|Elevated Coding Mutation Rate During the Reprogramming of Human Somatic Cells into Induced Pluripotent Stem Cells|
From Stem Cells
Recent studies in the field of induced pluripotent stem cells (iPSCs) through karyotypic (Taapken et al) and meta-analysis of gene expression data (Mayshar et al) have revealed aneuploidy, and also copy number analysis has detected large-scale sub-chromosomal aberrations (Laurent et al and Martins-Taylor et al) that arise upon prolonged passaging. Further, it is known that mutations not present in the parental cell of reprogramming arise during the reprogramming process (Gore et al and Hussein et al), but the proportion of mutations in iPSCs acquired due to the reprogramming process is unknown.
However, in a study in the March edition of Stem Cells, from the laboratory of Nizar N. Batada at the Ontario Institute for Cancer Research, Toronto, detailed coding sequence analysis of iPSCs and parental cell types has demonstrated that in vitro passaging contributes 7% to iPSC coding point mutation load and that 19% of the mutations pre-exist as rare mutations in the parental fibroblasts so suggesting that 74% of the mutations present in the iPSCs were acquired during cellular reprogramming (Ji and Ng et al). Moreover, the authors suggest the rate of mutation may be ninefold higher during reprogramming than the normal background mutation rate of the cultured fibroblasts. It is unclear if such enhanced mutational load influences the genomic stability and differentiation capacity of iPSC.
iPSCs were first generated from human primary neonatal foreskin fibroblasts using retroviruses encoding KLF4, MYC, OCT4, and SOX2 transgenes, using as similar a technique as possible with all samples. Five randomly selected iPSCs were picked, which all displayed the hallmarks of pluripotency, and were sequenced at passage 6 and 12 subsequent to the initial colony being picked at day 28 of the reprogramming process, alongside a sample of the parental fibroblasts. Exomic data (that is DNA-encoding protein coding) was generated using the Illumina Genome Analyzer IIx giving more than 60 million uniquely aligning reads per sample.
Through comparisons between the data for the parental fibroblast and the human reference NCBI build 36.1, unique variants, or mutations, could be identified in each iPSC line which specifically occurs during the reprogramming process. 59 synonymous (protein sequence changing) point mutations were discovered in the 5 passage 6 iPSC samples which were confirmed to be absent from the parental fibroblasts. To analyse the attainment of mutations during passaging, p6 iPSCs were compared to the p12 iPSCs. 60 mutations were found in the p12 iPSCs, with 56 of the 59 mutations in the p6 iPSCs persisting, with 4 mutations being classified as passaging induced mutations. The authors note that some of these mutations may have been present in the p6 iPSCs below the detection limit for the current technology. Accordingly, mutations may also be present in rare fibroblast subpopulation, although deep amplicon sequencing of the parental fibroblasts only found 8 of 46 randomly selected mutations at a rare frequency.
Mutations in the crucial p53 gene were then analysed, with no mutations in any of the 11 exons of TP53 found in any iPSC lines or the parental fibroblasts. The presence of functional p53 in the parental fibroblasts is of obvious importance as this should. Further, gene up and downstream (MDM2, CDKN2A, P21, and BCL2) also did not contain any mutations. Gene ontology analysis of the genes with mutations also failed to signify an enrichment of a specific biological process and no mutations in DNA repair genes were found. This suggests that the parental fibroblast line, and the iPSCs, contained no mutations in genes which would foster the presence or amplification of mutations.
This study suggests that coding point mutations generally arise during the reprogramming process and persist during passaging, and could have potentially deleterious effects. The authors note that this therefore may suggest that optimal reprogramming conditions to lower the mutational load are required so that iPSCs may be useful to regenerative medicine.
Gore A et al.
Hussein SM et al.
Ji J, Ng SH et al.
Laurent LC et al.
Martins-Taylor K et al.
Mayshar Y et al.
Taapken SM et al.