Strong Components of Epigenetic Memory in Cultured Human Fibroblasts Related to Site of Origin and Donor Age.

Differentiating pluripotent cells from fibroblast progenitors is a potentially transformative tool in personalized medicine. We previously identified relatively greater success culturing dura-derived fibroblasts than scalp-derived fibroblasts from postmortem tissue. We hypothesized that these differences in culture success were related to epigenetic differences between the cultured fibroblasts by sampling location, and therefore generated genome-wide DNA methylation and transcriptome data on 11 intrinsically matched pairs of dural and scalp fibroblasts from donors across the lifespan (infant to 85 years). While these cultured fibroblasts were several generations removed from the primary tissue and morphologically indistinguishable, we found widespread epigenetic differences by sampling location at the single CpG (N = 101,989), region (N = 697), "block" (N = 243), and global spatial scales suggesting a strong epigenetic memory of original fibroblast location. Furthermore, many of these epigenetic differences manifested in the transcriptome, particularly at the region-level. We further identified 7,265 CpGs and 11 regions showing significant epigenetic memory related to the age of the donor, as well as an overall increased epigenetic variability, preferentially in scalp-derived fibroblasts-83% of loci were more variable in scalp, hypothesized to result from cumulative exposure to environmental stimuli in the primary tissue. By integrating publicly available DNA methylation datasets on individual cell populations in blood and brain, we identified significantly increased inter-individual variability in our scalp- and other skin-derived fibroblasts on a similar scale as epigenetic differences between different lineages of blood cells. Lastly, these epigenetic differences did not appear to be driven by somatic mutation--while we identified 64 probable de-novo variants across the 11 subjects, there was no association between mutation burden and age of the donor (p = 0.71). These results depict a strong component of epigenetic memory in cell culture from primary tissue, even after several generations of daughter cells, related to cell state and donor age

Increased methylome distances within scalp-derived fibroblasts.

<p>Y-axis: methylome (Euclidean) distance between pairs of samples stratified by cell and tissue types. CD4T: CD4+ T-cell, NK: natural killer cell, Mono: monocyte, NeuN+: neuronal DLPC cell, NeuN-: non-neuronal DLPFC cell.</p

Regional DNA methylation changes manifest in the transcriptome.

<p>(A) Plot of the DNAm levels (proportion methylation) of an example significant DMR, which overlaps the gene SIM1. (B) Plot of the average DMR DNAm levels versus the expression level of <i>SIM1</i>, showing high positive correlation (p = 4.67x10⁻⁸).</p

Long-range DNA methylation changes manifest in the transcriptome.

<p>(A) Plot of the DNAm levels (proportion methylation) of a significant DNAm block overlapping genes in the <i>HOX</i> family. Y-axis: proportion DNAm levels, x-axis: genomic coordinates on chromosome 17. (B) Corresponding expression levels of the <i>HOX</i> genes within the DNAm block are more highly expressed in the scalp. Y-axis: log₂ transformed fragments per kilobase per million mapped (FPKM), x-axis: sampling location.</p

Representative patterns of age-associated changes in DNAm by sampling location.

<p>Each panel (A-H) shows mean adjusted expression levels versus age for each of eight clusters of location-specific age-related changes in DNAm levels. Y-axis: mean-centered DNAm levels, x-axis: sample age, p-value represents the statistical interaction between sampling location and age on DNAm levels. N: number of CpGs in the cluster. Vertical lines at each sample indicate +/- 3 times the standard error by cluster.</p

DNA methylation patterns in dura- and scalp-derived fibroblasts.

<p>(A) Histogram of difference in DNAm levels at CpGs/probes between scalp and dura derived fibroblasts (on the proportion methylation scale). (B) The first principal component (PC1) of the DNAm data plotted against fibroblast sampling location (scalp versus dura). (C) Example significant differentially methylated region (DMR) that overlaps the gene <i>RUNX3</i>, with DNAm levels on the y-axis and genomic coordinates on the x-axis. (D) Example significant DNAm block, with DNAm levels on the y-axis and genomic coordinates on the x-axis. Gene annotation panels in C and D are based on Ensembl annotation–dark blue represents exons and light blue represents introns.</p