2012 Fiscal Year Final Research Report
Screening for signaling pathways regulating human ES/iPS cell self-renewal
| Project/Area Number |
22603018
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
ケミカルバイオロジー
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| Research Institution | 独立行政法人医薬基盤研究所 |
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Principal Investigator |
KINEHARA Masaki 独立行政法人医薬基盤研究所, 難病・疾患資源研究部, 研究員 (70568451)
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| Co-Investigator(Kenkyū-buntansha) |
FURUE Miho 独立行政法人医薬基盤研究所, 難病・疾患資源研究部, 研究員リーダー (80257310)
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| Project Period (FY) |
2010 – 2012
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| Keywords | iPS細胞 / 多能性幹細胞 / 小分子化合物 / シグナル経路 / PKC |
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| Research Abstract |
The self-renewal of human pluripotent stem (hPS) cells including embryonic stem and induced pluripotent stem cells have been reported to be supported by various signal pathways. Among them, fibroblast growth factor-2 (FGF-2) appears indispensable to maintain self-renewal of hPS cells. However, downstream signaling of FGF-2 has not yet been clearly understood in hPS cells. In this study, we screened a kinase inhibitor library using a high-throughput alkaline phosphatase (ALP) activity-based assay in a minimal growth factor-defined medium to understand FGF-2-related molecular mechanisms regulating self-renewal of hPS cells. We found that in the presence of FGF-2, an inhibitor of protein kinase C (PKC), GF109203X (GFX), increased ALP activity. GFX inhibited FGF-2-induced phosphorylation of glycogen synthase kinase-3β (GSK-3β), suggesting that FGF-2 induced PKC and then PKC inhibited the activity of GSK-3β. Addition of activin A increased phosphorylation of GSK-3β and extracellular signal-regulated kinase-1/2 (ERK-1/2) synergistically with FGF-2. Intriguingly, functional gene analysis by RNA interference revealed that the phosphorylation of GSK-3β was reduced by siRNA of PKCδ, PKCε, and ζ, the phosphorylation of ERK-1/2 was reduced by siRNA of PKCε and ζ, and the phosphorylation of AKT was reduced by PKCε in hPS cells. Addition of GFX with a MEK inhibitor, U0126, in the presence of FGF-2 and activin A provided a long-term stable undifferentiated state of hPS cells even though hPS cells were dissociated into single cells for passage. This study untangles the cross-talk between molecular mechanisms regulating self-renewal and differentiation of hPS cells.
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[Journal Article] Protein Kinase C Regulates Human Pluripotent Stem Cell Self-Renewal2013
Author(s)
Masaki Kinehara, Suguru Kawamura, Daiki Tateyama, Mika Suga, Hiroko Matsumura, Sumiyo Mimura, Noriko Hirayama, Mitsuhi Hirata, Kozue Uchio-Yamada, Arihiro Kohara, Kana Yanagihara, Miho K. Furue
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Journal Title
PLOS ONE
Volume: 8(1)
Pages: e54122
DOI
Peer Reviewed
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