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TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells with gain of 20q11.21

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Abstract

Background

Despite highly effective machinery for the maintenance of genome integrity in human embryonic stem cells (hESCs), the frequency of genetic aberrations during in-vitro culture has been a serious issue for future clinical applications.

Method

By passaging hESCs over a broad range of timepoints (up to 6 years), the isogenic hESC lines with different passage numbers with distinct cellular characteristics, were established.

Result

We found that mitotic aberrations, such as the delay of mitosis, multipolar centrosomes, and chromosome mis-segregation, were increased in parallel with polyploidy compared to early-passaged hESCs (EP-hESCs) with normal copy number. Through high-resolution genome-wide approaches and transcriptome analysis, we found that culture adapted-hESCs with a minimal amplicon in chromosome 20q11.21 highly expressed TPX2, 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 stabilization, misaligned chromosomes, and polyploidy.

Conclusion

These studies suggest that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

Graphical abstract

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Data Availability

Source data are available from the corresponding authors upon request. The RNAseq results have been deposited to Gene Expression Omnibus (GEO) under accession number GSE167495 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE167495). The flow cytometry data were deposited in Flowrepository (https://flowrepository.org/id/FR-FCM-Z3W9).

Abbreviations

hESC:

Human embryonic stem cell.

hPSC:

Human pluripotent stem cell.

hiPSCs:

Human induced pluripotent stem cell.

Chr:

Chromosome.

EP:

Early-passage.

LP:

Late-passage.

DMSO:

Dimethyl sulfoxide.

TPX2:

Targeting protein for Xklp2.

aCGH:

Array comparative genomic hybridization.

CNV:

Copy number variation.

CIN:

Chromosome instability.

7-AAD:

7-Aminoactinomycin D.

MtOH:

Methanol.

EtOH:

Ethanol

PI:

Propidium iodide

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Acknowledgements

We thank Dr. Minh Dang Nguyen in University of Calgary for kindly providing TPX2 constructs.

Funding

This work including the design of the study and collection, analysis, and interpretation of data, was supported by a grant from the National Research Foundation of Korea (NRF) (NRF-2020M3A9E4037905 and NRF-2020R1A2C2005914) and Korean Fund for Regenerative Medicine funded by Ministry of Science and ICT, and Ministry of Health and Welfare (Grant number RS-2022–00070316), Republic of Korea.

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Author notes

  1. Ho-Chang Jeong and Young-Hyun Go these authors contributed equally.

Authors and Affiliations

  1. Department of Life Science, Sogang University, Seoul, 04107, Republic of Korea

    Ho-Chang Jeong

  2. Research Institute of Pharmaceutical Science, Seoul National University, Seoul, 08826, Republic of Korea

    Young-Hyun Go

  3. College of Pharmacy, Seoul National University, 1 Gwanak-ro Gwanak-gu, Seoul, 08826, South Korea

    Young-Hyun Go, Yun-Jeong Kim & Hyuk-Jin Cha

  4. Research Institute for Basic Science, Sogang University, 35 Baekbum ro, Mapo-gu, Seoul, 04107, South Korea

    Joong-Gon Shin & Hyoung Doo Shin

  5. Department of Biochemistry and Molecular Biology, Ajou University School of Medicine, Suwon, 16499, Republic of Korea

    Min-Guk Cho & Jae-Ho Lee

  6. Drug Information Research Institute, College of Pharmacy, Sookmyung Women’s University, Seoul, 04310, Republic of Korea

    Dasom Gwon & Chang-Young Jang

  7. Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, 04107, Republic of Korea

    Hyun Sub Cheong & Hyoung Doo Shin

  8. College of Pharmacy, Pusan Nati, onal University, Pusan, Korea

    Haeseung Lee

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Contributions

HJ.C conceived the overall study design, led the experiments and wrote the manuscript. HC.J and YH.G conducted the experiments, data analysis and wrote the first draft. JG.S and HD.S performed CGH array and exome sequencing and analyzed the data. HS.L analyzed the transcriptome data. YJ.K validated the experiments and generated cell lines. MG.C, D.G, HS.C, JH.L and CY.J performed cell cycle analysis and measured the spindle stability. All authors contributed to manuscript writing and revising and endorsed the final version of this manuscript.

Corresponding authors

Correspondence to Hyoung Doo Shin or Hyuk-Jin Cha.

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Jeong, HC., Go, YH., Shin, JG. et al. TPX2 Amplification-Driven Aberrant Mitosis in Culture Adapted Human Embryonic Stem Cells with gain of 20q11.21. Stem Cell Rev and Rep 19, 1466–1481 (2023). https://doi.org/10.1007/s12015-023-10514-4

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