Lineage commitment of conditionally immortalized bone marrow mesenchymal stromal cells from tetracycline-regulated SV40 large T-antigen transgenic mice [Elektronische Ressource] / Maria Rostovskaya

Maria Ferretti

Sujets

• Biologie

Informations

• Publié par: technische_universitat_dresden
• Publié le: 01 janvier 2010
• Nombre de lectures: 31
• Langue: English
• Poids de l'ouvrage: 20 Mo

Extrait

DRESDEN UNIVERSITY OF TECHNOLOGY
FACULTY OF MATHEMATICS AND NATURAL SCIENCES





LINEAGE COMMITMENT OF
CONDITIONALLY IMMORTALIZED
BONE MARROW MESENCHYMAL STROMAL CELLS
FROM TETRACYCLINE-REGULATED SV40 LARGE T-ANTIGEN
TRANSGENIC MICE



PHD THESIS

MARIA ROSTOVSKAYA



Dresden, July 2010

INTERNATIONAL MAX PLANCK RESEARCH SCHOOL
MAX PLANCK INSTITUTE OF MOLECULAR CELL BIOLOGY AND GENETICS


LINEAGE COMMITMENT OF
CONDITIONALLY IMMORTALIZED
BONE MARROW MESENCHYMAL STROMAL CELLS
FROM TETRACYCLINE-REGULATED SV40 LARGE T-ANTIGEN
TRANSGENIC MICE


D I S S E R T A T I O N


zur Erlangung des akademischen Grades

Doctor rerum naturalium
(Dr. rer. nat.)


vorgelegt

der Fakultät Mathematik und Naturwissenschaften
der Technischen Universität Dresden


von

ROSTOVSKAYA MARIA

geboren am 22.04.1981 in Ramenskoe, Russia



Gutachter: Prof. Dr. Francis Stewart
Prof. Dr. Austin Smith

Eingereicht am: 6. July, 2010

Tag der Verteidigung: 30. November, 2010
2

Abstract
Adult bone marrow contains a population of mesenchymal stem cells capable
to self-renew and to differentiate into haematopoietic-supportive stroma, osteo,
adipo- and chondrocytes. However, the identity of mesenchymal stem cells still
remains uncertain. The complex population of their descendants, bone marrow
mesenchymal stromal cells (BM MSCs), represents a model to study the principles of
differentiation and commitment into mesodermal lineages. The experiments using
BM MSCs are often hampered by their low proliferative capacity in vitro. In the
present study, we established conditionally immortalized BM MSCs from tetracycline-
regulated SV40 Large T-antigen transgenic mice. The identity of the conditionally
immortalized BM MSCs was confirmed by marker expression, ability to support
haematopoiesis and differentiation potential. The advantages of the conditional
immortalization are encompassed in (1) indefinite expansion of cell populations, (2)
possibility to perform cellular cloning and (3) prevention from spontaneous
differentiation.
We demonstrated the heterogeneity of BM MSCs and identified at least 6
types of progenitors within BM MSCs population based on their differentiation
potential (“OAC”, “OA”, “OC”, “AC”, “O”, “A”). A hypothetical model of BM MSC
hierarchy and the relationships between the progenitors has been proposed.
We observed that the Wnt/β-catenin signaling pathway and GSK3 activity
could modulate the efficiency of osteo- and adipogenic differentiation pathways, but
we didn’t find evidence that the lineage commitment of BM MSCs is determined by
Wnt.
We elucidated the mechanism of transcriptional regulation of the adipogenic
induction of BM MSCs in vitro. Our data revealed the key regulatory role of PPARγ1
during adipogenesis in BM MSCs. Furthermore, we assume that PPARγ1 is a
potential trigger of the adipogenic commitment of the BM MSCs progenitors. Finally,
the non-adipogenic BM MSCs progenitors were converted into the adipogenic
lineage using ectopical expression of the transcription factors C/EBPα, C/EBPβ and
C/EBPδ. Our findings provide a novel insight into the molecular mechanisms of BM
MSCs lineage commitment.
3


1. Introduction.............................................................................................. 8
1.1 Bone marrow mesenchymal stromal cells ...................................... 9
1.1.1. Bone marrow stroma structure and developmental origin.......................9
1.1.2. Identity of bone marrow mesenchymal stromal cells ............................11
1.1.3. Sources of mesenchymal stromal cells.................13
1.1.4. Isolation of bone marrow mesenchymal stromal cells...........................14
1.1.5. Markers of bone marrow mesenchymal stromal cells14
1.1.6. Differentiation of bone marrow mesenchymal stromal cells..................15
1.1.6.1. Adipogenic induction and molecular control of adipogenesis ....................15
1.1.6.2. Osteogenic induction and molecular control of osteogenesis20
1.1.6.3. Chondrogenic induction and molecular control of chondrogenesis ...........22
1.1.7. Plasticity of bone marrow mesenchymal stromal cells..........................23
1.1.8. Cell fate choice......................................................................................25
1.1.9. Heterogeneity of bone marrow mesenchymal stromal cells..................28
1.1.10. Subpopulations of BM MSCs and shifting of paradigms.....................29
1.1.10.1. Rapidly Self-renewing (RS) Cells and MSC heterogeneity......................29
1.1.10.2. Multipotent Adult Progenitor Cells (MAPCs) and the plasticity of BM MSCs
30
1.1.10.3. Very Small Embryonic-like Stem Cells (VSELs) and pluripotent lineages in
the adults 31
1.1.10.4. Marrow-Isolated Adult Multilineage Inducible (MIAMI) cells and
microenvironment.......................................................................................................33
1.1.10.5. Human MCAM-positive cells and pericyte concept..33
+ +1.1.10.6. PDGFRα Sca-1 (PαS) cells from mouse bone marrow and self-renewal
36
1.2. Cellular immortalization ................................................................. 37
1.2.1. Cellular senescence of human and mouse cells...38
1.2.2. Molecular regulators of cellular senescence.........39
1.2.2.1. Telomere maintenance..............39
1.2.2.2. Retinoblastoma pathway............40
1.2.2.3. p53 tumor suppressor ................................................................................41
1.2.3. Methods of cellular immortalization.......................42
1.2.4. Cellular immortalization by Simian Virus 40..........44
1.2.5. Conditional immortalization of mouse cells using tetracycline-regulated
SV40 Large T-antigen........................................................................................47
1.2.6. Immortalization of bone marrow mesenchymal stromal cells................50
1.2.6.1. Spontaneously immortalized stromal lines.50
1.2.6.2. Induced immortalization in stromal lines....................50
1.2.6.3. Conditionally immortalized stromal lines from transgenic mice..................51
2. Materials and methods.......................................................................... 52
2.1. General materials............ 52
2.1.1. Reagents...............................................................52
2.1.1.1. Cell culture media and supplements..........................52
2.1.1.2. Chemicals and dyes...................52
2.1.1.3. Other reagents (markers, inhibitors etc).....................................................53
2.1.1.4. Buffers and solutions..................................................53
2.1.1.5. Antibodies ..................................................................54
2.1.1.5.1. Primary antibodies...............54
2.1.1.5.2. Secondary antibodies...........55
2.1.1.6. Kits and enzymes.......................................................................................56
2.1.1.7. Primer sequences......................56
2.1.2. Materials................................................................................................57
2.1.3. Plasmids and constructs.......57
2.2. Transgenic mice............................................. 58
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2.3. Cell culture methods ...................................................................... 58
2.3.1. Isolation of bone marrow mesenchymal stromal cells (BM MSCs).......58
2.3.2. Basic procedures with BM MSCs..........................................................58
2.3.3. Induction of immortalization of the bone marrow mesenchymal stem
cells 59
2.3.4. Colony-forming units fibroblasts (CFU-F) assay ...................................59
2.3.5. Cell proliferation assay..........................................59
2.3.6. In vitro differentiation assays.59
2.3.6.1. Osteogenic differentiation..........59
2.3.6.2. Adipogenic differentiation...........................................60
2.3.6.3. Chondrogenic differentiation ......................................60
2.3.6.3.1. Differentiation in vitro...........60
2.3.6.3.2. Paraffin sectioning................61
2.3.6.3.3. Alcian Blue staining..............................................61
2.3.6.3.4. Immunohistological staining.................................61
2.3.7. Cobblestone-area forming assay (CAFC).............62
2.3.8. Transfection of conditionally immortalized MSCs .................................62
2.3.8.1. Electroporation of conditionally immortalized BM MSCs............................62
2.3.8.2. Lipofection of conditionally immortalized BM MSCs..62
2.3.9. Wnt-reporter assay................................................................................62
2.3.10. Establishment of the clonally derived conditionally immortalized BM
MSCs 63
2.3.10.1. Cellular cloning of conditionally immortalized BM MSCs .........................63
2.3.10.2. Manipulations with the clonally derived cells in the 96-well plates...........63
2.4. Flow cytometry analysis ...........