title: n-Butylidenephthalide (BP) maintains stem cell pluripotency by activating Jak2/Stat3 pathway and increases the efficiency of iPS cells generation.
abstract: 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.
authors.affiliation: Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan. spliu@mail.cmu.edu.tw
authors.forename: Shih-Ping
authors.initials: SP
authors.lastname: Liu
authors.forename: Horng-Jyh
authors.initials: HJ
authors.lastname: Harn
authors.forename: Ying-Jiun
authors.initials: YJ
authors.lastname: Chien
authors.forename: Cheng-Hsuan
authors.initials: CH
authors.lastname: Chang
authors.forename: Chien-Yu
authors.initials: CY
authors.lastname: Hsu
authors.forename: Ru-Huei
authors.initials: RH
authors.lastname: Fu
authors.forename: Yu-Chuen
authors.initials: YC
authors.lastname: Huang
authors.forename: Shih-Yin
authors.initials: SY
authors.lastname: Chen
authors.forename: Woei-Cherng
authors.initials: WC
authors.lastname: Shyu
authors.forename: Shinn-Zong
authors.initials: SZ
authors.lastname: Lin
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects / Q000235:genetics; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D016207:Cytokines / Q000378:metabolism; D030541:Databases, Genetic; D004622:Embryo, Mammalian / Q000166:cytology; D058732:Embryoid Bodies / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D053614:Janus Kinase 2 / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D010766:Phosphorylation / Q000187:drug effects; D010796:Phthalic Anhydrides / Q000494:pharmacology*; D039904:Pluripotent Stem Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics; D013194:Staining and Labeling
chemical_list: D015415:Biomarkers; D016207:Cytokines; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D050814:Octamer Transcription Factor-3; D010796:Phthalic Anhydrides; D055748:SOXB1 Transcription Factors; D050796:STAT3 Transcription Factor; C527783:Sox2 protein, mouse; D053614:Janus Kinase 2; C026105:butylidenephthalide