Abstract

Background: Cell-based therapy using pancreatic islet cells has emerged as a promising new approach for the treatment of diabetes mellitus. Along with the conventional usage of the purified islets for transplantation, our group has recently established a novel dissociated islet cell-based approach, named islet cell sheet transplantation (Transplantation 30: 5943, 2011). In this approach, individually dissociated islet cells are cultured on a specially-defined culture surface to form a thin and contiguous monolayered islet cell sheet. Since it is known that islet cells are susceptible to apoptotic process during the dispersed cell status, the present study conduced a gene transduction of an anti-apoptotic gene FNK, during the culture period for achieving higher cell survival as well as higher functionality. Methods: Fiber knob-modified adenoviral vectors expressing FNK or GFP under the control of CAG promoter, Ad-CA-K7-GFP, Ad-CA-K7-FNK, were prepared. Purified rat islet cells were dissociated into single islet cells, followed by their culturing on laminin-5-coated and temperature-responsive polymer-immobilized plastic dishes. Cells were incubated with the Ad-vectors at day 1 for 1 hr at a previously defined vector dose, MOI=10. Apoptosis was then induced by inoculating hydrogen peroxide into culture medium at 400μM. Results: After infection with Ad-CA-K7-GFP, approximately 90% of the dispersed-cultured islet cells were found to be gene-transduced. There were no Ad-vector infection-mediated cellular damages. In an another set of experiment, cells were infected with Ad-CA-K7-FNK, and hydrogen peroxide were inoculated on day 2 for the induction of apoptosis. FNK-transduced cultured islet cells showed significantly less caspase expressions as well as remarkable resistance to the apoptosis. Finally, the FNK-transduced islet cells could be harvested as an intact cell sheet format that was expected to produce higher levels of insulin. Conclusions:The present study described a new proof-of-concept approach to engineer apoptosis-resistant and highly functional islet cell construct for transplantation.

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