HPSC cells and their differentiated derivatives have been widely acclaimed to provide a much needed alternative supply of human cell types that closely resemble their in vivo counterparts. A recent study by Kevin Eggan and his colleagues1, finds 5% of 257 hPSC lines tested to have acquired oncogenic mutations. The bad news, WA09 (H9), the world’s most utilized hESC line and MShef10 (a GMP line) were found to have acquired oncogenic mutations. The good news, the study included nine Genea Biocells (GBC) lines in which NO mutations were found. This study provides a timely reminder that hPSC lines are as susceptible to mutation-driven genotype drift as many other cultured mammalian lines and that if the hope expressed above is to be realised , there is a need for much better quality control at the stages of hPSC derivation, expansion and differentiation.
It has long been appreciated that cells grown in culture often adapt to the changed environmental conditions, and that some naturally occurring genetic changes can be positively selected for. Human pluripotent stem cells (hPSC) are non-oncogenic cell lines that have been feted for two main reasons: a) their ability in cell culture to indefinitely replicate and maintain their pluripotent phenotype, and b) their ability to differentiate into desired cellular phenotypes on addition of specific triggering factors. These features have made them attractive candidates for use in cell therapy and drug discovery. Nonetheless, it should come as no surprise that during cell culture, mutations occur and positive selection (for growth) results in a drift away from the original genotype. It now appears that some of these acquired mutations are very similar to those that are seen in, and contributing to, cancer.
We have known for some time that aneuploidies and copy number variations have been seen during the progressive culture of some hESC lines2 More recently, in later passage hESC cells, deletions have been reported in the genomic region harboring the tumor suppressor gene TP53 which lead to its decreased expression3 . A functioning TP53 pathway is essential to the well being of many cell types and in over 50% human cancers one of the two TP53 alleles is mutated. These missense mutations occur in the DNA binding region of TP53 protein and through a dominant-negative effect, impede the functioning of the nonmutated TP53. In the newly published paper, Merkle et al1 analysed data from several hundred different hPSC lines and found that some of these lines accumulate the very same mutations prevalent in human cancers.
Using the NIH registry as a primary guide, these scientists first acquired 114 independent research hES lines along with a further 26 hESC lines prepared under GMP conditions. Genomic DNA was isolated and subjected to whole exome sequencing; the mean passage number of the lines was p18. Six mutations of the TP53 gene were found with a distribution that was mosaic, with allelic fractions ranging from 7-40%. This indicates that 14-80% of the cell populations harbored a mutation. Notably WA09 (H9), the world’s most utilized hESC line, and MShef10 (a GMP line) were among the afflicted lines identified. The authors were further able to show that these mutations likely were acquired and progressively accumulated during culture. Finally, they used published RNA sequencing data to screen for similar mutations in 104 hiPSC lines and found seven mutations mapping to the same TP53 DNA binding region.
This work constitutes a large and thorough investigation of genomic stability in cultured hPS cells. The authors were faced with the vexing issue that there was considerable divergence in the derivation and maintenance of many of the hESC lines before they obtained them. Whilst they did their best to wean all the cells on to a common culture platform, the impact of each line’s history remains unknown. In their historical analyses, authors looked but could not find correlations between the occurrence of mutations and choice of tissue culture media, cell passaging method, plate matrix, or culture supplements. They suggest research be carried out on better culture techniques, and that genome analysis be carried out at various key steps during the development, expansion, and use of chosen lines since these TP53 mutations could be highly problematical in cell therapy and arguably, if highly prevalent in a population of hPSC-derived, differentiated cells, could affect their value in drug discovery.
The message here is that popularity does not ensure quality. Too often studies are carried out using a small and limited selection of hESC lines, with WA09 dominating research usage. This choice is exacerbated by frequent omission of testing of the lines for their maintained pluripotency and genetic stability. Of the hundreds of hESC lines and no doubt now thousands of hiPSC lines in the world, how can one best assess suitability and applicability of lines? While the NIH and UKSCB databases assure the ethical derivation of lines, the lack of scrutiny on pluripotency and genetic characterization makes it difficult to tell the quality of lines within their databases. More recent databases, like hPSCreg and SCR lab resources, practice a higher level of scrutiny by peer reviewing characterization data towards approval and addition of lines to their collection. Still, this approval does not guarantee the quality of a received line. Researchers should employ or secure batch specific validation of hPSC lines and repeat this validation at key stages in their studies. This information should be shared with the reviewers. These steps should improve the quality and reproducibility of their findings.
Genea Biocells has 159 hPSC lines, 62 so far on the NIH registry. Genea Biocells has always been meticulous, not only in culture and expansion regimes, but batch specific QC including IF marker staining, CGH, STR and PluriTest. We would like to think that this level of internal scrutiny contributed to the absence of detectable TP53 mutations seen in all nine GBC lines examined in this recent paper, and is something that should be expected from all sources of hPSC lines.
Alan Colman May 2017
- Merkle et al (2017) Nature doi:10.1038/nature22312
- Adewumi et al. (2007) Nat. Biotechnol. 25, 803–816 (2007)
- Garitaonandia et al (2015) PLOS ONE DOI: 10.1371/journal.pone. 0118307