A pluripotent developmental state confers a low fidelity of chromosome segregation

Deng, Chenhui; Ya, Amanda; Compton, Duane A.; Godek, Kristina

doi:10.1016/j.stemcr.2022.12.008

Cited by 8 publications

(19 citation statements)

References 51 publications

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“…Human diploid nontransformed but immortalized cell lines such as RPE-1 are recognized for their high karyotype stability and low level of defective mitotic phenomena, including lagging chromosomes, acentric chromatin fragments and chromatin bridges 46 . Interestingly, the level of defective mitosis observed in our study during anaphase in LSCs grown in culture (< 4%) is comparable to that in RPE-1 cells grown in culture and scored during anaphase (< 5-6%) 46,47 . Similar percentages in LSCs and RPE-1 cells were observed for transiently multipolar mitotes, measured during metaphase (< 3% in both cases) 48 .…”

Section: Discussionsupporting

confidence: 80%

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Zekušić,

Mihica,

Skoko

et al. 2023

Preprint

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Background Limbal stem cells(LSCs) are crucial for the regeneration of the corneal epithelium in patients with limbal stem cell deficiency (LSCD). Thus, LSCs during cultivation in vitro should be in highly homogeneous amounts, while potency and expression of stemness without tumorigenesis would be desirable. Therefore, further characterization and safety evaluation of engineered limbal grafts is required to provide safe and high-quality therapeutic applications. Methods After in vitro expansion, LSCs undergo laboratory characterization in a single-cell suspension, cell culture, and in limbal grafts before transplantation. Through a clinically applicable protocol,the collected data on LSCs at passage 1 were summarized: identity (cell size, morphology); potency (yield, viability, population doubling time, colony-forming efficiency were analyzed by trypan blue staining); expression of putative stem cell markers by flow cytometry, immunofluorescence and immunohistochemistry. Then, mitotic chromosome stability and normal mitotic outcomes were explored by using live-cell imaging. Finally, impurities, bacterial endotoxins and sterility were determined. Results Expression of the stemness marker p63 in single-cell suspension and in cell culture showed high values by different methods. Limbal grafts showed p63-positive cells (78.7 ± 9.4%), Ki67 proliferation (41.7 ± 15.9%), while CK3 was negative. Impurity with 3T3 feeder cells and endotoxins was minimized. We presented mitotic spindles with a length of 11.40 ± 0.54 m and a spindle width of 8.05 ± 0.55 m as new characterization in LSC culture. Additionally, live-cell imaging of LSCs (n=873) was performed, and only a small fraction <2.5% of aberrant interphase cells was observed; 2.12 ± 2.10% of mitotic spindles exhibited a multipolar phenotype during metaphase, and 3.84 ± 3.77% of anaphase cells had a DNA signal present within the spindle midzone, indicating a chromosome bridge or lagging chromosome phenotype. Conclusion This manuscript provides, for the first time, detailed characterization of the parameters of fidelity of the mitotic process and mitotic spindle morphologies of LSCs used in a direct clinical application. Our data show that p63-positive CK3-negative LSCs grown in vitro for clinical purposes undergo mitotic processes with extremely high fidelity, suggesting high karyotype stability. This finding confirms LSCs as a high-quality and safe therapy for eye regeneration in humans.

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Section: Discussionsupporting

confidence: 80%

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Zekušić,

Mihica,

Skoko

et al. 2023

Preprint

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“…Since hESCs more closely resemble pluripotent ICM cells an open question is whether cell competition occurs between aneuploid and diploid TE cells. Subsequently as development progresses and developmental potential becomes restricted, mitotic error rates decrease 14 thus reducing the generation of aneuploid cells coupled with a switch to cell autonomous mechanisms that eliminate aneuploid cells (Figure 7B). One critical parameter in this model is the ratio of diploid to aneuploid cells during preimplantation development.…”

Section: Discussionmentioning

confidence: 99%

“…An inherent tolerance to aneuploidy combined with hESCs exhibiting a persistently high mitotic error rate 14 poses a conundrum in how hESCs maintain stable diploid populations for generations. Mechanistically, our data demonstrates that Myc- and p53-driven cell competition eliminates less fit aneuploid hESCs to preserve the genome integrity of the population (presumably a similar mechanism acts in induced PSCs) (Figure 7A).…”

Section: Discussionmentioning

confidence: 99%

“…This, coupled with a declining mitotic error rate as developmental potential becomes restricted, decreases the frequency of aneuploid embryonic cells to achieve genome stability and establish diploid embryos as development progresses. Models adapted from Deng et al, 2023 14 .…”

Section: Discussionmentioning

confidence: 99%

“…In combination with mitotic duration, anaphase errors were observed and scored. Criteria for scoring chromosome segregation errors was previously described 14 .…”

Section: Time-lapse Live-cell Fluorescence Imagingmentioning

confidence: 99%

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Cell Competition Eliminates Aneuploid Human Pluripotent Stem Cells

Ya,

Deng,

Godek

2024

Preprint

Self Cite

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Human pluripotent stem cells (hPSCs) maintain diploid populations for generations despite a persistently high rate of mitotic errors that cause aneuploidy, or chromosome imbalances. Consequently, to maintain genome stability, aneuploidy must inhibit hPSC proliferation, but the mechanisms are unknown. Here, we surprisingly find that homogeneous aneuploid populations of hPSCs proliferate unlike aneuploid non-transformed somatic cells. Instead, in mosaic populations, cell non-autonomous competition between neighboring diploid and aneuploid hPSCs eliminates less fit aneuploid cells. Aneuploid hPSCs with lower Myc or higher p53 levels relative to diploid neighbors are outcompeted but conversely gain a selective advantage when Myc and p53 relative abundance switches. Thus, although hPSCs frequently missegregate chromosomes and inherently tolerate aneuploidy, Myc- and p53-driven cell competition preserves their genome integrity. These findings have important implications for the use of hPSCs in regenerative medicine and for how diploid human embryos are established despite the prevalence of aneuploidy during early development.

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Culture‐acquired genetic variation in human pluripotent stem cells: Twenty years on

Vales,

Barbaric

2024

BioEssays

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Genetic changes arising in human pluripotent stem cells (hPSC) upon culture may bestow unwanted or detrimental phenotypes to cells, thus potentially impacting on the applications of hPSCs for clinical use and basic research. In the 20 years since the first report of culture‐acquired genetic aberrations in hPSCs, a characteristic spectrum of recurrent aberrations has emerged. The preponderance of such aberrations implies that they provide a selective growth advantage to hPSCs upon expansion. However, understanding the consequences of culture‐acquired variants for specific applications in cell therapy or research has been more elusive. The rapid progress of hPSC‐based therapies to clinics is galvanizing the field to address this uncertainty and provide definitive ways both for risk assessment of variants and reducing their prevalence in culture. Here, we aim to provide a timely update on almost 20 years of research on this fascinating, but a still unresolved and concerning, phenomenon.

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A pluripotent developmental state confers a low fidelity of chromosome segregation

Cited by 8 publications

References 51 publications

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

Cell Competition Eliminates Aneuploid Human Pluripotent Stem Cells

Culture‐acquired genetic variation in human pluripotent stem cells: Twenty years on

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