Single-cell RNA sequencing clarifies dermal fibroblast subset representation in vitro and reveals variable persistence of keloid disease-associated features

Abstract

In vitro models of scarring and fibrosis are essential to improve our understanding of disease mechanisms and ultimately develop much-needed effective therapeutic strategies. This is particularly true for keloids, the example of pathological scarring exploited in this study, as there is no animal model. Our emerging appreciation of fibroblast heterogeneity from single cell RNA sequencing (scRNA-seq) information leaves a knowledge gap about what is represented in typical fibroblast cultures. Specifically, it is important to know whether quantitative differences in fibroblast subtypes observed in pathological tissues are represented and/or whether disease-associated molecular alterations of subtypes are maintained. This study performed scRNA-seq on patient-matched keloid and normal adjacent dermis immediately following surgical removal, which was compared to sc- and bulk-RNA-seq on primary dermal fibroblast cultures from the same samples after 4+ passages. Freshly dissociated tissue showed anticipated differences in cell proportions in keloid versus normal skin; however, comparably for both tissue types, there was an assimilation of fibroblast subtypes after culture. Cultured cells clustered conspicuously from the original populations, with evidence of only minor heterogeneity persisting. Cells displayed, to varying degrees, elements of each of the original subset signatures, with FAP+/SFRP2+ mesenchymal features the strongest. Pseudo-bulk analysis of mesenchymal subpopulations ex vivo showed cell-intrinsic keloid versus normal skin transcriptional differences consistent with current disease understanding; however, only a subset of these persisted in vitro. Cell-cell communication analysis provides potential strategies to maintain specific cell populations and their in vivo phenotypes. As an example, we report that culture with ascorbic acid (stimulating cell-derived extracellular matrix) enriched the mesenchymal signature. The data presented herein provide resources supporting greater understanding of, and strategies to refine, essential human fibroblast culture models.