Low-grade chromosomal mosaicism in human somatic and embryonic stem cell populations

Abstract

Current knowledge on chromosomal mosaicism in human cell cultures is mostly based on cytogenetic banding methods. The recent development of high-resolution full-genome analysis methods applicable to single cells is providing new insights into genetic and cellular diversity. Here we study the genetic content of 92 individual human cells, including fibroblasts, amniocytes and embryonic stem cells (hESCs), using single-cell array-based comparative genomic hybridization (aCGH). We find that human somatic and embryonic stem cell cultures show significant fractions of cells carrying unique megabase-scale chromosomal abnormalities, forming genetic mosaics that could not have been detected by conventional cytogenetic methods. These findings are confirmed by studying seven clonal hESC sub-lines by aCGH. Furthermore, fluorescent in situ hybridisation reveals an increased instability of the subtelomeric regions in hESC as compared to somatic cells. This genetic heterogeneity may have an impact on experimental results and, in the case of hESC, on their potential clinical use.

Detecting mosaicism in trophectoderm biopsies: current challenges and future possibilities

“…Depending on different procedural aspects, reagent batch and parameters for quality control and data analysis, a varying false positive error rate can be expected from the application of a specific aCGH protocol on a single cell. For instance, one can be more stringent in the analysis and consider only arrays results with an SD ≤ 0.15, >80% included clones, signal-to-background ratio >3 and derivative log-ratio <0.2 and get a higher accuracy but a smaller sample size (Jacobs et al ., 2014), or less stringent and get more results but with lower reliability. Different versions of the analysis software and different protocols (dual channel array vs single channel array) may also impact performance.…”

“…All aCGH studies performed so far on blastomeres failed to report a primary validation on single cells, with the use of each specific protocol , to estimate the false positive error rate, or do not report such data on a sufficient number of samples (Jacobs et al ., 2014). …”

Random Mutagenesis, Clonal Events, and Embryonic or Somatic Origin Determine the mtDNA Variant Type and Load in Human Pluripotent Stem Cells

“…A significant amount of research has been devoted to this topic, proving that these cells undergo replicative stress (Jacobs et al., 2014, Jacobs et al., 2016, Lamm et al., 2016) and the cultures are frequently taken over by recurrent chromosomal abnormalities conferring an in vitro survival advantage (Amps et al., 2011, Avery et al., 2013, Nguyen et al., 2014, Merkle et al., 2017). In contrast, only a few studies provide some insight on the integrity of their mitochondrial genome, despite the important role mitochondria play in reprogramming and maintenance of the stem cell state (Van Blerkom, 2008, Lonergan et al., 2007).…”

DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation

“…There was a clear contrast between chromosome 19, which has the highest gene and CpG island density [26], and displayed negative waves with prominent losses affecting almost its entire length, and the similarly-sized chromosome 18, with the lowest gene density and one of the lowest CpG densities, which showed a mixed picture, with waves in either direction (S2A Fig). Chromosome 19 can be problematic on both aCGH [27] and single neuron whole genome amplification [28]. A loss of almost the whole chr19 had indeed been called in one sample by ADM2 with FZ on, but only at threshold 6.…”