Analysis of Scar Forming Fibroblasts Reveals Distinct Changes in Epigenetic Accessibility During Times of Phenotypic Transition
Alessandra Moore, MD, Ulrike Litzenburger, PhD, Clement Marshall, MD, Ryan Chase Ransom, BS, Heather desJardins-Parks, AB, Bryan Duoto, BS, Shamik Mascharak, AB, Leandra Barnes, AB, Elizabeth Brett, MS, Mike Hu, MD MS MPH, Howard Chang, PhD, H. Peter Lorenz, MD, Michael T. Longaker, MD MBA FACS.
Stanford University, Stanford, CA, USA.
Early fetal wounds heal by regeneration; an important but poorly understood phenomena. Understanding the fetal wound healing mechanism could achieve scarless healing in human patients. In 2015, our group proved that Engrailed-1 (En1) positive fibroblasts (EPFs) are responsible for all scar tissue deposition in adult and postnatal mice. Additionally, these cells appear around the time of phenotypic change from scarless (embryonic day 0-16) to scarring (embryonic day 18+) healing. Given Engrailed-1 positive fibroblasts (EPFs) and Engrailed-1 negative fibroblasts (ENFs) share a common precursor cell, we hypothesized that the EPFs accumulate epigenetic changes over time that result in their phenotypic transition and result in a permanent cellular phenotype.
Dorsal dermal fibroblasts from En1Cre/-; Rosa26mTmG/- mice were isolated at embryonic day (e)10, e16, e18, post-natal day (p)1, p30, and p30 wounded skin. EPFs and ENFs from these time points were sorted using Fluorescence-Activated Cell Sorting (FACS) and analyzed using the Assay for Transposase-Accessible Chromatin Using Sequencing (ATAC-seq). The data was then compared by time course analysis to generate a list of genes involved in fibrosis and to identify patterns of epigenetic change. E16 EPFs were then isolated by FACS and transplanted into a p1 host, and vice versa, to establish their intrinsic phenotype in vivo. Tissue was harvested 48 hours after transplant and analyzed using immunofluorescence to identify phenotypic differences based on cell type and microenvironment.
E10 fibroblasts are of a single lineage and were excluded from analysis. Time course analysis of e16-p30 EPFs and ENFs shows appropriate correlation between samples (Figure 1A). Principle Component Analysis shows p30 EPFs and ENFs being the most dissimilar, and EPFs from p30 are most like e16 EPFs (Figure 1B). Most epigenetic changes in the EPF lineage occur in embryonic development between e16 and e18, with fewer epigenetic changes occurring postnatally (significant peaks = 173 vs. 336, Figure 1C). These epigenetic changes are correlated with open promoter sequences at e18, which then by p1 appear to be closed (Figure 1D). In contrast, the ENF lineage accumulates increasing epigenetic changes from e18 and p30 (significant peaks = 88 vs. 545, Figure 1C). Lastly, reciprocal transplantation of e16 fibroblasts into a p1 host and vice versa reveal a significant difference in collagen overlap (2.13% versus 24.18%) and morphologic changes suggestive of quiescence versus reactivity (Figure 1E).
Our data suggest that fibroblast phenotype is highly cell intrinsic and based on the accumulation of epigenetic change. Epigenetic change correlates with the transition in healing phenotype, and localizes to promoter sequences. By using the CRISPR-Cas9 system in future experiments, we will delineate which genes associated with e18 open promoters are the master regulators of fibrosis. Intervention at these genes may allow for scarless healing in adults.
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