Effective culture conditions for the induction of pluripotent stem cells.
BACKGROUND
Induced pluripotent stem (iPS) cells, which are functionally comparable to embryonic stem (ES) cells, can be generated from mouse fibroblasts by expression of a defined set of transcription factors Oct4, Sox2, Klf4, and c-Myc. Since iPS cells are generated from somatic cells, they provide an invaluable source of pluripotent stem cells for cell transplantation therapy that does not present ethical problems. However, the reprogramming efficiency is extremely low, and optimal culture conditions for iPS cell derivation have not been clearly defined.


METHODS
To generate iPS cells efficiently, we tested 10 different culture conditions: DMEM supplemented with 15% fetal bovine serum (FBS), Knockout DMEM with 15% FBS from Invitrogen, Equitech, or HyClone, DMEM with 15% Knockout Serum Replacement (KSR), and Knockout DMEM with 10%, 15%, 20%, 25%, or 35% KSR. These media all contain 2 mM L-glutamine, 100 μM nonessential amino acids, 100 μM beta-mercaptoethanol, 1000 units ml⁻¹ leukemia inhibitory factor (LIF), 50 units ml⁻¹ penicillin, and 50 μg ml⁻¹ streptomycin.


RESULTS
Medium containing Knockout DMEM with 20% KSR permits efficient induction of iPS cells from both mouse embryonic fibroblasts (MEFs) and adult tail tip fibroblasts (TTFs). Mouse iPS cells generated in the condition express ES cell marker genes such as Oct4, Sox2, Rex1, and Nanog at levels comparable to those of ES cells. Furthermore, iPS cells derived form MEFs and adult TTFs can contribute to adult chimeras.


CONCLUSIONS
Our iPS cell induction efficiency is greater than that described in other reports.


CONCLUSIONS
These findings provide an important catalyst for examining different culture environments for the generation of iPS cells.
Okada
Minoru
M
Biomolecular Networks Laboratories, Department of Frontier Biosciences, Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
Oka
Masahiro
M
Yoneda
Yoshihiro
Y
D000818:Animals; D000943:Antigens, Differentiation / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D002478:Cells, Cultured; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism*; D000090062:Kruppel-Like Factor 4; D051379:Mice; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
D000943:Antigens, Differentiation; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D014157:Transcription Factors