Purification of cardiomyocytes derived from differentiated embryonic stem cells and study of the cytokines' effect on embryonic stem cell differentiation.

Leung, Sze Lee Cecilia.Thesis (M.Phil.)--Chinese University of Hong Kong, 2008.Includes bibliographical references (leaves 144-153).Abstracts in English and Chinese.Abstract --- p.iAbstract in Chinese (摘要） --- p.iiiAcknowledgements --- p.vTable of Content --- p.viAbbreviations --- p.xvChapter CHAPTER 1 --- INTRODUCTIONChapter 1.1 --- Stem cells --- p.1Chapter 1.1.1 --- Adult stem cells --- p.2Chapter 1.1.2 --- Embryonic stem cells --- p.2Chapter 1.1.3 --- Pros and cons of embryonic and adult stem cells --- p.5Chapter 1.1.4 --- Human embryonic stem cells (hESCs) --- p.6Chapter 1.1.5 --- Mouse embryonic stem cells (mESCs) --- p.7Chapter 1.1.6 --- Characteristics of ESC-derived cardiomyocytes --- p.7Chapter 1.2 --- Cardiovascular Diseases (CVD) --- p.9Chapter 1.2.1 --- Causes and statistics of CVD --- p.9Chapter 1.2.2 --- Current treatment for CVD --- p.10Chapter 1.2.3 --- Current hurdles of putting hESC-CMs into clinical use --- p.11Chapter 1.3 --- Myosin light chain2v --- p.13Chapter 1.4 --- Genetic-engineering of hESCs & their cardiac derivatives by lentiviral-mediate gene transfer --- p.14Chapter 1.5 --- Cytokines secretion during myocardial infarction --- p.15Chapter 1.6 --- Aims of the Project --- p.19Chapter 1.7 --- Significance of the Project --- p.19Chapter CHAPTER 2 --- MATERIALS AND METHODSChapter 2.1 --- Subcloning --- p.20Chapter 2.1.1 --- Amplification of MLC-2v --- p.20Chapter 2.1.2 --- Purification of DNA product --- p.21Chapter 2.1.3 --- Restriction enzyme digestion --- p.21Chapter 2.1.4 --- Ligation of MLC-2v promoter with DuetO 11 vector --- p.22Chapter 2.1.5 --- Transformation of ligation product into competent cells --- p.22Chapter 2.1.6 --- PCR confirmation of successful ligation --- p.23Chapter 2.1.7 --- Small-scale preparation of bacterial plasmid DNA --- p.23Chapter 2.1.8 --- Restriction enzyme digestions to reconfirm positive clones --- p.24Chapter 2.1.9 --- DNA sequencing of the cloned plasmid DNA --- p.25Chapter 2.1.10 --- Large-scale preparation of target recombinant expression vector --- p.25Chapter 2.2 --- Mouse Embryonic Fibroblast (MEF) Culture --- p.26Chapter 2.2.1 --- Derivation of MEF --- p.26Chapter 2.2.2 --- Mouse embryonic fibroblast cells culture --- p.27Chapter 2.2.3 --- Irradiation of mouse embryonic fibroblast --- p.28Chapter 2.3 --- HESC culture --- p.29Chapter 2.3.1 --- Thawing and Plating hESCs --- p.29Chapter 2.3.2 --- Splitting hESCs --- p.30Chapter 2.3.3 --- "Culture maintainence, selection and colony removal" --- p.31Chapter a) --- Distinguish differentiated and undifferentiated cells and coloniesChapter b) --- "Remove differentiated cells by ""Picking to Remove"""Chapter c) --- "Remove undifferentiated cells by ""Picking to Keep"""Chapter 2.3.4 --- Freezing hESCs --- p.31Chapter 2.3.5 --- Differentiation of hESCs --- p.32Chapter 2.3.6 --- "HESC culture on feeder free system, mTeSR TM1" --- p.34Chapter a) --- Preparation of mTeSRTMlChapter b) --- Preparation of BD MatrigelTM hESC-qualified Matrix aliquotsChapter c) --- Coating plates with BD MatrigelTM hESC-qualified MatrixChapter d) --- Human Embryonic stem cells culture in mTeSRTMlChapter 2.4 --- ES Cell Characterization (Chemicon Cat# SCR001) --- p.36Chapter 2.4.1 --- Alkaline Phosphatase Staining --- p.36Chapter 2.4.2 --- Immunofluorescence staining --- p.37Chapter 2.5 --- MESC culture --- p.38Chapter 2.5.1 --- Thawing and Plating mESCs --- p.38Chapter 2.5.2 --- Splitting mESCs --- p.38Chapter 2.5.3 --- Differentiation of mESCs --- p.39Chapter 2.5.4 --- To study the effects of cytokines on mESC differentiation --- p.40Chapter 2.6 --- Lentivirus (LV) Packaging --- p.41Chapter 2.6.1 --- Transfection of lentiviral vectors into HEK293FT cells --- p.41Chapter 2.6.2 --- LV titering --- p.42Chapter 2.7 --- MultipleTransduction --- p.43Chapter 2.8 --- Selection of transduced cells by hygromycin --- p.43Chapter 2.8.1 --- Determination of hygromycin selection dosage --- p.43Chapter 2.8.2 --- Selection of stable clones --- p.44Chapter 2.9 --- Isolation of green fluorescent cardiomyocytes derived from differentiated hESCs --- p.45Chapter 2.9.1 --- Collagenase digestion of embryoid bodies into single cells --- p.45Chapter 2.9.2 --- FACS --- p.46Chapter 2.10 --- Gene expression studyChapter 2.10.1 --- Primer design --- p.46Chapter 2.10.2 --- RNA extraction --- p.46Chapter 2.10.3 --- DNase Treatment --- p.47Chapter 2.10.4 --- Synthesis of Double-stranded cDNA from Total RNA --- p.47Chapter 2.10.5 --- Quantitative real-time PCR --- p.48Chapter 2.10.6 --- Quantification of mRNA expression --- p.49Chapter 2.11 --- Protein Expression study --- p.49Chapter 2.11.1 --- Crude protein extraction --- p.49Chapter 2.11.2 --- Quantitation of protein samples --- p.50Chapter 2.11.3 --- SDS-PAGE --- p.50Chapter 2.11.4 --- Western Blot --- p.51Chapter 2.11.5 --- Western blot luminal detection --- p.52Chapter 2.11.6 --- Quantification of protein expression --- p.52Chapter CHAPTER 3 --- PURIFICATION OF CARDIOMYOCYTES DERIVED FROM DIFFERENTIATED HESCsChapter 3.1 --- Subcloning --- p.57Chapter 3.1.1 --- Linearization of DuetO11 and excision of UBC promoter --- p.58Chapter 3.1.2 --- PCR cloning of MLC-2V --- p.59Chapter 3.1.3 --- Ligation of MLC-2v promoter to linearized DuetO11 --- p.60Chapter 3.1.3.1 --- Colony PCR to screen for positive clones --- p.61Chapter 3.1.3.2 --- Restriction digestion to confirm the success of ligation --- p.61Chapter 3.2 --- Lentivirus (LV) packaging --- p.62Chapter 3.2.1 --- Transfection --- p.63Chapter 3.2.2 --- LV titering --- p.64Chapter 3.3 --- HESC culture --- p.66Chapter 3.4 --- Multi-transduction of hESCs with LVs --- p.67Chapter 3.5 --- Differentiation after transduction --- p.69Chapter 3.6 --- Antibiotic selection --- p.71Chapter 3.6.1 --- Characterization of hESCs on feeder free system --- p.72Chapter 3.6.1.1 --- Alkaline Phosphatase (AP) staining --- p.72Chapter 3.6.1.2 --- Immunostaining with pluripotency marker --- p.73Chapter 3.6.2 --- Determination of hygromycin dosage by MTT assay --- p.74Chapter 3.6.3 --- HESCs after selection in feeder free system --- p.75Chapter 3.7 --- Differentiation of hESCs after selection --- p.76Chapter 3.8 --- FACS --- p.77Chapter 3.9 --- QPCR of cells after FACS --- p.80Chapter 3.9.1 --- Gene expression of Nkx2.5 --- p.81Chapter 3.9.2 --- Gene expression of c-Tnl --- p.82Chapter 3.9.3 --- Gene expression of c-TnT --- p.83Chapter 3.9.3 --- Gene expression of MLC-2v --- p.84Chapter CHAPTER 4 --- THE STUDY OF CYTOKINES' EFFECT ON MESC DIFFERENTIATIONChapter 4.1 --- mESC culture --- p.85Chapter 4.2 --- The effect of cytokines on the differentiation of mESCs --- p.86Chapter 4.2.1 --- Beating curves of mESCs treated with different concentrations of cytokines at differentiation day 2 to 6 before attachment --- p.88Chapter 4.2.2 --- qPCR to determine the cytokines' effect on the differentiation of mESCs --- p.94Chapter 4.2.2.1 --- The effect of IL-1α on the expression of cardiac specific genes --- p.95Chapter 4.2.2.2 --- The effect of IL-1β on the expression of cardiac specific genes --- p.98Chapter 4.2.2.3 --- The effect of IL-6 on the expression of cardiac specific genes --- p.101Chapter 4.2.2.4 --- The effect of IL-10 on the expression of cardiac specific genes --- p.104Chapter 4.2.2.5 --- The effect of IL-18 on the expression of cardiac specific genes --- p.107Chapter 4.2.2.6 --- The effect of TNF-α on the expression of cardiac specific genes --- p.110Chapter 4.2.3 --- Western blot analysis of the cytokines' effect on the differentiation of mESCs --- p.113Chapter 4.2.3.1 --- The effect of IL-lα on the abundance of cardiac specific proteins --- p.114Chapter 4.2.3.2 --- The effect of IL-1β on the abundance of cardiac specific proteins --- p.116Chapter 4.2.3.3 --- The effect of IL-6 on the abundance of cardiac specific proteins --- p.118Chapter 4.2.3.4 --- The effect of IL-10 on the abundance of cardiac specific proteins --- p.120Chapter 4.2.3.5 --- The effect of IL-18 on the abundance of cardiac specific proteins --- p.122Chapter 4.2.3.6 --- The effect of TNF-α on the abundance of cardiac specific proteins --- p.124Chapter CHAPTER 5 --- DISCUSSIONChapter 5.1 --- Purification of cardiomyocytes derived from differentiated hESCs --- p.127Chapter 5.2 --- Study on the effect of cytokines on mESC differentiation --- p.135Chapter 5.3 --- Conclusion --- p.142REFERENCES --- p.14