Sandoval Aaron Gabriel W, Maden Malcolm, Bates Lawrence E, Silva Jose C R
Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, CB2 0AW, UK.
Department of Biochemistry, University of Cambridge, Cambridge, CB2 1GA, UK.
Wellcome Open Res. 2022 Aug 18;7:215. doi: 10.12688/wellcomeopenres.18034.1. eCollection 2022.
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.
非洲刺毛鼠是一种用于无瘢痕再生研究的新兴哺乳动物模型，对其进一步研究可能推动再生医学领域的发展。从非洲刺毛鼠中分离多能干细胞将有助于转基因或嵌合动物的培育以及再生研究；然而，非洲刺毛鼠的生殖生物学特性尚未得到充分表征，这使得获取胚胎干细胞的工作变得复杂。因此，我们试图通过将体细胞重编程为多能性来生成非洲刺毛鼠诱导多能干细胞（iPSCs）。为了生成非洲刺毛鼠iPSCs，我们尝试采用在其他物种中建立的既定方案。我们利用PiggyBac转座子系统对非洲刺毛鼠成纤维细胞进行基因改造，使其在强力霉素诱导型TetON操纵子系统的控制下过表达山中伸弥重编程因子以及mOrange荧光蛋白。重编程因子的过表达导致非洲刺毛鼠成纤维细胞发生凋亡或衰老。当将SV40大T抗原（SV40 LT）添加到重编程混合物中时，非洲刺毛鼠细胞能够去分化为前iPSCs。尽管使用2iL培养条件诱导形成了类似非洲刺毛鼠多能干细胞的集落，但这些类似iPS的细胞缺乏多能性标记物表达，也无法形成胚状体。一种EOS - GiP系统在筛选真正的非洲刺毛鼠iPSCs方面未获成功；然而，在重编程混合物中加入Nanog以及在培养基中加入5 - 氮杂胞苷，使得能够生成几种幼稚多能性标记物表达增加的类似非洲刺毛鼠iPS的细胞。对非洲刺毛鼠细胞进行重编程存在重大障碍，需要未来的研究来确定非洲刺毛鼠特异性的重编程因子和/或培养条件要求。在非洲刺毛鼠去分化过程中对SV40 LT的需求可能表明肿瘤抑制途径在非洲刺毛鼠再生中起重要作用，并且非洲刺毛鼠可能具有未被报道的抗癌能力。
