title: A defined xeno-free and feeder-free culture system for the derivation, expansion and direct differentiation of transgene-free patient-specific induced pluripotent stem cells.
abstract: A defined xeno-free system for patient-specific iPSC derivation and differentiation is required for translation to clinical applications. However, standard somatic cell reprogramming protocols rely on using MEFs and xenogeneic medium, imposing a significant obstacle to clinical translation. Here, we describe a well-defined culture system based on xeno-free media and LN521 substrate which supported i) efficient reprogramming of normal or diseased skin fibroblasts from human of different ages into hiPSCs with a 15-30 fold increase in efficiency over conventional viral vector-based method; ii) long-term self-renewal of hiPSCs; and iii) direct hiPSC lineage-specific differentiation. Using an excisable polycistronic vector and optimized culture conditions, we achieved up to 0.15%-0.3% reprogramming efficiencies. Subsequently, transgene-free hiPSCs were obtained by Cre-mediated excision of the reprogramming factors. The derived iPSCs maintained long-term self-renewal, normal karyotype and pluripotency, as demonstrated by the expression of stem cell markers and ability to form derivatives of three germ layers both in vitro and in vivo. Importantly, we demonstrated that Parkinson's patient transgene-free iPSCs derived using the same system could be directed towards differentiation into dopaminergic neurons under xeno-free culture conditions. Our approach provides a safe and robust platform for the generation of patient-specific iPSCs and derivatives for clinical and translational applications.
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore. Electronic address: hflu@ibn.a-star.edu.sg.
authors.forename: Hong Fang
authors.initials: HF
authors.lastname: Lu
authors.affiliation: Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
authors.forename: Chou
authors.initials: C
authors.lastname: Chai
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore.
authors.forename: Tze Chiun
authors.initials: TC
authors.lastname: Lim
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore.
authors.forename: Meng Fatt
authors.initials: MF
authors.lastname: Leong
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore.
authors.forename: Jia Kai
authors.initials: JK
authors.lastname: Lim
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore.
authors.forename: Shujun
authors.initials: S
authors.lastname: Gao
authors.affiliation: Duke-NUS Graduate Medical School, Singapore 169857, Singapore; National Neuroscience Institute, Singapore 308433, Singapore.
authors.forename: Kah Leong
authors.initials: KL
authors.lastname: Lim
authors.affiliation: Institute of Bioengineering and Nanotechnology, 31 Biopolis Way, The Nanos, Singapore 138669, Singapore. Electronic address: awan@ibn.a-star.edu.sg.
authors.forename: Andrew C A
authors.initials: AC
authors.lastname: Wan
mesh_terms: D000328:Adult; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002454:Cell Differentiation; D049109:Cell Proliferation; D065150:Cellular Reprogramming; D059290:Dopaminergic Neurons / Q000166:cytology / Q000378:metabolism; D061252:Feeder Cells / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology; D005822:Genetic Vectors / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D016086:Lentivirus / Q000378:metabolism; D051379:Mice; D016513:Mice, SCID; D010300:Parkinson Disease / Q000473:pathology; D019076:Transgenes
keywords: Direct differentiation; Expansion; Laminin 521; Reprogramming; Xeno-free culture; iPSC