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.
(c) 2009 Wiley-Liss, Inc.