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

AbstractAlthough 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.

Download Full-text

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

10.21203/rs.3.rs-982680/v1 ◽

2021 ◽

Author(s):

Ho-Chang Jeong ◽

Young-Hyun Go ◽

Joong-Gon Shin ◽

Yun-Jeong Kim ◽

Min-Guk Cho ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Genome Integrity ◽

Mitotic Progression ◽

Genome Wide ◽

Spindle Dynamics ◽

Normal Copy Number

Abstract 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. 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 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, suggesting that the increased transcription of TPX2 in culture adapted hESCs could contribute to an increase in aberrant mitosis due to altered spindle dynamics.

Download Full-text

Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation

Science ◽

10.1126/science.1253799 ◽

2014 ◽

Vol 346 (6216) ◽

pp. 1529-1533 ◽

Cited By ~ 195

Author(s):

Kosuke Funato ◽

Tamara Major ◽

Peter W. Lewis ◽

C. David Allis ◽

Viviane Tabar

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Neural Progenitor Cells ◽

Embryonic Stem ◽

Cell System ◽

Genome Wide ◽

Regulator Genes

Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice.

Download Full-text

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

10.21203/rs.3.rs-626430/v1 ◽

2021 ◽

Author(s):

Yun-Jeong Kim ◽

Young-Hyun Go ◽

Ho-Chang Jeong ◽

Seong-Min Kim ◽

Hyun Sub Cheong ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Copy Number ◽

Embryonic Stem ◽

Aurora A Kinase ◽

High Tolerance ◽

Mitotic Stress

Abstract Genetic alterations have been reported in most human embryonic stem cells (hESCs) for decades. ‘Survival advantage,’ a typical trait acquired during long-term in vitro culture, results from induction of BCL2L1 upon frequent copy number variation (CNV) at locus 20q11.21 and is one of the strongest candidates associated with genetic alteration via escape from mitotic stress. However, the underlying mechanisms for BCL2L1 induction remain undefined. Furthermore, abnormal mitosis and ‘survival advantage’ frequently occurring in the late passage are respectively associated with the expression of TPX2 and BCL2L1, which are located in locus 20q11.21. In this study, we observed that 20q11.21 CNV was not sufficient for BCL2L1 induction and consequent survival traits in pairs of hESCs and human induced pluripotent stem cells (iPSCs) with normal and 20q11.21 CNVs. Inducible expression of TPX2 and basal TPX2 expression due to leakage of the inducible system in hESCs with normal copy number was sufficient to promote BCL2L1 expression and promoted high tolerance to mitotic stress. High Aurora A kinase activity by TPX2 stabilized YAP1 protein to promote YAP1 dependent BCL2L1 expression. Thus, a chemical inhibitor of Aurora A kinase and knockdown of YAP/TAZ significantly abrogated the aforementioned high tolerance to mitotic stress through BCL2L1 suppression. These results suggest that the collective expression of TPX2 and BCL2L1 from CNV at loci 20q11.21 and a consequent increase in YAP1 signaling would promote genome instability during long-term in vitro hESC culture.

Download Full-text

Faculty Opinions recommendation of INS(GFP/w) human embryonic stem cells facilitate isolation of in vitro derived insulin-producing cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.13984975.15442093 ◽

2012 ◽

Author(s):

Gordon Weir

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Insulin Producing Cells

Download Full-text

Faculty Opinions recommendation of CORTECON: a temporal transcriptome analysis of in vitro human cerebral cortex development from human embryonic stem cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.718478232.793498745 ◽

2014 ◽

Author(s):

Vijay Tiwari ◽

Johannes Jung

Keyword(s):

Stem Cells ◽

Cerebral Cortex ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Transcriptome Analysis ◽

Embryonic Stem ◽

Human Cerebral Cortex ◽

Cerebral Cortex Development

Download Full-text

Genome-wide Kinase-Chromatin Interactions Reveal the Regulatory Network of ERK Signaling in Human Embryonic Stem Cells

Molecular Cell ◽

10.1016/j.molcel.2013.04.030 ◽

2013 ◽

Vol 50 (6) ◽

pp. 844-855 ◽

Cited By ~ 61

Author(s):

Jonathan Göke ◽

Yun-Shen Chan ◽

Junli Yan ◽

Martin Vingron ◽

Huck-Hui Ng

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Regulatory Network ◽

Embryonic Stem ◽

Erk Signaling ◽

Chromatin Interactions ◽

Genome Wide

Download Full-text

Rapid Differentiation of Human Embryonic Stem Cells into Testosterone-Producing Leydig Cell-Like Cells In vitro

Tissue Engineering and Regenerative Medicine ◽

10.1007/s13770-021-00359-8 ◽

2021 ◽

Author(s):

Eun-Young Shin ◽

Seah Park ◽

Won Yun Choi ◽

Dong Ryul Lee

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Human Embryonic Stem Cells ◽

Embryonic Stem ◽

Male Hypogonadism ◽

Testosterone Levels ◽

Sequence Of Events ◽

Short Period ◽

Molecular Compounds

Abstract Background: Leydig cells (LCs) are testicular somatic cells that are the major producers of testosterone in males. Testosterone is essential for male physiology and reproduction. Reduced testosterone levels lead to hypogonadism and are associated with diverse pathologies, such as neuronal dysfunction, cardiovascular disease, and metabolic syndrome. LC transplantation is a promising therapy for hypogonadism; however, the number of LCs in the testis is very rare and they do not proliferate in vitro. Therefore, there is a need for an alternative source of LCs. Methods: To develop a safer, simple, and rapid strategy to generate human LC-like cells (LLCs) from stem cells, we first performed preliminary tests under different conditions for the induction of LLCs from human CD34/CD73 double positive-testis-derived stem cells (HTSCs). Based on the embryological sequence of events, we suggested a 3-step strategy for the differentiation of human ESCs into LLCs. We generated the mesendoderm in the first stage and intermediate mesoderm (IM) in the second stage and optimized the conditions for differentiation of IM into LLCs by comparing the secreted testosterone levels of each group. Results: HTSCs and human embryonic stem cells can be directly differentiated into LLCs by defined molecular compounds within a short period. Human ESC-derived LLCs can secrete testosterone and express steroidogenic markers. Conclusion: We developed a rapid and efficient protocol for the production of LLCs from stem cells using defined molecular compounds. These findings provide a new therapeutic cell source for male hypogonadism.

Download Full-text