Efficient Production of Bioactive Insulin from Human Epidermal Keratinocytes and Tissue-Engineered Skin Substitutes: Implications for Treatment of Diabetes

Abstract

Despite many years of research, daily insulin injections remain the gold standard for diabetes treatment. Gene therapy may provide an alternative strategy by imparting the ability to secrete insulin from an ectopic site. The epidermis is a self-renewing tissue that is easily accessible and can provide large numbers of autologous cells to generate insulin-secreting skin substitutes. Here we used a recombinant retrovirus to modify human epidermal keratinocytes with a gene encoding for human proinsulin containing the furin recognition sequences at the A-C and B-C junctions. Keratinocytes were able to process proinsulin and secrete active insulin that promoted glucose uptake. Primary epidermal cells produced higher amounts of insulin than cell lines, suggesting that insulin secretion may depend on the physiological state of the producer cells. Modified cells maintained the ability to stratify into 3-dimensional skin equivalents that expressed insulin at the basal and suprabasal layers. Modifications at the furin recognition sites did not improve proinsulin processing, but a single amino acid substitution in the proinsulin B chain enhanced C-peptide secretion from cultured cells and bioengineered skin substitutes 10- and 28-fold, respectively. These results suggest that gene-modified bioengineered skin may provide an alternative means of insulin delivery for treatment of diabetes.

Mechanistic study of endogenous skin lesions in diabetic rats

“…Our earlier studies revealed that insulin stimulates keratinocyte migration and proliferation and regulates keratinocyte differentiation (6,21,22). Moreover, previous studies suggest that hyperglycaemia and decreased insulin signals are involved in impairment of skin function (23–25). Therefore, the distorted keratinocyte phenotypes in diabetics may be a major factor in endogenous skin lesions and delayed wound healing.…”

Current treatment options and challenges in patients with Type 1 diabetes: Pharmacological, technical advances and future perspectives

“…Gene therapy has also been considered to achieve permanent restoration of insulin production [174]: studies in this field confirmed the possibility of obtaining ectopic insulin production from different cells, for example, keratinocytes or fibroblasts [175,176] using ex vivo gene transfer methods. Using in vitro techniques, gene transfer genetically modified cells in vitro, then they are transplanted into the subjects: in animal models, this approach allowed a secretion of insulin able to promote glucose uptake and normalize glycemia.…”

Magnetic Nanoparticles in Tissue Regeneration

“…This could be an aid to industrial fabrication of keratinocytes sheet in the 3D structure for skin tissue engineering. Some recent study have shown the genetic modification in the keratinocytes for overexpression of important protein encoding genes like high expression of platelet derived growth factor gene modified keratinocytes sheet for the high vasucalarization potential in vivo [164], overexpression of vascular endothelial growth factor using cationic liposomes for high reepithilization potential of keratinocytes with increased production of ECM [165], expression of human proinsulin gene for the active secretion of insulin from the skin tissue, which promote the uptake of glucose and could be used as an alternative technique for the tretment of diabetes [166].…”