title: Removal of reprogramming transgenes improves the tissue reconstitution potential of keratinocytes generated from human induced pluripotent stem cells.
abstract: Human induced pluripotent stem cell (hiPSC) lines have a great potential for therapeutics because customized cells and organs can be induced from such cells. Assessment of the residual reprogramming factors after the generation of hiPSC lines is required, but an ideal system has been lacking. Here, we generated hiPSC lines from normal human dermal fibroblasts with piggyBac transposon bearing reprogramming transgenes followed by removal of the transposon by the transposase. Under this condition, we compared the phenotypes of transgene-residual and -free hiPSCs of the same genetic background. The transgene-residual hiPSCs, in which the transcription levels of the reprogramming transgenes were eventually suppressed, were quite similar to the transgene-free hiPSCs in a pluripotent state. However, after differentiation into keratinocytes, clear differences were observed. Morphological, functional, and molecular analyses including single-cell gene expression profiling revealed that keratinocytes from transgene-free hiPSC lines were more similar to normal human keratinocytes than those from transgene-residual hiPSC lines, which may be partly explained by reactivation of residual transgenes upon induction of keratinocyte differentiation. These results suggest that transgene-free hiPSC lines should be chosen for therapeutic purposes.
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan k.igawa.derm@tmd.ac.jp takeda@mr-envi.med.osaka-u.ac.jp.
authors.forename: Ken
authors.initials: K
authors.lastname: Igawa
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Chikara
authors.initials: C
authors.lastname: Kokubu
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Kosuke
authors.initials: K
authors.lastname: Yusa
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Kyoji
authors.initials: K
authors.lastname: Horie
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Yoshimura
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Kaori
authors.initials: K
authors.lastname: Yamauchi
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Suemori
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Hiroo
authors.initials: H
authors.lastname: Yokozeki
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Masashi
authors.initials: M
authors.lastname: Toyoda
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Nobutaka
authors.initials: N
authors.lastname: Kiyokawa
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Okita
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Yoshitaka
authors.initials: Y
authors.lastname: Miyagawa
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Hidenori
authors.initials: H
authors.lastname: Akutsu
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Akihiro
authors.initials: A
authors.lastname: Umezawa
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan.
authors.forename: Ichiro
authors.initials: I
authors.lastname: Katayama
authors.affiliation: Department of Dermatology and Department of Social and Environmental Medicine, Graduate School of Medicine, Osaka University, Osaka, Japan; Department of Dermatology, Graduate School of Medicine, Tokyo Medical and Dental University, Tokyo, Japan; Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge, United Kingdom; Department of Embryonic Stem Cell Research, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan; Department of Reproductive Biology, Center for Regenerative Medicine, National Center for Child Health and Development, Tokyo, Japan k.igawa.derm@tmd.ac.jp takeda@mr-envi.med.osaka-u.ac.jp.
authors.forename: Junji
authors.initials: J
authors.lastname: Takeda
mesh_terms: D015139:Blotting, Southern; D002454:Cell Differentiation / Q000235:genetics; D065150:Cellular Reprogramming / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D007150:Immunohistochemistry; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D015603:Keratinocytes / Q000166:cytology*; D060888:Real-Time Polymerase Chain Reaction; D023822:Tissue Engineering / Q000379:methods*; D019076:Transgenes
keywords: Differentiation; Human; Induced pluripotent stem cell; Keratinocyte; Transgene