Probing the signaling requirements for naive human pluripotency by high-throughput chemical screening

  • Cell Rep. 2021 Jun 15;35(11):109233. doi: 10.1016/j.celrep.2021.109233.
Shafqat A Khan  1 ,  Kyoung-Mi Park  1 ,  Laura A Fischer  1 ,  Chen Dong  1 ,  Tenzin Lungjangwa  2 ,  Marta Jimenez  3 ,  Dominick Casalena  3 ,  Brian Chew  1 ,  Sabine Dietmann  1 ,  Douglas S Auld  4 ,  Rudolf Jaenisch  5 ,  Thorold W Theunissen  6
Affiliations
  • 1. Department of Developmental Biology and Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA.
  • 2. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA.
  • 3. Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA.
  • 4. Novartis Institutes for Biomedical Research, Cambridge, MA 02139, USA. Electronic address: [email protected].
  • 5. Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA; Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: [email protected].
  • 6. Department of Developmental Biology and Center of Regenerative Medicine, Washington University School of Medicine, St. Louis, MO 63110, USA. Electronic address: [email protected].
PMID: 34133938 DOI: 10.1016/j.celrep.2021.109233
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.
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