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Functional characterization of the PBX interacting protein (HPIP) in normal and malignant human haematopoiesis [Elektronische Ressource] / submitted by Pawandeep Kaur

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From the Department of Medicine III, Grosshadern Hospital, Ludwig-Maximilians-University, Munich Director: Prof. Dr. med. Wolfgang Hiddemann Functional characterization of the ‘PBX interacting protein’ (HPIP) in normal and malignant human haematopoiesis. Thesis submitted for a Doctoral Degree in Human Biology at the Faculty of Medicine, Ludwig-Maximilians-University, Munich, Germany Submitted by Pawandeep Kaur From Amritsar, India 2009 Aus der Medizinischen Klinik und Poliklinik III am Klinikum Großhadern der Ludwig-Maximilians-Universität München, Direktor: Prof. Dr. med. Wolfgang Hiddemann Funktionelle Charakterisierung des ‘PBX interagierenden Proteins’ (HPIP) in der normalen und malignen humanen Hämatopoese Dissertation zum Erwerb des Doktorgrades der Humanbiologie an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu München, Deutschland Vorgelegt von Pawandeep Kaur aus Amritsar, Indien 2009 Mit Genehmigung der Medizinischen Fakultät der Universität München Berichterstatter: Priv. Doz. Dr. Michaela Feuring - Buske Mitberichterstatter: Prof. Dr. Laurenz J. Würzinger Priv. Doz. Dr. Irmgard Bumeder Mitbetreuung durch den promovierten Mitarbeiter: Dekan: Prof. Dr. Dr. h.c. M. Reiser, FACR, FRCR Tag der mündlichen Prüfung: 10.02.2010 With permission from the Faculty of Medicine University of Munich Supervisor/Examiner: PD Dr.

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Published 01 January 2009
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From the Department of Medicine III, Grosshadern Hospital,
Ludwig-Maximilians-University, Munich
Director: Prof. Dr. med. Wolfgang Hiddemann


Functional characterization of the
‘PBX interacting protein’ (HPIP)
in normal and malignant human
haematopoiesis.


Thesis submitted for a Doctoral Degree in Human Biology
at the Faculty of Medicine, Ludwig-Maximilians-University,
Munich, Germany


Submitted by
Pawandeep Kaur
From
Amritsar, India
2009
Aus der Medizinischen Klinik und Poliklinik III am Klinikum
Großhadern der Ludwig-Maximilians-Universität München,
Direktor: Prof. Dr. med. Wolfgang Hiddemann


Funktionelle Charakterisierung des
‘PBX interagierenden Proteins’ (HPIP)
in der normalen und malignen humanen
Hämatopoese


Dissertation zum Erwerb des Doktorgrades der Humanbiologie
an der Medizinischen Fakultät der Ludwig-Maximilians-Universität zu
München, Deutschland


Vorgelegt von
Pawandeep Kaur
aus
Amritsar, Indien
2009

Mit Genehmigung der Medizinischen Fakultät
der Universität München




Berichterstatter: Priv. Doz. Dr. Michaela Feuring - Buske


Mitberichterstatter: Prof. Dr. Laurenz J. Würzinger
Priv. Doz. Dr. Irmgard Bumeder

Mitbetreuung durch den
promovierten Mitarbeiter:


Dekan: Prof. Dr. Dr. h.c. M. Reiser, FACR, FRCR


Tag der mündlichen Prüfung: 10.02.2010



With permission from the Faculty of Medicine University of
Munich




Supervisor/Examiner: PD Dr. Michaela Feuring - Buske


Co-Examiners: Prof. Dr. Laurenz J. Würzinger
Priv. Doz. Dr. Irmgard Bumeder

Co-Supervisor:


Dean: Prof. Dr. Dr. h.c. M. Reiser, FACR, FRCR


Date of submission:

Date of Oral Exam: 10.02.2010









Dedicated to

My parents

Gagan, Ankur, Kashvi, Gulraj, Aman

And my loving husband
EmmanuelIntroduction
Table of contents
1. Introduction
1.1. Haematopoiesis and development of blood cellular componenets
1.1.1. Human haematopoiesis 1
1.1.2. Classification of human haematopoietic stem cell. 3
1.1.3. The haematopoietic colony forming cell (CFC). 4
1.1.4. The haematopoietic inductive environment. 6
1.1.5. Homing, engraftment and repopulation of hHSCs. 6
1.1.6. Sources of haematopoietic stem cells. 7
1.1.7. Human CD34+ blood cells. 8
1.2. Developmental pathways in human haematopoiesis
1.2.1. Long term-HSCs in xenotransplant models. 8
1.2.2. The subsets of human lymphoid and myeloid progenitors. 8
1.2.3. Lineage Bias inHSCs. 9
1.3. Extrinsic and intrinsic factors affecting the haematopoiesis
1.3.1. Cytokines. 11
1.3.2. Cooperative interactions of cytokines. 12
1.3.3. Tyrosine kinase receptor and cell signalling. 14
1.3.4. The role of transcription factors. 15
1.4. The role of HOX and non-homeobox genes in haematopoiesis
1.4.1. The structure of HOXB4 and its co factors. 19
1.4.2. The expression of HOX in haematopoietic cells. 21
1.4.3. The TALE homeodomain protein family PBX. 22
1.4.4. The novel Haematopoietic PBX-interacting protein (HPIP). 22
1
Introduction
2. Aims of the study 23
3. Materials and methods 4
3.1. Materials
3.1.1. Buffers
3.1.1.1. Western Blot 24
3.1.1.2. Agarose gel electrophoresis 25
3.1.1.3. Stem cell sorting buffers 25
3.1.2. Mediums
3.1.2.1. Serum free medium 25
3.1.2.2. Haematopoietic cell washing medium 26
3.1.2.3. Lymphoid cell culture medium 26
3.1.2.4. Maintenance medium for feeder cells 26
3.1.3. Mammalian cell lines 26
3.1.4. The NOD/SCID mice 27
3.1.5. NOD/SCID mice related reagents and equipment 28
3.1.6. Bacterial Strain 28
3.1.7. Cytokines and antibodies 28
3.1.8. Commercial Kits 29
3.1.9. Umbilical cord blood cells, plasmids and molecular markers

31
3.1.10. Miscellaneous reagents 32
3.1.11. Software and machines 3
3.1.12. Microscopes 34
2
Introduction
3.2. Methods
3.2.1. Thawing, passage and freezing of mammalian cells 34
3.2.2. MSCV based retroviral vector 35
3.2.3. Mutagenesis 35
3.2.4. Generation of shRNA constructs 37
3.2.5. Establishment of stable PG13 packaging cell lines 42
3.2.6. Purification of umbilical cord blood CD34enr population of
cels 43
3.2.7. Feeders and co-cultures 44
3.2.8. Transient transduction of hCB CD34enr stem cell population

44
3.2.9. Detection of gene and protein expression 46
3.2.10. Sub cellular localization of proteins 47
3.2.11. Colony forming assays 48
3.2.12. Liquid Expansion cultures 49
3.2.13. B-Lymphoid progenitor cell assay 50
3.2.14. Bulk Human Long term culture initiating cell assay
(Bulk LTC-IC) 50
3.2.15. Fluorescence associated cell sorting 51
3.2.16. Xenotransplantation models 52
3.2.17. Analysis of BM of sacrificed mice 54
3.2.18. Generation of monoclonal antibody against HPIP-WT 54
3.2.19. Affymetrix gene chip expression analysis 54
3
Introduction
3.3. Analysis
3.1. Staistc 54
3.3.2. Gene expression profile 54
3.3.3. Confocal microscope 55
4. Results
4.1. Transduction efficiency measured on the human umbilical cord blood
derived CD34enr stem cell population 56
4.2. RNA and protein expression analysis 6
4.3. Sub cellular localization of constitutively expressed proteins

58
4.4. In vitro proliferation assay
4.4.1. Commitment and differentiation of tranduced cells 60
4.5. Colony forming assay (CFC)
4.5.1. Quantification of committed haematopoietic progenitor cells in vitro

63
4.5.2. CFC replating assay 66
4.6. In vitro B-Lymphoid progenitor assay 69
4.7. Long term culture initiating cell assay (LTC-IC) in vitro 73
4.8. Double transduction with HPIP-WT and HOXB4 wt vectors 75
4.9. Analysis of wt and mutant HPIP functional in the xenotransplantat
NOD/SCID model
4
Introduction
4.9.1. Engraftment of transduced hCB CB34+ cells in the bone
marrow of NOD/SCID mice
77
4.9.2. The assessment of lymphoid/myeloid ratio in vivo 79
4.9.3. Short term (hSTRC) and long term (hLTRC) repopulating
populations in NOD/SCID bone marrow 81
4.9.4. The effect of constitutively expressed wt and mutant HPIP on
the multilineage repopulation of human haematopoietic cells
in NOD/SCID xenotransplantation model

84
4.9.5. Limiting dilution assay (LDA) in NOD/SCID mice:
Quantification of SCID repopulating (SRC) cell frequency
86
4.10. Affymetrix differential gene expression profile 89
4.11. Gene expression profiling in subtypes of AML and ALL 94
5. Discusion 96
6. Sumary 107
7. Zusammenfassung 109
8. Refrences 112
9. Abreviatons 2
10. Acknowledgments 24
11. Curriculum Vitae
12. Publications from the work
5