| Summary: | 碩士 === 中國醫藥大學 === 基礎醫學研究所碩士班 === 99 === Abstract
The first induced pluripotent stem (iPS) cells were generated in 2006 from somatic cells by introducing Oct4, Sox2, c-Myc and Klf4. The original process was inefficient, and maintaining the pluripotency of embryonic stem (ES) and iPS cell cultures required leukemia induced factor (LIF) as an expensive reagent. Our goal is to find a pure compound that not only maintains ES and iPS cell pluripotency, but also increases iPS cell generation efficiency. From 15 candidate compounds we determined that 10 μg/ml n-Butylidenephthalide (BP), an Angelica sinensis extract, triggers up-regulation of Oct4 and Sox2 gene expression levels in MEF cells. We used ES and iPS cells treated with different concentrations of BP to test its usefulness for maintaining stem cell pluripotency. Results indicate higher expression levels of several stem cell markers in BP-treated ES and iPS cells compared to controls that did not contain LIF including alkaline phosphatase, SSEA1, and Nanog. Embryoid body formation and differentiation results confirm that BP containing medium culture is capable of maintaining ES cell pluripotency. Microarray analysis data identified PPAR, ECM and Jak-Stat signaling as the top three deregulated pathways. We subsequently determined that phospholation-Jak2 and phospholation-Stat3 protein levels increased following BP treatment, and that cytokines associated with the Jak2-Stat3 pathway were up-regulated. Last, we used pou5f1-GFP MEF cells to test iPS generation efficiency following BP treatment. Our data demonstrate the ability of BP to maintain stem cell pluripotency via the Jak2-Stat3 pathway by inducing cytokine expression levels, at the same time improving iPS generation efficiency.
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