Advances in human induced pluripotent stem (iPSC) technology have raised new questions regarding the effect of reprogramming on the phenotype and epigenetic profile of cells differentiated from them. We recently derived cells from iPSC showing characteristic properties of fibroblasts (iPDK) that were similar to human embryonic stem cell- (hESC) derived fibroblasts (EDK). The goal of the current study was to compare the DNA methylation profile of fibroblasts derived from iPSC to fibroblasts from both normal and chronic wound tissues, to determine if there is a DNA methylation signature characteristic of chronic wound fibroblasts (CWF).
CWFs were harvested from excess tissues procured during wound debridement, expanded in culture and banked. Methylation analysis was performed using Infinium Human Methylation27 BeadChip (Illumina Inc.), analyzing 27,578 individual CpG sites. DNA was extracted using the DNeasy kit and 1 μg of purified genomic DNA was bisulfite-converted using the EZ DNA Methylation Kit and hybridized to the Infinium Methylation Chip. Immunoprecipitation of methylated DNA was performed using a MeDIP methylation kit (Diagenode).
We demonstrate that EDK and iPDK fibroblasts shared a similar DNA methylation profile with adult fibroblasts, but distinct from other cell lineages. Initial analysis indicated that we can enrich methylated DNA using immunoprecipitation, to allow for screening of CWF samples. We are currently analyzing primary fibroblasts from non-healing wounds using an immunoprecipitation-based method to enrich for methylated DNA, and using NimbleGen technology to determine their DNA methylation profile.
When CWFs are compared to ESC- and iPSC-derived fibroblasts, we expect to find significant differences between their methylation profile at specific promoter sites of CWFs that may partially account for their non-healing phenotype. These studies will be an important step towards understanding the underlying epigenetic mechanisms of chronic wounds that will have implications for oral disease processes characterized by failure to heal.
Keywords: Bioengineering, Fibroblasts, Stem Cells, Tissue engineering and Wound healing