title: Sequential introduction of reprogramming factors reveals a time-sensitive requirement for individual factors and a sequential EMT-MET mechanism for optimal reprogramming.
abstract: Present practices for reprogramming somatic cells to induced pluripotent stem cells involve simultaneous introduction of reprogramming factors. Here we report that a sequential introduction protocol (Oct4-Klf4 first, then c-Myc and finally Sox2) outperforms the simultaneous one. Surprisingly, the sequential protocol activates an early epithelial-to-mesenchymal transition (EMT) as indicated by the upregulation of Slug and N-cadherin followed by a delayed mesenchymal-to-epithelial transition (MET). An early EMT induced by 1.5-day TGF-β treatment enhances reprogramming with the simultaneous protocol, whereas 12-day treatment blocks reprogramming. Consistent results were obtained when the TGF-β antagonist Repsox was applied in the sequential protocol. These results reveal a time-sensitive role of individual factors for optimal reprogramming and a sequential EMT-MET mechanism at the start of reprogramming. Our studies provide a rationale for further optimizing reprogramming, and introduce the concept of a sequential EMT-MET mechanism for cell fate decision that should be investigated further in other systems, both in vitro and in vivo.
authors.affiliation: CAS Key Laboratory of Regenerative Biology, South China Institute for Stem Cell Biology and Regenerative Medicine, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou 510530, China.
authors.forename: Xiaopeng
authors.initials: X
authors.lastname: Liu
authors.forename: Hao
authors.initials: H
authors.lastname: Sun
authors.forename: Jing
authors.initials: J
authors.lastname: Qi
authors.forename: Linli
authors.initials: L
authors.lastname: Wang
authors.forename: Songwei
authors.initials: S
authors.lastname: He
authors.forename: Jing
authors.initials: J
authors.lastname: Liu
authors.forename: Chengqian
authors.initials: C
authors.lastname: Feng
authors.forename: Chunlan
authors.initials: C
authors.lastname: Chen
authors.forename: Wen
authors.initials: W
authors.lastname: Li
authors.forename: Yunqian
authors.initials: Y
authors.lastname: Guo
authors.forename: Dajiang
authors.initials: D
authors.lastname: Qin
authors.forename: Guangjin
authors.initials: G
authors.lastname: Pan
authors.forename: Jiekai
authors.initials: J
authors.lastname: Chen
authors.forename: Duanqing
authors.initials: D
authors.lastname: Pei
authors.forename: Hui
authors.initials: H
authors.lastname: Zheng
mesh_terms: D000818:Animals; D015820:Cadherins / Q000378:metabolism; D002454:Cell Differentiation; D002478:Cells, Cultured; D065150:Cellular Reprogramming; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D058750:Epithelial-Mesenchymal Transition; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D057026:Induced Pluripotent Stem Cells / Q000378:metabolism / Q000473:pathology*; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D051379:Mice; D050814:Octamer Transcription Factor-3 / Q000378:metabolism*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism*; D011720:Pyrazoles / Q000494:pharmacology; D011725:Pyridines / Q000494:pharmacology; D055748:SOXB1 Transcription Factors / Q000378:metabolism*; D016212:Transforming Growth Factor beta / Q000378:metabolism; D014945:Wound Healing
chemical_list: D015820:Cadherins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D011720:Pyrazoles; D011725:Pyridines; C550621:RepSox; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D016212:Transforming Growth Factor beta; D049452:Green Fluorescent Proteins