In-vitro induction of embryonic stem cells into neural lineage through stromal cell-derived inducing activity.

• Other Authors: Fong, Shu Pan.
• Format: Others
• Language: English; Chinese
• Published: 2005
• Subjects: Cell differentiation; Embryonic stem cells; Cell culture > Technique; Embryonic Induction; Cell Differentiation; Cell Culture Techniques > methods
• Online Access: http://library.cuhk.edu.hk/record=b5892484; http://repository.lib.cuhk.edu.hk/en/item/cuhk-325397

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