Fig. 5: Epigenetic characterization of naïve and primed hPSCs.

a Expression of epigenetic regulators in naïve and primed hPSCs. Depiction of DNMT3L, MAGEB2, PRDM14, DNMT1, TDG, and EP300 epigenetic regulator transcript levels for H1 and H9 hESC lines analyzed by RT-qPCR. The aliquots of statically cultured, cells for each naïve (P4) and primed hPSC sample were collected for gene expression analysis before inoculating them into stirred suspension bioreactors. Those cells were counted as “NStat” and “PStat”. The aggregates of day 4 post-inoculation were collected for each naïve and primed hPSC sample for gene expression analysis. Expression was quantified relative to the housekeeping gene GAPDH and was normalized to statically cultured, primed hPSC (PStat) level (=1). All the cultures underwent fed-batch condition (60% media change, 48-h post-inoculation) for bioreactor cultures. The data presented are generated from inoculation density of 50,000 cells/mL. The data are represented as mean ± SEM (n = 3). **** = Adj-P < 0.0001, *** = Adj-P = 0.0002 and 0.0004, ** = Adj-P = 0.0021, * = Adj-P = 0.0291 using GraphPad Prism. Adj-P = adjusted p value. RQ = relative quantification. PStat = primed hPSCs cultured under static condition. PBior = primed hPSCs cultured under stirred suspension bioreactor condition. NStat = naïve hPSCs cultured under static condition. NBior = naïve hPSCs cultured under stirred suspension bioreactor condition. b Intensity and distribution of H3K27me3 foci in the nuclei of bioreactor-cultured, naïve and primed H9 hPSC aggregates. Representative confocal images were obtained after whole-mount immunofluorescence for H3K27me3 on day 4 post-inoculated naïve and primed hPSC aggregates. Alexa Fluor 488 was used as secondary antibody. Scale bars = 10 µm. Intensity and distribution of H3K27me3 foci were analyzed by Image J using Plot Profile analysis. Enlarged insets from selected nuclei per naïve and primed hPSC aggregates are shown on the right side. A symmetric midline was applied in naïve and primed hPSC aggregate’s nuclei (within the enlarged images) using Plot Profile analysis in Image J to measure H3K27me3 intensity and distribution foci along the indicated midline. 279 foci for naïve hPSC nuclei and 281 foci for primed hPSC nuclei were analyzed along the midline. The H3K27me3 distribution graphs show the intensities of foci along the midline within the enlarged images. The x-axis represents the position of analyzed foci along the midline and the y-axis reflects the intensity value of the analyzed foci along the midline. The indicated numbers (%) within the black boxes show the percentage of dots that are located above the indicated arbitrary red line (the percentage of dots located above 40).