The Aberrant DNA Methylation Profile of Human Induced Pluripotent Stem Cells Is Connected to the Reprogramming Process and Is Normalized During In Vitro Culture.

The potential clinical applications of human induced pluripotent stem cells (hiPSCs) are limited by genetic and epigenetic variations among hiPSC lines and the question of their equivalency with human embryonic stem cells (hESCs). We used MethylScreen technology to determine the DNA methylation profile of pluripotency and differentiation markers in hiPSC lines from different source cell types compared to hESCs and hiPSC source cells. After derivation, hiPSC lines compromised a heterogeneous population characterized by variable levels of aberrant DNA methylation. These aberrations were induced during somatic cell reprogramming and their levels were associated with the type of hiPSC source cells. hiPSC population heterogeneity was reduced during prolonged culture and hiPSCs acquired an hESC-like methylation profile. In contrast, the expression of differentiation marker genes in hiPSC lines remained distinguishable from that in hESCs. Taken together, in vitro culture facilitates hiPSC acquisition of hESC epigenetic characteristics. However, differences remain between both pluripotent stem cell types, which must be considered before their use in downstream applications

MethylScreen technology principles and data interpretation.

<p>(A) Representative qPCR standard curve for PAX6 obtained from 100, 50, 10, 1 and 0.1 ng of control DNA per reaction. (B₁) <i>UTF1</i> MethylScreen qPCR results for CCTL-12, IPSCF, CBIA-11, A549 and KG-1 genomes. Changes in c_t between enzyme treated and non-treated templates are depicted: Rs-R0 is represented by white columns (HhaI reaction), Rd-R0 is represented by grey columns (McrBC reaction), and Rsd-R0 is represented by black columns (both HhaI and McrBC reaction). (B₂) <i>UTF1</i> DNA methylation profile for five cell lines. c_t values from four restriction reactions (B₁) were converted to DNA methylation occupancy, expressed as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. (C) MethylScreen results obtained from sample mixtures with an increasing ratio of hypermethylated DNA (0–100%, x-axis). The colour legend is identical for (B₂), and the values are averages for <i>PAX6</i> and <i>TSPYL5</i> genes after serial mixing of CCTL-14 (unmethylated) and A549 (hypermethylated) samples and NDFs (unmethylated) and CCTL-14 (hypermethylated) samples, respectively.</p

DNA methylation profile of selected genes in hiPSCs source cells.

<p>DNA methylation occupancy is reported as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. The profile is shown for NDFs (A), ADFs (B), and PBMC CD34⁺ cells (C).</p

Gene expression levels in hPSCs and dermal fibroblasts, as determined by quantitative real-time PCR.

<p>Expression levels of eleven genes were compared i) among the hiPSC lines, hESC lines and dermal fibroblasts (NDFs and ADFs together) (A-C) and ii) within hiPSC lines between low and high passage numbers (D-F). Analysed hiPSC lines and the passage numbers are reported in the graph legend. The values are relative to the values from hESCs (set at 1). Values are the mean + SD, and asterisks indicate the significance between indicated samples: *p < 0.05; **p < 0.005; ***p < 0.0005.</p

hPSC lines used in this study.

<p>hPSC lines used in this study.</p

Hierarchical clustering of hPSC lines and dermal fibroblasts based on quantitative real-time PCR data.

<p>The colour-coded scale on the left of the picture indicates the expression value for each gene; low expression is represented by green, and high expression is represented by red. The values are relative to the values from CCTL-14 (set at 1).</p

DNA methylation profile of selected genes in hPSC lines.

<p>DNA methylation occupancy is reported as the percentage of unmethylated (UM), intermediately methylated (IM) and hypermethylated (HM) DNA. The profile is shown for hESC lines (A), hiPSC lines from NDFs (B), hiPSC lines from ADFs (C, D) and hiPSC lines from PBMC CD34⁺ cells (E, F) generated by STEMCCA lentivirus (STE), Sendai virus (SEN) and the episomal vector (EPI) reprogramming. Values are the averages from hPSC lines within the line group where appropriate.</p

PCR primers used for MethylScreen reactions.

<p>PCR primers used for MethylScreen reactions.</p

Additional file 2: Figure S2. of DNA double-strand breaks in human induced pluripotent stem cell reprogramming and long-term in vitro culturing

Histological staining of a teratoma. Cell types representative of the three germ layers were detected by histological analysis in the CBIA-7 hiPSC cell line at passage number 26. (A) Glandular structures with secretory cells (endoderm); (B) mesenchymal cells (mesoderm); (C) cells with melanin (ectoderm); (D) glomerulus-like cells (mesoderm). (PPTX 32103 kb