The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines.
BACKGROUND
Induced pluripotent stem (iPS) cells can be generated from somatic cells by introducing the four transcription factors Oct4, Sox2, Klf4, and c-Myc. Given that iPS cell technology may be useful for medical applications, the quality of iPS cells needs to be maintained during prolonged cultivation. However, it is unclear whether there are any differences in stability among different iPS clones.


METHODS
We infected mouse embryonic and adult fibroblasts with retroviruses encoding Oct4, Sox2, Klf4, c-Myc, and green fluorescent protein (GFP). We obtained embryonic stem (ES) cell-like colonies with silenced retroviral transgenes and divided these colonies into two groups: ES cell-like colonies that underwent retroviral silencing (i) on around day 14 (called early iPS) or (ii) on around day 30 (called late iPS), after infection. We compared morphology, proliferation efficiency, pluripotency marker expression, and karyotype between early iPS and late iPS cells.


RESULTS
Early iPS cells were more stable than late iPS cells. At passage 20, most of the early iPS clones maintained ES cell-like morphology, expressed pluripotency markers, and showed proliferation efficiency similar to ES cells. Furthermore, early iPS clones derived from both embryonic and adult fibroblasts gave rise to chimeras and could show germ line competency. In contrast, late iPS clones tended to lose their ES cell-like morphology and normal karyotype in prolonged culture.


CONCLUSIONS
Our results provide useful information on the efficient derivation of stable iPS cells that may be useful for germline transmission in mouse. This study suggests that early completion of full reprogramming allows for superior iPS cell generation.
Okada
Minoru
M
Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.
Yoneda
Yoshihiro
Y
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D004622:Embryo, Mammalian / Q000166:cytology; D053595:Embryonic Stem Cells / Q000166:cytology / Q000378:metabolism*; D005260:Female; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D020869:Gene Expression Profiling; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D007621:Karyotyping; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008813:Mice, Inbred ICR; D008856:Microscopy, Fluorescence; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000235:genetics / Q000378:metabolism; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055748:SOXB1 Transcription Factors / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D014161:Transduction, Genetic
D018398:Homeodomain Proteins; C000715468:Klf4 protein, mouse; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors; C489428:Myc protein, mouse; D000071317:Nanog Homeobox Protein; C475540:Nanog protein, mouse; D050814:Octamer Transcription Factor-3; C494174:Pou5f1 protein, mouse; D016271:Proto-Oncogene Proteins c-myc; D055748:SOXB1 Transcription Factors; C527783:Sox2 protein, mouse; D049452:Green Fluorescent Proteins