Single Cell Rna-seq Analyses Of Healthy Skin And Diabetic Ulcers Reveals Fundamentally Different Cell Type-specific Transcriptional Profiles
Georgios Theocharidis, Swati Bhasin, Konstantinos Kounas, Thanh Dinh, Barry Rosenblum, Manoj Bhasin, Aristidis Veves
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
BACKGROUND Fifteen percent of diabetes mellitus (DM) patients are expected to develop a diabetic foot ulcer (DFU) within their lifetime. DFUs represent a considerable burden for the healthcare system of both developed and developing countries. Chronic wounds, like DFUs, lack the linear progression from one classical wound healing phase to the next and are mainly characterized by the persistence of the inflammatory phase.
METHODS To gain further insight on the molecular mechanisms involved in DFU development we embarked on building the transcriptomic profile of DFUs by means of single cell RNA sequencing. Discarded specimens from foot surgeries of DM patients without ulcers (n=4), DM patients with DFU following debridement process (n=2) or healthy controls (n=4) were collected and a single cell suspension was achieved with enzymatic digestion and mechanical dissociation of the tissue. Particular care was taken to ensure high cell viability.
RESULTS Bioinformatics methods applied transcriptome correlation to identify distinct clusters of cells with different transcriptome states. The clusters were further annotated into specific cell types. A total of 4,142 cells were captured from the control samples, whilst 3,692 were captured from DM skin and 727 from DFUs. The most abundant cell type was as anticipated of mesenchymal origin - fibloblasts and adipocytes. Additional clusters were endothelial cells, either vascular or lymphatic, smooth muscle cells and immune cells with a clear division between T-cells and macrophages. Functions and pathways enrichment analysis of the macrophages’ transcriptomic profiles revealed TREM1 and NF-kB signaling as significantly dysregulated in non-healing DFUs, while master regulator analysis highlighted STAT3 as top activated and ESR1 as top inhbited gene.
CONCLUSIONS We present for the first time single cell RNA-Seq analysis of the lower extremity skin of healthy controls as well as DM patients and DFUs. Our findings provide a global view of the heterogeneity of DFU immunome and could help identify novel therapeutic targets.
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