Tumor suppressors inhibit reprogramming of African spiny mouse ( Acomys ) fibroblasts to induced pluripotent stem cells

• Published in: Wellcome open research 2022, Vol.7, p.215
• Main Authors: Sandoval, Aaron Gabriel W, Maden, Malcolm, Bates, Lawrence E, Silva, Jose C R
• Format: Article
• Language: English
• Subjects: Acomys; African spiny mouse; dedifferentiation; eng; induced pluripotent stem cell; regeneration; reprogramming
• ISSN: 2398-502X; 2398-502X
• DOI: 10.12688/wellcomeopenres.18034.1

The African spiny mouse ( ) is an emerging mammalian model for scar-free regeneration, and further study of could advance the field of regenerative medicine. Isolation of pluripotent stem cells from would allow for development of transgenic or chimeric animals and study of regeneration; however, the reproductive biology of is not well characterized, complicating efforts to derive embryonic stem cells. Thus, we sought to generate induced pluripotent stem cells (iPSCs) by reprogramming somatic cells back to pluripotency. To generate iPSCs, we attempted to adapt established protocols developed in . We utilized a PiggyBac transposon system to genetically modify fibroblasts to overexpress the Yamanaka reprogramming factors as well as mOrange fluorescent protein under the control of a doxycycline-inducible TetON operon system. Reprogramming factor overexpression caused fibroblasts to undergo apoptosis or senescence. When SV40 Large T antigen (SV40 LT) was added to the reprogramming cocktail, cells were able to dedifferentiate into pre-iPSCs. Although use of 2iL culture conditions induced formation of colonies resembling PSCs, these iPS-like cells lacked pluripotency marker expression and failed to form embryoid bodies. An EOS-GiP system was unsuccessful in selecting for bona fide iPSCs; however, inclusion of Nanog in the reprogramming cocktail along with 5-azacytidine in the culture medium allowed for generation of iPSC-like cells with increased expression of several naïve pluripotency markers. There are significant roadblocks to reprogramming cells, necessitating future studies to determine -specific reprogramming factor and/or culture condition requirements. The requirement for SV40 LT during dedifferentiation may suggest that tumor suppressor pathways play an important role in regeneration and that may possess unreported cancer resistance.