Stem cells have the ability to reproduce themselves for a long period and differentiate into specific morphological and functional cells. The stem cells are an important material in the developmental biology, genomics, and transgenic methods, as well as in potential clinical applications, gene therapy and tissue engineering. The pluripotent stem cells will be a valuable source in numerous functional degenerated pathologies. Therefore, the objective of this research program was to establish transgenic porcine embryonic stem (pES) cell lines which can express green fluorescent protein (GFP) report gene stably for tracking after transplantation. We also developed a directed differentiation of pES into neural lineages and applied in rat Parkinson¡¦s disease model. Although the establishment of pluripotent ES cell lines from domestic species is much more difficult than that in murine species, our results had successfully isolated and established pES cell lines from pre-implantation blastocysts. Furthermore, we established the novel GFP-expressing pES cell lines (pES/GFP+), which were obtained by electroporation- mediated transfection with exogenous GFP gene. These pES/GFP+ cells exhibited pluripotent markers including Oct-4, AP, SSEA-4, TRA-1-60, and TRA-1-81 as that of human ES cells. The strategy of directed neural differentiation was to culture pES with neurogenic stimulators such as retinoic acid (RA), sonic hedgehog (SHH), and fibroblast growth factor (FGF). Upon directed differentiation toward neural differentiation, these pES-derived cells exhibited typical neuronal morphology and expressed neural lineage-specific markers such as nestin, NFL, MAP2, GFAP, A2B5, TH, ChAT, and GABA. These results showed the pES cells had the potential to differentiate into neural lineages. When pES/GFP+ cells were transplanted into the SD rat¡¦s brain, and their survival and development was determined by the non-invasive In Vivo Imaging System (IVIS 50), and the invasive fibered confocal Cellvizio® Imaging System (Cellvizio®). The results showed that fluorescent signals from pES/GFP+ cells on the injection site of SD rats¡¦ brain could be detected through the experimental period of 3 months. The level of fluorescent signals detected in treatment groups was two folds above that of the control group. Besides, the functional behavior recovery analysis by amphetamine-induced rotation test indicated the PD rat grafted with pES/GFP+ cells and their derived neural progenitors showed no significant recovery of rotation rate in these two treatments because a progressively increased relative rotation through 3 months duration. However, the relative rotation of PD rats grafted with the pES/GFP+-derived mature neurons, showed a stably decrease relative rotation and resulted in a functional recovery from Parkinsonian behavioral defects. Following 3 months completion of behavioral analyses, PD rats were sacrificed for immunohistochemical analysis. In the section of injected site without tumorgenesis and showed the survival and dopaminergic differentiation of grafted pES/GFP+ derived cells when stained with anti-TH and anti-DA. To our knowledge, there have been no reports of establishing GFP-expressing pES cell lines. These novel pES/GFP+ cell lines established in this study might serve as a non-rodent model and could benefit to the studies involving ES cell transplantation, cell replacement therapy, tissue regeneration, and actual approach for pre-clinical research due to their traceable capacity.
| Identifer | oai:union.ndltd.org:NSYSU/oai:NSYSU:etd-0616109-191923 |
| Date | 16 June 2009 |
| Creators | Yang, Jenn-rong |
| Contributors | Long-sen Chang, Yow-ling Shiue, Lih-ren Chen, Kuang-hung Cheng, Lynn Ling-huei Huang |
| Publisher | NSYSU |
| Source Sets | NSYSU Electronic Thesis and Dissertation Archive |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lib.nsysu.edu.tw/ETD-db/ETD-search/view_etd?URN=etd-0616109-191923 |
| Rights | not_available, Copyright information available at source archive |
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