Isolation And Characterization Of Pericytes From Burn Eschar Tissues
Alexander Evdokiou1, Richard Bodnar2, Latha Satish1.
1Shriners Hospitals for Children-Cincinnati, Cincinnati, OH, USA, 2Veterans Affairs Medical Center, Pittsburgh, PA, USA.
BACKGROUND: Pericytes, a population of mesenchymal stem cells, that are capable of differentiating into fibroblast/myofibroblasts phenotype promoting fibrosis and scar contractures. We hypothesize that in the burn wound environment, pericytes dissociated from vessels are in a hyperactive state in response to the various factors secreted by activated immune cells. These hyperactive pericytes can become predisposed due to excessive PDGF in the wound environment and differentiate into myofibroblasts promoting excessive fibroplasia. METHODS: To test this hypothesis, we utilized a novel approach to isolate and culture pericytes from patients undergoing burn wound debridement. RESULTS: Discarded burn wound eschar tissues and normal skin tissues obtained from patients undergoing elective plastic surgery procedures were processed without enzyme treatment by gently scraping the tissues into pericytes specific-growth medium. Within 3-4 days, an outgrowth of cells was observed and pericytes were analyzed and sorted directly for CD146+/CD73+/CD105+/CD56-/CD34-/CD45-. FACS analyses showed increased percentage of pericytes (~67%) in burn eschar tissues as opposed to normal skin tissues (~21%). FACS sorted pericytes were allowed to grow and expand in vitro using pericyte-specific growth medium. Immunofluorescence studies using CD146 and anti-fibroblast antibodies confirmed the presence of pericytes and absence of fibroblasts. Immunohistochemistry revealed increased amount of CD146 positive cells in burn eschar tissues compared to normal skin tissue. RNA-seq studies are in progress to understand the transcriptomic changes of pericytes in burn wound environment and to focus on the genes that contribute to pericyte differentiation (PDGF, TGF-β/Smad 1/2, ADAM12, TWEAK, FN14, CD248 (endosialin), Wnt) to fibroblast/myofibroblasts phenotype. CONCLUSIONS: Results from these studies may provide clues to modify pericytes in the burn wound environment for better healing outcome in reducing scar contractures.
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