Genetic and Epigenetic Profiling Reveals EZH2-mediated Down Regulation of OCT-4 Involves NR2F2 during Cardiac Differentiation of Human Embryonic Stem Cells

Human embryonic (hES) stem cells are widely used as an in vitro model to understand global genetic and epigenetic changes that occur during early embryonic development. In-house derived hES cells (KIND1) were subjected to directed differentiation into cardiovascular progenitors (D12) and beating cardiomyocytes (D20). Transcriptome profiling of undifferentiated (D0) and differentiated (D12 and 20) cells was undertaken by microarray analysis. ChIP and sequential ChIP were employed to study role of transcription factor NR2F2 during hES cells differentiation. Microarray profiling showed that an alteration of about 1400 and 1900 transcripts occurred on D12 and D20 respectively compared to D0 whereas only 19 genes were altered between D12 and D20. This was found associated with corresponding expression pattern of chromatin remodelers, histone modifiers, miRNAs and lncRNAs marking the formation of progenitors and cardiomyocytes on D12 and D20 respectively. ChIP sequencing and sequential ChIP revealed the binding of NR2F2 with polycomb group member EZH2 and pluripotent factor OCT4 indicating its crucial involvement in cardiac differentiation. The study provides a detailed insight into genetic and epigenetic changes associated with hES cells differentiation into cardiac cells and a role for NR2F2 is deciphered for the first time to down-regulate OCT-4 via EZH2 during cardiac differentiation.


Supplementary table 1: Studies reporting the differentiation of ES cells into cardiac lineage and their transplantation DIFFERENTIATION STUDIES Method Differentiation technique and conditions Efficiency Reference
Embryoid Body (EB) formation hES cells placed in suspension cultures for 7-10 days to form EBs and replated on gelatine coated plates for 2 weeks 1% beating areas Kehat 12 Yang et al 6 Monolayered hES cells exposed to growth factors like Activin A (24 hrs.), BMP4 (4 days) and DKK1 (4 days) in RPMI-B27 medium; cultured for 20 days 60-80% beating areas on day 20 Pawani et al 7 Manipultion of WNT pathway ES or iPS cells subjected to WNT/beta-catenin signalling stimulation with GSK3 inhibitor followed by its suppression using specific inhibitors along with growth factors ike BMP4

Maintenance of human embryonic stem cells
In-house derived human ES cells were grown on Geltrex (Invitrogen, Carlsbad, CA, USA) coated plates in Stempro hESC SFM medium (Invitrogen, Carlsbad, CA, USA) supplemented with basic fibroblast growth factor (bFGF) (R & D Systems, MN, USA) at a concentration of 8ng/ml in a humidified atmosphere of 37 0 C with 5% CO 2 . Cells were subcultured every 5-7 days by detaching them from the geltrex-coated plates mechanically using cell lifter (Sigma Aldrich, MO, USA). Prior to directed differentiation into cardiac lineage, undifferentiated hES cells were analyzed at both transcript and protein levels for the expression of pluripotent markers by qPCR, immunofluorescence and flow cytometry (Spplementary figures [3][4][5].

Directed Differentiation
Undifferentiated human ES cells were subjected to a series of defined growth factors to direct the differentiation towards cardiac lineage by the protocol as reported previously by our group (Pawani et  specificity and homogeneity of the amplified product was confirmed by performing melt curve analysis at the end of each amplification cycle and also by electrophoresing the products on 2% agarose gel (Bangalore Genei). Human fetal and adult whole heart RNA were used to compare the maturity of the differentiated cells. The fold change was determined for each sample of different time points using the 2 -ΔΔCt method and was expressed relative to that of GAPDH which was used as an internal control. The expression level of each gene transcript is normalized to a value of 1.0 for undifferentiated cells. The error bars represent ± standard error of the mean (SEM). All the above results are an average of at least 3 biological replicates (Supplementary figure 3). Primers used are mentioned in Table 1.

Immunofluorescence
Undifferentiated human ES cells were subjected to immunostaining using specific primary antibodies to pluripotent markers. Briefly undifferentiated human ES cells were fixed with 4% paraformaldehyde (PFA) for 15 mins at room temperature followed by washing with 1X PBS plus 0.02% Tween 20 (Sigma Aldrich). Permeabilization for nuclear antigens was done with 0.3% triton X-100 (Sigma-Aldrich). Non-specific sites were blocked by blocking buffer containing 1X PBS plus 5% BSA (Sigma Aldrich) and 1% normal goat serum (Bangalore Genei, Bangalore, India) for 60mins at room

Microarray Data Validation
Immunofluorescence Microarray data was also validated at protein level by studying the immuno-expression of NKX2.5 and CTNT by confocal microscopy.
Immuno-staining was performed as described above using the primary antibodies specific for markers of hES derived cardiac progenitors and cardiomyocytes; rabbit anti-human NKX2. 5