Bioengineered Skin from Stem Cells for Treatment of Cutaneous Vesicant Injury

Sulfur mustard is a chemical warfare agent and potent vesicant that penetrates rapidly through the skin and causes prolonged injuries and incapacitation. Severe exposure to HD induces blistering skin reactions and significant loss of stem cell keratinocytes that are required for a continuous renewal of the epidermal cell layer. Therefore, HD injuries require long healing periods leaving significant cosmetic and/or functional deficits. We are developing bioengineered skin from embryonic stem cells for improved therapy for HD-induced skin damage. Since stem cell keratinocytes lack major histocompatibility class II antigens, they exhibit little immunogenicity and are suitable for allograft use. The multipotential nature of stem cells may be particularly valuable for skin damage where several cell types and growth factors are necessary for proper repair and regeneration. We differentiated embryonic stem cells to skin keratinocytes in a cellular matrix cultured in an air/liquid system to form the bioengineered skin, mimicking normal skin. The efficacy of bioengineered skin for HD-induced skin lesions was evaluated using a C57BL/6 mouse model and 2-chloroethylethyl sulfide (half-mustard or CEES). Mice were exposed to 3 μL neat CEES for 10 minutes, and at 48 h post exposure, the injured skin was excised and the site was cleaned with debridase. Next, the bioengineered skin was transferred directly to the wound and affixed with a non-adherent sterile gauze pad. The embryonic stem cell derived bioengineered skin exhibited growth and healing in 1 to 3 weeks. The edema decreased and skin contraction around the exposed area was minimal. In contrast, the CEES exposed animals not treated with bioengineered skin had a) no skin growth and b) obvious contraction of the skin in the injured area. We also evaluated cryo-technologies to preserve the bioengineered skin for immediate application in the battlefield and then assessed their efficacy in the CEES model. Our results demonstrate that for the first time, topically applied fresh or frozen bioengineered skin or skin keratinocytes from embryonic stem cells produce improved healing when applied 48 h after HD exposure.