Generation of Footprint-Free Canine Induced Pluripotent Stem Cells Using Auto-Erasable Sendai Virus Vector

Abstract

Canine induced pluripotent stem cells (ciPSCs) can be used in regenerative medicine. However, there are no reports on the generation of genome integration-free and completely exogenous gene-silenced (footprint free) ciPSCs that are tolerant to enzymatic single-cell passage. In this study, we reprogrammed canine embryonic fibroblasts using the auto-erasable replication-defective and persistent Sendai virus vector, SeVdp(KOSM)302L, and generated two ciPSC lines. The ciPSCs were positive for pluripotent markers, including alkaline phosphatase activity as well as OCT3/4, SOX2, and NANOG transcripts, and NANOG, stage-specific embryonic antigen-1, and partial TRA-1-60 protein expression, even after SeVdp(KOSM)302L removal. The ciPSCs were induced to differentiate into all the three germ layers as embryoid bodies in vitro and as teratomas in vivo. Furthermore, SeVdp(KOSM)302L-free ciPSCs maintained a normal karyotype even after repeated enzymatic single-cell passaging. Therefore, to our knowledge, for the first time, we demonstrated the generation of footprint-free and high-quality ciPSCs that can be passaged at the single-cell stage using enzymatic methods. Our method for generation of ciPSCs is a good step toward the development of clinical application of ciPSCs.

Use of a Sendai virus-based vector for effcient transduction of pinniped fbroblasts

“…Векторы на основе вируса Сендай эффективно заражают различные клетки млекопитающих и не встраиваются в геном, так как функционируют в цитоплазме (Li et al, 2000 Change of the fluorescence of fibroblasts transduced with CytoTuneEmGFR, % векторов -после первой трансдукции клетки приобрета ют резистентность к вирусу. Ранее с помощью векторов на основе вируса Сендай были получены ИПСК человека (Fusaki et al, 2009), макакикрабоеда (Coppiello et al, 2017), макакирезус (Lu et al, 2013), шимпанзе (Fugie et al, 2014, собаки (Tsukamoto et al, 2018) и мыши (Tucker et al, 2013). Описаны также ИПСКподобные колонии байкальской нерпы и сивуча (Борода и др., 2015), хотя из статьи неясно, использовали ли авторы векторы на основе ретровирусов или Сендай.…”

Oocytes, embryos and pluripotent stem cells from a biomedical perspective

“…In the subsequent work, researchers used mostly human or mouse OSKM reprogramming factors, occasionally with addition of LIN28 and NANOG (Whitworth et al, 2012), introduced using retroviral (Shimada et al, 2010;Koh et al, 2013;Baird et al, 2015) or lentiviral approaches (Lee et al, 2011;Luo et al, 2011;Whitworth et al, 2012;Nishimura et al, 2017;Gonçalves et al, 2017). Lastly, non-integrative Sendai virus have been attempted (Chow et al, 2017;Tsukamoto et al, 2018). Again, as earlier described for the pig, the silencing of the integrated transgenes in the canine iPSCs seems to represent a consistent problem and was only described in a few studies (Baird et al, 2015;Gonçalves et al, 2017).…”

“…The first information on potential canine iPSCs was reported some years after Yamanaka's breakthrough (Takahashi and Yamanaka, 2006), and the quest for deriving fully reprogrammed and stable canine iPSCs is still ongoing (Shimada et al, 2010;Lee et al, 2011;Luo et al, 2011;Whitworth et al, 2012;Koh et al, 2013;Baird et al, 2015;Nishimura et al, 2017;Gonçalves et al, 2017;Chow et al, 2017;Tsukamoto et al, 2018). In the first studies on canine iPSCs, canine reprograming factors were utilized for reprogramming (Shimada et al, 2010).…”

“…However, based on the expression of pluripotency markers, one can speculate that most of the generated cell lines represent a primed status, characterized by expression of markers such as SSEA4, TRA-1-60 and TRA-1-80 (Lee et al, 2011;Luo et al, 2011;Whitworth et al, 2012;Baird et al, 2015;Nishimura et al, 2017;Chow et al, 2017). Nevertheless, canine iPSCs expressing naïve pluripotency markers, like SSEA1, have also been described (Koh et al, 2013;Tsukamoto et al, 2018). The expression of pluripotency markers, however, may differ between species making it difficult to draw firm conclusions on the state of pluripotency just based upon such markers.…”

“…The expression of pluripotency markers, however, may differ between species making it difficult to draw firm conclusions on the state of pluripotency just based upon such markers. Overall, the potential canine iPSCs have been reported to show different combinations of classic pluripotency markers, such as OCT4, Nanog, SOX2, amongst others (for review, see Pessôa et al, in press), and some of these cells lines were also able to form teratomas (Lee et al, 2011;Whitworth et al, 2012;Koh et al, 2013;Gonçalves et al, 2017;Chow et al, 2017;Tsukamoto et al, 2018). Contribution of iPSCs to the development of chimeric embryos has, however, not been described so far.…”