DNA Methylation Dynamics in Human Induced Pluripotent Stem Cells over Time

Abstract

Epigenetic reprogramming is a critical event in the generation of induced pluripotent stem cells (iPSCs). Here, we determined the DNA methylation profiles of 22 human iPSC lines derived from five different cell types (human endometrium, placental artery endothelium, amnion, fetal lung fibroblast, and menstrual blood cell) and five human embryonic stem cell (ESC) lines, and we followed the aberrant methylation sites in iPSCs for up to 42 weeks. The iPSCs exhibited distinct epigenetic differences from ESCs, which were caused by aberrant methylation at early passages. Multiple appearances and then disappearances of random aberrant methylation were detected throughout iPSC reprogramming. Continuous passaging of the iPSCs diminished the differences between iPSCs and ESCs, implying that iPSCs lose the characteristics inherited from the parent cells and adapt to very closely resemble ESCs over time. Human iPSCs were gradually reprogrammed through the “convergence” of aberrant hyper-methylation events that continuously appeared in a de novo manner. This iPS reprogramming consisted of stochastic de novo methylation and selection/fixation of methylation in an environment suitable for ESCs. Taken together, random methylation and convergence are driving forces for long-term reprogramming of iPSCs to ESCs. iPSCs change to resemble ESCs via two phases: the transgene-dependent phase, in which the transcription factors act to transform somatic cells into pluripotent stem cells, and the transgene-independent phase, in which the transcription factors are silenced. In this study, we established human iPSCs derived from 5 different cell types by retroviral infection of the Yamanaka 4 factors, and we identified 8 novel epigenetic markers (SALL4, EPHA1, PTPN6, RAB25, GBP4, LYST, SP100, and UBE1L) by comprehensive DNA methylation analysis. The aberrant hyper-methylation in iPSCs occurred stochastically throughout the genome and decreased during the long-term iPSC reprogramming, suggesting that the aberrant stochastic hyper-methylation and their convergence are a direct cause of the transgene-independent phase of iPS reprogramming. These results favor the stochastic model of the Yamanaka model rather than the elite model. In addition, the stem cell–specific methylation states and the epigenetic difference between iPSCs and ESCs are useful indices for evaluating human iPSCs in therapeutic applications.