Embryonic stem (ES) cells are considered to exist in a ground state if shielded from differentiation triggers. Here we show that FGF4 and TGFβ signaling pathway inhibitors, designated R2i, not only provide the ground state pluripotency in production and maintenance of naïve ES cells from blastocysts of different mouse strains, but also maintain ES cells with higher genomic integrity following long-term cultivation compared with the chemical inhibition of the FGF4 and GSK3 pathways, known as 2i. Global transcriptome analysis of the ES cells highlights augmented BMP4 signaling pathway. The crucial role of the BMP4 pathway in maintaining the R2i ground state pluripotency is demonstrated by BMP4 receptor suppression, resulting in differentiation and cell death. In conclusion, by inhibiting TGFβ and FGF signaling pathways, we introduce a novel defined approach to efficiently establish the ground state pluripotency.
Embryonic stem cells ERK pathway Ground state pluripotency TGF-β pathway
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We thank the members of the Department of Stem Cells and Developmental Biology labs for their helpful suggestions and critical reading of the manuscript. We thank Behrouz Asgari for chimera formation and germline transmission. This study was funded by grants provided from Royan Institute and Iranian Council of Stem Cell Technology and the Iran National Science Foundation (INSF).
S. H., M. T., H. R. S. and H. B. designed all experiments and wrote the manuscript. S. H. and S. M. performed cell culture. M. T. and A. S. performed real-time PCR analysis. A. F. and M. P. operated in vivo experiments. N. M. and H. G. performed karyotype analysis. S. M. and G. S. H. performed western blot analysis. M. T., A. S., M. S., B. G. and M. J. A. designed and interpreted microarray analysis. G. H. S., S. M. and D. S. contributed to the overall design and writing of the article.
Conflict of Interest
The authors indicate no potential conflicts of interest.
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