Principles of stem cell biology and cancer: future applications and therapeutics. Edited by T. Regad, T. J. Sayers and R. C. Rees. John Wiley & Sons (2015)

1. Isolation and characterization of human embryonic stem cells and future applications in tissue engineering therapies

1.3. Stem cell quality and culture adaptation with reference to cancer

Epigenetics is the study of changes acting upon but not altering the DNA sequence, namely such mechanisms as imprinting, DNA methylation and histone modification to regulate gene expression. The epigenetic characterization of hESC lines, and other cell types, is much less established than genomic analysis, because of its higher level of complexity. While we have sequenced the whole human genome, we do not yet have the same understanding of our epigenome. There are hundreds of epigenomes for every genome, because every person has hundreds of cell types, each of which has different DNA modifications. Another reason is that epigenetic changes are dynamic: able to adapt to changes in the cellular environment over time. High-throughput methods for the analysis of a full set of methylations are now available, but the technology is still new and expensive, and it still needs to develop reliable references for hESCs.

Epigenetic mechanisms give specific cell types their identity by allowing only a subset of genes to be active. Faulty regulation in early embryonic development can result in embryo mortality or distort differentiation and should be evaluated and deselected from hESC cultures, if necessary.

Human ESCs are derived from an early blastocyst stage – a developmental time point at which cells are fragile because processes like X-chromosome inactivation (XCI) are still ongoing (van den Berg et al., 2009). Imprinting and XCI

Expression of a number of genes is necessarily mono-allelic, which means that one of the parental alleles needs to be silenced (in a process termed ‘imprinting’) for proper development to occur. For example, mono-allelic expression is important for X-linked genes and hence requires XCI in female embryos. With regards to imprinting and XCI, the gene dosage is important, and failure to properly silence one allele can result in lethality or developmental disorders. Several studies have indicated that prolonged hESC culture can affect the XCI pattern of pluripotent cell lines (Lengner et al., 2010; Tchieu et al., 2010; Nazor et al., 2012). Cell culture under low oxygen allows for the derivation of female ESC lines with two active X chromosomes, while normal oxygen will produce mixed cultures, indicating that the cell culture environment has a profound effect on XCI (Lengner et al., 2010). Consequently, if a certain X-linked expression is required for disease modelling, the activation or inactivation should be evaluated during ESC characterization. A PCR for X-inactive specific transcript (XIST) expression, which is expressed from the inactivated X chromosome, will give an initial idea of whether XCI has occurred and is maintained in a particular culture system. Methylation pattern

DNA methylation silences promoter regions and prevents gene expression where it is not required. New technologies have now started to evaluate genome-wide DNA methylation patterns and are building a reference map for ESCs. While many promoter regions are equally methylated and demethylated between ESC lines, other genes appear to be variably methylated (Bock et al., 2011). Processes that give rise to variation include underlying human variability, cell culture methods, the time point, the method of derivation and other stress factors. What seems clear is that these changes in methylation patterns are impacting the differentiation capacity of ESCs and could be used to predict their ability to differentiate along certain lineages (Bock et al., 2011). Hence, methylation analysis on promoter regions for genes that are important for lineage-specific differentiation can give important insight into the selection of a cell line for a specific purpose and may be included in the characterization of a line. Established methods such as methylation-specific polymerase chain reaction (MSP), pyrosequencing or array-based methylation analysis, together with a reference map, can give clues as to whether a particular cell line is able to differentiate towards all lineages equally. Histone modifications

Histones are proteins that package the DNA in eukaryotic cells and play a role in gene regulation, by rendering DNA active or inactive. They can be highly modified through various modifying enzymes and thereby affect gene regulation. For example, promoters occupied by a histone H3 lysine 4 trimethylation (H3K4me3) or histone H3 lysine 27 trimethylation (H3K27me3) are associated with gene activation and repression, respectively. Histone modifications can be affected by cell culture adaptation, and may lead to higher proliferation and differential expression of tumour suppressor genes with parallels to cancer cells. While analysis of histone modification is not commonly carried out for hESC characterization, they impact many genes that are linked to severe developmental disorders and cancers (Lund et al., 2013).


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