From the Department of Medicine III, Grosshadern Hospital, Clinical Cooperative Group “Leukemia’’ Ludwig-Maximilians-University, Munich Director: Prof. Dr. med. Wolfgang Hiddemann Role of the ABC transporter ABCG2 in human haematopoiesis Thesis Submitted for a Doctoral degree in Human Biology at the Faculty of Medicine Ludwig-Maximilians-University, Munich, Germany Submitted by Farid Ahmed From Patna, India 2007 With the permission from the Faculty of Medicine, University of Munich Supervisor/Examiner: Prof. Dr. med. Stefan Bohlander Co-examiners: Prof. Dr. Thomas Brocker Priv. Doz. Dr. Peter Nelson, Ph.D Prof. Dr. Arndt Borkhardt Co-Supervisor: PD Dr. Michaela Feuring-Buske Dean: Prof. Dr. med. Dietrich Reinhardt Viva voce held on: 23.01.2007 Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern der Universität München, Direktor: Prof. Dr. med. W.
FromtheDepartmentofMedicineIII,Grosshader’’nHospital,Clinical Cooperative Group “Leukemia Ludwig-Maximilians-University, Munich
Director: Prof. Dr. med. Wolfgang Hiddemann
Role of the ABC transporter ABCG2 in human haematopoiesis
Thesis Submitted for a Doctoral degree in Human Biology at the Faculty of Medicine Ludwig-Maximilians-University, Munich, Germany
Submitted by Farid Ahmed
From Patna, India
2007
With the permission from the Faculty of Medicine, University of Munich
Supervisor/Examiner: Co-examiners:Co-Supervisor:Dean: Viva voce held on:
Prof. Dr. med. Stefan Bohlander
Prof. Dr. Thomas Brocker
Priv. Doz. Dr. Peter Nelson, Ph.D
Prof. Dr. Arndt Borkhardt
PD Dr. Michaela Feuring-Buske
Prof. Dr. med. Dietrich Reinhardt
23.01.2007
Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern der Universität München, Direktor: Prof. Dr. med. W. Hiddemann
Die funktionelle Bedeutung des ATP bindenden Transportproteins ABCG2 für die Hämatopoese
Dissertationzum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München
vorgelegt von Farid Ahmed aus Patna, Indien 2007
der
Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Prof. Dr. med. Stefan Bohlan Mitberichterstatter: Prof. Dr. Thomas Brocker Priv. Doz. Dr. Peter Nelson, Prof. Dr. Arndt Borkhardt Mitbetreuung durch die promovierte Mitarbeiterin: PD Dr. Michaela Feuring-Bu Dekan: Tag der mündlichen Prüfung: 23.01.2007
Ph.D
ske
Prof. Dr. med. Dietrich Reinhardt
This work is dedicated to my grandfathers, late Dr. Fakhrul Hasan and late Hafiz Abdur-Rahman
Table of Contents
1Introduction ......................................................................................................... 41.1Haematopoiesis ........................................................................................... 41.1.1Haematopoietic Stem Cells................................................................... 41.1.2 5Origin of HSC........................................................................................1.1.3 ........................................................ 6Genetic programs specifying HSC1.1.4 ....................................................................... 8Characterization of HSC1.1.5 12 .........................................................Surface marker analysis of HSC1.1.6 14Other features of haematopoietic stem cells.......................................1.1.7Sources of HSC .................................................................................. 161.2ABC proteins.............................................................................................. 181.2.1 21Multidrug resistance 1 (ABCB1):.........................................................1.2.2ABCG2:............................................................................................... 211.2.3Organisation ofABCG2gene and regulation: ..................................... 221.2.4 ............................................................. 23Tissue distribution of ABCG2:1.2.5Multidrug resistance:........................................................................... 241.2.6ABCG2 is the molecular determinant of side population (SP) phenotype: ........................................................................................................ 251.2.7ABCG2 in stem cells: .......................................................................... 261.2.8Expression of ABCG2 in human cancers and the concept of cancer stem cells .......................................................................................................... 272Aim of the study. ............................................................................................... 303Materials & Methods ......................................................................................... 313.1Materials .................................................................................................... 313.1.1Plasmids ............................................................................................. 313.1.2Antibodies ........................................................................................... 313.1.3Primers ............................................................................................... 323.1.4 32Mammalian cell lines...........................................................................3.1.5 .................................................................................... 33Bacterial strain3.1.6 33The NOD-SCID Mice: .........................................................................3.1.7 33Mice related reagents and equipment:................................................3.1.8Commercial kits .................................................................................. 343.1.9Buffers and special medium................................................................ 353.1.10Microscopes........................................................................................ 363.1.11 ................................ 36Fluorescence activated cell sorting and analysis3.1.12Reagents ............................................................................................ 36
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3.1.13Cytokines ............................................................................................ 373.1.14Apparatus ........................................................................................... 373.2Methods ..................................................................................................... 383.2.1 ..........................................................................................Cell culture 383.2.2Freezing of mammalian cells .............................................................. 383.2.3 .................................................................................. 39Thawing of cells3.2.4ABCG2 cloning ................................................................................... 393.2.5Developing high titre producer cell lines ............................................. 403.2.6Purification of human UCB CD133+and CD34+ 40cells .........................3.2.7 41and transduction of human cells ................................Pre-stimulation 3.2.8 ............................................................................. 42Human CFC Assay3.2.9 44Liquid expansion assay.......................................................................3.2.10Human Long-Term Culture-Initiating Cell (LTC-IC) Assay .................. 453.2.11LTC-IC Limiting Dilution Analysis........................................................ 453.2.12B-cell progenitor assay ....................................................................... 473.2.13 47Transplantation into NOD/SCID mice .................................................3.2.14Intracellular staining with ABCG2 antibody ......................................... 483.2.15RNA and genomic DNA isolation and cDNA preparation.................... 483.2.16 49LM-PCR for detection of retroviral integration site ..............................Results .............................................................................................................. 514.1Isolation of human UCB CD133+cells ....................................................... 514.2Efficient retroviral transduction ofABCG2in human haematopoietic cells. 524.3Assessment of ABCG2 overexpression in retrovirally transduced cells ..... 534.4Constitutive expression ofABCG2increases the production of CFCin vitro 544.5ABCG2overexpression increases the replating potential of the CFCin vitro 564.6Constitutive expression ofABCG2does not change the expansion kinetics + of CD34 cells ....................................................................................................... 584.7Bulk LTC-IC assays demonstrate no increase in the output of the LTC-IC by overexpression ofABCG2.................................................................................... 594.8LTC-IC limiting dilution analysis ................................................................. 604.9Multilineage differentiation ofABCG2-transduced human CB cells in NOD/SCID mice.................................................................................................... 624.10 62 .........Detailed phenotypic analysis of human CB cells in NOD/SCID mice
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Inversion of the lymphoid-myeloid ratio......................................................
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Effect on HSC ............................................................................................ 66
4.13Constitutive expression ofABCG2 68does not change the SRC frequency ..
Blood contains several different cell types that can be classified into two main classes, lymphoid cells (T, B, and natural killer cells) and myeloid cells (granulocytes, monocytes, erythrocytes, and megakaryocytes). Each of these has a unique set of specialized properties and, in most cases, important life-supporting functions. All blood cells have a limited lifespan: several hours for granulocytes, several weeks for erythrocytes, and up to several years for memory T-cells. Each day the body produces billions of new cells to replace blood cells lost to normal turnover processes as well as to illness or trauma. A variety of homeostatic mechanisms allow blood cell production to respond quickly to stresses such as bleeding or infection and then return to normal levels when the stress is resolved. The highly orchestrated process of blood cell production and homeostasis is termed haematopoiesis(fig 1.1).
1.1.1 Haematopoietic Stem Cells
All blood cells are produced from a small common pool of pluripotent cells called haematopoietic stem cells (HSC) and oligo-potent progenitors by differentiation (Morrison et al., 1995). The production of mature blood cells from such a pluripotent HSC involves a highly regulated progression through successive stages as commitment to a specific lineage, terminal differentiation of lineage restricted progenitor, growth arrest and apoptosis. The expression of different molecules on the surface of haematopoietic progenitors permits the interaction with various regulatory elements present in their environment, which includes stromal cells, extracellular matrix molecules and soluble regulatory cytokines as growth and differentiation factors. HSC are endowed with two characteristics: they give rise to additional HSC through self-renewal and also undergo differentiation to progenitor cells that become variously committed to different haematopoietic lineages (Weissman, 2000). These processes are under the tight control of distinct genetic programs with specific nuclear factors playing a key role. Operationally, HSC are best described as those cells capable of reconstituting the haematopoietic system of a recipient individual.