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.
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_344397 |
| Date | January 2009 |
| Contributors | Yang, Tao, Chinese University of Hong Kong Graduate School. Division of Anatomical & Cellular Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, theses |
| Format | electronic resource, microform, microfiche, 1 online resource (xviii, 194 leaves : ill.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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