Non-viral expression of mouse Oct4, Sox2, and Klf4 transcription factors efficiently reprograms tadpole muscle fibers in vivo.
Adult mammalian cells can be reprogrammed into induced pluripotent stem cells (iPSCs) by a limited combination of transcription factors. To date, most current iPSC generation protocols rely on viral vector usage in vitro, using cells removed from their physiological context. Such protocols are hindered by low derivation efficiency and risks associated with genome modifications of reprogrammed cells. Here, we reprogrammed cells in an in vivo context using non-viral somatic transgenesis in Xenopus tadpole tail muscle, a setting that provides long term expression of non-integrated transgenes in vivo. Expression of mouse mOct4, mSox2, and mKlf4 (OSK) led rapidly and reliably to formation of proliferating cell clusters. These clusters displayed the principal hallmarks of pluripotency: alkaline phosphatase activity, up-regulation of key epigenetic and chromatin remodeling markers, and reexpression of endogenous pluripotent markers. Furthermore, these clusters were capable of differentiating into derivatives of the three germ layers in vitro and into neurons and muscle fibers in vivo. As in situ reprogramming occurs along with muscle tissue repair, the data provide a link between these two processes and suggest that they act synergistically. Notably, every OSK injection resulted in cluster formation. We conclude that reprogramming is achievable in an anamniote model and propose that in vivo approaches could provide rapid and efficient alternative for non-viral iPSC production. The work opens new perspectives in basic stem cell research and in the longer term prospect of regenerative medicine protocols development.
UMR CNRS 7221,Évolution des Régulations Endocriniennes,Département Régulations Développement et Diversité Mole´culaire,Muse´um National d’Histoire Naturelle, 75231 Paris, France.
Céline
C
Vivien
Pierluigi
P
Scerbo
Fabrice
F
Girardot
Karine
K
Le Blay
Barbara A
BA
Demeneix
Laurent
L
Coen
D000818:Animals; D054337:Cell Dedifferentiation; D049109:Cell Proliferation; D015870:Gene Expression; D018014:Gene Transfer Techniques; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D007814:Larva / Q000166:cytology / Q000378:metabolism; D051379:Mice; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000378:metabolism*; D050814:Octamer Transcription Factor-3 / Q000096:biosynthesis* / Q000235:genetics; D055748:SOXB1 Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014982:Xenopus laevis
C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse