The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines
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. How...
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| Published in | Biochimica et biophysica acta Vol. 1810; no. 2; pp. 226 - 235 |
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| Format | Journal Article |
| Language | English |
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01.02.2011
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| Abstract | 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.
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.
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.
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. |
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| AbstractList | 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.BACKGROUNDInduced 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.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.METHODSWe 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.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.RESULTSEarly 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.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.GENERAL SIGNIFICANCEOur 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. 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. 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. 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. 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. 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. GENERAL SIGNIFICANCE: 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. |
| Author | Yoneda, Yoshihiro Okada, Minoru |
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| Snippet | 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... BACKGROUND: Induced pluripotent stem (iPS) cells can be generated from somatic cells by introducing the four transcription factors Oct4, Sox2, Klf4, and c-Myc.... |
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| SubjectTerms | adults Animals Cell Line Cells, Cultured chimerism clones Embryo, Mammalian - cytology Embryonic Stem Cells - cytology Embryonic Stem Cells - metabolism Female fibroblasts Fibroblasts - cytology Fibroblasts - metabolism Gene Expression Profiling germ cells green fluorescent protein Green Fluorescent Proteins - genetics Green Fluorescent Proteins - metabolism Homeodomain Proteins - genetics Homeodomain Proteins - metabolism Induced Pluripotent Stem Cells - cytology Induced Pluripotent Stem Cells - metabolism Karyotyping Kruppel-Like Transcription Factors - genetics Kruppel-Like Transcription Factors - metabolism Male Mice Mice, Inbred C57BL Mice, Inbred ICR Microscopy, Fluorescence Nanog Homeobox Protein Octamer Transcription Factor-3 - genetics Octamer Transcription Factor-3 - metabolism Proto-Oncogene Proteins c-myc - genetics Proto-Oncogene Proteins c-myc - metabolism Retroviridae Retroviridae - genetics Reverse Transcriptase Polymerase Chain Reaction somatic cells SOXB1 Transcription Factors - genetics SOXB1 Transcription Factors - metabolism stem cells Time Factors transcription factors Transduction, Genetic transgenes |
| Title | The timing of retroviral silencing correlates with the quality of induced pluripotent stem cell lines |
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