2 Severe myeloablation provokes HSC activation 105
4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107
4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113
4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118
4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120
5 Discussion 123
5.1 Labels matter: What is a true HSC? 123
5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124
5.1.2 ES HSCs reside at the top of the hierarchy 126
5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127
5.1.4 The myeloid lineage diverges within phenotypic HSCs 130
5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132
5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132
5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135
5.3 Fundamental dynamics of label propagation are preserved in aged mice 136
5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138
5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139
5.4.2 Severe myeloablation provokes HSC activation 143
5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145
5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149
5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150
6 Conclusion 153
References 155
Acknowledgements 185
Appendices 187
Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189
Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190 / Die Blutbildung in erwachsenen Organismen wird durch eine komplexe, hierarchisch organisierte Kaskade von sich fortwährend differenzierenden Zellen aufrechterhalten, und hat ihren Ursprung in adulten h\'amatopoietischen Stammzellen (HSZs). HSZs wurden ausgiebig unter unphysiologischen Extrembedingungen (z.B. Transplantation) untersucht, in welchen sich viel über das maximale potential einer Zelle, aber wenig über ihr Verhalten in ungestörtem Zustand sagen lässt. Obwohl durch moderne \textit-Techniken mittlerweile h\'amatopoietische Differenzierung in ihrer ursprünglichen Umgebung beobachtet werden kann, bleiben etliche Fragen zum nativen Verhalten von HSZs offen. So ist es noch unklar, ob und in welchem Ausmaß HSZs überhaupt zur stationären Blutbildung beitragen, oder ob sie eine Reservepopulation darstellen, die im Notfall aktiviert werden kann. Da bis dato gebr\'auchliche Definitionen von HSZs verschiedene Sub-Populationen mit heterogener Funktionalität umfassen, sind die inkohärenten Ergebnisse wahrscheinlich mit einer anderen grundlegenden Frage verknüpft: Was ist die wahre Identit\'at der HSZ-Population, die an der Spitze der h\'amatopoetischen Hierarchie steht? Die Beantwortung dieser Fragen ist essenziell für ein besseres Verst\'andnis von fatalen humanen Krankheiten, wie beispielsweise Leuk\'amie oder aplastische An\'amie. Wir beschreiben einen integrativen Ansatz, der nicht-invasive experimentelle Methoden mit mathematischer Modellierung vereint, um die zugrundeliegenden Pfade und Differenzierungsmuster der station\'arer H\'amatopoese zu ergründen. Auf diese Weise können wir eine Population von Sca-1\textsuperscript{hi} und CD201(EPCR)\textsuperscript{hi} HSZs als die bisher unbekannte Apex-Population identifizieren, und zeigen, dass diese Zellen geringf\'ugig, aber kontinuierlich, zur nativen H\'amatopoese beitragen. Darüber hinaus klären wir die Architektur eines direkten thrombozytischen Differenzierungspfades, welcher unmittelbar von HSZs abzweigt und diese über eine neu definierte CD48\textsuperscript{-/lo} Megakaryozyten-Vorläuferpopulation direkt mit Thrombozyten ver- bindet. Schließlich liefern wir eine umfassende Analyse des dynamischen Differenzierungs- und Proliferationsverhaltens von hämatopoetischen Stamm- und Vorläuferzellen in prototypischen Stresssituationen, die Blutverlust, Infektion und Entzündung nachahmen. Wir zeigen, dass primitive Stammzellen nicht direkt zur Notfall-Hämatopoese beitragen und nur bei schwerer Myeloablation und chronischer Entzündung aktiviert werden. Ebenfalls hat das Training des angeborenen Immunsystems keinen Einfluss auf die Aktivität von HSZs bei wiederholter Stimulation. Unsere Ergebnisse belegen, dass primitive HSZs weder einen wesentlichen Beitrag zur stationären Blutbildung leisten, noch ein Reservoir für die Notfallversorgung darstellen. Wir stellen somit das derzeitige Dogma der Hämatopoese in Frage und argumentieren, dass die primäre Funktion der HSZ im erwachsenen Organismus noch zu entdecken ist.:Contents
Abstract iv
Zusammenfassung vii
List of Figures xii
List of Tables xiii
List of Abbreviations xv
1 Introduction 1
1.1 Hematopoiesis and the hematopoietic system 1
1.1.1 Developmental origins 1
1.1.2 The hematopoietic hierarchy 3
1.2 Hematopoietic stem cells 5
1.2.1 Characteristics of hematopoietic stem cells 7
1.2.2 Identification of hematopoietic stem and progenitor cells 7
1.2.3 HSC heterogeneity 11
1.3 Novel genetic tools to analyse native hematopoiesis 12
1.3.1 Conditional gene targeting 12
1.4 Hematopoietic stem cell behaviour in adult steady-state hematopoiesis 15
1.4.1 Models of native Hematopoiesis 16
1.4.2 Models of HSC quiescence 23
1.4.3 Aged hematopoiesis 25
1.5 Hematopoietic stem cell behaviour in stress response 26
1.5.1 Inflammation and Infection 27
1.5.2 Myeloablation 30
1.5.3 Innate immune training 32
2 Aim of Thesis 35
3 Material and Methods 37
3.1 Materials 37
3.1.1 Antibodies 37
3.1.2 Buffer and Solutions 38
3.1.3 Chemicals and Reagents 39
3.1.4 Cultivation Media 40
3.1.5 Kit Systems 41
3.1.6 Software 41
3.2 Mice 42
3.2.1 Animal housing and husbandry 42
3.2.2 Mouse strains 42
3.3 Genotyping of mice 43
3.4 Treatments of mice 46
3.4.1 Reporter induction 46
3.4.2 Hematopoietic stress induction 46
3.5 Hemograms 48
3.6 Transplantation and in vivo repopulation assays 49
3.6.1 Preconditioning and transplantation of recipients 49
3.6.2 Competitive repopulation assay 50
3.6.3 Limiting dilution assay 50
3.6.4 Transplantation into sublethally irradiated hosts 51
3.7 Cell preparation for transplantation and flow cytometry 51
3.7.1 Bone marrow 51
3.7.2 Splenocytes 51
3.7.3 Peripheral blood 51
3.7.4 Peritoneal cells 52
3.7.5 Cell depletion by magnetic activated cell sorting (MACS) 52
3.8 Flow Cytometry 53
3.8.1 Cell staining 54
3.8.2 Cell counting 55
3.8.3 Cell identification and gating 55
3.8.4 Chimerism analysis 57
3.8.5 Cell sorting 58
3.9 Cytokine detection assay 58
3.10 in vitro single cell expansion assay 59
3.11 Sequencing 61
3.11.1 Single cell RNA sequencing 61
3.11.2 Bulk RNA sequencing 62
3.11.3 Single cell and bulk transcriptome analysis 62
3.12 Mathematical modelling 62
3.13 Data Normalization 63
3.14 Statistical Analysis 64
4 Results 67
4.1 Identification of an apex HSC population 67
4.1.1 Fgd5 Cre-System preferentially labels primitive HSCs 68
4.1.2 ES HSCs reside at the top of the hierarchy 71
4.2 A combination of proliferation and differentiation modelling uncovers lineage trajectories 76
4.2.1 Iterative modelling reveals subtle, but continuous contribution of HSC to Steady state hematopoiesis 77
4.2.2 The myeloid lineage diverges within phenotypic HSCs 80
4.3 Fate mapping uncovers an alternative pathway of thrombopoiesis 82
4.3.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 82
4.3.2 CD48-stratified MkP subsets display variable thrombopoietic potential 88
4.3.3 Thrombopoietin signalling enhances platelet production via the direct pathway 92
4.4 Ageing modulates the dynamics of fate mapping 95
4.5 HSCs in times of crisis: Contribution to stress recovery 99
4.5.1 Faithful reporters are necessary to study HSPC behaviour during stress hematopoiesis 100
4.5.2 Severe myeloablation provokes HSC activation 105
4.5.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 107
4.5.4 Innate immune training does not alter HSC proliferation and differentiation 113
4.5.5 Compensation of blood cell loss is achieved without HSC contribution 118
4.5.6 Emergency hematopoiesis proceeds without activation of primitive Hematopoietic stem cells (HSCs) 120
5 Discussion 123
5.1 Labels matter: What is a true HSC? 123
5.1.1 Fgd5ZsGreen:CreERT2R26LSL-tdRFP fate mapping preferentially labels primitive HSCs 124
5.1.2 ES HSCs reside at the top of the hierarchy 126
5.1.3 Subtle, but continuous contribution of HSCs to steady state hematopoiesis 127
5.1.4 The myeloid lineage diverges within phenotypic HSCs 130
5.2 Fate mapping uncovers an alternative pathway of thrombopoiesis 132
5.2.1 CD201-/lo Sca-1lo HSCs feed thrombopoiesis via CD48-/lo MkPs 132
5.2.2 Thrombopoietin signalling enhances platelet production via the direct pathway 135
5.3 Fundamental dynamics of label propagation are preserved in aged mice 136
5.4 HSCs in times of crisis: are HSC a reserve population for emergency response? 138
5.4.1 Faithful reporters: Caveats and merits of using in situ models to study HSC activation 139
5.4.2 Severe myeloablation provokes HSC activation 143
5.4.3 Inflammation-induced emergency hematopoiesis only mildly amplifies HSC activity 145
5.4.4 Compensation of blood cell loss is achieved without HSC contribution 149
5.4.5 Innate immune training does not alter HSC proliferation and differentiation 150
6 Conclusion 153
References 155
Acknowledgements 185
Appendices 187
Anlage 1: Erklärungen zur Eröffnung des Promotionsverfahrens 189
Anlage 2: Erklärung über die Einhaltung der gesetzlichen Vorgaben 190
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:86454 |
| Date | 12 July 2023 |
| Creators | Munz, Clara Marie |
| Contributors | Wielockx, Benjamin, Roers, Axel, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | https://doi.org/10.1182/blood.2022018996, https://doi.org/10.1038/s41467-022-31914-z |
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