title: Optimal ratio of transcription factors for somatic cell reprogramming.
abstract: Somatic cell reprogramming is achieved by four reprogramming transcription factors (RTFs), Oct3/4, Sox2, Klf4, and c-Myc. However, in addition to the induction of pluripotent cells, these RTFs also generate pseudo-pluripotent cells, which do not show Nanog promoter activity. Therefore, it should be possible to fine-tune the RTFs to produce only fully pluripotent cells. For this study, a tagging system was developed to sort induced pluripotent stem (iPS) cells according to the expression levels of each of the four RTFs. Using this system, the most effective ratio (Oct3/4-high, Sox2-low, Klf4-high, c-Myc-high) of the RTFs was 88 times more efficient at producing iPS cells than the worst effective ratio (Oct3/4-low, Sox2-high, Klf4-low, c-Myc-low). Among the various RTF combinations, Oct3/4-high and Sox2-low produced the most efficient results. To investigate the molecular basis, microarray analysis was performed on iPS cells generated under high (Oct3/4-high and Sox2-low) and low (Oct3/4-low and Sox2-high) efficiency reprogramming conditions. Pathway analysis revealed that the G protein-coupled receptor (GPCR) pathway was up-regulated significantly under the high efficiency condition and treatment with the chemokine, C-C motif ligand 2, a member of the GPCR family, enhanced somatic cell reprogramming 12.3 times. Furthermore, data from the analysis of the signature gene expression profiles of mouse embryonic fibroblasts at 2 days after RTF infection revealed that the genetic modifier, Whsc1l1 (variant 1), also improved the efficiency of somatic cell reprogramming. Finally, comparison of the overall gene expression profiles between the high and low efficiency conditions will provide novel insights into mechanisms underlying somatic cell reprogramming.
authors.affiliation: Department of Cell Differentiation, The Sakaguchi Laboratory, School of Medicine, Keio University, Tokyo 160-8582, Japan. gonag@z2.keio.jp
authors.forename: Go
authors.initials: G
authors.lastname: Nagamatsu
authors.forename: Shigeru
authors.initials: S
authors.lastname: Saito
authors.forename: Takeo
authors.initials: T
authors.lastname: Kosaka
authors.forename: Keiyo
authors.initials: K
authors.lastname: Takubo
authors.forename: Taisuke
authors.initials: T
authors.lastname: Kinoshita
authors.forename: Mototsugu
authors.initials: M
authors.lastname: Oya
authors.forename: Katsuhisa
authors.initials: K
authors.lastname: Horimoto
authors.forename: Toshio
authors.initials: T
authors.lastname: Suda
mesh_terms: D000818:Animals; D054337:Cell Dedifferentiation; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000235:genetics; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D008822:Mice, Transgenic; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000235:genetics; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
chemical_list: C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D011518:Proto-Oncogene Proteins; D014157:Transcription Factors