Efficient Production of Bioactive Insulin from Human Epidermal Keratinocytes and Tissue-Engineered Skin Substitutes: Implications for Treatment of Diabetes PEDRO LEI, 1 ADEBIMPE OGUNADE, 1 KEITH L. KIRKWOOD, 2 SUZANNE G. LAYCHOCK, 3 and STELIOS T. ANDREADIS 1 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. INTRODUCTION D IABETES MELLITUS is a group of diseases characterized by an absolute or relative insulin deficiency and poor glucose control (hyperglycemia) in the blood. In type 1 di- abetes, also known as insulin-dependent diabetes mellitus (IDDM) or juvenile-onset diabetes mellitus, there is an ab- solute lack of insulin due to the progressive loss of pan- creatic b-cells in the islets of Langerhans. Destruction of pancreatic islet b-cells is governed in part by hereditary 1 and environmental factors such as viruses, toxins, dietary pro- teins, and stress. 2 In type 2 diabetes, insulin is present, but the signal for proper glucose uptake is lost because of nutri- tional or genetic factors. 3 Despite intense research efforts, daily insulin injections remain the gold standard for diabetes treatment, although the discomfort to patients may be sig- nificant. On the other hand, islet transplantation suffers from scarcity of donors, the need for long-term immunosuppres- sion, and short-term survival of transplanted cells because of recurrence of anti-islet cell autoimmunity. 4,5 Therefore, en- gineering alternative routes of physiologic insulin delivery is a major goal of diabetes research. Because type 1 diabetes results from lack of insulin pro- duction in the pancreas, it is reasonable to assume that se- cretion of insulin from ectopic sites might overcome the insulin deficit. Several cell types have been used to produce 1 Bioengineering Laboratory, Department of Chemical and Biological Engineering, University at Buffalo, The State University of New York, Amherst, New York; 2 Department of Periodontics and Oral Medicine, University of Michigan, Ann Arbor, Michigan; 3 Department of Pharmacology and Toxicology, University of Buffalo, The State University of New York, Amherst, New York. TISSUE ENGINEERING Volume 13, Number 8, 2007 # Mary Ann Liebert, Inc. DOI: 10.1089/ten.2006.0210 2119