CD44 as a hematopoietic stem cell marker [Elektronische Ressource] / von Rahul Singh

Ulrike Müller

Sujets

• Biologie

Informations

• Publié par: karlsruher_institut_fur_technologie
• Publié le: 01 janvier 2011
• Nombre de lectures: 67
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

CD44 as a Hematopoietic stem cell
marker



Zur Erlangung des akademischen Grades eines
DOKTORS DER NATURWISSENSCHAFTEN
(Dr. rer. nat.)

Fakultät für Chemie und Biowissenschaften
Karlsruher Institut für Technologie (KIT) – Universitätsbereich

vorgelegte
DISSERTATION
Von
Rahul Singh
aus
Indien
2011








Dekan: Prof. Dr. Stefan Bräse
Referent: Prof. Dr. Margot Zöller
Korreferent: Prof. Dr. Andrew Cato
Tag der mündlichen Prüfung: 15.04.2011

i
“Never give up on what you want to do. A person with real big dreams is more
powerful than all the facts”







To my loving parents and my family….














iiTable of Contents
Abbreviations………………………………………………………………………........1
Zusammenfassung……………………………………………………………………...4
1: Introduction………………………………………………………………………….6
1. Stem cells and Hematopoietic stem cells…………………………………..6
1.1 Definition of stem cells………………………………………………………...6
1.2 Hematopoietic stem cells and Hematopoiesis………………………………8
1.2.1 Hematopoietic stem cells……………………………………………...….8
1.2.2 Hematopoiesis………………………………………………………..…..10
1.3 Characterization of HSC…………………………………………………..…13
1.4 The importance of the HSC niche………………………………………..…15
1.5 The clinical demand on HSC…………………………………………….….17
1.6 Sources of HSC……………………………………………………………....18
1.6.1 Bone marrow & Mobilized peripheral blood…………………………...18
1.6.2 Umbilical cord blood……………………………………………………..18
1.6 3 Embryonic stem cells………………………………………………….…19
2. CD44 molecule………………………………………………………………....20
2.1 Structure ……………………………………………………………………....20
2.2 Hyaluron binding, amino terminal domain ………………………………….22
2.3 Stem structure………………………………………………………………….22
2.4 Transmembrane and cytoplasmic domain……………………………...…..23
2.5 CD44 functions…………………………………………………………......….25
2.5.1 Cell adhesion and migration……………………………………………..25
2.5.2 Interaction with the cytoskeleton……………………………...………...26
2.5.3 Interaction with cytokines, chemokines and enzymes………………...28
2.5.4 CD44 as a co-receptor and signalling……………………………...…...29
2.6 CD44 in hematopoietic stem cells……………………………………..…….35
3. AIM…………………………………………………………………………….......36
2: Materials and methods………………………………………………………........37
2.1 Materials………………………………………………………………….….....37
2.1.1 Animals……………………………………………………………….…..37
iii 2.1.2 Bacterial strain………………………………………………………….…37
2.1.3 Cell lines………………………………………………………..…….…...37
2.1.4 Primers………………………………………………………...…………..38
2.1.5 Primary antibodies…………………………………………………….....38
2.1.6 Secondary antibodies…………………………………………………....41
2.1.7 Inhibitors and enzymes……………………………………………….....42
2.1.8 Instruments…………………………………………………………….....42
2.1.9 Miscellaneous ……………………………………………………………44
2.1.10 Chemicals…………………………………………………………….....45
2.1.11 Buffers and solutions…………………………………………………..48
2.2 Methods…………………………………………………………………….…..49
2.2.1 Molecular Biology……………………………………………………....49
2.2.1.1 Chemical competent cells………………………………………….....49
2.2.1.2 Transformation………………………………………………………....49
2.2.1.3 Mini-prep…………………………………………………………….….49
2.2.1.4 Enzymatic restriction digestion……………………………….………50
2.2.1.5 Midi-prep……………………………………………………….……….50
2.2.1.6 RNA preparation, cDNA synthesis and amplification………….…..50
2.2.1.7 DNA gel electrophoresis………………………………………….…...51
2.2.2 Protein chemistry………………………………………………….……52
2.2.2.1 Antibody purification……………………………………………….…..52
2.2.2.2 Column preparation……………………………………………….…...52
2.2.2.3 Purification of the CD44 recombinant soluble molecule……….…..53
2.2.2.4 Biorad assay………………………………………………………....…53
2.2.2.5 Pull down assay …………………………………………………….….53
2.2.2.6 SDS-PAGE………………………………………………………….…..54
2.2.2.7 Immunoprecipitation …………………………………………….….....54
2.2.2.8 Western blotting………………………………………………….……..55
2.2.2.9 Silver staining …………………………………………………….….…55
2.2.3 Cell biology………………………………………………………….…...55
2.2.3.1 Cell culture………………………………………………………...........55
iv 2.2.3.2 Magnetic beads separation…………………………………………..56
2.2.3.3 Flow cytometry………………………………………………………...57
2.2.3.4 Apoptosis assay…………………………………………………….…58
2.2.3.5 Proliferation assay…………………………………………………….58
2.2.3.6 Phosphatase assay…………………………………………………...58
2.2.3.7 Sucrose gradient centrifugation …………………………………….59
2.2.3.8 Cytosol, nuclei and mitochondrial preparation……………………..59
2.2.3.9 Transfection of adherent cells ……………………………………….59
2.2.4 Animal Experiment ………………………………………………….…60
2.2.4.1 Long term bone marrow culture (LTBMC)…………………………..60
2.2.4.2 Reconstitution and tumor cell injection……………………………...60
2.2.5 Statistical analysis……………………………………………………..60
3: Results………………………………………………………………………………..61
Part-I The importance of CD44 in the crosstalk between HSC
and bone marrow stroma………………………………………………61
1. CD44 expression, associating molecules & ligands on HSC
& niche cells………………………………………………………………….61
1.1 CD44 expression on BMC and LTBMC stroma cells…………………..61
1.1.1 RT-PCR profiling on BMC subpopulations…………………………....65
1.1.2 Adhesion molecule profile on BMC & LTBMC stroma ………….......67
1.1.3 Cytokines & cytokine/chemokine receptor expression on
BMC & LTBMC stroma………………………………………………...68
1.2 CD44 associating molecules in HSC & LTBMC stroma cells………....69
1.2.1 CD44 co-expression with stem cells, progenitor cells and
leukocyte markers on BMC/HSC & LTBMC stroma……………….…69
1.2.2 CD44 co-expression with cytokine/chemokine receptor on
BMC/HSC and LTBMC stroma………………………………………....72
1.2.3 CD44 co-expression with adhesion molecules on BMC/HSC
and LTBMC stroma………………………………………………………74
1.2.4 CD44 associating molecules on BM, HSC & LTBMC stroma……….76
1.3 Potential ligands on HSC and stroma cells for CD44 and CD44v…….78
v1.3.1 Generation of soluble recombinant CD44s & CD44v7…………….…78
1.3.2 Pulldown assay…………………………………………………………...80
1.4 Functional consequences of HSC CD44 interaction with
LTBMC stroma………………………………………………………………82
1.4.1 Role of HSC CD44 interaction in the maintenance
of quiescence……………………………………………………………..82
1.4.2 Role of HSC CD44 interaction in the apoptosis resistance……….83
Part-II The importance of CD44 in the crosstalk between leukemia
and the bone marrow stroma…………………………………………...86
2.1 Anti-panCD44 interferes with hematopoiesis and thymoma growth...86
2.2 Anti-CD44 promotes thymoma cell apoptosis…………………………89
2.3 Apoptosis induction, loss of mitochondrial membrane polarization
and caspase-9 activation…………………………………………….…...91
2.4 Anti-CD44 induces ERK inhibition through PP2A………………….....94
2.5 ERK1/2 inhibition promotes apoptosis and caspase-9 cleavage…....98
4: Discussion……………………………………………………………….…………..101
4.1 CD44 expression in HSC and bone marrow stroma cells……….….....101
4.2 CD44 associating molecule in HSC and bone marrow stroma cells....102
4.3 Functional consequences of HSC CD44 interaction with bone
marrow stroma cells………………………………………………….…….102
4.4 CD44 activities in leukemic cells……………………………...………...103
4.5 T lymphoma cells are less CD44-dependent than hematopoietic
progenitors…………………………………………………………………….....104
4.6 A CD44 antibody blockade promotes death receptor-independent
leukemic T cell apoptosis…………………………………………………..…..104
4.7 Anti-CD44-promoted apoptosis proceeds via CD44 redistribution and
PP2A association…………………………………………………………….…105
4.8 CD44-associated PP2A promotes mitochondrial membrane de-
stabilization via ERK1/2 dephosphorylation………………………………....105
5: Summary………………………………………………………………………….….107
6: References……………………………………………………………………….…..109
Acknowledgements………………………………………………………………..…..127
Curriculum vitae………………………………………………………….…………….128
viFigures
Part I
Figure 1A Expression of stem cells, progenitors and mature leukocyte
marker in BMC/HSC……………………………………………………….…62
Figure 1B Expression of stem cells, progenitors and mature leukocyte
marker in LTBMC- stroma…………………………………………………...63
Figure 1C Expression of CD44 and CD44v in BMC sub populations…………….…63
Figure 1D Expression of CD44 and CD44v in LTBMC-stroma and S17 cells……...64
Figure 1E RT-PCR for BM and LTBMC-Stroma…………………………………….…65
Figure 1F RT-PCR of separated BMC subpopulations…………………………….…66
Figure 1G Integrin expression profile on BMC, HSC and LTBMC-stroma cell……..67
Figure 1H Expression of cell adhesion molecule and selectins on BMC,
HSC and LTBMC-stroma cell…………………………………………….….67
Figure 1I cytokines and cytokines receptors expression on BMC, HSC and
LTBMC-stroma cells……………………………………………………….….68
Figure 2A CD44 co-expression with stem cells, progenitors and mature
leukocyte markers on BMC……………………………………………….