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

Abstract

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.

TPX2 Induces Mitotic Survival via BCL2L1 Induction Through YAP1 Protein Stabilization in Human Embryonic Stem Cells

“…2A). Among the genes in the 20q11.21 locus, TPX2, which was previously shown to induce aberrant mitosis in culture-adapted hESCs [17], was concurrently induced with BCL2L1 expression. Similarly, BCL-xL protein level was closely associated with TPX2 protein level in P3 and P4 hESCs where the survival trait was evident (Fig.…”

“…We have previously demonstrated that TPX2, located at locus 20q11.21 along with BCL2L1, would be a putative driver for abnormal mitosis [17]. Using iTPX2-hESCs, cell death under mitotic stress (e.g., Noc) was determined after TPX2 induction by doxycycline (Dox).…”

“…3D and E). Higher TPX2 in P4 hESCs than P1 hESCs occurred constantly regardless of cell cycle phase [17]. To further assess whether high TPX2 expression was responsible for the resistance to mitotic stress, TPX2 was transiently depleted by siRNA#3 (Fig.…”

“…Aurora-A stabilizes the YAP1 protein Previously, Aurora A, a mitotic kinase strongly associated with TPX2 for spindle assembly, was shown to phosphorylate and stabilize YAP1 in cancer [9,36]. Constant activation of Aurora-A, which corresponded with TPX2 induction regardless of cell cycle phase, was a distinct cellular phenotype of P3 and P4 hESCs [17]. P4 hESCs with high Aurora-A (determined by phospho-Aurora A) and TPX2 activities exhibited high protein levels of BCL-xL and YAP1 (Fig.…”

“…A recent study revealed that escape from mitotic cell death during mitotic errors due to this acquired survival trait (either induction of BCL2L1, or dominant mutation of NOXA, a pro-apoptotic factor) leads to aneuploidy [9]. In this context, a targeting protein for Xklp2 (TPX2) also located in 20q11.21 was suggested to be the putative driver for abnormal mitosis by disrupting spindle dynamics [17]. However, despite the signi cance of 'survival advantage' in the phenotypic changes of culture-adapted hESCs and even aneuploidy, the molecular mechanisms underlying this phenomenon are not fully understood except for BCL2L1 induction.…”