Abstract
Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. To assess the genetic risks induced by culturing, we determined all mutations in individual human stem cells by whole genome sequencing. Individual pluripotent, intestinal, and liver stem cells accumulate 3.5 ± 0.5, 7.2 ± 1.1 and 8.3 ± 3.6 base substitutions per population doubling, respectively. The annual in vitro mutation accumulation rate of adult stem cells is nearly 40-fold higher than the in vivo mutation accumulation rate. Mutational signature analysis reveals that in vitro induced mutations are caused by oxidative stress. Reducing oxygen tension in culture lowers the mutational load. We use the mutation rates, spectra, and genomic distribution to model the accumulation of oncogenic mutations during typical in vitro expansion, manipulation or screening experiments using human stem cells. Our study provides empirically defined parameters to assess the mutational risk of stem cell based therapies.
Highlights
Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine
The resultant genome-wide mutation spectra and distribution have been shown to provide novel insights into the activity of specific mutational and DNA repair processes in adult stem cells in vivo and CRISPR/Cas9-edited organoids[23,24,25]. We have adapted this approach to systematically measure the mutational impact of in vitro culture on individual cells of three human stem cell types that were chosen for their relevance in pharmacology, toxicology, and regenerative medicine: pluripotent stem cells (PSCs), liver ASCs, and intestinal ASCs
Clones were established for one pluripotent embryonic stem cell line, two induced PSC lines, two liver ASC lines, and two intestinal ASC lines
Summary
Genetic changes acquired during in vitro culture pose a risk for the successful application of stem cells in regenerative medicine. Multiple studies have identified recurrent genomic alterations that originate in routinely cultured human ASCs and PSCs7–20 These studies suggest that the genomic integrity of stem cells is reduced in culture, the majority of these studies have been performed on bulk cultures with methods that have limited resolution. Bioinformatic analyses are performed that identify with high confidence those mutations (single-nucleotide variants, indels, copy number alterations, structural variants (SVs), and aneuploidies) present in the original cell and filter out germline variants and subclonal mutations that occurred after the single-cell step[21] With this method, high noise rates of in vitro amplification approaches are avoided, which enables the reliable detection of less than a hundred mutations per genome[22]. We used the measured quantitative and qualitative mutational characteristics to model genome-wide mutation accumulation and perform genetic risk assessments associated with in vitro and in vivo applications of stem cells
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