Abstract
For the first time, two types of hybrid cells with embryonic stem (ES) cell-like and fibroblast-like phenotypes were produced through the fusion of mouse ES cells with fibroblasts. Transcriptome analysis of 2,848 genes differentially expressed in the parental cells demonstrated that 34–43% of these genes are expressed in hybrid cells, consistent with their phenotypes; 25–29% of these genes display intermediate levels of expression, and 12–16% of these genes maintained expression at the parental cell level, inconsistent with the phenotype of the hybrid cell. Approximately 20% of the analyzed genes displayed unexpected expression patterns that differ from both parents. An unusual phenomenon was observed, namely, the illegitimate activation of Xist expression and the inactivation of one of two X-chromosomes in the near-tetraploid fibroblast-like hybrid cells, whereas both Xs were active before and after in vitro differentiation of the ES cell-like hybrid cells. These results and previous data obtained on heterokaryons suggest that the appearance of hybrid cells with a fibroblast-like phenotype reflects the reprogramming, rather than the induced differentiation, of the ES cell genome under the influence of a somatic partner.
Highlights
Cell fusion with embryonic stem (ES) cells is a powerful tool for restoring pluripotency in somatic cells[1,2,3,4,5]
We used tau-green fluorescent protein (GFP) ES cells cultured in standard ES cell medium without 2i (PD0325901 and CHIR99021), and after fusion with m5S fibroblasts, we observed the formation of 50 primary HATand puromycin-resistant colonies: 15 colonies with an ES cell-like phenotype and 35 colonies with a fibroblast-like phenotype
These results suggest that the presence or absence of 2i in ES cell medium prior to cell fusion does not affect the prevalence of primary colonies with fibroblast-like phenotypes over ES cell-like phenotypes
Summary
Cell fusion with embryonic stem (ES) cells is a powerful tool for restoring pluripotency in somatic cells[1,2,3,4,5]. This article is dedicated to the detailed characterization of a set of ES cell-like and fibroblast-like hybrid cells obtained through the fusion of mouse ES cells with m5S fibroblasts as a somatic partner Both types of hybrid cells had stable near-tetraploid karyotypes and a ratio of the parental genomes close to 1:1. The transcriptome analysis revealed that the sets of genes involved in the establishment of both phenotypes of hybrid cells were different, both types of hybrid cells had similar ratios of activated or silenced genes and genes with intermediate and “novel” expression These data and previous our data[12] obtained on heterokaryons suggest that the observed alternative manifestation of the parental genomes in two types of hybrid cells reflects the bidirectional reprogramming of the parental genomes
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