Hybrid cells generated by fusion of embryonic stem cells with di-and tetraploid fibroblasts have alternative parental phenotypes

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Hybrid cells generated by fusion of embryonic stem cells (ESCs) with diploid somatic cells have the phenotype and level of pluripotency that are comparable to those of ESCs, including the capacity for forming chimeras [1, 2]. The basis of the dominance of the ESC genome in the hybrid cells is reprogramming of genome of the diploid somatic partner (fibroblast, thymocyte, etc.), which converts the profile of the activities of its genes into the profile similar to that of ESCs. The character of gene expression of the pluripotent partner in the hybrid cells of this type slightly differs from its character in the parental ESCs [3‐5]. This unidirectional dominance of the ESC genome over the somatic genome is an unusual phenomenon, because both genomes are in the same nucleus, where they may exchange trans -regulating signals. As a first step in the study of the phenomenon of dominance of the ESC phenotype in the hybrid cells, we decided to evaluate the effect of the ploidy of cells of the somatic partner on the phenotype of hybrid cells by comparison of the characteristics of hybrids generated by fusion of di- and tetraploid fibroblasts with diploid ESCs. The experiments performed allowed us to discover alternative dominance of parental genomes in the hybrid cells, which depends on the ploidy of the somatic partner: the cell hybrids formed by a diploid ESC and a diploid fibroblast had the characteristics of an ESC and, conversely, hybrid cells formed by a diploid ESC and a tetraploid fibroblast had the characteristics of a fibroblast. Deciphering of the mechanisms of the all-or-nothing “switching” between the dominances of the two genomes may help to control this process. Cultures of diploid fetal fibroblasts were obtained by the standard method [6] from 12-day-old embryos of DD/c mice (strain dMEF) and C57Bl/6-I(I)1RK mice with a heterozygous double insertion in chromosome 1, which was previously described in [7] (the iMEF strain). A primary culture of embryonic fibroblasts proliferates for a limited time period. However, in rare cases, the cells of primary cultures obtain the capacity for unlimited proliferation, which is frequently accompanied by tetraploidy. These cells were used to obtain tetraploid dtMEF fibroblasts from the dMEF strain and itMEF cells from the iMEF strain, the proportion of cells with a tetraploid set of chromosomes reaching 95%. In the cell hybridization experiments, we used ESC line E14Tg2aSc4TP6.3 with a deficient activity of hypoxanthine phosphoribosyltransferase (HPRT) and transformed with a construct that contained the genes of green fluorescent protein (GFP) and puromycin resistance [8]. To induce cell fusion, growing diploid or tetraploid fibroblasts (about 70% of the monolayer) were covered with ESCs at a ratio of 1 : 1; within 1 h, the growth medium was removed, and the cells were treated for 1.5