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Stem Cells Dev. 2018 Nov 15;27(22):1577-1586. doi: 10.1089/scd.2018.0084. Epub 2018 Oct 11.

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

Author information

1
1 Department of Advanced Pathobiology, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Izumisano, Japan .
2
2 Institute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine , Stanford, California.
3
3 Division of Stem Cell Therapy, Institute of Medical Science, University of Tokyo , Tokyo, Japan .
4
4 Department of Integrated Structural Biosciences, Graduate School of Life and Environmental Sciences, Osaka Prefecture University , Izumisano, Japan .
5
5 Biotechnology Research Institute for Drug Discovery, National Institute of Advanced Industrial Science and Technology (AIST) , Tsukuba, Japan.
6
6 Laboratory of Gene Regulation, Faculty of Medicine, University of Tsukuba , Tsukuba, Japan .

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.

KEYWORDS:

Sendai virus; canine; induced pluripotent stem cells; integration free; single-cell passaging

PMID:
30215317
DOI:
10.1089/scd.2018.0084
[Indexed for MEDLINE]

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