Nonviral Minicircle Generation of Induced Pluripotent Stem Cells Compatible with Production of Chimeric Chickens

Chickens are vitally important in numerous countries as a primary food source and a major component of economic development. Efforts have been made to produce transgenic birds through pluripotent stem cell [primordial germ cells and embryonic stem cells (ESCs)] approaches to create animals with improved traits, such as meat and egg production or even disease resistance. However, these cell types have significant limitations because they are hard to culture long term while maintaining developmental plasticity. Induced pluripotent stem cells (iPSCs) are a novel class of stem cells that have proven to be robust, leading to the successful development of transgenic mice, rats, quail, and pigs and may potentially overcome the limitations of previous pluripotent stem cell systems in chickens. In this study we generated chicken (c) iPSCs from fibroblast cells for the first time using a nonviral minicircle reprogramming approach. ciPSCs demonstrated stem cell morphology and expressed key stem cell markers, including alkaline phosphatase, POU5F1, SOX2, NANOG, and SSEA-1. These cells were capable of rapid growth and expressed high levels of telomerase. Late-passage ciPSCs transplanted into stage X embryos were successfully incorporated into tissues of all three germ layers, and the gonads demonstrated significant cellular plasticity. These cells provide an exciting new tool to create transgenic chickens with broad implications for agricultural and transgenic animal fields at large.