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

Embryonic stem (ES) cells, which possess proliferating and differentiating abilities, are a potential source of cells for regenerative medicine. Nowadays, the challenge in using ES cells for developmental biology and regenerative medicine has been to direct the wide differentiation potential towards the derivation of a specific cell fate. This study is aimed to establish a simple and efficient method to derive ES cells into neural lineage cells and examine the safety and efficacy of derived cells in a mouse ischemic stroke model. To explore the underlying mechanisms responsible for lineage commitment of stem cells, Notch signaling and serotonin responses are also studied. / In a non-contact coculture system, mouse ES cells (D3 and E14TG2a) were cocultured with the stromal cells MS5 for eight days. On the other hand, human ES cells (H9 and H14) were directly cocultured with MS5 in a contact manner for two weeks. Derived cells were further propagated in a serum-free medium and selected subsequently in a differentiating medium. The cell viability, numbers, phenotypes and lineage-specific gene expression profile were evaluated at stages of induction, propagation and selection. / In vivo, behavioral assessments of ischemic mice after transplantation of mouse ES cell derivatives revealed a significant improvement in spatial learning and memory ability as compared to ischemic mice without cell therapy. Histology of brain sections of transplanted mice demonstrated the migration of BrdU+ cells to the CA1 region of the hippocampus, which was evident of both an increase of pyramidal neuron density and normalized morphology. Teratoma development was found in one out of 17 transplanted mice. / MS5 was noted to express genes encoding neurotrophins and neuroprotective factors. Functional tests showed that MS5 exerted neurotrophism on neuroblastoma cell lines (SK-N-AS, SH-SY5Y, and SK-N-MC) and ES cells. The numbers of viable cells and the proportion of neural subtypes derived from ES cells at three stages of the culture system were significantly higher than those of the control cultures without MS5 induction, respectively. MS5 cocultures generate a relatively higher yield of neural lineage cells but select against the mesodermal and endodermal lineage derivatives. Together with non-contact MS5 coculture, serotonin could further increase the proportion of neural precursors and accelerate maturation of neural progenitor cells in a synergistic manner. During the induction phase with non-contact MS5 coculture, the Notch inhibitor could significantly decrease the number of derived neural precursors and instigate non-neural differentiation. With the supplement of the Notch inhibitor, serotonin could neither promote the expression of neuroectodermal genes nor enhance the proportion of neural precursors in MS5-cocultured ES cells. Notably, in the propagation of undifferentiated human ES cells, Notch signaling was also found to play an active role in maintaining cell survival. / The Notch inhibitor (gamma-secretase inhibitor) and serotonin were supplemented into induction cultures to investigate the roles of Notch signaling and the neurotransmitter serotonin in neural differentiation. For in vivo study, mouse ES cell-derived cells were labeled with BrdU and implanted onto the caudate putamens of mice having undergone transient occlusion of bilateral common carotid arteries and reperfusion to induce cerebral ischemia. Spatial learning and memory ability of transplanted mice were assessed in a water maze system. Histological assessment was also conducted on brain sections of mice three weeks post transplant to examine the migration and homing of implanted cells. / This study describes a simple and efficient differentiation protocol to derive mouse ES cells and human ES cells into neural lineage cells. Derived cells appear to significantly improve cognitive functions in a mouse ischemic stroke model. Data of the study suggest that MS5 cells may exert a neurotrophic effect on ES cells. With MS5 coculture, serotonin synergistically promotes neural commitment and facilitates maturation of derived neural precursors in ES cell cultures. In contact coculture with MS5, Notch signaling is shown to play a role in the directed neural differentiation of human ES cells, whereas in maintenance culture, Notch signaling is also important to cell survival of human ES cells. Thus, Notch signaling through cell-cell interaction may explain, at least partially, the difference between mouse ES cells and human ES cells in cell growth ability when seeded at low cell densities. / Yang Tao. / Adviser: Ho Keung Ng. / Source: Dissertation Abstracts International, Volume: 70-09, Section: B, page: . / Thesis submitted in: November 2008. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 161-194). / 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.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains ix, 148 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 132-148).

Thesis (doctoral)--Lund University, 1995.

Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains xiii, 152 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 141-149).

Thesis (doctoral)--Lund University, 1995.

Embryonic stem cells (ESCs) are a unique cell population that can differentiate into all three embryonic germ layers (endoderm, mesoderm, and ectoderm), rendering them an invaluable cell source for studying the molecular mechanisms of embryogenesis. Signaling molecules that direct tissue patterning during embryonic development are secreted by ESC aggregates, known as embryoid bodies (EBs). As many of these signaling proteins interact with the extracellular matrix (ECM), manipulation of the ESC extracellular environment provides a means to direct differentiation. ECM components, such as glycosaminoglycans (GAGs), play crucial roles in cell signaling and regulation of morphogen gradients during early development through binding and concentration of secreted growth factors. Thus, engineered biomaterials fabricated from highly sulfated GAGs, such as heparin, provide matrices for manipulation and efficient capture of ESC morphogens via reversible electrostatic and affinity interactions. Ultimately, biomaterials designed to efficiently capture and retain morphogenic factors offer an attractive platform to enhance the differentiation of ESCs toward defined cell types. The overall objective of this work was to examine the ability of microparticles synthesized from both synthetic and naturally-derived materials to enhance the local presentation of morphogens to direct ESC differentiation. The overall hypothesis was that microparticles that mimic the ECM can modulate ESC differentiation through sequestration of endogenous morphogens present within the EB microenvironment.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Biochemistry / Master / Master of Philosophy

published_or_final_version / abstract / Pathology / Doctoral / Doctor of Philosophy