Abstract
SUMMARYDerivation and stable maintenance of porcine induced pluripotent stem cells (piPSCs) is challenging. We herein systematically analyzed two piPSC lines, derived by lentiviral transduction and cultured under either leukemia inhibitory factor (LIF) or fibroblast growth factor (FGF) conditions, to shed more light on the underlying biological mechanisms of porcine pluripotency. LIF‐derived piPSCs were more successful than their FGF‐derived counterparts in the generation of in vitro chimeras and in teratoma formation. When LIF piPSCs chimeras were transferred into surrogate sows and allowed to develop, only their prescence within the embryonic membranes could be detected. Whole‐transcriptome analysis of the piPSCs and porcine neonatal fibroblasts showed that they clustered together, but apart from the two pluripotent cell populations of early porcine embryos, indicating incomplete reprogramming. Indeed, bioinformatic analysis of the pluripotency‐related gene network of the LIF‐ versus FGF‐derived piPSCs revealed that ZFP42 (REX1) expression was absent in both piPSC‐like cells, whereas it was expressed in the porcine inner cell mass at Day 7/8. A second striking difference was the expression of ATOH1 in piPSC‐like cells, which was absent in the inner cell mass. Moreover, our gene expression analyses plus correlation analyses of known pluripotency genes identified unique relationships between pluripotency genes in the inner cell mass, which are to some extent, in the piPSC‐like cells. This deficiency in downstream gene activation and divergent gene expression may be underlie the inability to derive germ line‐transmitting piPSCs, and provides unique insight into which genes are necessary to achieve fully reprogrammed piPSCs. 84: 229–245, 2017. © 2016 Wiley Periodicals, Inc.
Highlights
The development of mouse (Takahashi and Yamanaka, 2006) and human induced pluripotent stem cells (Takahashi et al, 2007; Yu et al, 2007) ignited the hope that patient-specific pluripotent stem cells would soon be used for therapy (Singh et al, 2015)
A lentiviral construct in which expression of the porcine sequences of Octamerbinding transcription factor 4 (OCT4), Sex-determining region Y-box 2 (SOX2), transcription factor (c-MYC), and Kru€ppel-like factor 4 (KLF4) are under the control of a doxycycline-inducible TetO promoter (TetOpOSMK) (Fig. S1) was simultaneous transduced with a second lentivirus carrying the reverse tetracycline-controlled transactivator gene, which allows for doxcycyclne-dependent activation of the pluripotency factors, into porcine neonatal fibroblasts derived from piglets ubiquitously expressing the green fluorescent protein ‘‘Venus’’ (Garrels et al, 2012)
A comprehensive literature search of predicted gene functions resulted in 22 genes that we considered potentially important for the pluripotency of the porcine inner cell mass (Table 1), and include genes encoding transcription factors, growth factor receptors, membrane proteins involved in cellÀcell interactions, as well as proteins involved in phosphoinositide 3-kinase/ AKT, transforming growth factor beta, and Wnt signaling ÀÀall of which have previously been shown to play a role in pluripotency and differentiation (Watanabe et al, 2006; Watabe and Miyazono, 2009; Holland et al, 2013)
Summary
The development of mouse (Takahashi and Yamanaka, 2006) and human induced pluripotent stem cells (iPSCs) (Takahashi et al, 2007; Yu et al, 2007) ignited the hope that patient-specific pluripotent stem cells would soon be used for therapy (Singh et al, 2015). The pig is considered an excellent model for several devastating human diseases, such as diabetes, cardiovascular, and nervous system diseases. Miniature pigs were successfully used as a cell-replacement model for amyotrophic lateral sclerosis (ALS), as they were able to receive human spinal stem cells (Boulis et al, 2011; Glass et al, 2012). A number of genetically modified pigs have been produced as potential disease models that faithfully recapitulate human disease characteristics (Flisikowska et al, 2014). Further stymying the efficiency of establishing genetic pig models, a considerable proportion of cloned piglets exhibit epigenetic abnormalities, resulting in a perinatal mortality rate of approximately 50% (Schmidt et al, 2015)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
