Fig. 2

Culture and identification of purified undifferentiated putative porcine mGSCs. a The putative mGSCs were grown on mouse embryonic fibroblast feeder cells, and images were taken on days 7, 14, and 21 of culture. Two distinct stem cell-like clusters, termed as C1 and C2, appeared at day 14 of culture and became more obvious at day 21 of culture. b A graph shows the fluctuations in total cell numbers in the mGSC culture, revealing an increase in cell number toward the end of the culture period. c Clusters of C1 and C2 were separated and collected for AP activity analysis and compared with porcine-induced pluripotent stem cells (piPSCs) as a positive control. d Images of immunofluorescence analysis show expression of OCT4, NANOG, VASA, and PGP9.5 in the C1 and C2; nuclei were stained by Hoechst 33,342 (blue). e RT-PCR was performed to analyze the expression of pluripotent and germline genes in the C1 and C2. f A schematic indicates the differentially methylated regions (DMRs) of Oct4, Nanog, and H19 used for analysis in the C1 and C2. g A diagram shows the bisulfite sequencing results for each gene. Each line represents an individual clone sequencing result, and each circle represents one CpG site. The solid circles indicate a methylated CpG, whereas an empty circle indicates an unmethylated CpG. h Quantitative PCR was performed to show the relative mRNA expression levels of reprogramming factors (Oct4, Sox2, Nanog, Lin28, c-Myc, Klf4, and Tert) between the C1 and piPSCs (n = 3). The data are expressed as the mean ± SEM. *P < 0.05