@article{4989c70f1ae94ccea48e17e04994f028,
title = "Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening",
abstract = "Naive human embryonic stem cells (hESCs) have been isolated that more closely resemble the pre-implantation epiblast compared to conventional “primed” hESCs, but the signaling principles underlying these discrete stem cell states remain incompletely understood. Here, we describe the results from a high-throughput screen using ∼3,000 well-annotated compounds to identify essential signaling requirements for naive human pluripotency. We report that MEK1/2 inhibitors can be replaced during maintenance of naive human pluripotency by inhibitors targeting either upstream (FGFR, RAF) or downstream (ERK1/2) kinases. Naive hESCs maintained under these alternative conditions display elevated levels of ERK phosphorylation but retain genome-wide DNA hypomethylation and a transcriptional identity of the pre-implantation epiblast. In contrast, dual inhibition of MEK and ERK promotes efficient primed-to-naive resetting in combination with PKC, ROCK, and TNKS inhibitors and activin A. This work demonstrates that induction and maintenance of naive human pluripotency are governed by distinct signaling requirements.",
keywords = "DNA methylation, FGFR signaling pathway, X chromosome reactivation, high-throughput screening, naive pluripotency, pre-implantation development, primed pluripotency, signal transduction, transposable elements, trophoblast",
author = "Khan, {Shafqat A.} and Park, {Kyoung mi} and Fischer, {Laura A.} and Chen Dong and Tenzin Lungjangwa and Marta Jimenez and Dominick Casalena and Brian Chew and Sabine Dietmann and Auld, {Douglas S.} and Rudolf Jaenisch and Theunissen, {Thorold W.}",
note = "Funding Information: We thank the Genome Technology Access Center at Washington University School of Medicine for assistance with RNA sequencing and whole-genome bisulfite analysis, and the Cytogenetics and Molecular Pathology Laboratory, Department of Pathology and Immunology, Washington University School of Medicine, for G-banded karyotyping. All experiments involving hESCs were approved by the Institutional Biological and Chemical Safety Committee and Embryonic Stem Cell Research Oversight Committee at Washington University School of Medicine. We thank Angela Bowman for critical reading of the manuscript. This work was supported by the National Institutes of Health (NIH) Director's New Innovator Award (DP2 GM137418) and grants from the Children's Discovery Institute (CDI-LI-2019-819), the Shipley Foundation Program for Innovation in Stem Cell Science, and the Edward Mallinckrodt, Jr. Foundation to T.W.T. Federal NIH/NIGMS funds were not used to develop 3D models of early human development. T.W.T. and D.S.A. conceived and designed the high-throughput screens for modulators of naive human pluripotency under supervision of R.J. T.W.T. T.L. M.J. D.C. and D.S.A. conducted the screens. Multi-parametric image analysis was performed by D.S.A. S.A.K. further defined the activity of the selected hit compounds during maintenance and induction of naive human pluripotency under supervision of T.W.T. K.-m.P. L.A.F, C.D. and B.C. assisted S.A.K. with tissue culture and molecular biology experiments. S.D. performed bioinformatic analyses. S.A.K. and T.W.T. analyzed and interpreted the results and wrote the paper with input from D.S.A. and R.J. R.J. is a co-founder of Fate Therapeutics, Fulcrum Therapeutics, and Omega Therapeutics and an advisor to Dewpoint Therapeutics. T.W.T. S.A.K. and R.J. are co-inventors on a patent application related to naive hPSCs. Funding Information: We thank the Genome Technology Access Center at Washington University School of Medicine for assistance with RNA sequencing and whole-genome bisulfite analysis, and the Cytogenetics and Molecular Pathology Laboratory, Department of Pathology and Immunology, Washington University School of Medicine, for G-banded karyotyping. All experiments involving hESCs were approved by the Institutional Biological and Chemical Safety Committee and Embryonic Stem Cell Research Oversight Committee at Washington University School of Medicine. We thank Angela Bowman for critical reading of the manuscript. This work was supported by the National Institutes of Health (NIH) Director{\textquoteright}s New Innovator Award ( DP2 GM137418 ) and grants from the Children's Discovery Institute ( CDI-LI-2019-819 ), the Shipley Foundation Program for Innovation in Stem Cell Science , and the Edward Mallinckrodt, Jr. Foundation to T.W.T. Federal NIH/NIGMS funds were not used to develop 3D models of early human development. Publisher Copyright: {\textcopyright} 2021 The Authors",
year = "2021",
month = jun,
day = "15",
doi = "10.1016/j.celrep.2021.109233",
language = "English",
volume = "35",
journal = "Cell Reports",
issn = "2211-1247",
number = "11",
}