Abstract
Maintenance of a euploid ES cell genome during ex-vivo expansion and differentiation to hematopoietic cells is essential for their safe use in therapeutic intervention and regenerative medicine. It is estimated that about half of all spontaneous mutations that occur in cultured murine ES cells are attributable to chromosomal non-disjunction and chromosome loss. This type of mutation is essentially undetectable in somatic cell cultures of comparable culture age. The mechanisms responsible for this difference between ES and somatic cells are unknown, but are likely related to the absence of a rigorous G1 cell cycle checkpoint and other peculiarities in ES cell cycle regulation compared to somatic cells. The fate of aneuploid ES cells during differentiation has not been systematically studied. Here, we report a remarkable tolerance for aneuploidy/polyploidy in murine ES cells in-vitro when challenged by mitotic stress that is induced by failed cell division followed by mitotic slippage resulting in stable, cycling, tetraploid/polyploid ES cell lines (oscillating between 4N and 8N). We present evidence supporting the idea that canonical apoptosis is uncoupled from mitotic checkpoints in undifferentiated ES cells in contrast to somatic cells and embryoid body (EB) cells. We also present evidence suggesting similar behavior occurs in human ES cell cultures. Uncoupling was associated with low levels of the pro-apoptotic form of phospho-BAD (p-ser128) combined with high expression of anti-apoptotic Survivin in ES compared to EB cells. This suggests uncoupling from mitotic checkpoints may be related to a heightened apoptotic threshold in ES cells compared to EB/somatic cells. Interestingly, culture of ES cells under hypoxic conditions also generated polyploid cells. The polyploid ES cells did not appear to be trophoblastic cells because they continue to express SSEA-1 and other markers of pluripotency. However, we demonstrate that (re-)coupling occurs very early in the differentiation process because only diploid ES cells contribute to EB formation while tetraploid cells do not when EBs are generated from tetraploid/diploid-mosaic cultures. This is, to our knowledge, the first evidence that there may be a potent in-vitro barrier to cells with numerical chromosomal aberrations from contributing to differentiated cells to be used in therapeutic settings. Finally, using a conditional oct-4 knock-down ES cell line, we demonstrate that the self-renewal regulating transcription factor, oct-4, is essential for maintaining the aneuploidy/polyploidy tolerant state. We conclude that aneuploidy/polyploidy-tolerance in pluripotent ES cells in-vitro is an expected occurrence when viewed within the context of ex-vivo ES cell culture where apoptotic culling of cells with chromosomal aberrations, which normally occurs in vertebrate embryos during the peri-implantation period in-vivo, has been artificially interrupted by ES cell derivation. This behavior likely contributes significantly to spontaneous aneuploidization in murine and human ES cell cultures. These data not only lend insight into mechanisms of aneuploidy in ES cell cultures but may also have implications for mechanisms of aneuploidy during tumorigenesis.
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