Studies on some factors critical for the development of pancreatic progenitor cells derived from human fetal pancreas.

Ng, Ka Yan.Thesis (M.Phil.)--Chinese University of Hong Kong, 2011.Includes bibliographical references (leaves 179-204).Abstracts in English and Chinese.Abstract --- p.I摘要 --- p.IVPublications --- p.VIIAcknowledgements --- p.VIIITable of contents --- p.IXList of figures --- p.XVList of tables --- p.XVIIList of abbreviations --- p.XVIIIChapter Chapter 1 --- General IntroductionChapter 1.1 --- The Pancreas --- p.2Chapter 1.1.1 --- Anatomy of Pancreas --- p.2Chapter 1.1.2 --- The Exocrine Pancreas --- p.4Chapter 1.1.3 --- The Endocrine Pancreas --- p.5Chapter 1.1.3.1 --- Structure of Islets --- p.5Chapter 1.1.3.2 --- "Functions of α-, β-, y-, ð-, Σ-and PP-cells in Islets" --- p.7Chapter 1.1.4 --- Overview of Pancreas Development --- p.9Chapter 1.1.4.1 --- Organ Morphology --- p.10Chapter 1.1.4.2 --- Cyto-differentiation --- p.12Chapter 1.1.4.3 --- Control by Transcriptional Factors --- p.14Chapter 1.1.5 --- Postnatal Pancreas Development and Regeneration --- p.18Chapter 1.1.5.1 --- Proliferation of Pre-existing β-cells --- p.19Chapter 1.1.5.2 --- Neogenesis from Precursor Cells --- p.20Chapter 1.1.5.3 --- Transdifferentiation of other Cells --- p.20Chapter 1.2 --- Diabetes Mellitus --- p.22Chapter 1.2.1 --- Pathophysiology of Diabetes Mellitus and Current Treatments --- p.24Chapter 1.2.1.1 --- Type I Diabetes Mellitus --- p.24Chapter 1.2.1.2 --- Type II Diabetes Mellitus --- p.25Chapter 1.2.1.3 --- Gestational Diabetes --- p.27Chapter 1.2.1.4 --- Secondary Diabetes --- p.28Chapter 1.3 --- Stem Cell therapy --- p.29Chapter 1.3.1 --- Stem Cell --- p.29Chapter 1.3.1.1 --- Mesenchymal Stem Sell --- p.31Chapter 1.3.1.2 --- Embryonic Stem Cell --- p.35Chapter 1.3.1.3 --- Induced Pluripotent Stem Cell --- p.36Chapter 1.3.2 --- Islets Engineering --- p.37Chapter 1.3.2.1 --- Genetic Modification --- p.37Chapter 1.3.2.2 --- Directed Differentiation --- p.38Chapter 1.3.2.3 --- Microenvironment --- p.38Chapter 1.3.2.4 --- In vivo Regeneration --- p.39Chapter 1.3.2.5 --- Cell Fusions --- p.40Chapter 1.3.2.6 --- Combinatory Treatments --- p.40Chapter 1.4 --- The Vitamin A & Vitamin D System --- p.42Chapter 1.4.1 --- The Vitamin A --- p.42Chapter 1.4.2 --- Vitamin A Metabolism --- p.44Chapter 1.4.3 --- Roles of vitamin A in Pancreatic Development --- p.46Chapter 1.4.4 --- The Vitamin D --- p.48Chapter 1.4.5 --- Vitamin D Metabolism --- p.49Chapter 1.4.6 --- Metabolic Functions of Vitamin D in Islets --- p.51Chapter 1.4.7 --- Cod Liver Oil --- p.53Chapter 1.4.8 --- Interactions between Vitamin A and Vitamin D --- p.53Chapter 1.5 --- The Relations of Liver and Pancreas Development --- p.55Chapter 1.5.1 --- Endoderm Induction for Hepatic and Pancreatic Differentiation of ESCs --- p.55Chapter 1.5.2 --- Bipotential Precursor Population within Embryonic Endoderm --- p.56Chapter 1.5.3 --- Pancreatic Islets Promote Mature Liver Hepatocytes Proliferation --- p.57Chapter 1.5.4 --- Transdifferentiation --- p.57Chapter 1.5.5 --- Transplantation in Liver Niche Promotes Maturation of Insulin-Producing Cells --- p.60Chapter 1.5.6 --- Neuronal Relay from the Liver to Pancreatic --- p.61Chapter 1.5.7 --- Development of Islets in the Nile Tilapia --- p.62Chapter 1.6 --- The Insulin-like Growth Factor-I (IGF1) --- p.64Chapter 1.6.1 --- IGF1 System --- p.64Chapter 1.6.2 --- IGF 1 Regulation --- p.65Chapter 1.6.3 --- Roles of IGF 1 in Pancreatic Development and Regeneration --- p.68Chapter 1.7 --- Aims and Objectives of Study --- p.70Chapter Chapter 2 --- General Materials and MethodsChapter 2.1 --- Pancreatic progenitor cells (PPCs) and liver stromal cells (LSCs) isolation and cell culture --- p.72Chapter 2.1.1 --- Tissue procurement --- p.72Chapter 2.1.2 --- PPC and LSC culture --- p.72Chapter 2.1.3 --- "Treatments of vitamin A, vitamin D and IGF 1" --- p.76Chapter 2.1.4 --- "Cell culture of Caco-2, HepG2 and DU-145" --- p.76Chapter 2.2 --- Induction of Islet-like Cell Clusters (ICCs) Differentiation --- p.77Chapter 2.2.1 --- In vitro Directed Differentiation --- p.77Chapter 2.2.2 --- In vitro LSC Microenvironment --- p.77Chapter 2.3 --- RNA Expression Detection --- p.79Chapter 2.3.1 --- RNA isolation --- p.79Chapter 2.3.2 --- Reverse Transcription --- p.79Chapter 2.3.3 --- Polymerase Chain Reaction (PCR) --- p.80Chapter 2.3.4 --- Realtime PCR --- p.81Chapter 2.4 --- Immunocytochemistry --- p.83Chapter 2.5 --- Western Blotting --- p.85Chapter 2.5.1 --- Protein extraction and quantification --- p.85Chapter 2.5.2 --- Western Blotting --- p.85Chapter 2.6 --- Enzyme-linked Immunosorbent Assay (ELISA) --- p.87Chapter 2.6.1 --- Detection of cell viability --- p.87Chapter 2.6.2 --- Detection of cell proliferation --- p.87Chapter 2.6.3 --- Measurement of Cell death --- p.88Chapter 2.6.4 --- Measurement of IGF 1 level in condition medium --- p.89Chapter 2.6.5 --- Measurement of glucose induced insulin secretion --- p.90Chapter 2.7 --- Regeneration model --- p.92Chapter 2.7.1 --- Regeneration model in neonatal-STZ rat --- p.92Chapter 2.7.2 --- Change in IGF1 expression in pancreas and liver --- p.92Chapter 2.8 --- Statistical Data Analysis --- p.93Chapter Chapter 3 --- Vitamin D and vitamin A receptor expression and the proliferative effects of ligand activation of these receptors on the development of pancreatic progenitor cells derived from human fetal pancreas. (Stem Cell Rev. 2011;7:53-63)Chapter 3.1 --- Abstract --- p.95Chapter 3.2 --- Introduction --- p.97Chapter 3.3 --- Materials and Methods --- p.101Chapter 3.3.1 --- Fetal Tissue Procurement --- p.101Chapter 3.3.2 --- Culture of Pancreatic Progenitor Cells --- p.101Chapter 3.3.3 --- RNA Expression Analysis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) --- p.102Chapter 3.3.4 --- Western Blot Analysis --- p.103Chapter 3.3.5 --- Immunocytochemstry --- p.105Chapter 3.3.6 --- PPC Proliferation Assays --- p.106Chapter 3.3.7 --- PPC Cell Death Assays --- p.107Chapter 3.3.8 --- Statistical Data Analysis --- p.108Chapter 3.4 --- Results --- p.110Chapter 3.4.1 --- "Expression and Localization of RAR, VDR and RXR, CYP26 and CYP24 in PPCs" --- p.110Chapter 3.4.2 --- Incubation of PPC with atRA Enhances PPC Viability due to Increased Proliferation and Anti-apoptosis --- p.111Chapter 3.4.3 --- Incubation of PPCs with Calcitriol Enhances Viability due to Increased Proliferation --- p.111Chapter 3.4.4 --- Both atRA and Calcitriol Induce Up-regulation of both the RAR and the VDR but not the RXR --- p.112Chapter 3.4.5 --- Combination Treatment with atRA and Calcitriol on Cell Viability and NGN3 Expression --- p.112Chapter 3.5 --- Discussion --- p.114Chapter Chapter 4 --- Human fetal liver stromal cell co-culture enhances the growth and differentiation of pancreatic progenitor cells into islet-like cell clusters (In submission to Gastroenterology)Chapter 4.1 --- Abstract --- p.128Chapter 4.2 --- Introduction --- p.129Chapter 4.3 --- Materials and Methods --- p.133Chapter 4.3.1 --- Use of human and animal tissues --- p.133Chapter 4.3.2 --- "Cell preparation, characterizations and Differentiation" --- p.133Chapter 4.3.3 --- Examination of PPC growth and ICC differentiation and functions with LSC co-culture --- p.133Chapter 4.3.3 --- Identification of growth factors and investigation of their effects --- p.134Chapter 4.3.4 --- Statistical Analysis --- p.135Chapter 4.4 --- Results --- p.136Chapter 4.4.1 --- "Isolation, Culture and Characterizations of LSCs" --- p.136Chapter 4.4.2 --- Establishment of LSC co-culture system --- p.136Chapter 4.4.3 --- LSC co-culture enhances PPC-derived ICC differentiation --- p.137Chapter 4.4.4 --- Differential expression of mRNA for cytokines and growth factors between 1st and 2nd trimester LSCs --- p.138Chapter 4.4.5 --- Characterization of IGF 1 receptors in PPCs and the effects of exogenous IGF1 on PPC growth and ICC differentiation --- p.139Chapter 4.4.6 --- Neutralizing antibodies against IGF1R inhibit ICC differentiation --- p.140Chapter 4.5 --- Discussion --- p.142Chapter 4.6 --- Supplementary Materials and Methods --- p.147Chapter 4.6.1 --- Cell Preparation and culture --- p.147Chapter 4.6.2 --- In Vitro ICC differentiation --- p.148Chapter 4.6.3 --- RNA expression analysis --- p.149Chapter 4.6.4 --- Immunocytochemistry --- p.149Chapter 4.6.5 --- PPC viability and cell count assays --- p.150Chapter 4.6.6 --- IGF1 and insulin ELISA --- p.151Chapter 4.6.7 --- Western blotting analysis --- p.152Chapter 4.6.8 --- Neonatal streptozotocin regeneration model --- p.153Chapter Chapter 5 --- General Discussion and Future StudiesChapter 5.1 --- General Discussion --- p.165Chapter 5.1.1 --- Proliferative effects and enhance expression of NGN3 by vitamin A and vitamin D on PPC --- p.166Chapter 5.1.2 --- Induction of PPC derived ICCs by LSCs --- p.169Chapter 5.1.3 --- Potential effects of liver stroma derived IGF1 on PPC derived ICCs differentiation --- p.172Chapter 5.1.4 --- Significance of islet engineering in the management of diabetes --- p.174Chapter 5.1.5 --- Conclusions --- p.176Chapter 5.2 --- Future Studies --- p.177Chapter Chapter 6 --- ReferenceReference --- p.18