title: Efficient generation of integration-free human induced pluripotent stem cells from keratinocytes by simple transfection of episomal vectors.
abstract: Keratinocytes represent an easily accessible cell source for derivation of human induced pluripotent stem (hiPS) cells, reportedly achieving higher reprogramming efficiency than fibroblasts. However, most studies utilized a retroviral or lentiviral method for reprogramming of keratinocytes, which introduces undesirable transgene integrations into the host genome. Moreover, current protocols of generating integration-free hiPS cells from keratinocytes are mostly inefficient. In this paper, we describe a more efficient, simple-to-use, and cost-effective method for generating integration-free hiPS cells from keratinocytes. Our improved method using lipid-mediated transfection achieved a reprogramming efficiency of ∼0.14% on average. Keratinocyte-derived hiPS cells showed no integration of episomal vectors, expressed stem cell-specific markers and possessed potentials to differentiate into all three germ layers by in vitro embryoid body formation as well as in vivo teratoma formation. To our knowledge, this represents the most efficient method to generate integration-free hiPS cells from keratinocytes.
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA;
authors.forename: Yulan
authors.initials: Y
authors.lastname: Piao
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA; Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, Australia;
authors.forename: Sandy Shen-Chi
authors.initials: SS
authors.lastname: Hung
authors.affiliation: O'Brien Institute and University of Melbourne, Department of Surgery, St. Vincent's Hospital, Australia;
authors.forename: Shiang Y
authors.initials: SY
authors.lastname: Lim
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA; Centre for Eye Research Australia, Department of Ophthalmology, University of Melbourne, Australia;
authors.forename: Raymond Ching-Bong
authors.initials: RC
authors.lastname: Wong
authors.affiliation: Laboratory of Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA; Department of Systems Medicine, Sakaguchi Laboratory, Keio University School of Medicine, Shinjuku, Tokyo, Japan KoM@z7.keio.jp.
authors.forename: Minoru S H
authors.initials: MS
authors.lastname: Ko
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D065150:Cellular Reprogramming; D018920:Coculture Techniques; D058732:Embryoid Bodies / Q000378:metabolism; D061252:Feeder Cells; D005347:Fibroblasts / Q000378:metabolism; D018507:Gene Expression Regulation, Developmental; D005822:Genetic Vectors; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism*; D015603:Keratinocytes / Q000378:metabolism*; D051379:Mice; D010957:Plasmids / Q000235:genetics*; D013724:Teratoma / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods*
chemical_list: D014157:Transcription Factors
keywords: Derivation of human induced pluripotent stem cells; Episomal vectors; Integration-free; Keratinocytes; Lipid mediated transfection