Analysis of protein-coding mutations in hiPSCs and their possible role during somatic cell reprogramming

Recent studies indicate that human-induced pluripotent stem cells contain genomic structural variations and point mutations in coding regions. However, these studies have focused on fibroblast-derived human induced pluripotent stem cells, and it is currently unknown whether the use of alternative somatic cell sources with varying reprogramming efficiencies would result in different levels of genetic alterations. Here we characterize the genomic integrity of eight human induced pluripotent stem cell lines derived from five different non-fibroblast somatic cell types. We show that protein-coding mutations are a general feature of the human induced pluripotent stem cell state and are independent of somatic cell source. Furthermore, we analyse a total of 17 point mutations found in human induced pluripotent stem cells and demonstrate that they do not generally facilitate the acquisition of pluripotency and thus are not likely to provide a selective advantage for reprogramming

Identification of a specific reprogramming-associated epigenetic signature in human induced pluripotent stem cells

Generation of human induced pluripotent stem cells (hiPSCs) by the expression of specific transcription factors depends on successful epigenetic reprogramming to a pluripotent state. Although hiPSCs and human embryonic stem cells (hESCs) display a similar epigenome, recent reports demonstrated the persistence of specific epigenetic marks from the somatic cell type of origin and aberrant methylation patterns in hiPSCs. However, it remains unknown whether the use of different somatic cell sources, encompassing variable levels of se- lection pressure during reprogramming, influences the level of epigenetic aberrations in hiPSCs. In this work, we characterized the epigenomic integrity of 17 hiPSC lines derived from six different cell types with varied reprogramming efficiencies. We demonstrate that epigenetic aberrations are a general feature of the hiPSC state and are independent of the somatic cell source. Interestingly, we observe that the reprogramming efficiency of somatic cell lines inversely correlates with the amount of methylation change needed to acquire pluripotency. Additionally, we determine that both shared and line- specific epigenetic aberrations in hiPSCs can directly translate into changes in gene expression in both the pluripotent and differenti- ated states. Significantly, our analysis of different hiPSC lines from multiple cell types of origin allow us to identify a reprogramming- specific epigenetic signature comprised of nine aberrantly methyl- ated genes that is able to segregate hESC and hiPSC lines regardless of the somatic cell source or differentiation state

Somatic coding mutations in human induced pluripotent stem cells

Defined transcription factors can induce epigenetic reprogramming of adult mammalian cells into induced pluripotent stem cells. Although DNA factors are integrated during some reprogramming methods, it is unknown whether the genome remains unchanged at the single nucleotide level. Here we show that 22 human induced pluripotent stem (hiPS) cell lines reprogrammed using five different methods each contained an average of five protein-coding point mutations in the regions sampled (an estimated six protein-coding point mutations per exome). The majority of these mutations were non-synonymous, nonsense or splice variants, and were enriched in genes mutated or having causative effects in cancers. At least half of these reprogramming-associated mutations pre-existed in fibroblast progenitors at low frequencies, whereas the rest occurred during or after reprogramming. Thus, hiPS cells acquire genetic modifications in addition to epigenetic modifications. Extensive genetic screening should become a standard procedure to ensure hiPS cell safety before clinical use

The Genetics and Epigenetics of Induced Pluripotent Stem Cells

The ability to induce pluripotency in human adult somatic cells by defined transcription factor expression is a revolutionary prospect in regenerative medicine. This discovery has the potential to both open new research avenues for diseases in tissue types that are difficult to obtain and to revolutionize medicine through the use of patient-derived replacement tissue. However, questions remain about the safety and efficacy of these induced pluripotent stem cells (iPSCs). Because iPSC generation protocols tend to be low efficiency, require derivation from adult tissue, often utilize viral transfection, force the expression of known oncogenes, and involve a large number of rapid cell divisions during reprogramming, it was thought that the iPSC genome itself might contain some genetic mutation. Additionally, the progenitor cell type used for iPSC derivation seemed to cause some differentiation pathways to be more highly favored, indicating that iPSCs might possess some sort of "epigenetic memory" of their progenitor state. Thanks to modern advances in high throughput sequencing, we were able to assess the genomic and epigenomic state of induced pluripotent stem cells, and thus determine if iPSCs could be used in either a clinical or a research context. We demonstrate that induced pluripotent stem cells contain a large number of point mutations across their genome regardless of donor age, time in culture, progenitor cell type, or reprogramming method. While a majority of these mutations arise due to rare progenitor mutations becoming fixed through clonal selection during reprogramming, approximately 43% arise either during the reprogramming step or during iPSC expansion. We additionally show that, in addition to epigenetic memory of the progenitor cell state and aberrant DNA methylation, nearly all iPSC lines carry a unique reprogramming-specific epigenetic signature that remains even after further differentiation and impacts gene expression in iPSC-derived cells. Taken together, these results demonstrate that iPSCs must still overcome major hurdles prior to their widespread clinical use. Rigorous work towards establishing clinical safety standards for genetic and epigenetic integrity in pluripotent-derived therapies will be essential before the promise of induced pluripotency can be fully realize

Whole genome sequencing analysis of human iPSC clones after Cas9- and TALEN-mediated homologous recombination at the AAVS1 locus

<p>Summary of WGS of four human iPSC clones after Cas9-mediated or TALEN-mediated homologous recombination. The sequencing results were compared to parental BC1 iPSCs.</p

The Presenilin-1 ΔE9 Mutation Results in Reduced γ-Secretase Activity, but Not Total Loss of PS1 Function, in Isogenic Human Stem Cells

Presenilin 1 (PS1) is the catalytic core of γ-secretase, which cleaves type 1 transmembrane proteins, including the amyloid precursor protein (APP). PS1 also has γ-secretase-independent functions, and dominant PS1 missense mutations are the most common cause of familial Alzheimer’s disease (FAD). Whether PS1 FAD mutations are gain- or loss-of-function remains controversial, primarily because most studies have relied on overexpression in mouse and/or nonneuronal systems. We used isogenic euploid human induced pluripotent stem cell lines to generate and study an allelic series of PS1 mutations, including heterozygous null mutations and homozygous and heterozygous FAD PS1 mutations. Rigorous analysis of this allelic series in differentiated, purified neurons allowed us to resolve this controversy and to conclude that FAD PS1 mutations, expressed at normal levels in the appropriate cell type, impair γ-secretase activity but do not disrupt γ-secretase-independent functions of PS1. Thus, FAD PS1 mutations do not act as simple loss of PS1 function but instead dominantly gain an activity toxic to some, but not all, PS1 functions

Targeted bisulfite sequencing reveals changes in DNA methylation associated with nuclear reprogramming

Current DNA methylation assays are limited in the flexibility and efficiency of characterizing a large number of genomic targets. We report a method to specifically capture an arbitrary subset of genomic targets for single-molecule bisulfite sequencing for digital quantification of DNA methylation at single-nucleotide resolution. A set of ~30,000 padlock probes was designed to assess methylation of ~66,000 CpG sites within 2,020 CpG islands on human chromosome 12, chromosome 20, and 34 selected regions. To investigate epigenetic differences associated with dedifferentiation, we compared methylation in three human fibroblast lines and eight human pluripotent stem cell lines. Chromosome-wide methylation patterns were similar among all lines studied, but cytosine methylation was slightly more prevalent in the pluripotent cells than in the fibroblasts. Induced pluripotent stem (iPS) cells appeared to display more methylation than embryonic stem cells. We found 288 regions methylated differently in fibroblasts and pluripotent cells. This targeted approach should be particularly useful for analyzing DNA methylation in large genomes.Molecular and Cellular Biolog

Whole-genome sequencing in autism identifies hot spots for de novo germline mutation

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans. PaperFlick

Non-invasive early detection of cancer four years before conventional diagnosis using a blood test.

Early detection has the potential to reduce cancer mortality, but an effective screening test must demonstrate asymptomatic cancer detection years before conventional diagnosis in a longitudinal study. In the Taizhou Longitudinal Study (TZL), 123,115 healthy subjects provided plasma samples for long-term storage and were then monitored for cancer occurrence. Here we report the preliminary results of PanSeer, a noninvasive blood test based on circulating tumor DNA methylation, on TZL plasma samples from 605 asymptomatic individuals, 191 of whom were later diagnosed with stomach, esophageal, colorectal, lung or liver cancer within four years of blood draw. We also assay plasma samples from an additional 223 cancer patients, plus 200 primary tumor and normal tissues. We show that PanSeer detects five common types of cancer in 88%&nbsp;(95% CI: 80-93%) of post-diagnosis patients with a specificity of 96% (95% CI: 93-98%), We also demonstrate that PanSeer detects cancer in 95%&nbsp;(95% CI: 89-98%) of asymptomatic individuals who were later diagnosed, though future longitudinal studies are required to confirm this result. These results demonstrate that cancer can be non-invasively detected up to four years before current standard of care