DNA methylation levels of selected genes in human pluripotent stem cell lines intended for hematopoietic differentiation.
|Fakulta / Pracoviště MU|
|Popis||Generation of hematopoietic stem cells from human pluripotent stem cells (hPSCs) promise many applications in regenerative medicine. Nevertheless, the limitation is variable differentiation efficiency of hPSCs reasoned by abberant molecular signature and epigenetic status of developing hPSCs. We evaluated DNA methylation status of selected genes in several hPSC lines to determine if this DNA modification is associated with the variabilty of hPSC lines and with their hematopoietic differentation potential. Using MethylScreen technology, promotor DNA methylation was analysed in human embryonic stem cell (hESC) lines (CCTL-12, CCTL-14) and human induced pluripotent stem cell (hiPSC) lines derived from human fibroblasts (STENF, IPSCF, AM13). The gene panel included markers for pluripotent cells (UTF1, SOX2), embryonic germ layers (Brachyury T, FOXA2, PAX6) and blood progenitors (SOX17, RUNX1, GATA2, CD34) and genes with described DNA methylation variability in hPSCs (TCERG1L, TSPYL5). In hESC lines all analysed genes were found to be unmethylated with the exception of fully methylated TSPYL5. For hiPSC lines, STENF and IPSCF showed decreased TSPYL5 methylation level and IPSCF was further characterised by intermediate methylation of UTF1. The highest level of DNA methylation was found in AM13, in addition to full methylation of TSPYL5, up to 20 percent of hypermethylation was detected for other five genes. In conclusion, it was demonstrated that there are differences in promotor DNA methylation of selected genes between hiPSC and hESC lines. Differences were detected both in residual DNA methylation of somatic genes and in DNA methylation level of UTF1 and TSPYL5, which suggests these two genes as putative markers linked to the pluripotency state. The association of DNA methylation status in hPSC lines with their ability to differentiate into blood progenitors will be further studied.|