Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

Abstract

Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

Disrupting biological sensors of force promotes tissue regeneration in large organisms

“…However, these previous small animal studies were limited in scope and unable to fully examine the plethora of cellular signaling pathways, which are altered in response to mechanical stress disruption. Recent advances in single-cell transcriptomics have increased our ability to explore heterogeneous cellular responses, such as those associated with modulation of mechanical forces 35 .…”

Dermal Drivers of Injury-Induced Inflammation: Contribution of Adipocytes and Fibroblasts

“…Diabetic fibroblasts exhibit decreased proliferation, adhesion, and migration as well as altered MMP expression and ECM production [ 218 , 219 ]. This fibroblast dysregulation hinders fibrosis and results in significant delays in dermal repair [ 29 , 32 , 220 ]. Mounting evidence supports that impaired responsiveness and an exaggerated pro-inflammatory phenotype in fibroblasts contributes to reduced macrophage recruitment shortly after injury and subsequent persisting inflammation at later time points ( Figure 2 ).…”

“…In addition to in vitro studies, analysis of chronic wounds demonstrates that fibroblasts can support persistent inflammation. Specifically, fibroblasts within chronic diabetic ulcers exhibit diminished heterogeneity that favors populations that are enriched for inflammation-related gene expression [ 29 , 32 ]. The elevated pro-inflammatory state can also be observed across different populations of fibroblasts, as diabetic ulcer papillary fibroblasts have elevated IL11, IL24, MMP1, and MMP3 expression and another identified fibroblast cluster is enriched with IL6 , MMP12, and prostaglandin endoperoxide synthase 2 ( PTGS2 ) expression [ 32 ].…”

“…The elevated pro-inflammatory state can also be observed across different populations of fibroblasts, as diabetic ulcer papillary fibroblasts have elevated IL11, IL24, MMP1, and MMP3 expression and another identified fibroblast cluster is enriched with IL6 , MMP12, and prostaglandin endoperoxide synthase 2 ( PTGS2 ) expression [ 32 ]. Locked in this pro-inflammatory state, diabetic fibroblasts are anti-angiogenic and antifibrotic with reduced transcription of growth factors and genes involved in proliferation and collagen organization [ 29 , 32 ]. This anti-angiogenic and antifibrotic polarization is epigenetically-encoded and maintained by diabetic fibroblasts after repeated passages in culture [ 230 ].…”

“…Frequently, diseases associated with impaired wound healing do not properly activate early inflammatory pathways or do not fully resolve inflammation, and therefore do not successfully progress into the proliferative phase. A delayed or incomplete transition from the inflammatory phase to the proliferative phase is associated with the persistence of inflammatory neutrophils and macrophages [ 27 , 28 , 29 ], contributing to chronic or non-healing wounds. These hard-to-treat wounds pose a significant medical challenge; as their prevalence has steadily increased over time and only modest therapeutic advancements have come from animal studies [ 30 , 31 ].…”