title: Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming.
abstract: Fair comparison of reprogramming efficiencies and in vitro differentiation capabilities among induced pluripotent stem cell (iPSC) lines has been hampered by the cellular and genetic heterogeneity of de novo infected somatic cells. In order to address this problem, we constructed a single cassette all-in-one inducible lentiviral vector (Ai-LV) for the expression of three reprogramming factors (Oct3/4, Klf4 and Sox2). To obtain multiple types of somatic cells having the same genetic background, we generated reprogrammable chimeric mice using iPSCs derived from Ai-LV infected somatic cells. Then, hepatic cells, hematopoietic cells and fibroblasts were isolated at different developmental stages from the chimeric mice, and reprogrammed again to generate 2nd iPSCs. The results revealed that somatic cells, especially fetal hepatoblasts were reprogrammed 1200 times more efficiently than adult hepatocytes with maximum reprogramming efficiency reaching 12.5%. However, we found that forced expression of c-Myc compensated for the reduced reprogramming efficiency in aged somatic cells without affecting cell proliferation. All these findings suggest that the Ai-LV system enables us to generate a panel of iPSC clones derived from various tissues with the same genetic background, and thus provides an invaluable tool for iPSC research.
authors: Japan Science Technology Agency, ERATO, Nakauchi Stem Cell and Organ Regeneration Project, Tokyo, Japan. tomoyama@ims.u-tokyo.ac.jp
authors: Tomoyuki
authors: T
authors: Yamaguchi
authors: Sanae
authors: S
authors: Hamanaka
authors: Akihide
authors: A
authors: Kamiya
authors: Motohito
authors: M
authors: Okabe
authors: Mami
authors: M
authors: Kawarai
authors: Yukiko
authors: Y
authors: Wakiyama
authors: Ayumi
authors: A
authors: Umino
authors: Tomonari
authors: T
authors: Hayama
authors: Hideyuki
authors: H
authors: Sato
authors: Youn-Su
authors: YS
authors: Lee
authors: Megumi
authors: M
authors: Kato-Itoh
authors: Hideki
authors: H
authors: Masaki
authors: Toshihiro
authors: T
authors: Kobayashi
authors: Satoshi
authors: S
authors: Yamazaki
authors: Hiromitsu
authors: H
authors: Nakauchi
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D049109:Cell Proliferation; D004318:Doxycycline / Q000494:pharmacology; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D005821:Genetic Techniques; D005822:Genetic Vectors; D006412:Hematopoietic Stem Cells / Q000166:cytology; D022781:Hepatocytes / Q000166:cytology; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology*; D000090062:Kruppel-Like Factor 4; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008957:Models, Genetic; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D051381:Rats; D013997:Time Factors
chemical_list: C000715467:KLF4 protein, human; C000715468:Klf4 protein, mouse; C000715469:Klf4 protein, rat; D000090062:Kruppel-Like Factor 4; D016271:Proto-Oncogene Proteins c-myc; D004318:Doxycycline