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

Wellcome Open Research

article

Aaron Gabriel W. Sandoval¹  Malcolm Maden³  Lawrence E. Bates¹  Jose C.R. Silva¹ 

[1] Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge;Department of Biochemistry, University of Cambridge;Department of Biology & UF Genetics Institute, University of Florida;MRC Human Genetics Unit, Institute of Genetics and Cancer, University of Edinburgh;Guangzhou Laboratory, Guangzhou International Bio Island, Guangzhou 510005, Guangdong Province

关键词: African spiny mouse;    Acomys;    regeneration;    reprogramming;    induced pluripotent stem cell;    dedifferentiation;    SV40 Large T antigen;    tumor suppressor;   

DOI  :  10.12688/wellcomeopenres.18034.1

学科分类：内科医学

来源: Wellcome

【 摘 要 】

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

【 授权许可】

CC BY