TPX2 Amplification-Driven Aberrant Mitosis in Long-Term Cultured Human Embryonic Stem Cells

Although human embryonic stem cells (hESCs) are equipped with highly effective machinery for the maintenance of genome integrity, the frequency of genetic aberrations during long-term in vitro hESC culture has been a serious issue that raises concerns over their safety in future clinical applications. By passaging hESCs over a broad range of timepoints, we found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in the late-passaged hESCs (LP-hESCs) in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs). Through high-resolution genome-wide approaches and by following transcriptome analysis, we found that LP-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2 (targeting protein for Xklp2), a key protein for governing spindle assembly and cancer malignancy. Consistent with these findings, the inducible expression of TPX2 in EP-hESCs reproduced aberrant mitotic events, such as the delay of mitotic progression, spindle stability, misaligned chromosomes, and polyploidy. This data suggests that the amplification and increased transcription of the TPX2 gene at 20q11.21 could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

Culture conditions for human embryonic stem cells

Human embryonic stem cell (hESC) lines have been derived and cultured in variable conditions. The idea behind derivation of hESC lines is to use them in human cell transplantation after differentiation, but already now these cells are widely used for research purposes. Despite similarities among the established lines, important differences have been reported between them, and it has been difficult to compare the results obtained using different lines. Recent optimization of hESC culture conditions has moved from cultures on mouse embryonic fibroblasts (MEFs) in fetal bovine serum-containing medium towards feeder-free culture methods using more defined animal substance-free cultures. The aim has been to establish robust and cost-effective systems for culturing these cells and eliminate the risk of infection transmitted by animal pathogens and immunoreactions caused by animal substances in cell cultures before clinical treatment. It is important to take these modifications into account when carrying out research using these cells. It is known that culture conditions influence gene expression and, hence, probably many properties of the cells. Optimization and standardization of culture methods is needed for research as well as for clinical purposes.