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.

A practical guide to induced pluripotent stem cell research using patient samples

“…11,12 One must use caution when analyzing epigenetic aberrations in imprinting disease iPSCs because the process of iPSC generation is associated with epigenetic dynamics that may bias interpretation. 13 However, iPSCs with in vitro multipotency have been an invaluable tool to clarify molecular mechanisms as a simulator of developmental defects. 14,15 PRACTICAL ADVICE BEFORE YOU BEGIN These exemplary cases certainly make us feel hopeful that we can apply patient iPSCs to various diseases.…”

“…In addition to these standard quality controls, profiling of RNA expression, DNA methylation and glycans can be added for monitoring when necessary. 10,13,36,37 These comprehensive analyses would also elucidate pathogenic states such as aberrant genomic methylation and gene expression of patient iPSCs.…”

“…To circumvent this, commonly available iPSCs from healthy donors may be used for comparison. MRC5-derived (fetal lung fibroblast) iPSCs have been utilized as a control in several previous reports, 13,[37][38][39][40][41] and can be obtained from the public bank. If MRC5-iPSCs do not demonstrate pathogenic phenotypes that disease iPSCs do under the same experimental condition, MRC5-iPSCs would serve as a practical control.…”

Promise and challenges of human iPSC-based hematologic disease modeling and treatment

“…A study using human iPSCs derived from pancreatic islet beta cells has shown that these iPSCs can be distinguished by their epigenetic profiles from other pluripotent stem cells and that they had increased ability to differentiate into insulinproducing cells [46]. On the other hand, studies from both mouse and human iPSCs have suggested that the epigenetic memory is likely to be observed at early passages of iPSCs and can be erased through continuous passaging [45,47]. Functional studies have also shown that with improved differentiation conditions, iPSCs of diverse origins can be differentiated into functional cell types such as neural cells and hepatic cells at comparable efficiencies even though they can be distinguished by epigenetic profiles [48,49].…”

Induced Pluripotent Stem Cell Technology and Direct Conversion: New Possibilities to Study and Treat Parkinson’s Disease

“…This approach enabled us to perform an extensive comparison of iPSC-derived DA neurons with primary mDA neurons (including varying developmental stages) as far as their genetic and epigenetic profiles are concerned. We particularly focused on DNA methylation, since reprogramming of somatic cells towards pluripotent cells and subsequent differentiation into DA neurons must entail massive changes in DNA methylation patterns in specific genomic loci [47][48][49][50]. This type of characterization, which was based on the ability to analyze purified Pitx3-GFP DA neurons, enabled us to determine a close similarity in terms of DNA methylation patterns between iPSC-derived DA neurons and primary DA neurons and provides novel insight in cell type specific de novo methylation during in vitro differentiation (Roessler et al, manuscript in preparation).…”