ES cell extract-induced expression of pluripotent factors in somatic cells.
Reprogramming of somatic cells was induced by ES cell-free extract. The system relied on the transient uptake of regulatory components from a nuclear and cytoplasmic extract derived from ES cells by the nucleus of a reversibly permeabilized NIH3T3 cell. NIH3T3 cells were permeabilized by streptolysin O (SLO). Reprogramming cell-free extracts were prepared by repeatedly freezing and thawing ES cells in liquid nitrogen. After incubation in the extract for 1 hr, permeabilized NIH3T3 cells were resealed by CaCl(2) and continually cultured for weeks to assess expression of ES cell specific markers. As we observed using FACS and fluorescence microscope, the optimal SLO concentration for permeabilizing NIH3T3 cells was 25 U. After 2 weeks of culture, the treated NIH3T3 cells began to express Nanog, c-Myc, Klf4, and 6 weeks later Oct4 was detectable. However, Sox2 was detected only after 8 weeks of culture. Differentiated somatic cells could be reprogrammed in ES extract in vitro, which provides a new approach to decreasing differentiation levels in somatic cells without disturbing the DNA sequences.
Xu
Yan-Ning
YN
Department of Histology and Embryology, Harbin Medical University, Nangang District, Harbin 150081, Heilongjiang Province, China.
Guan
Na
N
Wang
Zhen-Dong
ZD
Shan
Zhi-Yan
ZY
Shen
Jing-Ling
JL
Zhang
Qing-Hua
QH
Jin
Lian-Hong
LH
Lei
Lei
L
D000818:Animals; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation; D002457:Cell Extracts / Q000737:chemistry / Q000494:pharmacology; D065150:Cellular Reprogramming; D015536:Down-Regulation / Q000187:drug effects*; D053595:Embryonic Stem Cells / Q000737:chemistry* / Q000502:physiology; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects / Q000502:physiology; D018398:Homeodomain Proteins / Q000378:metabolism; D000090062:Kruppel-Like Factor 4; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000502:physiology*; D016271:Proto-Oncogene Proteins c-myc / Q000378:metabolism; D055748:SOXB1 Transcription Factors / Q000378:metabolism; D013997:Time Factors
D015415:Biomarkers; D002457:Cell Extracts; 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