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Single Cell RNA-Seq Reveals Molecular Signatures Of Heterogeneous Populations Of Dendritic Cells And Macrophages In Murine Wound Healing And Hypertrophic Scarring
Dominic Henn1,2, Mimi R. Borrelli, MBBS1, Kellen Chen, PhD1, Michael Januszyk, MD, PhD1, Jagannath Padmanabhan, PhD1, Clark A. Bonham1, Michael T. Longaker, MD, MBA, FACS1,3, Geoffrey C. Gurtner, MD, FACS1.
1Hagey Laboratory for Pediatric and Regenerative Medicine, Division of Plastic and Reconstructive Surgery, Stanford University, Stanford, CA, USA, 2Department of Hand, Plastic and Reconstructive Surgery, Ludwigshafen Trauma Center, Heidelberg University, Ludwigshafen, Germany, 3Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA.

PURPOSE:
Defining cellular sub-populations within the skin is critical to understand wound healing and scarring and develop novel therapeutic approaches. Dendritic cells (DC) and macrophages (MF) are mediators of the inflammatory response to skin injury and are involved in tissue homeostasis, wound healing and fibrosis. However, their cellular sub-populations in the skin remain poorly characterized. Here, we use single-cell RNA sequencing to analyze sub-populations of DCs and MFs in murine excisional wounds and hypertrophic scars over time.
METHODS:
Cells were isolated from stented excisional wounds in C57/BL6J mice 7 and 14 days after wounding as well as from a murine hypertrophic scar (HTS) model 14 days after external mechanical loading. Unwounded skin and scars without mechanical loading served as controls. DCs and MFs were enriched in single cell suspensions using flow cytometry, gating for live, CD45+ cells and excluding B cells, T cells, NK cells and granulocytes. Cells were further processed for droplet-based microfluidic single cell analysis using the 10X Genomics platform, in which individual cells are barcoded, lysed, and sequenced. Read depth was 50,000 reads per cell. Data were log-normalized and partitioned using UMAP based density mappings.
RESULTS:
We identified 7 distinct clusters of DCs, MFs and tissue monocytes. Two MF sub-populations (MHCII+,CD11cLo,CD11b+,CD103- and MHCII+,CD115+F4/80Int) were most abundant in excisional wounds, whereas HTS and control scars exhibited an enrichment of CD103-CD11b+CD24+ DCs and tissue monocytes which were specifically enriched in HTS. Unwounded skin showed an abundance of CD11c+CD11b+ DCs and B220-F4/80Lo,CD115+MHCII+ macrophages. An enrichment of transcriptomic signatures of hypertrophy, fibrosis, and cytokine signaling was found in DC and MF subpopulations which were identified in wounds and HTS.
CONCLUSION:
Our study demonstrates that wound healing and scarring induces a high degree of heterogeneity among sub-populations of DCs and MFs and thus expands our knowledge on cellular diversity of these cell types identifying novel targets for the development of therapies for chronic wounds and hypertrophic scars.


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