Human Haemopoietic Progenitor Cell Mobilization Michael John Watts A thesis submitted to the University of London for the degree of Doctor of Philosophy 1999 The Department of Haematology University College London Medical School University College London 98, Chenies Mews LONDON WC1E6HX ProQuest Number: U642271 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest U642271 Published by ProQuest LLC(2015). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 Abstract The advent of recombinant growth factors in the late 1980's has ushered in a new era of haematopoietic progenitor cell (HPC) therapy by facilitating the mobilization of bone marrow progenitors into the circulation where they can be collected in large numbers by apheresis. The work of this thesis has defined the minimal and optimal CD34+ cell threshold requirements for engraftment. The frequency of poor mobilization was noted and risk factors determined. It was demonstrated that poor mobilization was usually a feature of bone marrow damage rather than a specific mobilization defect. In addition, studies in normal volunteers indicated that there was wide inter-individual variation in G-CSF induced progenitor cell mobilization which was not due to G-CSF pharmacodynamic variability. The clinical feasibility of CD34+ cell purification was determined and its clinical limitations documented. These purified cells were used clinically to demonstrate that the increased numbers of T-cells and monocytes in a peripheral blood stem cell (PBSC) transplant compared to bone marrow did not account for the more rapid haematological recovery that occurs with PBSC. These studies answer specific clinically relevant questions and form a platform for future studies in "graft engineering". Acknowledgements The work contained in this thesis represents 5 years of sometimes intensive but always satisfying effort. I am very grateful to Professor David Linch and Dr Tony Goldstone for their confidence in me (which exceeded my own) to play a major part in the successful development of the peripheral blood stem cell program at University College London Hospitals. This work began with my predecessor, Mark Jones and his wife Sally with our first PBSC transplant at UCLH in 1992. Above all David's intellectual input and clarity of vision has taught me that the most likely route to successful study is a focussed approach which is contrary to my natural inclination of aimless enthusiast. I would also like to pay tribute to the conscientious work of Angela Sullivan who joined me in August 1994 as the clinical demands of the blood stem cell program was rising exponentially. When she left to conduct two successful in vivo expansion experiments of her own, resulting in two baby boys I was fortunate to find Stuart Ings. Stu has been a mainstay of the stem cell laboratory, particularly as my thesis came closer to completion. His reward of course is more work and his own PhD registration last September. I would also like to thank all of the daycare specialist nurses without whom most of the clinical audits would have been impossible and my colleagues in haematology for their unstinting support. Finally, my wife and three children who have complained about my absence at home may hopefully get a chance to complain about my presence at home. Mike Watts, December 1998 Table of Contents Page Title page 1 Abstract 2 Acknowledgements 3 Table of contents 4 List of figures 8 List of tables 10 List of abbreviations IZ List of work published during the course of this thesis 13 Chapter 1 Introduction 1.0.1 Bone marrow the “seed bed” of the blood 15 1.0.2 The bone marrow and the Bomb 18 1.0.3 Early marrow transplantation 18 1.0.4 Improvements in survival 19 1.0.5 The growth of autologous transplantation 20 1.0.6 Stem cell sources 20 1.0.7 "Graft engineering" 21 1.0.8 Transplantation and this thesis 22 1.0.9 Circulating stem cells 23 1.1.0 Possible mechanisms of HPC mobilization 24 1.1.1 Monitoring of PBSC Collection and Transplantation 31 1.1.2 Clonogenic functional assays 32 1.1.3 Flow cytometric CD34+ cell measurement 33 1.1.4 Progenitor threshold requirements 35 1.1.5 Apheresis timing 35 1.1.6 Optimisation of Progenitor Cell Mobilization 36 1.1.7 Post PBSCT Growth Factors 41 1.1.8 Multiple High Dose Therapies 42 1.1.9 Use of Venesection Instead of Apheresis 42 1.2.0 Use of cytoklne-prlmed bone marrow 43 1.2.1 Future Developments 44 1.2.2 CONCLUSION 45 2 General Methods and Materials Page 2.0.1 "General purpose" tissue culture medium 46 2.0.2 Sample handling of apheresis products 46 2.0.3 Cytocentrifuge preparations 46 2.0.4 Viability counts 47 2.0.5 Fiow cytometry for CD34+ cell counts 47 2.0.6 Alkaline Phosphatase-anti-Aikaline Phosphatase (APAAP staining) 49 2.0.7 Manual counting of APAAP stained CD34+ cells 49 2.0.8 Esterase staining 51 2.0.9 Ciinicai CD34+ ceil purification 52 2.1.0 Methyicellulose colony forming cell (CFC) assays 52 2.1.1 In-house methyicellulose media 52 2.1.2 Recombinant growth factors added 54 2.1.3 Commercial methocellulose media 54 2.1.4 Plating concentrations 54 2.1.5 Piiot vial thaw method for CFC 55 2.1.6 Colony counting 55 2.1.7 Mononuclear cell preparation (MNC) and CFC/ml 56 2.1.8 Apheresis CFC dose caiculations 57 2.1.9 Cytokine Assays 58 2.2.0 Data handling and statistical analysis 59 Chapter 3 Progenitor Threshold Requirements for Haematological Recovery 3.0 INTRODUCTION 60 3.1 PATIENTS STUDIED 62 3.2 RESULTS 63 3.2.1 Overali incidence of engraftment delays 63 3.2.2 MNC dose and engraftment 64 3.2.3 CD34-h cell dose and engraftment 64 3.2.4 GM-CFC dose and engraftment 65 3.2.5 Correlation between CD34+ cells and GM-CFC collected at 70 apheresis 3.2.6 Effect of post infusion G-CSF on haematoiogical recovery 72 3.3 DISCUSSION 74 Chapter 4 Factors Predicting for Mobilization Efficiency 4.0 INTRODUCTION 77 4.1.0 PATIENTS AND METHODS 78 4.1.2 PBSC mobiiization and coilection 79 4.1.2 Statisticai analysis of data 80 4.2.0 RESULTS 80 4.2.1 Toxicity of Mobiiization regimen 80 4.2.2 Effect of G-CSF type and leukapheresis machine on PBSC yieids. 80 4.2.3 Factors Predicting for PBSC Yieid 83 4.2.4 Proportion of patients achieving different coilection thresholds 84 4.3 DISCUSSION 87 Chapter 55 Variability of Progenitor Numbers Mobilized by G-CSF in Normal Subjects Page 5.0 INTRODUCTION 90 5.1 METHODS AND MATERIALS 92 5.1.1 Volunteers 92 5.1.2 Study design 92 5.1.3 Progenitor Ceii Measurements 93 5.1.4 Serum G-CSF Assay 94 5.2 RESULTS : 94 5.2.1 Baseline Values 94 5.2.2 White Blood Ceii count 94 5.2.3 Progenitor Ceii Counts 94 5.2.4 Pharmacokinetics 95 5.2.5 individual Variability 96 5.2.6 Adverse events 96 5.3 DISCUSSION 102 Chapter 66 The Role of Back up Bone Marrow for “Poor Mobilizers” 6.0 INTRODUCTION 106 6.1 PATIENTS AND METHODS 108 6.1.1 Patients studied 108 6.1.2 Mobiiization regimens 108 6.1.3 PBSC Harvesting 109 6.1.4 Bone Marrow Han/esting 110 6.1.5 Cryopreservation 110 6.1.6 Progenitor ceii assays 110 6.1.6 High dose therapy regimens 111 6.1.7 Haemopoietic Recovery 112 6.2 RESULTS ; 112 6.2.1 Overaii haematoiogical recovery 112 6.2.2 Haematological recovery of the poorest mobiiizers 112 6.2.3 Back-up bone marrow use 114 6.3 DISCUSSION 119 Chapter 7 Purification Page 7.0 INTRODUCTION 123 7.1 METHODS AND MATERIALS 124 7.1.1 Patients studied 124 7.1.2 Conditioning 124 7.1.3 PBSC mobiiization and coilection 125 7.1.4 Purified CD34+ cell cryopreservation 125 7.1.5 CD34+ ceii purification protocols 126 7.1.6 Processing modifications 126 7.1.7 Progenitor evaluation 127 7.1.8 Caiculations used to assess ceil yieids 128 7.2 RESULTS : 128 7.2.1 Progenitor ceil purity and recovery (yieids) 128 7.2.2 Assessment of Yield variability 132 7.2.3 Effects of the pre-column washing steps 132 7.2.4 Cionogenicity of CD34+ purified ceils 134 7.2.5 Haematoiogical recovery after infusion of purified CD34+ ceils 135 7.2.6 Single versus multiple harvests 136 7.3 DISCUSSION 148 Chapter 88 Accessory Cells do not contribute to Serum G-CSF or IL-6 Cytokine Levels nor to Rapid Haematological Recovery post PBSCT 8.0 INTRODUCTION 152 8.1 METHODS AND MATERIALS 152 8.2.1 Patient groups 152 8.2.2 PBSC mobilization and coilection 153 8.2.3 CD34+ ceii selection 154 8.2.4 Progenitor evaluation 154 8.2.5 Accessory ceii depletion 155 8.2.6 Cytokine assays 155 8.2.7 Statisticai analysis of data 156 8.3 RESULTS : 156 8.3.1 CD34+ ceil purification and accessory cell depletion 156 8.3.2 Cytokine profiles following stem ceil infusions 159 8.3.3 Haematological recovery following infusion of purified cells 162 8.4 DISCUSSION 165 Chapter 99 Discussion and Conclusions 167 References 182 Appendix I 205 List of Figures Chapter 1 Figure Page 1.1 Clonogenic assays and antigens associated with differentiation of 17 Haemopoietic progenitor cells 1.2 Normal bone marrow structure showing sinusoidal blood supply and 26 egress into the circulation of mature blood cells.