Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR

Assou, Saïd; Girault, Nicolas; Plinet, Mathilde; Bouckenheimer, Julien; Sansac, Caroline; Combe, Marion; Mianné, Joffrey; Bourguignon, Chloé; Fieldès, Mathieu; Ahmed, Engi; Commes, Thérèse; Boureux, Anthony; Lemaı̂tre, Jean-Marc; Vos, John de

doi:10.1016/j.stemcr.2019.12.004

Cited by 62 publications

(38 citation statements)

References 11 publications

(15 reference statements)

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“…Genetic integrity is crucial for cellular therapeutics. Many other studies reported a high number of genetic abnormalities in human pluripotent stem cell (hPSC) lines, which often arise recurrently [39]. In fact, all three iPSC clones used in this study revealed chromosomal duplications and deletions, which were passed on to the corresponding iMSCs.…”

Section: Discussionmentioning

confidence: 61%

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

et al. 2020

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Background: The use of mesenchymal stromal cells (MSCs) for research and clinical application is hampered by cellular heterogeneity and replicative senescence. Generation of MSC-like cells from induced pluripotent stem cells (iPSCs) may circumvent these limitations, and such iPSC-derived MSCs (iMSCs) are already tested in clinical trials. So far, a comparison of MSCs and iMSCs was particularly addressed in bulk culture. Despite the high hopes in cellular therapy, only little is known how the composition of different subclones changes in these cell preparations during culture expansion. Methods: In this study, we used multicolor lentiviral genetic barcoding for the marking of individual cells within cell preparations. Based on this, we could track the clonal composition of syngenic MSCs, iPSCs, and iMSCs during culture expansion. Furthermore, we analyzed DNA methylation patterns at senescence-associated genomic regions by barcoded bisulfite amplicon sequencing. The proliferation and differentiation capacities of individual subclones within MSCs and iMSCs were investigated with limiting dilution assays. Results: Overall, the clonal composition of primary MSCs and iPSCs gradually declined during expansion. In contrast, iMSCs became oligoclonal early during differentiation, indicating that they were derived from few individual iPSCs. This dominant clonal outgrowth of iMSCs was not associated with changes in chromosomal copy number variation. Furthermore, clonal dynamics were not clearly reflected by stochastically acquired DNA methylation patterns. Limiting dilution assays revealed that iMSCs are heterogeneous in colony formation and in vitro differentiation potential, while this was even more pronounced in primary MSCs. Conclusions: Our results indicate that the subclonal diversity of MSCs and iPSCs declines gradually during in vitro culture, whereas derivation of iMSCs may stem from few individual iPSCs. Differentiation regimen needs to be further optimized to achieve homogeneous differentiation of iPSCs towards iMSCs.

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Section: Discussionmentioning

confidence: 61%

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

et al. 2020

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“…In addition, the G56R-CRISPR2 and -3 lines differentiated into the three germ layers, as determined by IF analysis of AFP expression for endoderm, SMA for mesoderm, and glial fibrillary acidic protein (GFAP) for ectoderm ( Figure 4 C). Moreover, we tested genetic stability of the G56R-CRISPR2 and -3 iPSC lines by a digital PCR test of the copy number variant (CNV) of the most commonly rearranged chromosomal regions reported in iPSCs [ 34 ]. A CNV of 2 was detected for the autosomes, and a CNV of 1 for the X chromosome, in both lines indicating that the G56R-CRISPR2 and -3 iPSCs were genetically stable ( Figure 4 D).…”

Section: Resultsmentioning

confidence: 99%

Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3

Diakatou

Dubois

Erkilic

et al. 2021

IJMS

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Retinitis pigmentosa (RP) is an inherited retinal dystrophy that causes progressive vision loss. The G56R mutation in NR2E3 is the second most common mutation causing autosomal dominant (ad) RP, a transcription factor that is essential for photoreceptor development and maintenance. The G56R variant is exclusively responsible for all cases of NR2E3-associated adRP. Currently, there is no treatment for NR2E3-related or, other, adRP, but genome editing holds promise. A pertinent approach would be to specifically knockout the dominant mutant allele, so that the wild type allele can perform unhindered. In this study, we developed a CRISPR/Cas strategy to specifically knockout the mutant G56R allele of NR2E3 and performed a proof-of-concept study in induced pluripotent stem cells (iPSCs) of an adRP patient. We demonstrate allele-specific knockout of the mutant G56R allele in the absence of off-target events. Furthermore, we validated this knockout strategy in an exogenous overexpression system. Accordingly, the mutant G56R-CRISPR protein was truncated and mis-localized to the cytosol in contrast to the (peri)nuclear localizations of wild type or G56R NR2E3 proteins. Finally, we show, for the first time, that G56R iPSCs, as well as G56R-CRISPR iPSCs, can differentiate into NR2E3-expressing retinal organoids. Overall, we demonstrate that G56R allele-specific knockout by CRISPR/Cas could be a clinically relevant approach to treat NR2E3-associated adRP.

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“…Trilineage differentiation potential confirms iPSC pluripotency. Importantly, accumulation of common chromosomal abnormalities should be closely monitored as aneuploidy (most frequently of chromosomes 12, 17, 20, and X) is common ( Assou et al., 2020 ; Taapken et al., 2011 ). At a minimum, G-banded chromosome analysis should be carried out immediately after iPSC derivation, after each cell expansion for banking, and after every 5–10 passages during culture ( Martins-Taylor et al., 2011 ; McIntire et al., 2020 ).…”

Section: Main Textmentioning

confidence: 99%

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders

et al. 2021

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Reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) and their differentiation into neural lineages is a revolutionary experimental system for studying neurological disorders, including intellectual and developmental disabilities (IDDs). However, issues related to variability and reproducibility have hindered translating preclinical findings into drug discovery. Here, we identify areas for improvement by conducting a comprehensive review of 58 research articles that utilized iPSC-derived neural cells to investigate genetically defined IDDs. Based upon these findings, we propose recommendations for best practices that can be adopted by research scientists as well as journal editors.

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Recurrent Genetic Abnormalities in Human Pluripotent Stem Cells: Definition and Routine Detection in Culture Supernatant by Targeted Droplet Digital PCR

Cited by 62 publications

References 11 publications

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

Genetic barcoding reveals clonal dominance in iPSC-derived mesenchymal stromal cells

Allele-Specific Knockout by CRISPR/Cas to Treat Autosomal Dominant Retinitis Pigmentosa Caused by the G56R Mutation in NR2E3

Balancing serendipity and reproducibility: Pluripotent stem cells as experimental systems for intellectual and developmental disorders

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