Concise Review: Assessing the Genome Integrity of Human Induced Pluripotent Stem Cells: What Quality Control Metrics?

Abstract

Human induced pluripotent stem cells (hiPSCs) have the potential to differentiate virtually into any cell type in unlimited quantities. Therefore, they are ideal for in vitro tissue modeling or to produce cells for clinical use. Importantly, and differently from immortalized and cancer cell lines, the hiPSC genome scrupulously reproduces that of the cell from which they were derived. However, hiPSCs can develop genetic abnormalities during reprogramming or prolonged cell culture, such as aneuploidies or oncogenic mutations (e.g., in TP53). Therefore, hiPSC genome integrity must be routinely monitored because serious genome alterations would greatly compromise their usefulness or safety of use. Here, we reviewed hiPSC genome quality control monitoring methods and laboratory practice. Indeed, due to their frequency and functional consequences, recurrent genetic defects found in cultured hiPSCs are inacceptable and their appearance should be monitored by routine screening. Hence, for research purposes, we propose that the genome of hiPSC lines should be systematically screened at derivation, at least by karyotyping, and then regularly (every 12 weeks) during experiments, for instance with polymerase chain reaction-based techniques. For some specific applications, such as research on aging, cell cycle, apoptosis or cancer, other tests (e.g., TP53 mutation detection) should also be included. For clinical use, in addition to karyotyping, we advise exome sequencing. STEM CELLS 2018; 00:000-000 SIGNIFICANCE STATEMENTThe landscape of genomic abnormalities found in human induced pluripotent stem cells has been extensively described. The present Review discusses how to classify these abnormalities, the tools for their detection, and the quality metrics that could be used for decision-making, both for research and clinical applications. This study also suggests some simple recommendations for the minimum genome integrity checks that are needed when working with pluripotent stem cells.

Long-term single-cell passaging of human iPSC fully supports pluripotency and high-efficient trilineage differentiation capacity

“…Genomic integrity was assessed by integration PCR and karyotyping as reprogramming and cultivation processes are recognized as the main causes for the alterations found in genome. [9] Because of the potential hazardous effects of reprogramming vectors, [30] several integration-free methods to generate iPSC were developed but, in comparison, the use of episomal plasmid vectors for reprogramming presents to its advantage a much increased efficiency. [19,25] By using plasmids pEB-C5, pEB-TG and Epi5, we were able to successfully generate hiPSC and PCR confirmed no vector integration into host DNA whatsoever.…”

“…[2,3] To overcome this issue, several groups committed to the search of conditions that could support single-cell passaging [4][5][6][7][8] but, despite the rapid progress, there is still a lack of straightforward standard protocols for iPSC cultivation that discuss the effects of combining different novelty biotechnologies as well as the effects when culturing PSC for a long time. Indeed, several recent reports present lacks of information about hiPSC genetic integrity [9], in special, after long-term cultivation in vitro, [10] even though many studies suggest abnormalities to be progressively favored by suboptimal culture conditions such as single-cell passaging [11] or high-cell density cultures. [12] Once long-term maintenance in culture seems likely to promote self-renewal [10,13] and limit differentiation through progressive selection of genetic variants, [14] the assessment and validation of PSC cultivation protocols is mandatory to support reproducibility in differentiations.…”

Patient-Derived Stem Cell Models in SPAST HSP: Disease Modelling and Drug Discovery

“…Development of non-integrating vectors for reprogramming have obviated some of the off-target effects of early methods of iPS genetic reprogramming, but reprogramming methods are still inefficient, laborious, expensive, and time-consuming posing real challenges to application in multiple labs and in multiple patients. As a consequence of the reprogramming process and prolonged cell culture, iPS cells can develop a wide range of variations, including aneuploidy and chromosomal aberrations, single nucleotide variations, and sub-chromosomal copy number variations [49,50,51]. Differentiation of iPS cells into neurons can be problematic: efficiency was low and variable between iPS cell lines that were generated by the same reprogramming method, different reprogramming methods, and between iPS cell lines that are generated from the same parental fibroblast cell line [52].…”

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7

“…To add to the complexity, specific stimuli are required to direct iPSC to re‐differentiate to progenitor cells of the lineage of interest. In addition, any remaining iPSC poses risk of teratoma formation following implantation . These issues have limited the therapeutic value of iPSC technology in tissue regeneration.…”

“…In addition, any remaining iPSC poses risk of teratoma formation following implantation. 1 These issues have limited the therapeutic value of iPSC technology in tissue regeneration.…”