Single cell transcriptome sequencing of diabetic foot skin
Chronic wounds, including diabetic foot ulceration and venous ulcers, are a major problem that is associated with significant morbidity and mortality and financial cost. Recent studies by our unit and elsewhere have shown that chronic inflammation is one of the major factors that is associated with impaired wound healing2-4. Wound healing requires a well-coordinated action by numerous cell types, including neutrophils, monocyte/macrophages, fibroblasts, endothelial cells and keratinocytes. However, there is considerable lack of understanding of the molecular physiology of each of these cells that could lead to understanding of the pathophysiology of impaired wound healing. In this application, we propose to perform single-cell transcriptome sequencing from forearm and foot skin biopsies from healthy subjects and diabetic patients with foot ulcer. Our main will be to evaluate gene expression in various cell types and also explore possible differences in various body sites using highly robust drop-seq sequencing approach that allow evaluation of transcriptomic state of thousands of human cells in single experiment by measuring 4,000-7,000 genes. Finally, we will explore possible differences between diabetic and non-diabetic patients. Successful completion of this application will not only contribute to the development of a reference atlas of skin cells transcriptome state but can also play a major role in the development of new therapeutic approaches.
Proteomic and Transcriptomic Single Cell Analysis In DFU Patient
Numerous cell types are involved in the pathogenesis of impaired wound healing in Diabetic Foot Ulceration (DFU). Previous studies in our unit have focused on endothelial and vascular smooth cells, endothelial progenitor cells, inflammatory cells, fibroblasts, macrophages and mast cells in both animal and human diabetes studies. In ongoing studies, we have also performed transcriptome and epigenome profiling on bulk foot and forearm tissue from patients with healed and non-healed DFU. Finally, we have recently generated single cell transcriptome profile from foot skin of DM and non-DM subjects and diabetic mice. Overall, our data indicate: A: Animal Studies: 1) Inflammation is increased in the pre-injured skin, which is characterized by infiltration of inflammatory cells, increased M1/M2 macrophage ratio and increased expression of inflammatory cytokines; 2) Global transcriptomic and systems biology studies identified pathways and master gene regulators related to inflammation as the major markers associated with improved wound healing; B: Human Studies: 1) Proinflammatory changes in the skin that confirm the association of chronic inflammation with impaired healing of diabetic foot ulcers (DFU); 2) Transcriptomic analysis identified considerable similarities in the pathways and master gene regulators associated with wound healing in DM mice. More specifically, the integrative systems biology analysis indicated key role of inflammation & immune pathways such as TREM1 signaling and dendritic cell maturation and associated MRs (e.g. CD40, IL1, IL6, NF?B) in wound healing; 3) Single cell transcriptome analysis in DFU patients identified activation of highly connected cohesive network of inflammatory and angiogenesis related master regulators (e.g., STAT3, IL15, HIF1?). Furthermore, similar analysis in DM patients without DFU identified activation of additional master regulators (e.g. SMAD3, CD44, TGF?). Overall, our data support the currently accepted consensus that inflammation plays an important role in the development of impaired diabetic wound healing. However, to further understand the pathophysiology of impaired diabetic wound healing, it is mandatory not only to understand the transcriptional state of individual cells at the skin and blood level but also their proteome state. A combined understanding of single cell transcriptome and proteome levels has the potential to greatly enhance our understanding in an agnostic way, as required by the current Collaborative Pilot Program, regarding the interaction of individual cells in the expression of various genes and production of proteins associated with wound healing. In this application, we propose to compare single cell transcriptome and proteome profiling of cells from forearm and foot skin biopsies and blood from healthy, non-DM subjects and DM patients with healed and non-healed DFU. Our main aim will be to evaluate differences in gene expression in various cell types and how this is related to protein expression. We hypothesize that diabetic patients with impaired wound healing will have aberrant gene and protein expression that will lead to a chronic inflammation stage that precludes linear progression to the next phases of wound healing. To this end, we first propose to evaluate single cell gene expression changes in foot and forearm skin specimens and blood immune cells in patients with healed and non-healed DFU, DM patients with no DFU and healthy, non DM patients. We will employ scRNA-seq to perform targeted single cell transcriptome profiling and identify differences with special emphasis on inflammatory and immune pathways. We then propose to evaluate protein expression in the same skin and blood specimens. We will evaluate single cell protein expression. More specifically, we will evaluate the expression of proteins know to be expressed in specific cells and involved in the wound healing process. In addition, in an agnostic way, we will compare the expression of the most highly expressed proteins among the various groups.