Development of an all-in-one inducible lentiviral vector for gene specific analysis of reprogramming
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 a...
Saved in:
Published in | PloS one Vol. 7; no. 7; p. e41007 |
---|---|
Main Authors | , , , , , , , , , , , , , , |
Format | Journal Article |
Language | English |
Published |
United States
Public Library of Science
18.07.2012
Public Library of Science (PLoS) |
Subjects | |
Online Access | Get full text |
Cover
Loading…
Summary: | 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. |
---|---|
Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 Current address: Institute of Innovative Science and Technology, Tokai University, Kanagawa, Japan Conceived and designed the experiments: TY. Performed the experiments: TY SH AK MO MK YW AU TH HS YSL MKI HM. Analyzed the data: TY. Contributed reagents/materials/analysis tools: TY SH. Wrote the paper: TY TK SY. Final approval of the manuscript: TY HN. |
ISSN: | 1932-6203 1932-6203 |
DOI: | 10.1371/journal.pone.0041007 |