Reversible dual inhibitor against G9a and DNMT1 improves human iPSC derivation enhancing MET and facilitating transcription factor engagement to the genome

<div><p>The combination of defined factors with small molecules targeting epigenetic factors is a strategy that has been shown to enhance optimal derivation of iPSCs and could be used for disease modelling, high throughput screenings and/or regenerative medicine applications. In this study, we showed that a new first-in-class reversible dual G9a/DNMT1 inhibitor compound (CM272) improves the efficiency of human cell reprogramming and iPSC generation from primary cells of healthy donors and patient samples, using both integrative and non-integrative methods. Moreover, CM272 facilitates the generation of human iPSC with only two factors allowing the removal of the most potent oncogenic factor cMYC. Furthermore, we demonstrated that mechanistically, treatment with CM272 induces heterochromatin relaxation, facilitates the engagement of OCT4 and SOX2 transcription factors to OSKM refractory binding regions that are required for iPSC establishment, and enhances mesenchymal to epithelial transition during the early phase of cell reprogramming. Thus, the use of this new G9a/DNMT reversible dual inhibitor compound may represent an interesting alternative for improving cell reprogramming and human iPSC derivation for many different applications while providing interesting insights into reprogramming mechanisms.</p></div

Analysis of H3K9me2 and H3K4me3 levels after CM272 treatment.

<p>ChIP-qPCR at different promoter regions of pluripotency and MET associated genes. (A and B) H3K9me2 ChIP-qPCR analysis in CM272-treated cells before OSKM induction. (C and D) H3K4me3 ChIP-qPCR analysis in CM272-treated cells 5 days after OSKM induction. Means and SD of three independent experiments are represented. ND: not-determined. Two-way ANOVA with Bonferroni post-tests to compare replicate means was used. * p<0.05, ** p<0.01, *** p<0.001, ns: not significative.</p

Reversible dual G9a/DNMT inhibitor activity in BJ cells.

<p>(A) Proliferation assay of OSKM-infected BJ cells measured by MTS after 48h incubation with increasing concentrations of CM272 (left) or CM579 (right). GI₅₀ is denoted. (B) Cell count at different times after treatment with CM272 (left) or CM579 (right) at the indicated doses. Error bars denote SD of three independent experiments. Two-way ANOVA with Bonferroni post-tests to compare replicate means was used. (C) Analysis and quantification of H3K9me2 levels 48h after treatment of BJ cells with different doses of CM272 and BIX-01294. (D) Time course analysis and quantification of H3K9me2 levels after CM272 treatment. (E) H3K9me2 levels 24h and 48h after CM272 withdrawal. WB images are representative of at least three independent experiments. Kruskal-Wallis with Dunn’s test as a post-hoc was used. * p<0.05, ** p<0.01, *** p<0.001.</p

iPSCs resemble pluripotent features.

<p>(A) Representative images of the characteristic morphology of pluripotent cells, AP⁺ staining and immunofluorescence of indicated pluripotency markers in a stablished iPSC cell line generated in the presence of CM272. (B) Expression levels measured by qPCR of indicated pluripotency-associated genes in a stablished iPSC cell line generated in the presence of CM272. Error bars denote SD of three independent measurements. (C) Bisulfite sequencing of NANOG and POU5F1 (OCT4) promoter regions in a stablished iPSC cell line generated in the presence of CM272. BJ cells and methylated DNA were used as controls. (D) Hematoxylin and eosin staining shows a teratoma from an iPSC cell line generated in the presence of CM272 containing multiple tissues, including tissues from the three germ layers (ectoderm, endoderm and mesoderm). Results for other iPSC lines are summarized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190275#pone.0190275.s006" target="_blank">S3 Table</a>. (E) Heatmap showing principal pluripotency and lineage specific markers differentially expressed in iPSC after trilineage differentiation.</p

Analysis of transcription factor binding at specific promoter regions after CM272 treatment.

<p>(A) POU5F1 and (B) SOX2 binding at promoter regions of genes located at refractory OSKM-binding regions (NANOG, DPPA4, GDF3 and ZFP42) and genes associated to early reprogramming (LIN28A, CDH2, SNAI2 and SOX2) analyzed by ChIP-qPCR in CM272-treated cells 48h after OSKM induction. OSKM-infected BJ cells without CM272 treatment (mock) were used as controls. Means and SD of three independent experiments are represented. Two-way ANOVA with Bonferroni post-tests to compare replicate means was used. * p<0.05, ** p<0.01, *** p<0.001, ns: not significative.</p

Reversible dual G9a/DNMT inhibitor activity increases reprogramming efficiency.

<p>(A) Schematic representation of the reprogramming process. (B) Quantification of AP⁺ colonies at day 30 of cell reprogramming in cells treated with CM272 at the indicated times related to doxycycline induction. Mock indicates no CM272 treatment. Error bars denote SD of three independent experiments. (C, D and E) Quantification of AP⁺ colonies at day 30 of cell reprogramming in cells treated with BIX-01294 or CM272 at the indicated doses (C), in primary cells treated with CM272 (D), and in BJ cells infected with the indicated mixtures of TFs (E). Fold increase over mock is indicated. Mock indicates no CM272 treatment. Circles ADSCs; Squares fibroblasts; Triangles PH1-fibroblasts. Kruskal-Wallis with Dunn’s test as a post-hoc was used. * p<0.05, ** p<0.01, *** p<0.001, ns: not significative.</p

CM272 treatment does not affect specifically the reprogramming program.

<p>(A) Gene expression analysis of type I IFN response (OAS1, MX1 and MX2) and cell cycle (CCNA2, CCNB2 and CDK1) related genes after CM272 treatment. (B, C and D) Analysis of expression by qPCR of pluripotency associated genes (B), MET/EMT associated genes (C), and lineage related genes (D) after CM272 treatment. Means and SD of three independent experiments are represented. Two-way ANOVA with Bonferroni post-tests to compare replicate means was used. *** p<0.001. Not significative differences were observed in pluripotency, MET/EMT or lineage related genes.</p

Kinetics of gene expression after OSKM induction in CM272 treated BJ cells.

<p>(A and B) Gene expression analysis of MET, EMT and pluripotency associated genes in CM272 treated cells at different time points after OSKM induction. OSKM-infected BJ cells without CM272 treatment (mock) were used as controls. (C) Endogenous SOX2 expression at the indicated times after doxycycline induction in OSKM-infected BJ cells treated with CM272. OSKM-infected BJ cells without CM272 treatment (mock) were used as controls. Means and SD of three independent experiments are represented. Two-way ANOVA with Bonferroni post-tests to compare replicate means was used. * p<0.05, ** p<0.01, *** p<0.001, ns: not significative.</p