2013 Fiscal Year Research-status Report
Small Bioactive Molecules for Orchestrating Embryonic Stem Cell Differentiation
Project/Area Number |
25870365
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Research Category |
Grant-in-Aid for Young Scientists (B)
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Research Institution | Kyoto University |
Principal Investigator |
PERRON AMELIE 京都大学, 物質ー細胞統合システム拠点, 研究員 (40525862)
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Project Period (FY) |
2013-04-01 – 2015-03-31
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Keywords | Embryonic / Regenerative medicine / Transcription factors / Hes1 / Oscillations / Stem cells / Gro/TLE |
Research Abstract |
FY2013, the focus of the research has been to develop small organic molecule tools for modulating the Notch pathway through the transcription factor Hes1 (hairy and enhancer of split-1). As the function of Hes1 largely depends on its interaction with the corepressor Gro/TLE, we aimed at developing small bioactive organic molecules based around the WRPW peptide motif of Hes1 to competitively block Gro/TLE binding to Hes1 in order to ultimately inhibit Hes1 from exerting a negative feedback effect on its own expression. Such negative feedback inhibition should lead to sustained expression of Hes1 expression, promoting homogenous differentiation of embryonic stem (ES) cells which will be useful as a strategy for increasing the efficacy of cell transplants and improve the outcome of regenerative medicine treatments. Since the WRPW peptide motif of the transcriptional repressor Hes1 is constituted of 2 tryptophan residues which each comprise an indole group, a chemical library derived from small organic molecules containing indole moieties (Tripos Receptor Research Ltd.) was screened using a cell-based transcriptional assay. Out of 1806 compounds, 3 compounds were selected as hit compounds as they were able to block Hes1-mediated repression. These compounds were then validated using a EGFP-based assay to investigate their effect in single cells by confocal microscopy. Among the 3 hits compounds, 1 of them (i.e. D8C) was also able to block Hes1 oscillations both in mammalian cells and in mouse presomitic mesoderm (PSM) preparations.
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Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
The research has been progressing rather smoothly as all of the objectives FY2013 were achieved. a) Compound Screening Procedure: The compound screening procedure using a Luciferase-base transcriptional assay developed in the laboratory allowed us to isolate 3 hit compounds from the indole-based chemical library of small organic molecules. Although the FRET-based assay mentioned in the grant application did not work out due to spectral bleed through between the 2 chomophores, a substitute ELISA-based competition assay was developed to investigate whether the hit compounds were able to decrease Hes1 interaction with Gro/TLE. Preliminary results indicate that D8C was able to do so in transfected HEK 293 cells. b) Validation Procedure: Out of these 3 hits, D8C was also validated as a HES1 modulator as it was able to increase the protein expression levels of Hes1 in HEK293 cells transfected with EGFP expressed under the control of Hes1 promoter. The typical oscillation pattern of Hes1 was also affected by D8C as revealed by electron confocal microscopy in single cells. D8C was also tested in embryos wherein the effect of D8C on Hes1 oscillations in PSM preparations was investigated.
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Strategy for Future Research Activity |
a) Structure-Affinity Relationship (SAR) studies: Several D8C derivatives will be synthetized by organic synthesis to analyse the commonalities among the different chemical groups for further optimization of the hit compound. b) Target Identification Procedure: Once that the D8C derivative with the highest activity will be determined, a biotinylated version of the compound will be synthesized for target identification purpose using a pull-down assay visualised by Western blotting analysis. c) Testing of Compounds in ES cells: We are planning on investigating the effect of the selected compounds on ES cell differentiation. The work will be done in collaboration with Prof. Ryoichiro Kageyama’s laboratory (Kyoto University). Briefly we will investigate the effect of small bioactive organic molecules on the cell fate of choice of ES cells during differentiation after removal of both LIF and feeder cells, a condition known to induce all three germ layers. mRNA levels of neural (Mash1 and Tuj-1) and mesodermal (Brachyury) markers will be analyzed by quantitative real-time PCR to determine the fate preference of ES cells. Marker protein expression will also be investigated by immunocytochemistry.
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Expenditure Plans for the Next FY Research Funding |
研究開始当初に計画していた大型機器の購入を次年度にしたため。 JuLi - Smart Fluorescent Cell Analyserの購入。
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