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

Nature Methods volume 15, pages 732–740 (2018)Cite this article

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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.

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Fig. 1: Attenuated MEK1/2 inhibition maintains naive pluripotency in hESCs.
Fig. 2: MEK1 inhibition is sufficient to sustain naive pluripotency in 5i/LAF.
Fig. 3: Transcriptome and proteome analysis of hESCs cultured in modified conditions.
Fig. 4: hESCs cultured in m5i/LAF are hypomethylated and have two active X chromosomes.
Fig. 5: Naive hESCs cultured in m5i/LAF retain a more stable karyotype.

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Acknowledgements

We thank T.W. Theunissen and R. Jaenisch for primed WIBR3 hESCs, the MGH next-generation sequencing facility for technical assistance, and members of the Hochedlinger lab for discussions. This work was supported by MGH (K.H.), the NIH (R01 HD058013 and P01 GM099134 to K.H.; P01 GM099134 to K.P.; GM67945 to S.P.G.), the Gerald and Darlene Jordan Chair in Regenerative Medicine (K.H.), EMBO (long-term fellowship #ALTF 1143-2015 to B.D.S.), UCLA (UCLA Broad Stem Cell Research Center–Rose Hills Foundation Training Award to S.S.; UCLA Dissertation Year Fellowship to A.S.; UCLA Broad Stem Cell Research Center (to K.P.); the David Geffen School of Medicine (to K.P.); the Jonsson Comprehensive Cancer Center (to K.P.)), the Howard Hughes Medical Institute (Faculty Scholar grant to K.P.), and JSPS KAKENHI (JP16K15489 and JP16H02465 to Y.T.).

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Authors and Affiliations

  1. Department of Molecular Biology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Bruno Di Stefano, Justin Brumbaugh, Aaron J. Huebner & Konrad Hochedlinger

  2. Center for Regenerative Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Bruno Di Stefano, Justin Brumbaugh, Aaron J. Huebner & Konrad Hochedlinger

  3. Cancer Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

    Bruno Di Stefano, Justin Brumbaugh, Aaron J. Huebner, Keiko Shioda, Toshi Shioda & Konrad Hochedlinger

  4. Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA, USA

    Bruno Di Stefano, Justin Brumbaugh, Aaron J. Huebner, Kendell Clement, Hongcang Gu, Alexander Meissner & Konrad Hochedlinger

  5. Harvard Stem Cell Institute, Cambridge, MA, USA

    Bruno Di Stefano, Justin Brumbaugh, Aaron J. Huebner, Kendell Clement, Hongcang Gu, Alexander Meissner & Konrad Hochedlinger

  6. Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan

    Mai Ueda & Yasuhiro Takashima

  7. David Geffen School of Medicine, Department of Biological Chemistry, Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, Jonsson Comprehensive Cancer Center, and Molecular Biology Institute, University of California, Los Angeles, Los Angeles, CA, USA

    Shan Sabri, Anna Sahakyan & Kathrin Plath

  8. Broad Institute of MIT and Harvard, Cambridge, MA, USA

    Kendell Clement, Hongcang Gu & Alexander Meissner

  9. Department of Cell Biology, Harvard Medical School, Boston, MA, USA

    Katie J. Clowers, Alison R. Erickson & Steven P. Gygi

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  1. Bruno Di Stefano
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Contributions

B.D.S. and K.H. conceived the study and wrote the manuscript. B.D.S., M.U., J.B., A.J.H., A.S., K.P., and Y.T. performed the experiments and analyzed the data. S.S. performed the bioinformatics analysis. K.C., H.G., and A.M. performed and analyzed the reduced-representation bisulfite sequencing experiments. K.S. and T.S. performed and analyzed the whole-genome sequencing experiments. K.J.C., A.R.E., and S.P.G. performed and analyzed the proteomics experiments.

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Correspondence to Konrad Hochedlinger.

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Integrated supplementary information

Supplementary Figure 1 hESCs cultured in m5i/LAF are pluripotent.

(a) Flow cytometric analysis of the proportion of ΔPE OCT4GFP+ WIBR3 hESCs at p8 cultured in 4i/LAF or 4i/LAF medium supplemented with 0.3 μM, 0.5 μM, 0.6 μM or 0.8 μM of PD03. (b) Representative bright field and fluorescence images of naïve WIBR3 hESCs grown in 4i/LAF and supplemented with 0.5 or 0.3 μM of PD03 (scale bar: 100 μm). (c) Flow cytometric analysis showing homogenous expression of the ΔPE OCT4GFP transgene in hESCs cultured in primed medium, 5i/LAF or m5i/LAF conditions for 6 passages. (d) Table listing the different primed hESC lines used in this study and their efficiency of conversion to a naïve state in 5i/LAF or m5i/LAF. (e) Representative bright field images of UCLA1 and UCLA17 hESC converted into naïve cells in 4i/LAF medium supplemented with the indicated PD03 concentration at P8 (scale bar: 100 μm). (f) Representative immunofluorescence images showing protein expression of KLF4, KLF17, NANOG and STELLA in UCLA9 hESCs grown in 4i/LAF supplemented with either 1 or 0.5 μM of PD03 (scale bar: 50 μm, right panels). (g) Section of teratomas obtained from primed or re-primed UCLA-1 hESCs stained with hematoxilin/eosin, showing differentiation into the three germ layers (scale bar: 100 μm).

Supplementary Figure 2 hESCs cultured in t2iLGöY require strong MEK inhibition to maintain naive pluripotency.

(a) Outline of experiment (top panel). Representative flow cytometric analysis for ΔPE OCT4GFP expression using naïve WIBR3 hESCs grown in 5i/LAF (PD03 1 μM) and t2iLGöY (PD03 1 μM) (lower left panel). (b) Quantitative RT-PCR analysis of KLF4, TFCP2L1, REX1, KLF2, KLF17 and DPPA5 expression in 5i/LAF (PD03 1 μM) and t2iLGöY (PD03 1 μM) cultured hESCs (lower right panel). Error bars indicate mean ± s.d., n = 3 biologically independent samples (cell cultures). (c) Representative microscope images of naïve hESCs cultured in t2iLGöY (scale bar: 100 μm). (d) Representative flow cytometry data for H9 (EOS-GFP) cells at 1 and 2 weeks after resetting with KLF2 and NANOG overexpression in t2iLGöY at the indicated concentrations of PD03. (e) Outline of experiment. (f) Representative bright field images of UCLA4 hESCs converted into naïve cells in 4i/LAF medium and supplemented with the indicated MEKi at P3 (scale bar: 100 μm).

Supplementary Figure 3 Alternative MEK inhibitors maintain naive pluripotency in hESCs cultured in t2iLGöY.

(a) Clonal colony formation assay of hESCs cultured in t2iLGöY supplemented with the indicated concentration of MEKi. Shown are representative images of alkaline phosphatase-positive hESC colonies. (b) Quantification of alkaline phosphatase-positive hESC colonies shown in Supplementary Figure 2A. (c) Quantitative RT-PCR analysis of OCT4 and NANOG expression using indicated samples. Error bars indicate mean ± s.d., n = 3 biologically independent samples (cell cultures). (d) Daily quantification of cell numbers during the expansion of naive hESCs in t2iLGöY for 5 passages.

Supplementary Figure 4 Effect of attenuated MEK inhibition on mRNA expression levels.

(a) PCA analysis of RNA-seq data for the indicated hESC samples (P10) using all expressed genes (n = 20435 genes). (b) THY-1 and DPPA5 expression level for samples indicated in the PCA analysis in Supplementary Figure 4a. (c) Quantitative RT-PCR analysis of REX1, KLF2 and DPPA5 expression using UCLA17 hESCs cultured under the indicated conditions. (d) KLF4 RNA expression levels in UCLA4 hESCs cultured in primed, 5i/LAF, m5i/LAF and 4i/LAF-TAK-733 conditions. (e) Reactome Pathway analysis of upregulated genes (fc>2, p<0.05, n = 179) in human naïve ESCs cultured in m5i/LAF conditions relative to 5i/LAF conditions.

Supplementary Figure 5 Effect of attenuated MEK inhibition on protein expression levels.

(a) A heatmap for all proteins detected by proteomics in the indicated samples. (b) A correlation plot comparing mRNA to protein expression in UCLA4 hESCs cultured in the indicated conditions. n = 8286 total points for which the correlation is computed. (c) Correlation plots for protein vs mRNA expression in primed and naïve hESCs lines in 5i/LAF (upper panel) and m5i/LAF (lower panel). n = 5 independent hESC line cultured in 5i/LAF and in m5i/LAF (UCLA1, 4, 5, 9 and WIBR3), n = 8271 total points for which the correlation is computed. (d) Venn diagrams showing the overlap between differentially expressed RNAs and proteins in primed and naïve hESCs. (e) MBNL1 RNA and protein expression levels. Error bars indicate mean ± s.d. (n = 3 independent hESC lines (UCLA4, UCLA5, WIBR3)), statistical significance was determined using a two-tailed unpaired Student’s t-test (P = 0.2587, P = 0.3085; P = 0.0078, P = 0.0159).

Supplementary Figure 6 Effect of attenuated MEK inhibition on DNA methylation levels.

(a) Heatmap for Z-scaled transposable element (TE) expression in primed and naïve samples (P10). (b) Heatmap showing methylation levels at the indicated genomic regions. (c) PCA analysis of methylation data (n = 95239 1kb tiles) for the indicated hESC samples (P10). Each dot represents one hESC line cultured in the indicated condition. (d) PCA analysis of methylation data at promoter regions (n = 18588 promoters) for the indicated hESC samples (P10). Each dot represents one hESC line cultured in the indicated condition. (e) Heatmap showing methylation levels at imprinting control regions (ICRs). (f) Differentially methylated regions between human naïve samples cultured in 5i/LAF and m5i/LAF. (g) Bar plot showing enrichment for SINE and LINE elements in the hypermethylated regions identified in Supplementary Figure 6f.

Supplementary Figure 7 Effect of attenuated MEK inhibition on X chromosome inactivation status.

(a) Representative RNA FISH images for primed and naïve UCLA5 at P16, detecting XIST and nascent transcription foci of UTX and HUWE1 (scale bar: 10 μm). (b) Representative RNA FISH images for primed and naïve UCLA1 at P16, detecting XIST and nascent transcription foci of UTX and HUWE1 (scale bar: 10 μm). Note that UCLA1 hESCs are often monoallelic for UTX. (c) Table summarizing the HUWE1 status in the different primed and naïve hESC lines analyzed.

Supplementary Figure 8 Effect of MEK inhibition on chromosome stability.

(a) Clonal colony formation assay of WIBR3 hESCs cultured in 5i/LAF or m5i/LAF. Shown are representative images (left) and quantification (right) of alkaline phosphatase-positive hESC colonies. Error bars indicate mean ± s.d. (n = 3 biologically independent samples). Statistical significance was determined using a two-tailed unpaired Student’s t-test (P = 0.0001). (b) Representative flow cytometric analysis of Annexin V expression in naïve WIBR3 hESCs cultured in 5i/LAF or m5i/LAF at p16. (c) Clonal colony formation assay of hESCs cultured in t2iLGöY (-Dox) supplemented with the indicated concentration of PD03. Shown are quantification (left) and representative images (right) of alkaline phosphatase-positive hESC colonies. Error bars indicate mean ± s.d. (n = 3 biologically independent samples (cell cultures)). Statistical significance was determined using a two-tailed unpaired Student’s t-test (P = 0.0034). (d) Karyotype analysis of human WIBR3 ESCs cultured in primed medium. (e) Karyotype analysis of human WIBR3 ESCs cultured in naive medium. (f) Summary of karyotyping results from primed and naïve WIBR3 hESC lines (n = 20 cells per sample). (g) Chromosomal copy number analysis by whole-genome sequencing. Black arrows indicate trisomy and deletions.

Supplementary Figure 9 Effect of attenuated MEK inhibition on protein phosphorylation levels.

(a) Western blot analysis showing pERK, ERK and ACTIN protein levels in UCLA4 naïve cells cultured with PD03 at 1 and 0.5 μM. Full scans of the western blots are in Supplementary Figure 10. (b) Phosphorylation levels of ERK2 assessed by phosphoproteomics in UCLA4 naïve cells cultured with PD03 at 1 and 0.5 μM, TAK-733 and t2iLGöY. (c) Venn diagram showing overlap for hyperphosphorylated proteins (>2-fold) in UCLA4 and UCLA9 hESCs cultured in m5i/LAF vs 5i/LAF. (d) Gene ontology analysis of hyperphosphorylated proteins from overlap (n = 164) in Supplementary Figure 9c. (e) Gene ontology analysis of hyperphosphorylated proteins (>2-fold, n = 115) in UCLA4 naïve cells cultured in t2iLGöY vs 5i/LAF. (f) Graphical summary of results.

Supplementary Figure 10

Uncut gels for Supplementary Fig. 9a.

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Supplementary Figures 1–10

Reporting Summary

Supplementary Protocol

Reduced MEK inhibition preserves genomic stability in naive human ESCs

Supplementary Table 1

Proteins detected in primed and naive hESCs by mass spectrometry

Supplementary Table 2

Phosphoproteomic analysis of primed and naive hESCs

Supplementary Table 3

qRT-PCR primers

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Di Stefano, B., Ueda, M., Sabri, S. et al. Reduced MEK inhibition preserves genomic stability in naive human embryonic stem cells. Nat Methods 15, 732–740 (2018). https://doi.org/10.1038/s41592-018-0104-1

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