Integrated spatial multi-omics reveals fibroblast fate during tissue repair

In the skin, tissue injury results in fibrosis in the form of scars composed of dense extracellular matrix deposited by fibroblasts. The therapeutic goal of regenerative wound healing has remained elusive in part because principles of fibroblast programming and adaptive response to injury remain incompletely understood. Here, we present a multimodal -omics platform for the comprehensive study of cell populations in complex tissue, which has allowed us to characterize the cells involved in wound healing across both time and space. We employ a stented wound model that recapitulates human tissue repair kinetics and multiple Rainbow transgenic lines to precisely track fibroblast fate during the physiologic response to injury. Through integrated analysis of single cell chromatin landscapes and gene expression states, coupled with spatial transcriptomic profiling, we are able to impute fibroblast epigenomes with temporospatial resolution. This has allowed us to define the mechanisms controlling cell fate during migration, proliferation, and differentiation following tissue injury and thereby reexamine the canonical phases of wound healing. We show that wounding triggers a polyclonal proliferation of tissue-resident, mechano-responsive fibroblasts, the subpopulations of which inhabit spatially-distinct regions within the wound and harbor distinct activities. These findings have broad implications for the study of tissue repair in complex organ systems. Overall design: Mouse wound tissue transcriptomes were profiles using RNA bulk and single cell RNA sequencing, single cell ATAC sequencing, and Visium spatial transcriptomics. For bulk RNA sequencing, RNA was obtained from mouse inner wound region and outer wound region, as well as uninjured control fibroblasts at post-operative day (POD) #7 and #14, as well as inner wound region and outer wound region fibroblasts from POD #14 treated with a small molecule FAK inhibitor. For single-cell RNA sequencing, FACS-isolated Rainbow mouse inner wound region and outer wound region fibroblasts harvested at 3 timepoints over the course of wound healing (POD #2, #7, and #14) and single cell sorted for plate-seq. For single-cell ATAC sequencing, Rainbow mouse wound fibroblasts were FACS-isolated from inner and outer wound regions at the same 3 timepoints as single-cell RNA sequencing. For Visium spatial transcriptomics, mouse wound tissue sections were mounted and sequenced from POD #2, #7, and #14 as well as uninjured skin.