UC San Diego

Genomic mutations pose a serious risk to the health of individuals both in terms of somatic cells and in stem cells used for clinical and research applications. Here we outline novel approaches for studying genome mutations in the context of human induced pluripotent stem cells and neurons. To definitively establish the extent of reprogramming-associated mutations, we utilize the fact that a single fibroblast can give rise to two iPSC colonies separated by at most two divisions. Comparison of the two colonies allows us to distinguish mutations present in the original cell (those found in both colonies) from mutations arising during the reprogramming process. We find on average 150-450 single nucleotide variants per iPSC line, with iPSCs derived by episomal method being significantly more mutated than iPSCs derived with lentivirus. Further, we find that the mutations from episomal reprogramming show unique signatures compared to lentiviral and somatic reprogramming methods. We also find that reprogramming does not contribute significant number of structural variant or mobile element insertion classes of mutation. In the context of neurons, we utilize somatic cell nuclear transfer to reprogram rod photoreceptors from young and aged mice , allowing us to amplify single neuronal genomes without error-prone PCR methods. We show that these neurons accumulate 20-40 SNVs per year, and that these mutations are enriched in nucleotiode contexts that implicate APOBEC deaminase, as well as potential regions of somatic hypermutation.