Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells

Abstract

Human embryonic stem cells (hESCs) can be captured in a primed state in which they resemble the postimplantation epiblast, or in a naive state where they resemble the preimplantation epiblast. Naive-cell-specific culture conditions allow the study of preimplantation development ex vivo but reportedly lead to chromosomal abnormalities, which compromises their utility in research and potential therapeutic applications. Although MEK inhibition is essential for the naive state, here we show that reduced MEK inhibition facilitated the establishment and maintenance of naive hESCs that retained naive-cell-specific features, including global DNA hypomethylation, HERVK expression, and two active X chromosomes. We further show that hESCs cultured under these modified conditions proliferated more rapidly; accrued fewer chromosomal abnormalities; and displayed changes in the phosphorylation levels of MAPK components, regulators of DNA damage/repair, and cell cycle. We thus provide a simple modification to current methods that can enable robust growth and reduced genomic instability in naive hESCs.

Figure 1.. Attenuated MEK1/2 inhibition maintains naïve pluripotency in hESCs.

(a) PD0325901 (PD03) titration strategy (upper panel). Representative phase and fluorescence images of WIBR3 ΔPE OCT4GFP hESCs at P8 grown in the indicated media (lower panel). Scale bar 250 μm. (b) Flow cytometric analysis of the proportion of ΔPE OCT4GFP⁺ cells after reversion of WIBR3 primed hESCs to a naïve state. (c) Representative bright field and immunofluorescence images for P9 UCLA4 hESCs cultured in the indicated media (scale bar: 100 μm, left panel; 50 μm, right panels). (d) Flow cytometric analysis of CD75 and THY-1 protein expression levels in hESC lines cultured as indicated.

Figure 2.. MEK1 inhibition is sufficient to sustain naïve pluripotency in 5i/LAF.

(a) Alternative MEK inhibitors used in this study. (b) Flow cytometric analysis of the proportion of WIBR3 ΔPE OCT4GFP⁺ hESCs cultured as indicated. (c) Representative immunofluorescence images of UCLA4 hESCs grown in the indicated media (P8) (scale bar: 100 μm). (d) Flow cytometric analysis of the proportion of ΔPE OCT4GFP⁺ cells after primed-to-naive conversion of WIBR3 hESCs in 5i/LAF supplemented with the indicated MEKi.

Figure 3.. Transcriptome and proteome analysis of hESCs cultured in modified conditions.

(a) PCA analysis of RNA-seq data for the indicated hESCs samples (P10) using the top 500 most variable genes. (b) Heatmap for selected primed and naïve pluripotency genes. (c) Unsupervised hierarchical clustering of proteomic samples (P18). (d) Heatmap for selected primed and naïve pluripotency proteins.

Figure 4.. hESCs cultured in m5i/LAF are hypomethylated and have two active X chromosomes.

(a) Violin plot representation of HERVK expression (n=64) in hESCs expanded in the indicated culture conditions (P10). For primed, naïve PD03 1μM and naïve PD03 0.5 μM n=5 independent hESC lines (UCLA1, 4, 5, 9 and WIBR3), for naïve Cobimetanib 0.5 μM, Refametinib, TAK-733 and t2iLGöY n=1 (UCLA4). (b) Global methylation analysis of primed and naive hESCs by RRBS analysis using violin plot representation. (c) Representative RNA FISH images for primed and naïve hESCs at P16, detecting the indicated transcripts. (scale bar: 10 μm). (d) Quantification of XIST RNA FISH patterns.

Figure 5.. Naïve hESCs cultured in m5i/LAF retain a more stable karyotype.

(a) Cell numbers during the expansion of naive hESCs in the indicated conditions. Error bars indicate mean ± s.d. (n=3 biologically independent samples). (b) Flow cytometric analysis for γH2AX⁺ cells using primed UCLA1 hESCs. Error bars indicate mean ± s.d. (n=3 biologically independent samples), naïve UCLA1 hESCs cultured in 5i/LAF (n=4 independent clones) or m5i/LAF (n=4 independent clones). Statistical significance was determined using a two-tailed unpaired Student’s t-test (P=0.0005). (c) Representative karyotypes at p10 of hESCs converted and cultured as indicated. Red boxes highlight chromosomal abnormalities. (d) Chromosomal copy number analysis by whole-genome sequencing. Black arrows indicate abnormalities. (e) Phosphorylation levels at the indicated residues assessed by phosphoproteomics in UCLA4 cells cultured as shown. (f) Gene ontology analysis of hyperphosphorylated proteins (>1.5-fold, n=826) in UCLA4 hESCs cultured as shown.