iPSCs and fibroblast subclones from the same fibroblast population contain comparable levels of sequence variations

Abstract

Genome integrity of induced pluripotent stem cells (iPSCs) has been extensively studied in recent years, but it is still unclear whether iPSCs contain more genomic variations than cultured somatic cells. One important question is the origin of genomic variations detected in iPSCs-whether iPSC reprogramming induces such variations. Here, we undertook a unique approach by deriving fibroblast subclones and clonal iPSC lines from the same fibroblast population and applied next-generation sequencing to compare genomic variations in these lines. Targeted deep sequencing of parental fibroblasts revealed that most variants detected in clonal iPSCs and fibroblast subclones were rare variants inherited from the parental fibroblasts. Only a small number of variants remained undetectable in the parental fibroblasts, which were thus likely to be de novo. Importantly, the clonal iPSCs and fibroblast subclones contained comparable numbers of de novo variants. Collectively, our data suggest that iPSC reprogramming is not mutagenic.iPSCs | fibroblasts | reprogramming | genomic variation | exome sequencing G enomic integrity of human induced pluripotent stem cells (iPSCs) is an unresolved question and a crucial issue for iPSCs-based therapeutics. To understand the scope of acquired genetic changes that occur during iPSC generation, previous studies have compared single nucleotide variants (SNVs), copy number variations (CNVs), and chromosomal rearrangements in iPSCs to donor somatic cells or embryonic stem cells (ESCs) using various assays, including SNP array and next-generation sequencing methods (1-6). Whole genome or exome sequencing (WGS/ WES) of parental and iPSC pairs have demonstrated that there are, on average, 6-12 coding SNVs per iPSC line (1, 7-9) with varying theories regarding when these SNVs arose in the iPSCs. Most reports attributed de novo SNVs or CNVs in iPSCs to reprogramming-induced stress or long-term in vitro culture (2,3,7,8,(10)(11)(12)(13)(14). However, several studies also suggested that many of the "de novo" variants were either inherited rare preexisting variations in the founder source cells or benign variants regardless of reprogramming method used (1,9,15,16).To determine whether iPSCs are inherently more likely to accumulate mutations and further elucidate the origin of genomic variants present in iPSCs, we have undertaken a unique approach by establishing clonal fibroblast subclones and iPSCs from the same fibroblast population to directly assess whether the reprogramming process leads to more mutations by next-generation sequencing. De novo mutations, which were not detected in the starting pooled parental fibroblasts, were identified in each daughter cell line, both fibroblast subclones and iPSCs. These putative de novo mutation sites were then deep sequenced in the parental fibroblast population to determine whether they were present at low frequency as mosaic variants. In addition, highresolution single nucleotide polymorphism (SNP) arrays were used to search for de novo CNVs in both fibrobla...

Insights into the Mutational Burden of Human Induced Pluripotent Stem Cells from an Integrative Multi-Omics Approach

“…Previous studies that examined the genomic integrity of iPSCs have mainly used SNP arrays (International Stem Cell et al, 2011; Laurent et al, 2011; Panopoulos et al, 2017; Taapken et al, 2011), but this approach only allows the detection of relatively large copy-number alterations (CNAs, > 50 kb). A number of studies using exome or whole-genome sequencing (WGS) technologies have shown that somatic single nucleotide variants (SNVs), and small insertion and deletion (indel) mutations in iPSC lines, are predominantly derived from the parental cell rather than arising during the reprogramming process (Cheng et al, 2012; Gore et al, 2011; Kwon et al, 2017; Lo Sardo et al, 2017; Rouhani et al, 2016). Of note, the functional impact of somatic CNAs, SNVs and indels has not yet been examined in detail.…”

Need for high-resolution Genetic Analysis in iPSC: Results and Lessons from the ForIPS Consortium Authors

“…Recent studies suggest that most variants identified in iPSC, but absent from the donor germline, are already present in a subpopulation of the cells of origin. 12,13,15 We also show extensive somatic mosaicism in the parental fibroblast cultures as a source for fixed somatic variants in iPSCs ( Figure 5F). Considering the data regarding passaging, we propose that random genetic drift induced by colony picking from poly-/oligoclonal cell cultures and not positive selection is a major cause of somatic variation in iPSC clones ( Figure 1C).…”

“…Several reports have shown considerable load of SNVs in iPSC. [12][13][14][15] Here, we describe the ForIPS stem cell biobank resource, a national consortium with the primary goal to establish iPSC technologies to study molecular and cellular mechanisms involved in neurological disorders like PD. We present our approach to a stringent genetic workup, including conventional karyotyping, genetic fingerprinting and CMA in all cell samples.…”

Mutational impact of culturing human pluripotent and adult stem cells

“…Maintenance of genomic integrity in culture is a prerequisite for the successful application of stem cells, as unwanted mutations may influence drug responses and toxicology measurements or might lead to oncogenic transformation following transplantation. However, multiple studies have identified recurrent genomic alterations that originate in routinely cultured human adult and pluripotent stem cell lines [2][3][4][5][6][7][8][9][10][11][12][13][14][15] . Although 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.…”