Propensity of murine embryonic stem cell differentiation in presence of Fam40b RNAi and chemopreventive agents [Elektronische Ressource] / Vilas S. Wagh

De
Publié par

Aus dem Zentrum Physiologie und Pathophysiologie Der Universität zu Köln Institut für Neurophysiologie Geschäftsführender Direktor: Universitätsprofessor Dr. med. J. Hescheler Propensity of Murine Embryonic Stem Cell Differentiation in Presence of Fam40b RNAi and Chemopreventive Agents Inaugural-Dissertation zur Erlangung der Doktorwürde eines doctor rerum medicinalium der Hohen Medizinischen Fakultät Der Universität zu Köln Vorgelegt von VILAS S. WAGH Aus Nagpur, MS, Indien Promoviert am 22. Juni 2011 Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln im Juni 2011 Dekanin/Dekan: Universitätsprofessor Dr. med. Dr. h.c.Th. Krieg 1. Berichterstatterin/Berichterstatter: Professor Dr. rer. nat. A. Sachinidis 2. terstatter: Universitätsprofessor Dr. Med. K. Brockmeier Erklärung Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als solche kenntlich gemacht. Bie der Auswahl und Auswertung des Materials sowie bei der Herstellung des Manuskriptes habe ich Unterstützungsleistungen von folgenden Personen erhalten: Prof . Dr. Agapios Sachinidis. Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht beteiligt.
Publié le : samedi 1 janvier 2011
Lecture(s) : 124
Tags :
Source : DIGITOOL.HBZ-NRW.DE:1801/WEBCLIENT/DELIVERYMANAGER?PID=4221229
Nombre de pages : 98
Voir plus Voir moins


Aus dem Zentrum Physiologie und Pathophysiologie
Der Universität zu Köln
Institut für Neurophysiologie
Geschäftsführender Direktor: Universitätsprofessor Dr. med. J. Hescheler


Propensity of Murine Embryonic Stem Cell Differentiation in Presence of
Fam40b RNAi and Chemopreventive Agents


Inaugural-Dissertation zur Erlangung der Doktorwürde
eines doctor rerum medicinalium
der Hohen Medizinischen Fakultät
Der Universität zu Köln








Vorgelegt von
VILAS S. WAGH
Aus Nagpur, MS, Indien

Promoviert am 22. Juni 2011




























Gedruckt mit Genehmigung der Medizinischen Fakultät der Universität zu Köln im
Juni 2011
Dekanin/Dekan: Universitätsprofessor Dr. med. Dr. h.c.Th. Krieg
1. Berichterstatterin/Berichterstatter: Professor Dr. rer. nat. A. Sachinidis
2. terstatter: Universitätsprofessor Dr. Med. K. Brockmeier


Erklärung

Ich erkläre hiermit, dass ich die vorliegende Dissertationsschrift ohne unzulässige
Hilfe Dritter und ohne Benutzung anderer als der angegebenen Hilfsmittel angefertigt
habe; die aus fremden Quellen direkt oder indirekt übernommenen Gedanken sind als
solche kenntlich gemacht.

Bie der Auswahl und Auswertung des Materials sowie bei der Herstellung des
Manuskriptes habe ich Unterstützungsleistungen von folgenden Personen erhalten:
Prof . Dr. Agapios Sachinidis.

Weitere Personen waren an der geistigen Herstellung der vorliegenden Arbeit nicht
beteiligt. Insbesondere habe ich nicht die Hilfe einer Promotionsberatein/eines in
Anspruch genommen. Dritte haben von mir weder unmittelbar noch mittelbar
geldwerte Leistungen für Arbeiten erhalten, die im Zuzammenhang mit dem Inhalt
der vorgelegten Dissertationsschrift stehen.

Die Dissertationsschrift wurde von mir bisher weder im Inland noch im Ausland in
gleicher oder ähnlicher Form einer anderen Prüfungsbehöhrde vorgelegt.

Köln, den 10. Nov 2010

ACKNOWLEDGEMENTS

First and the foremost, I would like to express my sincere gratitude to my
supervisor, Prof. Agapios Sachinidis whose encouragement, guidance and support
from the initial to the final levels enabled me to develop an understanding of the
subject. I would like to thank very much Prof. Sachinidis for having given me the
opportunity to carry out my research in his laboratory and his wisdom, knowledge
and commitment to the highest standard inspired and motivated me.

I am heartily thankful to my institute director Prof. Jürgen Hescheler, for his
advice and valuable suggestions during my doctoral thesis work. Besides, I owe a
great many thanks to a great many people who helped and supported me during this
work, its is to them that I owe my deepest gratitude to-

• Drs Michael Doss and Johannes Winkler, for finding me suitable for this
position, short listing me, and their continuous support during my stay
• Mrs. Rita Altenburg, Susan Rohani, Broses and Cornelia Böttinger for her
kind laboratory assistance
• All institutional faculty members (Prof. Toni Schneider, Dr. Tomo Saric) for
their advice and suggestion
• Dr. Kurt Pfannkuche for his everyday small chats, buildup my knowledge for
molecular biology techniques, I owe him a special thanks
• Kesavan Meganathan for his help, encouragement and above all believing in
me, I express my deepest thanks to him for extending his support and care
• Rabea Niemann, Martin Lehmann, Matthias Matzkies, Manoj Gupta, Azra
Fatima, Vivek Peche, Kayalvizhi Radhakrishnan, Suresh Kumar, for their
friendship and support during my stay here, I must state that in contact with
whom I shared great companionship
• My lab colleagues and friends Drs. Shiva Potta, Shuhua Chen, John Antony Gasper, Qamaruddin Lund, Dimitry Spitkovsky, Ignatius Gunnaselli, Sania,
Karthick Natarajan, Raja Sahito, Snehal Bhalekar, Praveen Devulpally and
Narshima Telugu, for their helpful scientific discussions
• Prof. Toni Schneider for his advice for our lab safety instruction and care
• Mrs. Suzanne Wood, Mrs. Elke Lieske and Mr. Daniel Cieszkowski for their
kind administrative support
• Mr. Frank Stassen and Mr. Döwelling for his excellent computer support
• Mr. Metzner and his team for the technical assistance
• Project-granting agency “European Community” (FunGenES, CRYSTAL,
ESNATS) I gained a lot of thought provoking new ideas and excellent
challenges from the experts in the fields of embryonic stem cell
• Somesh Sharma, Kalpana Joshi, P.M. Patel, Rajesh Pandya, Irfan Kaji and
Pravin Mahajan, with whom I was associated for many years and learned
lessons in drug-discovery
• Smita Jagtap, my wife, without whom this effort would have been worth
nothing. Your love, support and constant patience have taught me so much
about sacrifice, discipline and compromise-even if there were times when you
said “I told you so”
• My mother, my brother Vijay Wagh, my sister-in-law Subhangi and my niece
Srushti, who always encouraged and believed in me, in all my endeavors and
who so lovingly and unselfishly cared for me always
Lastly I would like to thank generically all those who contributed to this work.
All those who helped to built the fundamentals for this work, who contributed to the
development of new ideas, who motivated and inspired me and who offered their
support and friendship. Be assured that I feel obliged to you and have thought long
about you and the appropriate way of expressing my gratitude to you

I dedicated this dissertation to my father late Shree Sampatrao Wagh.
- My sincere thanks-
[Vilas S. Wagh] TABLE OF CONTENTS
ABBREVIATIONS ...................................................................................................... 1
1. INTRODUCTION .................................................................................................... 2
1.1. Embryonic stem cells......................................................................................... 2
1.2. Developmental and functional studies in cardiomyogenic process ................... 2
1.3. Chemopreventive agents and differentiation ..................................................... 5
2. OBJECTIVE ............................................................................................................. 7
3. MATERIAL AND METHODS................................................................................ 8
3.1. Materials ............................................................................................................ 8
3.2. Methods............................................................................................................ 11
3.2.1. Methodologies pertaining to Fam40b RNAi .................................................... 11
3.2.1.1. Culture of ES cell and embryoid body assay..................................... 11
3.2.1.2. siRNA Transient Transfection of ES Cells by Lipofectamine........... 12
3.2.1.3. Fam40b shRNA vector constructs..................................................... 13
3.2.1.4. Transfection of vectors Into Undifferentiated ES Cells and Generation
of Stable shFam40b sub lines ......................................................................... 15
3.2.1.5. RNA Isolation, Reverse Transcription, RT-PCR, and Real Time PCR
......................................................................................................................... 15
3.2.1.6. Microarray analysis............................................................................ 16
3.2.1.7. Gene ontology.................................................................................... 17
3.2.1.8. Western Blot and Immunofluorescence............................................. 17
3.2.1.9. Flow cytometry and light microscopy ............................................... 18
3.2.2. Methodologies pertaining mES cell differentiation in presence of
chemopreventive agents.............................................................................................. 18
3.2.2.1. Compounds, Embryonic stem Cells lines and culture ....................... 18
3.2.2.2. Induction of ES cell Differentiation................................................... 19
3.2.2.3. Measurement of growth inhibition of cells........................................ 20
3.2.2.4. Measurement of ES cell differentiation inhibition............................. 20
3.2.2.5. RNA isolation and cDNA synthesis .................................................. 21
3.2.2.6. Detection of tissue specific genes by semi quantitative RT-PCR and
quantitative PCR ............................................................................................. 21
3.2.2.7. Expression of tissue specific proteins analyzed by western blot and
immunofluorscence......................................................................................... 22
3.2.2.8. Data analysis ...................................................................................... 23
4. RESULTS ............................................................................................................... 24
4.1. Fam40b antisense RNA expression in murine embryonic stem cells leads to
loss of the cardiogenesis phenotype........................................................................ 24
4.1.2. Fam40b locus, EST tissue expression and transcript expression in neonatal
organs.......................................................................................................................... 24
4.1.2 Fam40b expression in differentiation ES cells .................................................. 31
4.1.3. Transient knockdown of Fam40b leads to reduced cardiogenic phenotype..... 32
4.1.4. shRNA specifically reduce Fam40b in CGR8 cells resulting in stable
knockdown sub line .................................................................................................... 35
4.1.5. ES cell with Fam40b knockdown represses cardiomyogenic phenotype......... 37 4.1.6 Fam40b knockdown represses a subset if mesoderm and cardiomyocytes gene
during ES cell differentiation...................................................................................... 38
4.1.7. Gene ontology characterization ........................................................................ 41
4.1.8. Investigation of DET in knock down clone ...................................................... 42
4.2. Effect of chemo-preventive agents on differentiation of mouse embryonic stem
cells ......................................................................................................................... 45
4.2.1 Identification of the three germ linage and pluripotency makers during
differentiation of embryoid bodies.............................................................................. 45
4.2.2 Estimation of cytotoxicity and embryo toxicity pertaining to functional
properties associated with generation of cardiomyocytes from embryonic stem cells47
4.2.3. Inducible effect of EGCG on the directional differentiation of embryonic stem
cells into cardiomyocytes............................................................................................ 52
4.2.4. Directed formation of ectodermal and neuroectodermal lineages by curcumin
upon ES cell differentiation ........................................................................................ 57
4.2.5. Effect of quercetin on ES cell differentiation ................................................... 59
5. DISCUSSION AND CONCLUSION..................................................................... 61
5.1 Fam40b, a novel regulator of cardiomyogenesis.............................................. 61
5.2 Effect of chemo-preventive agents on differentiation of mouse embryonic stem
cells ......................................................................................................................... 63
SUMMARY................................................................................................................ 67
ZUSAMMENFASSUNG ........................................................................................... 68
7. BIBLIOGRAPHY................................................................................................... 69
8. ANNEXURES ........................................................................................................ 81
TEILPUBLICATIONE............................................................................................... 89
CURRICULUM VITAE............................................................................................. 90


1

ABBREVIATIONS

Dpc-days postcoitum; ESCs- Embryonic Stem cells; nt- Nucleotide; EBs-Embryoid bodies; d:day
(s); B-Luria Bertani; LIF-Leukemia Inhibitory Factor; BMP-2:Bone morphogenetic protein-2;
TGF-Transforming growth factor; aMHC-a-Myosin Heavy Chain (Myh6); EGFP-Enhanced green
fluorescent protein; PCR-Polymerase Chain Reaction; RT-PCR-Reverse Transcriptase-
Polymerase chain reaction; cDNA-Complimentary deoxy ribonucleic acid; DNA-Deoxy
ribonucleic acid; RNA-Ribonucleic acid; RNAi-Ribonucleic acid interference; siRNA-small
interfering RNA; qPCR-Qunatitative PCR; FACS-Flourescence Activated Cell Sorting; FBS-
Featal Bovine Serum; LIF- Leukemia inhibitory factor; DMSO- Dimethyl Sulfoxide; PBS-
Phosphate Buffered Saline; EST-expressed sequence tag ; DEU- digital expression levels; GMEM
-Glasgows buffered minimal essential medium ; IMDM- Iscove's modified Dulbecco's Medium;
ECACC- European Collection of Cell Cultures; aRNA- Amplified RNA; DET -differentially
expressed transcripts; Database for Annotation, Visualization and Integrated Discovery (DAVID;
expressed sequence tag or EST; TBST- Tris-Buffered Saline Tween-20; PVDF- Polyvinylidene
fluoride; HRP- horseradish peroxidase; ECL- Enhanced Chemiluminescence; MI-myocardial
infarction; COX-2- cyclooxygenase-2; MMP-9- matrix metalloproteinase-9; DNA-
deoxyribonucleic acid; H O -hydrogen peroxide; EGCG- (-)-epigallocatechin-3-gallate; CVD- 2 2
cardiovascular diseases; PDGF- platelet derived growth factor; PDGF-R- platelet derived growth
factor-receptor; ROS- reactive oxidative species; CHD- coronary heart disease; HSP70- heat shock
protein 70; NF- κB- nuclear factor- κB; MAPK- mitogen activated protein kinase; AP-1- activator
protein-1 EGFR- epidermal growth factor receptor; IL-1- interleukin-1; IL-6- interleukin-6;
ICAM-1- intercellular adhesion molecule-1; VCAM-1- vascular cell adhesion molecule-1; GSH-
Glutathione; NPSH- Non-protein sulfhydryls; g-GCS- g-glutamyl cysteine synthetase; ODC-
ornithine decarboxylase; NMBA- N-nitrosomethylbenzylamine; ISO- isoproterenol; IAP- inhibitor
of apoptosis; TLR- Toll-like receptor; NO- nitric oxide; VSMCs- vascular smooth muscle cells


2
1. INTRODUCTION

1.1. Embryonic stem cells
Embryonic stem cell research has become a realm for basic research and regenerative
medicine in these recent years of development. The characteristic features of self-renewal and
pluripotency are two key elements making them a unique research tool for studying gene function
and its potential use in cell replacement therapy. Self-renewal is the ability of ES cells to be
maintained in a proliferative culture condition state for prolonged periods of time, whereas
pluripotency is the ability of ES cells to differentiate into any organ specific cell type (1, 2)(Figure
1). ES cells are able to differentiate into the three primitive layers (endoderm, mesoderm and
ectoderm) of the organism, including the germline.


Figure 1. Embryonic stem cells (ESCs) have the potential to give rise to all tissues and cell types
in the body. Depending upon defined culture conditions and the presence of specific growth factors
(Adapted from http://www.sydneyivfstemcells.com/)

1.2. Developmental and functional studies in cardiomyogenic process
In mice embryo, heart is the first organ to function derived from mesoderm approximately
on 7.5dpc the cardiac crescent migrate ventrally to form a linear heart tube and gains contractile

Soyez le premier à déposer un commentaire !

17/1000 caractères maximum.