Risk of genotoxicity in human iPS cell-based therapeutics

by Alexey Bersenev on December 8, 2012 · 1 comment

in embryonic/iPS, reviews

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We wrote a lot about risks associated with potential therapeutic application of iPS cells. Generation of iPS cells linked to significant genomic instability and acquisition of mutations. The recent review, published in Molecular Therapy, nicely summarizes the current status of the problem of iPS cell genotoxicity. This is the best review on the topic! Because it’s not openly accessible, I’m going to go through the major points of the review here.

What is genotoxicity?

Genotoxicity is a term used to refer to heritable and potentially toxic or deleterious effects on a cell’s genetic material.

What predisposes cells to acquire genotoxicity?

  • prolonged cell culture;
  • stemness (high expression of pluripotency genes);
  • use of intergating viral vectors as gene carriers (insertional genotoxicity/ mutagenesis);
  • genetic modifications for therapeutic purpose.

All of these factors could be directly applied to iPS cell generation.
Today we have significant evidence for high genomic instability of human iPS cells. The authors cited recent reports and highlighted the differences with embryonic stem cells:

The human iPSCs showed a similar frequency of chromosome 12 duplications compared with human ESCs. However, unlike human ESCs, no aberration involving chromosome 17 was detected in human iPSCs.

How can we assess genomic instability and what is the best method? You can see all current methodologies in the following scheme:
(Adapted from: Mol Ther doi: 10.1038/mt.2012.255, modified).

Each method has advantages and disadvantages. For example, G-band karyotyping – well established, widely available, cheap, but has low resolution:

… standard karyotyping did not reveal any abnormalities, the subclones possessed subkaryotypic abnormalities identified by aCGH.

Molecular karyotyping via array-based comparative genomic hybridization (aCGH) or single-nucleotide polymorphism (SNP) arrays have higher resolution but poor sensitivity for detecting minor subclones compared with traditional karyotyping, and cannot be used to detect mosaicism because these assays read out the pooled hybridization of genomes from many cells.

Sequencing technologies have very high resolution and good sensitivity, but costly and required bioinformatics support:

Gore et al. performed exome sequencing of human iPSCs and their parental fibroblasts. A total of 124 mutations were found in 22 otherwise karyotypically normal human iPSC lines, with an average of five protein-coding point mutations in the regions sampled per each line.

Unresolved issue:

… it still remains unclear whether reprogramming itself is a mutagenic process and moreover what the biological consequences of the described abnormalities might be. The studies published to date reported conflicting data, suggesting that in this nascent field, lab to lab variability in culture conditions or passaging procedures could potentially account for the inconsistent results.

For both clinical and “disease in a dish” applications, it will be important for the field to agree upon a standardized approach to characterization, both initially and with prolonged culture. In addition, epigenetic or functional instability of iPSCs are also of concern, and should be carefully evaluated along with genotoxicity.

How different reprogramming methods impact iPS cell genotoxicity?

To date, available data suggests that the reprogramming method itself, whether integrating or nonintegrating, does not impact on reprogramming-associated genotoxicity.

How to minimize iPS cell genotoxicity risk:

  • Standardization of genetic assessment of iPS cells;
  • Developing new preclinical models for evaluation of safety. The authors proposed a nonhuman primate model and validated high-throughput in vitro assay.
  • Suicide gene (safety switch) strategies. It was recently described for ES cells and iPS cells.

Finally, the authors called for collaboration between researchers, cell product developers and regulators in order to advance the use of iPS cells in medicine.

This review is highly recommended!

{ 1 comment… read it below or add one }

Paul Knoepfler December 10, 2012 at 11:34 pm

Great piece Alexey.
I’m one of the biggest fans of iPS cells in the world, but rushing them to the clinic is unwise. We still have so much to learn about these exciting cells. If I point that out, people in the stem cell field often get cranky.
Paul

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