Abstract
SUMMARYNaive 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.
Highlights
A major objective in stem cell research is to devise in vitro culture conditions for pluripotent stem cells (PSCs) that recapitulate specific stages of embryonic development
Similar to our prior study, we used naive human embryonic stem cells (hESCs) that were generated with doxycycline (Dox)-inducible transgenes driving exogenous KLF2 and NANOG transgenes (Theunissen et al, 2014)
We designed a multi-parametric data analysis (MPDA) algorithm to analyze these images, computing a Mahalanobis distance score between each compound and the active (+DOX) and negative (–DOX) control wells based on features that included area, compactness, and fluorescence intensity of individual object regions (Figure 1B)
Summary
A major objective in stem cell research is to devise in vitro culture conditions for pluripotent stem cells (PSCs) that recapitulate specific stages of embryonic development. The use of MEK and GSK3 inhibitors and leukemia inhibitory factor (2i/LIF) captures mouse embryonic stem cells (ESCs) in a ‘‘naive’’ state of pluripotency that closely corresponds to the pre-implantation epiblast at embryonic day (E) 4.5 (Boroviak et al, 2015; Ying et al, 2008). This naive state of pluripotency contrasts with the ‘‘primed’’ pluripotent state observed in mouse epiblast stem cells (EpiSCs), which aligns more closely with the anterior primitive streak of the late-gastrula stage embryo (Brons et al, 2007; Kojima et al, 2014; Tesar et al, 2007). Conventional hPSCs exhibit some primate-specific features that are not observed in either mouse ESCs or EpiSCs, such as expression of N-cadherin at colony boundaries (Nakanishi et al, 2019)
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