Fong Shu Pan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 147-167). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.i / LIST OF PUBLICATIONS --- p.ii / ABSTRACT --- p.iii / ABSTRACT [IN CHINESE] --- p.vii / TABLE OF CONTENT --- p.ix / LISTS OF FIGURES --- p.xv / LIST OF TABLES --- p.xxi / LIST OF ABBREVATIONS --- p.xxii / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Embryonic stem (ES) cells --- p.1 / Chapter 1.2 --- Stem cell plasticity --- p.5 / Chapter 1.2.1 --- Differentiation and trans-differentiation of lineage-restricted stem cells --- p.5 / Chapter 1.2.1.1 --- Multilineage differentiation in-vitro --- p.5 / Chapter 1.2.1.2 --- Trans-differentiation --- p.6 / Chapter 1.2.2 --- Prospective applications of stem cells --- p.7 / Chapter 1.2.2.1 --- Basic research on development --- p.7 / Chapter 1.2.2.2 --- Study of human disease --- p.7 / Chapter 1.2.2.3 --- Cancer research --- p.7 / Chapter 1.2.2.4 --- Drug screening --- p.8 / Chapter 1.2.2.5 --- Cell therapy --- p.8 / Chapter 1.3 --- Neuro-degenerative diseases and cell therapy --- p.9 / Chapter 1.3.1 --- Neuro-degenerative diseases --- p.9 / Chapter 1.3.2 --- Neuro-regeneration --- p.10 / Chapter 1.3.3 --- Cell sources for neuro-regenerative therapy --- p.11 / Chapter 1.3.3.1 --- Comparison of stem cells --- p.11 / Chapter 1.3.3.2 --- Stem cells in neuro-regenerative therapy --- p.12 / Chapter 1.4 --- In-vitro derivation into neural lineage --- p.17 / Chapter 1.4.1 --- In-vitro induction strategies available --- p.17 / Chapter 1.4.1.1 --- Chemical agents --- p.18 / Chapter 1.4.1.1.1 --- Retinoic acid (RA) --- p.18 / Chapter 1.4.1.1.2 --- Ascorbic acid --- p.19 / Chapter 1.4.1.2 --- Growth factors/cytokines --- p.19 / Chapter 1.4.1.2.1 --- Neurotrophins --- p.20 / Chapter 1.4.1.2.2 --- Stimulants --- p.20 / Chapter 1.4.1.2.3 --- Signalling molecules --- p.21 / Chapter 1.4.1.3 --- Culture Selection --- p.23 / Chapter 1.4.1.3.1 --- Conditions --- p.23 / Chapter 1.4.1.3.2 --- Medium --- p.23 / Chapter 1.4.1.4 --- Transfection of regulator genes using viral vector --- p.24 / Chapter 1.4.1.5 --- Stromal cell-derived inducing activity (SDIA) --- p.26 / Chapter Chapter 2 --- Aims --- p.28 / Chapter 2.1 --- Hypothesis and study objectives --- p.28 / Chapter 2.1.1 --- Soliciting an optimal method for ES cell propagation --- p.28 / Chapter 2.1.2 --- Pursuing alternative SDIA --- p.29 / Chapter Chapter 3 --- Materials and Methods --- p.33 / Chapter 3.1 --- Chemicals and Reagents --- p.33 / Chapter 3.1.1 --- Cell Culture --- p.33 / Chapter 3.1.2 --- Immunohistochemistry and staining --- p.35 / Chapter 3.1.3 --- Molecular Biology --- p.36 / Chapter 3.2 --- Consumable --- p.37 / Chapter 3.3 --- Cell lines --- p.39 / Chapter 3.3.1 --- Feeder cells --- p.39 / Chapter 3.3.1.1 --- Primary mouse embryonic fibroblasts --- p.39 / Chapter 3.3.1.2 --- STO --- p.39 / Chapter 3.3.1.3 --- L Cells --- p.40 / Chapter 3.3.1.4 --- L-Wnt-3A Cells --- p.40 / Chapter 3.3.1.5 --- C17.2 --- p.40 / Chapter 3.3.2 --- ES cells --- p.41 / Chapter 3.3.2.1 --- ES-D3 --- p.41 / Chapter 3.3.2.2 --- ES-E14TG2a --- p.41 / Chapter 3.4 --- In-house prepared solutions --- p.42 / Chapter 3.4.1 --- "Stock solution of Insulin, Transferrin, Selentine (ITS) Supplement" --- p.42 / Chapter 3.4.2 --- Enriched Knock-Out Dulbecco's Modified Eagle's Medium (KO DMEM) --- p.42 / Chapter 3.4.3 --- Mitomycin C solution --- p.42 / Chapter 3.4.4 --- Gelatin solution 0.1% --- p.42 / Chapter 3.4.5 --- p-mercaptoethanol solution --- p.43 / Chapter 3.4.5.1 --- (3-mercaptoethanol solution 0.1M --- p.43 / Chapter 3.4.5.2 --- P-mercaptoethanol solution 0.1M --- p.43 / Chapter 3.4.5.3 --- p-mercaptoethanol solution 0.1M for preparation of culture medium --- p.43 / Chapter 3.4.6 --- ALL-trans retinoic acid --- p.43 / Chapter 3.4.6.1 --- ALL-trans retinoic acid stock solution 0.01M --- p.43 / Chapter 3.4.6.2 --- ALL-trans retinoic acid working solution lμM --- p.43 / Chapter 3.4.7 --- Paraformaldehyde solution 4% (PFA) --- p.44 / Chapter 3.4.8 --- TritoxX-100 solution --- p.44 / Chapter 3.4.8.1 --- Tritox X-100 solution 3% --- p.44 / Chapter 3.4.8.2 --- Tritox X-100 solution 0.3% --- p.44 / Chapter 3.4.9 --- Popidium iodide solution lug/mL (PI) --- p.44 / Chapter 3.4.10 --- Geneticin solution --- p.45 / Chapter 3.4.10.1 --- Geneticin solution 50mg/mL --- p.45 / Chapter 3.4.10.2 --- Geneticin solution 5mg/mL --- p.45 / Chapter 3.4.11 --- Poly-L-ornithine solution --- p.45 / Chapter 3.4.12 --- Laminin solution --- p.45 / Chapter 3.4.13 --- Maintenance medium for cell feeders --- p.46 / Chapter 3.4.14 --- Mitomycin C inactivation medium --- p.46 / Chapter 3.4.15 --- Freezing medium --- p.46 / Chapter 3.4.16 --- Propagation medium for ES cells --- p.47 / Chapter 3.4.16.1 --- Serum-based propagation medium for ES cells --- p.47 / Chapter 3.4.16.2 --- Serum-free propagation medium for ES cells --- p.47 / Chapter 3.4.16.3 --- Serum-free induction medium for ES cells --- p.48 / Chapter 3.4.16.3.1 --- Serum-free induction medium 1 --- p.48 / Chapter 3.4.16.3.2 --- Serum-free induction medium II --- p.48 / Chapter 3.4.16.3.3 --- Serum-free induction medium III --- p.48 / Chapter 3.5 --- Equipments --- p.49 / Chapter 3.6 --- Methods --- p.50 / Chapter 3.6.1 --- Cell Culture --- p.50 / Chapter 3.6.1.1 --- Preparation of round cover-slips --- p.50 / Chapter 3.6.1.2 --- Gelatinization of tissue culture wares --- p.51 / Chapter 3.6.1.3 --- Poly-L-ornithine and laminin coating --- p.51 / Chapter 3.6.1.4 --- Thawing frozen cells --- p.51 / Chapter 3.6.1.5 --- Passage of adherent culture --- p.52 / Chapter 3.6.1.6 --- Cell count --- p.52 / Chapter 3.6.1.7 --- Cytospin --- p.53 / Chapter 3.6.1.8 --- Cell viability test --- p.53 / Chapter 3.6.1.9 --- Cryopreservation --- p.53 / Chapter 3.6.1.10 --- Preparation of primary mouse embryonic fibroblast (PMEF) --- p.54 / Chapter 3.6.1.11 --- Mitomycin C inactivation of feeder cells --- p.55 / Chapter 3.6.1.12 --- Gamma irradiation of various feeders --- p.55 / Chapter 3.6.1.13 --- Preparation of CM from feeder cells --- p.56 / Chapter 3.6.1.14 --- Propagation of ES cells in serum-based medium --- p.56 / Chapter 3.6.1.15 --- Propagation of ES cell in serum-free medium --- p.56 / Chapter 3.6.1.16 --- Neural differentiation using all-trans retinoic acid --- p.57 / Chapter 3.6.1.17 --- Stromal cells-derived inducing activity --- p.58 / Chapter 3.6.1.18 --- BrdU labeling of the cell products --- p.59 / Chapter 3.6.2 --- Molecular analysis --- p.60 / Chapter 3.6.2.1 --- RNA extraction --- p.60 / Chapter 3.6.2.2 --- RNA quantitation --- p.60 / Chapter 3.6.2.3 --- Reverse Transcription of the First Strand complementary DNA --- p.61 / Chapter 3.6.2.4 --- Polymerase chain reaction --- p.61 / Chapter 3.6.2.5 --- RNA Integrity Check --- p.66 / Chapter 3.6.2.6 --- Electrophoresis and visualization of gene products --- p.66 / Chapter 3.6.3 --- Immunofluoresent staining --- p.66 / Chapter 3.6.4 --- In-vivo studies --- p.69 / Chapter 3.6.4.1 --- Induction of cerebral ischaemia in mice --- p.69 / Chapter 3.6.4.2 --- Transplantation --- p.69 / Chapter 3.6.4.3 --- Assessment of learning ability and memory --- p.70 / Chapter 3.6.5 --- Histological analysis --- p.70 / Chapter 3.6.5.1 --- Animal sacrifice for brain harvest --- p.70 / Chapter 3.6.5.2 --- Cryosectioning --- p.71 / Chapter 3.6.5.3 --- Paraffin sectioning --- p.71 / Chapter 3.6.5.4 --- Haematoxylin and eosin staining --- p.72 / Chapter 3.7 --- Data analysis --- p.73 / Chapter Chapter 4 --- Results --- p.74 / Chapter 4.1 --- ES cell maintenance --- p.74 / Chapter 4.1.1 --- Serum effect --- p.74 / Chapter 4.1.2 --- Feeder effect --- p.79 / Chapter 4.1.3 --- Serum-free and feeder-free condition --- p.86 / Chapter 4.1.4 --- Overall effect --- p.89 / Chapter 4.2 --- ES cell Induction --- p.91 / Chapter 4.2.1 --- Retinoic acid --- p.91 / Chapter 4.2.2 --- Stromal cell-derived inducing activity --- p.96 / Chapter 4.2.2.1 --- Molecular characterization of candidate stromal cells --- p.96 / Chapter 4.2.2.2 --- Direct contact co-culture --- p.98 / Chapter 4.2.2.3 --- Non-contact co-culture --- p.100 / Chapter 4.2.2.4 --- Cultures in CM --- p.109 / Chapter 4.3. --- ES cell Differentiation --- p.115 / Chapter 4.4 --- In vivo study of ES cell-derived cell products --- p.117 / Chapter 4.4.1 --- Animal preparation --- p.117 / Chapter 4.4.2 --- Cell preparation --- p.117 / Chapter 4.4.3 --- Cell implantation --- p.117 / Chapter 4.4.4 --- Behaviour Monitoring --- p.121 / Chapter 4.4.5 --- Histology of cell-implanted brain --- p.125 / Chapter Chapter 5 --- Discussion --- p.129 / Chapter Chapter 6 --- Conclusion --- p.144 / References --- p.147

During development of the nervous system, formation of specific connections between nerve cells depends on the stability of growing axons to reach appropriate target cells and form synapses. In culture, hippocampal neurons form numerous synapses by developing axonal and dendritic extensions. To elucidate principles of neuronal signaling and network establishment, creation of neuronal networks in which connectivity and pathways can be experimentally controlled is of great interest. In the present study we used a microcontact printing technique to control and study neurite outgrowth of hippocampal neurons in vitro. My preliminary results show that hippocampal neurons follow the microcontact printed pattern of poly-D-lysine (PDL). In doing so, neurons retain their morphology with normal subcellular distribution of various cell adhesion and synaptic molecules. However, the distribution of various axonal or dendrite components is altered. Hence we have developed a system in which isolated axons and dendrites align with inputs from very few neurons. With this technique we intend to study axon-dendrite communications on a spatially restricted and defined substrate.

No description available.

In recent years, three dimensional (3D)-culture technique has emerged as a very popular approach to reconstruct tissue architectures and develop experimental models for studying epithelial cancers. However, 3D culture models of prostate epithelial cells to mimic prostate cancer development are relatively rare, making it highly desirable to develop and characterize novel 3D culture models suitable for studying prostate cancer. Recently, epithelial-mesenchymal transition (EMT) has emerged as an important mechanism for cancer cell invasion. The zinc finger transcriptional factor Snail as a key regulator of EMT has been found to contribute to aggressive progression in many types of neoplasms. Even though several studies corroborated that EMT is implicated in prostate cancer, the expression patterns of Snail in normal prostate and prostate cancer, and the functional role of Snail in prostate cancer as well as its relation with EMT are still unknown. Based on this background, my major efforts were to establish a 3D culture model of human prostatic epithelial cells with structural and functional relevance to prostate gland and to employ this model to study the functional role of Snail in the prostate cancer. / When embedded in Matrigel for 3D culture, BPH-1 cells developed into growth-arrested acinar structures with a hollow lumen. Ultrastructural examination of BPH-1 spheroids by electricon microscopy indicated that BPH-1 spheroids displayed a polarized differentiation phenotype. Immunoflurescence analysis of polarized epithelial markers further confirmed that BPH-1 spheroids were polarized. In contrast, tumorigenic BPH-1CAFTD cells exhibited disorganized and continuously proliferating structures in Matrigel, with polarized epithelial markers randomly diffused or completely lost. In addition, BPH-1 CAFTD cells displayed significantly higher invasive capacity in comparison to BPH-1 cells by transwell invasion assay. Moreover, LY294002 treatment of BPH-1CAFTD1 and BPH-1CAFTD3 cells in 3D cultures resulted in impaired cell proliferation as evidenced by reduced colony size and decreased Ki-67 index, and western blot analysis showed that cyclin D1 protein levels were significantly decreased, while p21 protein levels were slightly up-regulated in LY294002-treated 3D cultures. Additionally, LY94002 significantly decreased the invasive capacity of BPH-1CAFTD1 and BPH-1CAFTD3 cells. Interestingly, LY294002 treatment completely reverted the disorganized non-polar 3D structures of BPH-1CAFTD1 cells to well-organized polarized spheroid structures in Matrigel, but failed to restore the polarized differentiation in 3D cultures of BPH-1CAFTD3 cells, which still formed compact aggregates as shown by confocal immunofluorescence analysis. Snail protein was barely detected in the epithelial cells of human benign prostatic tissue but significantly elevated as nuclear protein in primary prostate cancer and bone metastatic specimens by immunohistochemical analysis. Snail transcript levels were weakly expressed in a majority of nonmalignant prostatic epithelial cell lines, while markedly increased in almost all tested cancer cell lines. Snail expression induced a morphological switch to more scattered and spindle-shaped appearance in BPH-1 and BPH-1CAFTD1 cells in 2D culture, and immunofluorescence analysis of several EMT specific markers indicated that Snail-expressing cells underwent EMT. In 3D contexts, Snail-expressing cells developed into more disorganized structures with many cords or protrusions, with a concurrent EMT change as evidenced by reduced E-cadherin and increased vimentin expression. In addition, Snail expression augmented the invasive capacities in both BPH-1 cells and BPH-lCAFTD1 cells, but did not significantly affect the migratory capacities. Snail expression enhanced the MMP2 activity in BPH-1 cells and promoted both MMP-2 and MMP-9 activities in BPH-1CAFTD1 cells. Moreover, Snail expression enhanced anchorage-independent growth capability in BPH-1 cells, but failed to initiate tumor formation in nude-mice. Lastly, Snail expression induced a dramatic increase in FoxC2 and SPARC transcripts but a marked decrease in claudin-1 and p63 transcripts. / Chu, Jianhong. / Adviser: Franky Chan Leung. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3448. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 143-166). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.