Supplementary Materialsijms-20-05428-s001. indicating that the steady genomic imprinting state in somatic cells Igfbp4 could be changed after pluripotential reprogramming. Reprogramming somatic cells into pluripotent stem cells is a useful strategy for understanding epigenetic changes and the nature of pluripotency [23]. Previously, Troxacitabine (SGX-145) we generated parthenogenetic induced pluripotent stem cells (piPSCs) by reprogramming parthenogenetic neural stem cells (pNSCs). The piPSCs displayed typical na?ve pluripotency, including the ability to form a germ line chimera [24], and exhibited different imprinting patterns from those of pNSCs, which display parthenogenetic imprinting patterns, characterized by completely unmethylated paternally imprinted genes (and and and were expressed at lower levels in EpiSCs and pEpiSCs than in ESCs and pESCs (Figure 2c), whereas primed pluripotency genes, such as and (may be expressed at higher levels in parthenogenetic cells. However, we cannot explain why. Open in a separate window Figure 2 Pluripotency and differentiation potential of pEpiSCs, EpiSCs, parthenogenetic embryonic stem cells (pESCs), and ESCs. (a,b) Immunocytochemistry using anti-Oct4 and anti-Nanog antibodies in pEpiSCs, EpiSCs, pESCs, and ESCs. All parthenogenetic and biparental pluripotent stem cell types expressed the core pluripotency markers Oct4 (a) and Nanog (b). Nuclei were stained with DAPI (blue). Scale bars represent 200 m. (c) Real-time RT-PCR analysis of pEpiSCs, EpiSCs, pESCs, and ESCs for the expression of na?ve and primed pluripotency-related genes. Data are presented as the mean SEM for = 3 independent experiments. *** and and and are adjacent and have a common differentially methylated region (DMR) whose DNA methylation patterns regulate their expression. Although both and are paternally methylated, is only actively expressed from the paternal allele, whereas is only actively expressed from the maternal allele; thus, is a paternally-imprinted paternally-expressed gene and is a paternally-imprinted maternally-expressed gene. Conversely, and are maternally-imprinted paternally-expressed genes. First, we Troxacitabine (SGX-145) investigated the expression of the paternally (and and and were expressed at higher levels in pEpiSCs than in EpiSCs (22.11- and 2.03-fold, respectively), whereas maternally imprinted genes and were expressed at higher levels in EpiSCs than in pEpiSCs (2.52- and 93.01-fold, respectively). Open in a separate window Figure 3 Expression and DNA methylation status of imprinted genes in parthenogenetic and biparental pluripotent stem cells. Analyses of imprinted gene expression and bisulfite genome sequencing. (a) Real-time RT-PCR analysis of pEpiSCs, EpiSCs, pESCs, and ESCs for paternally (and Troxacitabine (SGX-145) and and were expressed at higher levels in pEpiSCs than in EpiSCs (~22.11- and 2.03-fold, respectively), whereas and = 3 independent experiments. *** and in pEpiSCs (~57.14%) was much lower than that in EpiSCs (~84.28%), whereas similar DNA methylation levels were observed in (76% vs. 82%, respectively). Black and white circles represent methylated and unmethylated CpGs, respectively. (c) Bisulfite DNA sequencing analysis of paternally imprinted genes (and and differentially methylated regions (DMRs) were almost completely methylated in pEpiSCs; however, the EpiSCs displayed a differentially methylated pattern (composed of completely methylated and completely unmethylated alleles) typical of imprinted genes in somatic cells. Black and white circles represent methylated and unmethylated CpGs, respectively. We also investigated the DNA methylation status of DMRs in the imprinted genes of EpiSCs and pEpiSCs (Figure 3b,c). As reported previously [27], the DMR methylation status of did not exactly correspond with its expression levels (Figure 3a,b); however, the DNA methylation levels of were considerably lower in the pEpiSCs (~42.85%) than in the EpiSCs (~84.28%), corresponding with a 22.11-fold difference in expression levels. Clear differences were also observed in the expression levels of and and were completely unmethylated, whereas those of and were completely methylated, which is the typical pattern Troxacitabine (SGX-145) observed in parthenogenetic imprinted genes (Figure S1). pEpiSCs displayed distinctly different DNA methylation patterns in the DMRs of and and showing a lesser change in the DNA methylation levels (Figure S1). The pEpiSC imprinting patterns should not have changed from the typical parthenogenetic patterns of pNSCs if the primed pluripotent state did not change the imprinted genes. Thus, these results suggest that both na?ve and primed pluripotent stem cells have an unstable imprinting status and display a tendency to lose typical DNA methylation patterns in imprinted genes..