|More Me: More iPSC?|
Original article from STEM CELLS
Widespread epigenetic remodelling is acknowledged as being a critical process in the reprogramming of somatic cells to induced pluripotent stem cells (iPSCs), and the incomplete erasure of the epigenetic signature of somatic cells (“epigenetic memory”) can influence their differentiation properties (Kim et al and Polo et al). While many comparisons have found iPSCs and embryonic stem cells (ESCs) to be very similar, stochastic differences and differences shared between independent iPSC lines have been observed (Bock et al, Lister et aland Ohi et al). However, within these comparisons, no studies have analysed the differences in epigenetic patterns in iPSCs derived from somatic cells which have different reprogramming efficiencies. Researchers from the laboratories of Manel Esteller and Juan Carlos Izpisua Belmonte now propose that higher reprogramming efficiency correlates with the hypermethylation of tissue-specific genes rather than with a more permissive pluripotency gene network through their studies in reprogramming human keratinocytes, fibroblasts and their comparison to hESCs (Barrero and Berdasco et al).
Initial unsupervised clustering of CpG methylation status separated differentiated primary tissues from iPSCs reprogrammed from fibroblasts (FiPS), foreskin keratinocytes (KiPS) and plucked hair keratinocytes (hKiPS) and ESCs, which all clustered together revealing a high level of similarity to the methylation status of iPSCs and ESCs. However, detailed analysis of methylation events found that while ESCs and KiPS cells were characterized by the highest number of hypermethylated probes, FiPS cells exhibited only around half of the amount of hypermethylated probes observed in the rest of the pluripotent cells, suggesting incomplete epigenetic reprogramming. The authors suggest that this reflects both memory of epigenetic status of cell of origin and aberrant methylation during reprogramming.
Two of the most prominent regions of difference were associated with the HIST1H3C gene and the TCERG1L gene. HIST1H3C is located in the HIST1 cluster which is generally hypomethylated in somatic cells, but while the KiPS demonstrated hypomethylation here, the FiPS cells and ESCs did not, and the FiPS cells consistently showed hypermethylation along the HIST1 cluster than the rest of the cell lines analyzed. The TCERG1L gene is hypomethylated in ESCs, keratinocytes and fibroblasts, but is the study found that it was hypermethylated in KiPS and FiPS cells, and correlated negatively with gene expression levels.
Focusing on changes at CpGs which occur after reprogramming, 556 differentially methylated regions (DMRs) between iPS cells and cell of origin were found with gain of methylation in the iPSCs observed to be a more common event. However, sites of hypomethylation correlated significantly to genes with bivalent domains in hESCs; most bivalent genes were hypomethylated in ESCs and somatic cells while iPSCs exhibited higher levels of DNA methylation at some of these genes, a phenomenon more obvious in FiPS cells. Bivalent domains are generally associated with developmental genes, and so over-methylation events may modulate iPS cell differentiation in affected cell lines (Kim et al).
Overall, gain in DNA methylation is more prominent in KiPS cells than in FiPS cells, making KiPS cells more similar to ESCs. Regions that become hypermethylated after reprogramming generally show low CpG content and are associated with tissue-specific genes (Fouse et al and Meissner et al). Keratinocytes share a high level of hypermethylated tissue-specific genes with ESCs, as compared to fibroblasts, and this may contribute to the higher efficiency of keratinocyte reprogramming. However, hypomethylation at pluripotency associated genes may also allow easier activation during reprogramming. Subsequent analysis of genes associated with pluripotency found a cluster of pluripotency genes whose differential expression in pluripotent and somatic cells is clearly regulated by DNA methylation, and of these keratinocytes have higher methylation levels in these pluripotency genes indicating that the higher reprogramming efficiency does not correlate with a more permissive chromatin environment at these genes. To further test the hypothesis of higher methylation being associated with reprogramming, cells which have been noted for relative “ease” of reprogramming (foetal and extra-embryonic cells) were analysed. These experiments also found that cells which reprogram more efficiently share more hypermethylated genes with ESCs, again demonstrating the link between hypermethylation and reprogramming.
In conclusion, a positive correlation between DNA hypermethylation in areas with low CpG content and efficiency of reprogramming has been demonstrated. DNA methylation patterns in iPSCs reprogrammed from keratinocytes are more similar to ESCs than those reprogrammed from fibroblasts mostly due to the presence of genes that fail to acquire high levels of DNA methylation in FiPS cells. Low CpG content is often found at regions containing tissue specific genes and suggest that the silencing of differentiation genes, through increases in DNA methylation, is a very important step in reprogramming. Supporting this hypothesis are studies that demonstrate that ESC-like DNA hypermethylation patterns are only fully established at later stages of iPSC reprogramming (Koche et al) and that miRNA mediated silencing of differentiation genes can reprogram somatic cells to pluripotency (Anokye-Danso et al).
Does this mean that fibroblasts are an inherently flawed source of somatic cells for the reprogramming process? Although there are differences at the DNA methylation level with correlative changes in gene expression, perhaps the functionality - or lack thereof, will be the final test of iPSCs?
Anokye-Danso F et al.
Barrero MJ et al.
Bock C et al.
Fouse SD et al.
Kim K et al.
Koche RP et al.
Lister R et al.
Meissner A et al.
Ohi Y et al.
Polo JM et al.
STEM CELLS correspondent Stuart Atkinson reports on those studies appearing in current journals that are destined to make an impact on stem cell research and clinical studies.