Abstract
The pig is recognized as a valuable model in biomedical research in addition to its agricultural importance. Here we describe a means for generating skeletal muscle efficiently from porcine induced pluripotent stem cells (piPSC) in vitro thereby providing a versatile platform for applications ranging from regenerative biology to the ex vivo cultivation of meat. The GSK3B inhibitor, CHIR99021 was employed to suppress apoptosis, elicit WNT signaling events and drive naïve-type piPSC along the mesoderm lineage, and, in combination with the DNA methylation inhibitor 5-aza-cytidine, to activate an early skeletal muscle transcription program. Terminal differentiation was then induced by activation of an ectopically expressed MYOD1. Myotubes, characterized by myofibril development and both spontaneous and stimuli-elicited excitation-contraction coupling cycles appeared within 11 days. Efficient lineage-specific differentiation was confirmed by uniform NCAM1 and myosin heavy chain expression. These results provide an approach for generating skeletal muscle that is potentially applicable to other pluripotent cell lines and to generating other forms of muscle.
Highlights
One of the main driving forces for pursuing studies on porcine pluripotent stem cells has been to create tissue precursors in vitro that might be used to explore engraftment procedures in an animal model with close physiological and morphological similarity to the human[1]
As with naive mouse embryonic stem cells (ESC)[31], naïve pluripotent stem cells (piPSC) culture was supported by a medium (Renewal Medium, renewal medium (RM)-1; Supplementary Table 1) containing CHIR99021/PD032591/PD173074 and leukemia inhibitory factor (LIF) under a 5% O2, 5% CO2, 90% N2 atmosphere
To identify conditions that might permit differentiation from the ground state, piPSC colonies were dissociated to single cells and sub-cultured on a poly-D-lysine, laminin and gelatin (PLG) substratum in RM-1 containing DOX in order to maintain expression of the two reprogramming genes, which are essential for self-renewal in this cell line
Summary
One of the main driving forces for pursuing studies on porcine pluripotent stem cells has been to create tissue precursors in vitro that might be used to explore engraftment procedures in an animal model with close physiological and morphological similarity to the human[1]. True embryonic stem cells from agriculturally important large animals, including the pig, have proven difficult to generate, and piPSC, which are readily created, have only rarely been used for directed differentiation purposes[2]. From the earliest stages of embryonic development through terminal differentiation, skeletal myogenesis is directed by CTNNB1 (β-catenin) and GSK3B (glycogen synthase kinase-3β). Like T, expression of a premyogenic transcription factor, PAX3, is directed by WNT signaling[10,11,12,13] and is CTNNB1-activated[14,15,16]. (c) Western blot detection of full-length CPP32 (~32 kD procaspase 3a) and the large cleaved fragment (~17 kD cleaved-caspase 3a) prior to (0 h) and following (12–48 h) differentiation. (g) Western blot detection of full-length and cleaved CPP32 following 42 h differentiation ±CHIR99021, as indicated.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
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.
