ABSTRACT
Human embryonic stem cells (hESC) show great promise for clinical and research applications, but their well-known proneness to genomic instability hampers the development to their full potential. Here, we demonstrate that medium acidification linked to culture density is the main cause of DNA damage and genomic alterations in hESC grown on feeder layers, and this even in the short time span of a single passage. In line with this, we show that increasing the frequency of the medium refreshments minimizes the levels of DNA damage and genetic instability. Also, we show that cells cultured on laminin-521 do not present this increase in DNA damage when grown at high density, although the (long-term) impact on their genomic stability remains to be elucidated. Our results explain the high levels of genome instability observed over the years by many laboratories worldwide, and show that the development of optimal culture conditions is key to solving this problem.
Subject(s)
Cell Culture Techniques/methods , DNA Damage , Embryonic Stem Cells/cytology , Genomic Instability , Cell Line , Embryonic Stem Cells/drug effects , Embryonic Stem Cells/metabolism , Humans , Laminin/pharmacologyABSTRACT
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.
Subject(s)
Amnion/metabolism , Chromosomes, Human/genetics , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Mosaicism , Amnion/cytology , Cell Line , Cells, Cultured , Comparative Genomic Hybridization , Humans , In Situ Hybridization, FluorescenceABSTRACT
Cultured human embryonic stem (hES) cells have a known predisposition to aneuploidy of chromosomes 12, 17 and X. We studied 17 hES cell lines by array-based comparative genomic hybridization (aCGH) and found that the cells accumulate other recurrent chromosomal abnormalities, including amplification at 20q11.21 and a derivative chromosome 18. These genomic changes have a variable impact at the transcriptional level.