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

Abstract

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.

Derivation of chicken induced pluripotent stem cells tolerant to Newcastle disease virus-induced lysis through multiple rounds of infection

“…Chicken iPSCs were derived as previously described [16]. ciPSCs had typical characteristics of a stem cell fate: large nucleus to cytoplasm ratio, large nucleoli, positive cytochemical reaction for PAS and Alkaline Phosphatase, and expression of embryonic stem cell markers as shown by RT-PCR and immunocytochemistry [16].…”

“…iPSC technology has been successfully applied to mammalian species, including humans [9–12], and it has garnered success with avian species as well, such as chickens and quails [13–15]. Chicken induced pluripotent stem cells (ciPSCs) display characteristics indicative of a stem cell state including morphological and functional characteristic [13, 14, 16]. ciPSCs have demonstrable alkaline phosphatase enzymatic activity, and positive cytochemical staining for periodic acid-schiff (PAS) [13, 14, 16], consistent with stem cell staining characteristics [17].…”

“…Chicken induced pluripotent stem cells (ciPSCs) display characteristics indicative of a stem cell state including morphological and functional characteristic [13, 14, 16]. ciPSCs have demonstrable alkaline phosphatase enzymatic activity, and positive cytochemical staining for periodic acid-schiff (PAS) [13, 14, 16], consistent with stem cell staining characteristics [17]. As the most stringent proof of pluripotency, ciPSCs can be used to generate chimeric birds by transplantation into the embryo at early stages of embryogenesis, as shown with chicken-quails and chicken-chicken chimeras [13, 14].…”

Improvement of Chicken Primordial Germ Cell Maintenance In Vitro by Blockade of the Aryl Hydrocarbon Receptor Endogenous Activity

“…Therefore, a lot of research has been conducted either to study PGCs and improve their culture (Han, 2009;Nakamura et al, 2013) or to develop new approaches and offer alternative protocols for transgenesis (Yu et al, 2014). Findings of a positive effect of AHR antagonists on the maintenance of hematopoietic stem cells in culture (Boitano et al, 2010;Carlin et al, 2013;Gasiewicz et al, 2014) led us to investigate the action of a-naphthoflavone on the in vitro expansion of chicken PGCs.…”

<b>Estudo do potencial de pluripotência de células-tronco equinas derivadas de tecido adulto e cordão umbilical submetidas à reprogramação induzida geneticamente (células iPS)</b>

“…Portanto, as células mesenquimais estudadas neste projeto foram analisadas conforme o descrito na literatura BARBERINI et al, 2014;De SCHAUWER et al, 2014), levando em conta os anticorpos específicos para equinos disponíveis no mercado. (LIU et al, 2008;WU et al, 2010), ovinos (BAO et al, 2011;LIU et al, 2012), bovinos MALAVER-ORTEGA et al, 2015), frango (YU et al, 2014), cães (KOH; PIEDRAHITA, 2015), caprinos (TAI et al, 2015) e equinos (NAGY et al, 2011;LEE et al, 2016 no prelo) 4 , entre outros.…”