Spatial transcriptomics of healing and non-healing diabetic foot ulcers
Summary:Diabetic foot ulcers (DFUs) are a devastating complication of diabetes. To better understand the molecular mechanisms and cell types implicated in DFU healing, we used NanoStringâ€™s GeoMx Digital Spatial profiling platform on DFU tissue sections and compared gene expression of areas within the same ulcer as well as between patients who in 12 weeks following surgery healed their DFU (Healers, N=2) vs those who did not (Non-Healers, N=2).
Overall design:The spatial transcriptome profiling was performed using NanoString芒鈧劉s GeoMx Digital Spatial profiling platform on unfixed frozen 5 脦录m tissue sections. Samples were processed as follows: 1) 10% neutral buffered formalin fixation overnight, 2) target retrieval (1X Tris EDTA, pH 9.0 for 20 min), 3) proteinase K digestion (1 脗碌g/mL for 15 min), 4) post-fixation (10% NBF, Tris-glycine stop buffer), 5) in-situ hybridization overnight with the GeoMx Cancer Transcriptome Atlas probe panel (1800-plex), 6) stringent washes (50:50 formamide/4X SSC), and 7) fluorescent antibody/marker (aSMA, Clone: 1A4, Abcam; CD45, Clone: 2B11+PD7/26, Novus; PanCK, Clone: AE1/AE3, Novus) incubation, 1 hour at room temperature. Sections were then loaded onto the GeoMx脗庐 Digital Spatial Profiler. For profiling, circular regions of interest (ROIs), approximately 500 脦录m in diameter, located within the ulcers or in neighboring non-ulcerated tissue were selected to include high concentrations of CD45+ immune cells in close proximity to vessels (aSMA+ structures). After ROI selection, the GeoMx instrument illuminated each ROI separately with UV light to cleave, aspirate, and deposit the oligonucleotides from the hybridized ISH probes for downstream sequencing into a 96-well plate.
59 days, 50 days
Citation:Theocharidis, Georgios et al. “Single cell transcriptomic landscape of diabetic foot ulcers.” Nature communications vol. 13,1 181. 10 Jan. 2022, doi:10.1038/s41467-021-27801-8
Submission date: 2021-02-03Update date: 2021-10-30
Georgios Theocharidis; Aristidis Veves
AccessionsGEO Series Accessions: GSE166120