Defining Hypo-Methylated Regions of Stem Cell-Specific Promoters in Human iPS Cells Derived from Extra-Embryonic Amnions and Lung Fibroblasts

Abstract

BackgroundHuman induced pluripotent stem (iPS) cells are currently used as powerful resources in regenerative medicine. During very early developmental stages, DNA methylation decreases to an overall low level at the blastocyst stage, from which embryonic stem cells are derived.Therefore, pluripotent stem cells, such as ES and iPS cells, are considered to have hypo-methylated status compared to differentiated cells. However, epigenetic mechanisms of “stemness” remain unknown in iPS cells derived from extra-embryonic and embryonic cells.Methodology/Principal FindingsWe examined genome-wide DNA methylation (24,949 CpG sites covering 1,3862 genes, mostly selected from promoter regions) with six human iPS cell lines derived from human amniotic cells and fetal lung fibroblasts as well as two human ES cell lines, and eight human differentiated cell lines using Illumina's Infinium HumanMethylation27. A considerable fraction (807 sites) exhibited a distinct difference in the methylation level between the iPS/ES cells and differentiated cells, with 87.6% hyper-methylation seen in iPS/ES cells. However, a limited fraction of CpG sites with hypo-methylation was found in promoters of genes encoding transcription factors. Thus, a group of genes becomes active through a decrease of methylation in their promoters. Twenty-three genes including SOX15, SALL4, TDGF1, PPP1R16B and SOX10 as well as POU5F1 were defined as genes with hypo-methylated SS-DMR (Stem cell-Specific Differentially Methylated Region) and highly expression in iPS/ES cells.Conclusions/SignificanceWe show that DNA methylation profile of human amniotic iPS cells as well as fibroblast iPS cells, and defined the SS-DMRs. Knowledge of epigenetic information across iPS cells derived from different cell types can be used as a signature for “stemness” and may allow us to screen for optimum iPS/ES cells and to validate and monitor iPS/ES cell derivatives for human therapeutic applications.

Glycome Diagnosis of Human Induced Pluripotent Stem Cells Using Lectin Microarray

“…Cells-Endometrium (UtE) (29), placental artery endothelium (PAE) (30), and amnion (AM) (31,32) were independently established. UtE, AM, and MRC5 cells were maintained in the POWEREDBY10 medium (MED SHIROTORI CO., Ltd.).…”

“…Immunocytochemistry-Immunocytochemical analysis was performed as described previously (29,33,38). Human iPSCs were fixed with 4% paraformaldehyde in PBS for 10 min at 4°C.…”

Noncanonical NOTCH Signaling Limits Self-Renewal of Human Epithelial and Induced Pluripotent Stem Cells through ROCK Activation

“…All experiments involving human cells and tissues were performed in line with Tenets of the Declaration of Helsinki. Human iPS cell lines, MRC-hiPSCs and UtE-hiPSCs, were established from MRC-5 fetal lung fibroblasts (43) and UtE1104 endometrium-derived cells (44), respectively, via procedures described by Takahashi et al (45) with slight modification (46,47). Human iPS cells were maintained in iPSellon medium (Cardio Incorporated, Osaka, Japan) supplemented with 10 ng/ml recombinant human basic fibroblast growth factor (bFGF) (Wako Pure Chemical Industries, Ltd., Osaka, Japan) in the presence of irradiated mouse embryonic fibroblast (MEF) feeders.…”

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

“…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.…”