Feeder-free culture of human pluripotent stem cells drives MDM4-mediated gain of chromosome 1q.

Culture-acquired variants in human pluripotent stem cells (hPSCs) hinder their applications in research and clinic. However, the mechanisms that underpin selection of variants remain unclear. Here, through analysis of comprehensive karyotyping datasets from over 23,000 hPSC cultures of more than 1,500 lines, we explored how culture conditions shape variant selection. Strikingly, we identified an association of chromosome 1q gains with feeder-free cultures and noted a rise in its prevalence in recent years, coinciding with increased usage of feeder-free regimens. Competition experiments of multiple isogenic lines with and without a chromosome 1q gain confirmed that 1q variants have an advantage in feeder-free (E8/vitronectin), but not feeder-based, culture. Mechanistically, we show that overexpression of MDM4, located on chromosome 1q, drives variants' advantage in E8/vitronectin by alleviating genome damage-induced apoptosis, which is lower in feeder-based conditions. Our study explains condition-dependent patterns of hPSC aberrations and offers insights into the mechanisms of variant selection.

G-Banded Karyotyping of Human Pluripotent Stem Cell Cultures.

Acquired chromosomal abnormalities may occur during the reprogramming and culture of human pluripotent stem cells (hPSCs). Therefore, it is required that regular testing of genetic integrity be conducted. G-banded karyotyping is a widely used genetic assay that is capable of detecting chromosomal abnormalities. Karyotyping of hPSC cultures can be a challenging undertaking for inexperienced investigators; here, we provide detailed procedures for karyotyping, including sample preparation and analysis, as well as the interpretation of hPSC karyotype results.

Genomic Stability Testing of Pluripotent Stem Cells.

Pluripotent stem cell (PSC) cultures are subjected to selective pressures that can result in acquisition and expansion of recurrent genetic abnormalities at any time. These recurrent abnormalities enhance the variant cells harboring them with a competitive advantage over wild-type cells. Variant cells can eventually supplant wild-type cells entirely and become fixed in culture. Such variants can impact the efficacy of PSCs in research and clinical applications. Therefore, routine genomic characterization is required for reliable and effective use of PSCs. In this article we describe the capabilities and limitations of several assays commonly used for assessing PSC genomic stability. Based on this analysis, we provide a recommendation for integrating assays into a comprehensive testing regimen that maximizes coverage while minimizing cost. © 2020 by John Wiley & Sons, Inc.

Generation of seven induced pluripotent stem cell lines from neonates of different ethnic backgrounds.

Seven human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts from three neonatal individuals using non-integrative reprogramming. Most control iPSCs are derived from adults, so these iPSCs meet the need for control iPSCs from young individuals. Donors were from different ethnicities and these lines provide unique genetic profiles. All iPSCs have normal karyotypes, express stem cell markers, and exhibit pluripotency, as assessed by capacity to differentiate into three germ layers. These lines are valuable to study human development, as age-matched controls for disorder-specific iPSCs, and as platforms for gene editing to control for age and ethnicity.

Genetic correction and analysis of induced pluripotent stem cells from a patient with gyrate atrophy.

Gene-corrected patient-specific induced pluripotent stem (iPS) cells offer a unique approach to gene therapy. Here, we begin to assess whether the mutational load acquired during gene correction of iPS cells is compatible with use in the treatment of genetic causes of retinal degenerative disease. We isolated iPS cells free of transgene sequences from a patient with gyrate atrophy caused by a point mutation in the gene encoding ornithine-δ-aminotransferase (OAT) and used homologous recombination to correct the genetic defect. Cytogenetic analysis, array comparative genomic hybridization (aCGH), and exome sequencing were performed to assess the genomic integrity of an iPS cell line after three sequential clonal events: initial reprogramming, gene targeting, and subsequent removal of a selection cassette. No abnormalities were detected after standard G-band metaphase analysis. However, aCGH and exome sequencing identified two deletions, one amplification, and nine mutations in protein coding regions in the initial iPS cell clone. Except for the targeted correction of the single nucleotide in the OAT locus and a single synonymous base-pair change, no additional mutations or copy number variation were identified in iPS cells after the two subsequent clonal events. These findings confirm that iPS cells themselves may carry a significant mutational load at initial isolation, but that the clonal events and prolonged cultured required for correction of a genetic defect can be accomplished without a substantial increase in mutational burden.