碩士 / 國立中興大學 / 生命科學系所 / 102 / The accumulation of β-amyloid (Aβ), produced by endoproteolysis of the amyloid precursor protein (APP), results in amyloid plaques formation and is the cytopathological hallmark in the patient’s brain of Alzheimer’s disease (AD). To generate the AD cell model by in vitro differentiation of pluripotent stem cells, initially, we attempt to use APP-transgenic human embryonic stem cells (hESCs) as a platform for studying amyloid plaques associated diseases. Dual Oct4 and α-Tubulin promoters driving the neomycin-2A-green fluorescent protein (NeoGFP) and APP protein, respectively, are cloned into the PiggyBac vector for the establishment of APP-mutants overexpressing hESCs. Expression of APP mutants under α-Tubulin promoter was confirmed in HEK293T cells. Although the efficacy of both promoters was valid in individual vectors, the Oct4 promoter conjugated with α-Tubulin promoter in single PiggyBac vector were dramatically repressed for the expression of NeoGFP. This low expression of NeoGFP hampers the rapid selection and establishment of APP-transgenic ESCs. Alternatively, induced pluripotent stem cells (iPSCs) from Down syndrome patients (T21 iPSCs) have been shown to produce robust neural cells and to faithfully recapitulate the Aβ accumulation in vitro. Using our novel BiSF neural differentiation method, we successfully differentiated the T21 iPSCs into Nestin+ and Sox1+ neuroepithelial precursor cells on day 15 (D15) and mature TuJ1+ neurons on D21. Importantly, significant BTA-1+ Aβ plaques and Aβ42 accumulation were observed in the T21 iPSC-derived neurons on D30, but not in the differentiated normal-karyotype iPSCs. These results emphasize that differentiating neurons from T21 iPSCs can recapitulate the cytopathological features of AD and the BiSF method can rapidly steer the neural differentiation of T21 iPSCs and produce the cell model of AD efficiently.
| Identifer | oai:union.ndltd.org:TW/102NCHU5105010 |
| Date | January 2014 |
| Creators | Pei-Ying Chen, 陳姵穎 |
| Contributors | Hong-Lin Su, 蘇鴻麟 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 41 |
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