Single-Cell Sequencing Reveals Why Diabetic Foot Ulcers Refuse to Heal

Diabetic foot ulcers are among the most feared complications of diabetes, and for good reason. According to the International Diabetes Federation, roughly 537 million adults worldwide were living with diabetes in 2021, a figure projected to reach 783 million by 2045. Of those affected, approximately 15 percent are at high risk of developing diabetic foot disease, and once an ulcer forms, the outlook can be grim. In China alone, more than one million new ulcer cases emerge each year, with an amputation rate of 21.3 percent. Studies cited in a recent review indicate that five-year mortality among patients with diabetic foot ulcers approaches 50 percent, and survival three years after amputation falls below that same threshold. Against this backdrop, a team of researchers led by Shuhan Yang and Yifeng Zhao at the Second Affiliated Hospital of Chongqing Medical University has published a comprehensive review in Frontiers in Clinical Diabetes and Healthcare arguing that single-cell RNA sequencing, a technology that reads the genetic activity of individual cells rather than averaging signals across whole tissue, is poised to transform how scientists understand and treat these stubborn wounds.

The core problem with diabetic foot ulcers is that they are not single diseases. They arise from a convergence of peripheral neuropathy, ischemia and hypoxia, and recurrent infection, all superimposed on a metabolic environment warped by chronic hyperglycemia. Sensory neuropathy strips patients of the protective pain reflexes that warn of minor trauma, while motor neuropathy deforms the foot and redistributes pressure onto vulnerable tissue. Autonomic dysfunction dries the skin, cracking the protective barrier and inviting bacterial entry. Meanwhile, prolonged high blood sugar drives atherosclerosis in the large vessels of the leg and damages the microcirculation, starving tissue of oxygen and nutrients. The result is a wound microenvironment in which matrix metalloproteinases run rampant, collagen synthesis lags, advanced glycation end products accumulate, and inflammation refuses to resolve. Conventional diagnostic tools, from the Ankle-Brachial Index to MRI and transcutaneous oxygen pressure measurement, capture the macroscopic consequences of this cascade but say little about the cellular actors driving it.

This is where single-cell RNA sequencing enters the picture. Unlike bulk transcriptomic methods, which measure average gene expression across thousands of mixed cells and thereby mask the contributions of rare subpopulations, scRNA-seq profiles each cell individually. The review’s authors explain that this resolution allows researchers to identify distinct cellular subtypes, map ligand-receptor interactions between cell populations using computational tools such as CellChat and iCELLNET, and reconstruct the pathological evolution of a wound at unprecedented depth. When integrated with spatial transcriptomics, which preserves the physical location of cells within tissue, and proteomics, which validates transcriptomic predictions at the protein level, the approach yields a multidimensional picture that bulk sequencing simply cannot match. The technology has already proven its worth in oncology, where it uncovered exhausted CD8-positive T cell signatures in hepatocellular carcinoma and identified macrophages and dendritic cells as key mediators in colorectal cancer, insights that have guided immunotherapy development.

Applied to diabetic foot ulcers, single-cell sequencing has produced its most striking findings in the immune compartment. Macrophages, the versatile scavengers and regulators of tissue repair, emerge as central players whose behavior is fundamentally corrupted in the diabetic wound. In healthy healing, pro-inflammatory macrophages dominate early to fight infection and clear debris, then give way to pro-resolving counterparts that secrete anti-inflammatory mediators such as C1QA, C1QB, C1QC and CD163, promoting tissue remodeling and angiogenesis. In diabetic wounds, this transition stalls. The review highlights that the histone demethylase JMJD3, an epigenetic regulator, activates the JAK1/3-STAT3 signaling axis and amplifies NF-kappa-B-mediated release of inflammatory cytokines including tumor necrosis factor and interleukins, effectively trapping macrophages in a pro-inflammatory state. Inhibition of JMJD3 in macrophages has been shown to alleviate inflammation and improve wound repair in both streptozotocin-induced type 1 and db/db type 2 diabetic mouse models, positioning the enzyme as a promising immunomodulatory target.

Beyond macrophages, single-cell atlases have exposed the heterogeneity of T lymphocytes in diabetic foot ulcers. Vu and colleagues found marked increases in naive T cells and CD8-positive T cells, resolving them into ten and seven distinct subclusters respectively. Cheng and colleagues reported upregulation of IL-17 signaling genes in CD8-positive T cells, alongside reduced expression of regulatory genes such as TSC22D3 and PIK3IP1, driving excessive activation and local inflammation. Regulatory T cells, which normally suppress inflammation through IL-10 and TGF-beta secretion, are significantly depleted in diabetic ulcers compared with normal wounds, weakening a critical brake on Th1 and Th17-driven immune responses. Natural killer T cells and gamma-delta T cells are enriched, contributing to a self-reinforcing cycle of infection, inflammation and impaired repair. Neutrophils, too, display remarkable diversity, with Retnlg-positive, Lcn2-positive, Wfdc21-positive and Mmp8-positive subsets identified, some of which peak during early ulcer formation and decline during healing, suggesting distinct roles in initiating versus resolving inflammation.

The stromal side of the wound tells an equally compelling story. Fibroblasts, the workhorses of extracellular matrix production and wound contraction, exhibit profound heterogeneity in diabetic ulcers. Buechler and colleagues generated a single-cell fibroblast atlas classifying lineages into general steady-state cells, specialized steady-state cells, and disturbance-specific activated cells. Januszyk and colleagues compared fibroblasts from diabetic and non-diabetic ulcers across ethnic groups, uncovering subpopulations with varied fibrotic potentials. A key mechanistic discovery came from Ku and colleagues, who showed that dysregulation of the IGF-1-SP1-CD248 pathway impairs fibroblast migration and delays healing. Healing-associated fibroblasts characterized by high expression of MMP1, MMP3, MMP11, HIF1A, CHI3L1 and TNFAIP6 concentrate in the central granulation bed, where they drive matrix remodeling and angiogenesis. Senescent, pro-inflammatory fibroblasts accumulate at the wound margin and avascular base, sustaining chronic inflammation and blocking closure. Spatial transcriptomics confirms that this dysfunctional spatial distribution, rather than transcriptional change alone, is a core determinant of refractory ulcer progression.

Vascular endothelial cells fare no better. Single-cell profiling of the public dataset GSE165816 revealed that core genes including CCND1, ENO1, HIF1A and SERPINE1 are significantly downregulated in endothelial cells of diabetic foot ulcers, undermining cell cycle regulation, hypoxic response and microcirculatory homeostasis. Two characteristic subsets emerge: functionally reparative endothelial cells marked by SLCO2A1 and CYP1A1, which are enriched in granulation tissue with healing potential, and senescent dysfunctional cells that predominate in refractory wounds, secreting anti-angiogenic factors such as thrombospondin-1 and pigment epithelium-derived factor. Hyperglycemia and hypoxia jointly suppress the Nrf2 antioxidant pathway while persistent NF-kappa-B activation floods the wound with pro-inflammatory mediators, creating a vicious cycle of endothelial injury, ischemia and senescence. Even keratinocytes are implicated, with quiescent BC-2 subsets failing to support re-epithelialization and senescent epidermal cells expressing p16INK4a and p21Cip1 losing proliferative and migratory capacity.

Perhaps most surprisingly, the review highlights the role of sensory neurons in coordinating repair. Lu and colleagues demonstrated that nociceptive neurons expressing calcitonin gene-related peptide engage RAMP1 receptors on neutrophils, monocytes and macrophages, reducing immune cell recruitment, accelerating apoptosis, enhancing phagocytosis and polarizing macrophages toward pro-resolving phenotypes. Loss of NaV1.8-positive sensory neurons impairs skin closure and muscle regeneration, providing a cellular-level explanation for why neuropathy and poor healing are so tightly linked. This neuroimmune axis, invisible to conventional bulk analyses, exemplifies the kind of mechanistic insight that single-cell approaches are uniquely positioned to deliver.

The translational implications are substantial. The review identifies MMP1, MMP3, MMP11, JMJD3 and HIF1A as valuable biomarkers and therapeutic targets, validated across multiple independent human cohorts and animal models. Computational drug-target network analysis has yielded seven potential core therapeutic targets and twenty candidate small-molecule drugs with translational potential. Single-cell signatures may eventually allow diabetic foot ulcers to be classified into inflammation-dominant, angiogenesis-deficient and matrix repair-impaired molecular subtypes, guiding individualized anti-inflammatory, pro-angiogenic or matrix-remodeling strategies. Yet significant barriers remain. Most studies rely on small, cross-sectional samples; db/db and streptozotocin mouse models cannot fully replicate the neuropathy, arterial occlusion and mixed infections characteristic of human disease; and sequencing costs, lack of standardized pipelines and insufficient prospective validation continue to limit clinical adoption. The authors argue that future progress will require stratified, longitudinal human cohorts, multi-omics integration combining spatial transcriptomics, proteomics and epigenomics, and rigorous preclinical validation of targets such as JMJD3 and STING. If those challenges can be met, single-cell technology may finally convert one of diabetes’ most intractable complications from a clinical guessing game into a data-driven precision discipline.

Subject of Research: Application of single-cell RNA sequencing to diabetic foot ulcer pathogenesis, biomarker discovery and translational research

Article Title: Single-cell RNA sequencing in diabetic foot ulcers: pathogenesis, biomarkers, and translational potential

Article References: Yang, S., Wang, S., Yang, R., Xu, K., Yang, X., & Zhao, Y. (2026). Single-cell RNA sequencing in diabetic foot ulcers: pathogenesis, biomarkers, and translational potential. Frontiers in Clinical Diabetes and Healthcare, 7, Article 1833678. https://doi.org/10.3389/fcdhc.2026.1833678

Image Credits: AI Generated

DOI: 10.3389/fcdhc.2026.1833678

Keywords: diabetic foot ulcer, single-cell RNA sequencing, macrophage polarization, wound healing, JMJD3, biomarkers, angiogenesis, fibroblasts, spatial transcriptomics, precision medicine, diabetes complications, IGF-1-SP1-CD248 pathway