Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan.
PLoS One. 2012;7(9):e44024. doi: 10.1371/journal.pone.0044024. Epub 2012 Sep 7.
In 2006, induced pluripotent stem (iPS) cells were generated from somatic cells by introducing Oct4, Sox2, c-Myc and Klf4. The original process was inefficient; maintaining the pluripotency of embryonic stem (ES) and iPS cell cultures required an expensive reagent-leukemia induced factor (LIF). 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 the 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 was capable of maintaining ES cell pluripotency after six time passage. Microarray analysis data identified PPAR, ECM, and Jak-Stat signaling as the top three deregulated pathways. We subsequently determined that phosphorylated Jak2 and phosphorylated Stat3 protein levels increased following BP treatment and suppressed with the Jak2 inhibitor, AG490. The gene expression levels of cytokines associated with the Jak2-Stat3 pathway were also 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.
2006 年，通过引入 Oct4、Sox2、c-Myc 和 Klf4，从体细胞中产生了诱导多能干细胞（iPS）。最初的过程效率低下；维持胚胎干细胞（ES）和 iPS 细胞培养的多能性需要昂贵的试剂白血病诱导因子（LIF）。我们的目标是找到一种不仅能维持 ES 和 iPS 细胞多能性，而且还能提高 iPS 细胞生成效率的纯化合物。从 15 种候选化合物中，我们确定 10µg/ml 的正丁烯基苯酞（BP），一种当归提取物，能触发 MEF 细胞中 Oct4 和 Sox2 基因表达水平的上调。我们使用不同浓度 BP 处理的 ES 和 iPS 细胞来测试其维持干细胞多能性的用途。结果表明，与不含 LIF 的对照组相比，BP 处理的 ES 和 iPS 细胞中几种干细胞标志物的表达水平更高，包括碱性磷酸酶、SSEA1 和 Nanog。胚状体形成和分化结果证实，含有 BP 的培养基培养物能够在六次传代后维持 ES 细胞的多能性。微阵列分析数据确定了 PPAR、ECM 和 Jak-Stat 信号通路为前三个失调的通路。随后，我们确定 BP 处理后磷酸化 Jak2 和磷酸化 Stat3 蛋白水平增加，Jak2 抑制剂 AG490 抑制。与 Jak2-Stat3 通路相关的细胞因子的基因表达水平也上调。最后，我们使用 pou5f1-GFP MEF 细胞测试 BP 处理后 iPS 生成效率。我们的数据表明，BP 通过诱导细胞因子表达水平来维持干细胞多能性的能力，同时提高 iPS 生成效率。
