This article describes the engineering of human induced pluripotent stem cells to report a cellular response to oxidative stress, which is a common (but not universal) result of cells being exposed to a toxicant. It then describes the use of these cells to generate renal organoids that will report oxidative stress, and the use of these organoids to screen a panel of compounds for nephrotoxicity. This application is intended as an illustration; in principle, other organoids could be made from these cells, and other cell lines could be made to report different common markers of cellular stress (for example, the release of pro-inflammatory signals).
© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

Embryonic stem (ES) cells are pluripotent stem cells that can produce all cell types of an organism. ES cells proliferate rapidly and are thought to experience high levels of intrinsic replication stress. Here, by investigating replication fork dynamics in substages of S phase, we show that mammalian pluripotent stem cells maintain a slow fork speed and high active origin density throughout the S phase, with little sign of fork pausing. In contrast, the fork speed of non-pluripotent cells is slow at the beginning of S phase, accompanied by increased fork pausing, but thereafter fork pausing rates decline and fork speed rates accelerate in an ATR-dependent manner. Thus, replication fork dynamics within the S phase are distinct between ES and non-ES cells. Nucleoside addition can accelerate fork speed and reduce origin density. However, this causes miscoordination between the completion of DNA replication and cell cycle progression, leading to genome instability. Our study indicates that fork slowing in the pluripotent stem cells is an integral aspect of DNA replication.
© 2024. The Author(s).

SUMMARY The genetic integrity of pluripotent stem cells (PSC) is integral to their applications in research and therapy, but it is compromised by frequent development of copy number variations. Little is known about the basis of the variable genomic integrity among different PSC lines. Here we identify aneuploidies using RNA-seq and proteomics data from a panel of mouse embryonic stem cell (mESC) lines derived from 170 Diversity Outbred mice. We identified 62 lines with detectable aneuploid subpopulations and a subset of originally XX lines that lost one Chromosome X (XO). Strikingly, a much lower proportion of XX lines were aneuploid, compared to XY or XO lines. Two single-cell RNA-seq data sets demonstrated that aneuploid XY DO mESC also show lower Chromosome X gene expression and that XY mESC accumulate higher aneuploid proportions in culture than isogenic XX lines. Possible mechanisms for this protective effect of X chromosome dosage include our discovery that the lines with two active X Chromosomes have a higher expression of 2-cell-like state genes, and differential expression of X-linked tumor suppressor genes associated with DNA damage response. Highlights First genetic analysis of predisposition to aneuploidy in pluripotent stem cell cultures Chromosomal regions duplicated in aneuploid mouse embryonic stem cells are syntenic with regions overrepresented in human pluripotent stem cell lines bearing recurrent genetic abnormalities X-Chromosome dosage strongly influences susceptibility to aneuploidy in mouse embryonic stem cells and to a lesser degree in human pluripotent stem cells XX mouse embryonic stem cell lines show a higher proportion of cells in 2 cell-like state and higher expression of tumor suppressor genes associated with DNA damage response

Autosomal dominant polycystic kidney disease (ADPKD) is a dominant genetic disorder caused primarily by mutations in the PKD1 gene, resulting in the formation of numerous cysts and eventually kidney failure. However, there are currently no gene therapy studies aimed at correcting PKD1 gene mutations. In this study, we identified two mutation sites associated with ADPKD, c.1198 (C > T) and c.8311 (G > A), which could potentially be corrected by adenine base editor (ABE). The correction efficiencies of different ABE variants were tested using the HEK293T-PKD1 c.1198 (C > T) and HEK293T-PKD1 c.8311 (G > A) reporter cell lines. We then generated induced pluripotent stem cells (iPSCsmut/WT) from the peripheral blood mononuclear cells (PBMCs) of the heterozygous patient to develop a disease cell model. Since the iPSCsmut/WT did not exhibit a typical disease phenotype in stem cell status, differentiation into kidney organoids in vitro led to the expression of kidney organ-specific marker proteins. Stimulation of cAMP signaling with forskolin resulted in cystic expansion of renal epithelial tissue in iPSCmut/WT-derived kidney organoids, resembling the cystic phenotype observed in ADPKD patients. However, kidney organoids differentiated from ABE-corrected iPSCs did not display the cystic phenotype. Furthermore, we used a dual AAV split-ABEmax system as a therapeutic strategy and achieved an average editing efficiency of approximately 6.56% in kidney organoids. Overall, this study provides a framework for gene therapy targeting ADPKD through ABE single-base editing, offering promising prospects for future therapeutic interventions.
© 2024. The Author(s).

The power and scope of disease modeling can be markedly enhanced through the incorporation of broad genetic diversity. The introduction of pathogenic mutations into a single inbred mouse strain sometimes fails to mimic human disease. We describe a cross-species precision disease modeling platform that exploits mouse genetic diversity to bridge cell-based modeling with whole organism analysis. We developed a universal protocol that permitted robust and reproducible neural differentiation of genetically diverse human and mouse pluripotent stem cell lines and then carried out a proof-of-concept study of the neurodevelopmental gene DYRK1A. Results in vitro reliably predicted the effects of genetic background on Dyrk1a loss-of-function phenotypes in vivo. Transcriptomic comparison of responsive and unresponsive strains identified molecular pathways conferring sensitivity or resilience to Dyrk1a1A loss and highlighted differential messenger RNA isoform usage as an important determinant of response. This cross-species strategy provides a powerful tool in the functional analysis of candidate disease variants identified through human genetic studies.