Drug-resistant colonies expressing red fluorescent selleck compound protein and green fluorescent protein appeared on day 2, as illustrated in Fig. 1A. After 2 weeks of selection, the double-drug-resistant, dual-fluorescent colonies of ES-Hepa hybrids established a flattened and less compact appearance (Fig. 1B). Analysis of chromosome spreads and DNA content from 20 colonies indicated that the hybrid contained a near-tetraploid chromosome complement of 80 and 4n DNA content, demonstrating that the stable cell hybrid contained both the ES and the cancer cell chromosomes in a single cell (Fig. 1, C and D). The pluripotent genes Oct4, Nanog, Sox2, and Rex-1, which were silenced in Hepa1�C6 cells, were increased to a level similar to those of ES cells after fusion (Fig.

1E), and the tissue-specific genes Ttr and Alb in the ES-Hepa hybrids were obviously abolished compared with those in Hepa1�C6 cells (Fig. 1E). Also, the expression level of tumor-related genes, such as Bcl2, Bad, Bax, Cdkn1a, Rassf1, c-fos, Bmi1, Ezh2, and Eed in the ES-Hepa hybrids were similar to that of ES cells, except for the c-Jun oncogene (Fig. 1F). These results suggested that the ES-Hepa hybrids had ES-like potency, and the Hepa1�C6 hybrid counterparts lost their gene expression pattern. FIGURE 1. Generation of the ES-Hepa hybrid cells. A, schematic illustration of hybrid generation. ES cells and the Hepa1�C6 cells were stably transfected with drug-resistant and fluorescent markers and then induced to fuse by the addition of polyethylene … We adopted RT-PCR and real-time PCR to examine the expression level of p16INK4a in ES, Hepa1�C6, and ES-Hepa hybrid cells.

The p16INK4a gene was expressed in the ES and ES-Hepa hybrid cells, whereas it was silenced in the Hepa1�C6 cells (Fig. 2A). To confirm the reactivation of silenced p16INK4a in Hepa1�C6 cells, we measured the allelic expression of p16INK4a by using RNA fluorescence in situ hybridization. In the GSK-3 ES-Hepa hybrids, four dot signals were obvious per nuclei; these cells were in sharp contrast with the lack of signal in the Hepa1�C6 cells, demonstrating the reactivation of silenced p16INK4a in Hepa1�C6 cells by cell fusion (Fig. 2B). Further, we examined the SNP in the transcribed region of p16INK4a in ES, Hepa1�C6, and ES-Hepa hybrid cells. In this research, an adenine residue in the ES cells genome was different from a guanine residue in the Hepa1�C6 genome. Sequencing results of exon2 of p16INK4a in the ES-Hepa hybrids showed mixed transcription sequences originating from both ES and Hepa1�C6 cells, demonstrating the reactivation of p16INK4a derived from Hepa1�C6 cells (Fig. 2C).