Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome

Abstract

Induced pluripotent stem cells (iPSC) have been generated from somatic cells by introducing reprogramming factors. Integration of foreign genes into the host genome is a technical hurdle for the clinical application. Here, we show that Sendai virus (SeV), an RNA virus and carries no risk of altering host genome, is an efficient solution for generating safe iPSC. Sendai-viral human iPSC expressed pluripotency genes, showed demethylation characteristic of reprogrammed cells. SeV-derived transgenes were decreased during cell division. Moreover, viruses were able to be easily removed by antibody-mediated negative selection utilizing cell surface marker HN that is expressed on SeV-infected cells. Viral-free iPSC differentiated to mature cells of the three embryonic germ layers in vivo and in vitro including beating cardiomyocytes, neurons, bone and pancreatic cells. Our data demonstrated that highly-efficient, non-integrating SeV-based vector system provides a critical solution for reprogramming somatic cells and will accelerate the clinical application.

Reprogramming Mouse Fibroblasts with piggyBac Transposons

“…Therefore, a significant effort was put into finding reprogramming methods that had no transgene insertion left in the genome of the reprogrammed cells. In 2009-2010, numerous novel methods were published, including removable transposon-based transgene delivery (Woltjen et al 2009), nonintegrating virus transduction (Sendai [Fusaki et al 2009] or adenovirus [Zhou and Freed 2009]), episomal transfection (Yu et al 2007), and protein and modified RNA transduction (Zhou and Freed 2009;Warren et al 2010).…”

Reprogramming and Carcinogenesis—Parallels and Distinctions

“…Reprogramming has been successfully achieved by Fusaki and colleagues using Sendai virus-based delivery of Yamanaka factors (non-integrating RNA-virus) (Fusaki et al, 2009 Minicircle vectors-based reprogramming was for the first time described in 2010 (Jia et al, 2010). Minicircle vectors are cicular, nonviral DNA elements that are generated by PhiC31 integrase-based intramolecular recombination from parental plasmids that contain sequences of interest (i.e.…”

Direct reprogramming of somatic cells: an update

“…The first effort used F-deficient Sendai virus particles to induce pluripotency in somatic cells (Dowey et al, 2012;Fusaki et al, 2009). iPSCs produced using this method must be sub-cultured for 10-20 passages to remove the excess virus particles and to make virusfree iPSC lines.…”