Molecular characterization of human adipose tissue-derived stem cells.

Ng, Wing Chi Linda.Thesis (M.Phil.)--Chinese University of Hong Kong, 2007.Includes bibliographical references (leaves 120-142).Abstracts in English and Chinese.Abstract --- p.iAcknowledgement --- p.ivPublications --- p.vAbbreviations --- p.viTable of Contents --- p.viiiList of Tables --- p.xiiiList of Figures --- p.xivChapter CHAPTER 1 --- INTRODUCTION --- p.1Chapter 1.1 --- Stem Cells --- p.1Chapter 1.1.1 --- Definition of Stem Cells --- p.1Chapter 1.1.2 --- Different Origins of Stem Cells --- p.2Chapter 1.1.3 --- Challenges and Importance of Stem Cell Research --- p.5Chapter 1.2 --- Adult Mesenchymal Stem Cells --- p.7Chapter 1.2.1 --- Characteristics of Adult Mesenchymal Stem Cells --- p.7Chapter 1.2.2 --- Adipose Tissue as an Alternate Source of MSCs --- p.8Chapter 1.2.3 --- Adipose Tissue Versus Bone Marrow as a Source of MSCs --- p.10Chapter 1.3 --- Adipose Tissue-derived Stem Cells (ATSCs) --- p.11Chapter 1.3.1 --- Cell Surface Marker Characteristic of ATSCs --- p.11Chapter 1.3.2 --- Global Gene Expression Profile of ATSCs --- p.14Chapter 1.3.3 --- Immunomodulatory Effect of ATSCs --- p.15Chapter 1.3.4 --- Proliferation Capacity of ATSCs --- p.17Chapter 1.3.5 --- Multilineage Differentiation of ATSCs --- p.18Chapter 1.3.5.1 --- Differentiation Capability of ATSCs : Adipogenesis --- p.18Chapter 1.3.5.2 --- Osteogenesis --- p.19Chapter 1.3.5.3 --- Skeletal and Smooth Muscle Myogenesis --- p.21Chapter 1.3.5.4 --- Cardiomyogenesis --- p.23Chapter 1.3.5.5 --- Chondrogenesis --- p.24Chapter 1.3.5.6 --- Neurogenesis --- p.27Chapter 1.4 --- Signaling Pathways in Stem Cells --- p.31Chapter 1.4.1 --- Wnt Signaling --- p.31Chapter 1.4.2 --- Notch Signaling --- p.33Chapter 1.4.3 --- Signaling Pathway of the TGF-β Superfamily --- p.34Chapter 1.5 --- Pathways Controlling Chondrogenesis --- p.36Chapter 1.6 --- MicroRNA --- p.39Chapter 1.6.1 --- MicroRNA - A Novel Gene Regulator --- p.39Chapter 1.6.2 --- Biogenesis of MicroRNAs --- p.40Chapter 1.6.3 --- Post-transcriptional Repression by MicroRNAs --- p.43Chapter 1.6.4 --- Role of MicroRNAs in Development --- p.45Chapter 1.6.5 --- MicroRNAs in Stem Cell Differentiation --- p.46Chapter 1.6.5.1 --- MicroRNA Expression Profile in ESCs --- p.46Chapter 1.6.5.2 --- Lineage Differentiation --- p.47Chapter 1.7 --- Project Aims --- p.52Chapter 1.8 --- Significance of Study --- p.53Chapter Chapter 2 --- Materials and Methods --- p.54Chapter 2.1 --- Sample Collection --- p.54Chapter 2.2 --- Isolation and Culture of ATSCs --- p.54Chapter 2.3 --- Measurement of Cell Growth --- p.55Chapter 2.4 --- Effect of Estrogen Treatment on ATSC Proliferation --- p.55Chapter 2.5 --- Multilineage Differentiation of ATSCs --- p.55Chapter 2.5.1 --- Chondrogenic Differentiation --- p.56Chapter 2.5.2 --- Neural Differentiation --- p.56Chapter 2.6 --- Immunocytochemical Analysis of Surface Markers and Lineage Specific Markers --- p.57Chapter 2.7 --- Alcian Blue Staining --- p.58Chapter 2.8 --- RNA Extraction --- p.58Chapter 2.9 --- Reverse Transcription --- p.59Chapter 2.10 --- Quantitative Real-time Polymerase Chain Reaction --- p.59Chapter 2.11 --- Statistical Analysis of Real-time PCR Data --- p.61Chapter 2.12 --- MicroRNA Profiling --- p.61Chapter 2.12.1 --- Reverse Transcription --- p.62Chapter 2.12.2 --- Quantitative Real-time Polymerase Chain Reaction --- p.62Chapter 2.13 --- mRNA Target Prediction of MicroRNA --- p.63Chapter 2.14 --- MicroRNA Knockdown Assay --- p.63Chapter 2.15 --- MicroRNA Over-expression Assay --- p.64Chapter 2.15.1 --- Vector Amplification --- p.64Chapter 2.15.1.1 --- Transformation --- p.64Chapter 2.15.1.2 --- Purification of Plasmid DNA --- p.65Chapter 2.15.1.3 --- Confirmation of Construct Insertion --- p.66Chapter 2.15.2 --- Transfection of Plasmid and Establishment of MicroRNA Precursor Expressing Cell Lines --- p.66Chapter 2.16 --- Gene Expression Microarry --- p.67Chapter 2.16.1 --- Preparation of Amplification and Labeling Reaction --- p.67Chapter 2.16.2 --- Purification of the Labeled/Amplified RNA --- p.68Chapter 2.16.3 --- RNA Fragmentation --- p.68Chapter 2.16.4 --- Hybridization --- p.69Chapter 2.16.5 --- Array Washing and Scanning --- p.69Chapter 2.16.6 --- Statistical Analysis of Microarray Data --- p.69Chapter CHAPTER 3 --- RESULTS --- p.71Chapter 3.1 --- Isolation and Characterization of ATSCs --- p.71Chapter 3.2 --- ATSCs Exhibited Multilineage Differentiation --- p.75Chapter 3.2.1 --- Chondrogenic Differentiation --- p.75Chapter 3.2.2 --- Expression of Chondrogenic Markers --- p.76Chapter 3.2.3 --- Neural Differentiation --- p.80Chapter 3.2.4 --- Expression of Neural Markers --- p.83Chapter 3.3 --- Effect of Donor's Reproductive Status on the Proliferation and Differentiation Capacity of ATSCs --- p.83Chapter 3.3.1 --- Expression of Stem Cell Makers --- p.86Chapter 3.3.2 --- Cell Proliferation Assay --- p.86Chapter 3.3.3 --- Differentiation Capacity of ATSCs --- p.89Chapter 3.4 --- Effect of E2 Treatment on the Proliferation Rate of ATSCs --- p.89Chapter 3.5 --- MicroRNA --- p.91Chapter 3.5.1 --- MicroRNA Expression Profile of Undifferentiated and Chondrogenic Differentiated ATSCs --- p.91Chapter 3.5.2 --- Clustering Analysis Identified MicroRNAs Segregate with ATSCs --- p.91Chapter 3.5.3 --- Identification of Differentially Expressed MicroRNAs in Chondrogenic-induced ATSCs --- p.95Chapter 3.5.4 --- mRNA Target Prediction for miR-199a --- p.97Chapter 3.6 --- Correlating MicroRNA Expression and mRNA Levels: Clues to MicroRNA Function --- p.97Chapter 3.6.1 --- Effect ofmiR-199a RNAi in Phenotypic Changes of Chondrogenic-induced ATSCs --- p.97Chapter 3.6.2 --- Identification of Potential Target Genes by Microarray Analysis of ATSCs with miR-199a Over-expression and Knockdown --- p.102Chapter CHAPTER 4 --- DISCUSSION --- p.104Chapter CHAPTER 5 --- CONCLUSIONS --- p.115APPENDICES --- p.117REFERENCES --- p.12