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The potential use of human embryonic stem cells (hESC) is
an exciting but controversial area in medicine today. In concept, hESC cells
might be able to repair and/or regenerate damaged tissues and replace injured
cells. It is often assumed that after harvesting, these cells are genetically
stable, even though they must be expanded substantially through repeated cell
division to generate enough cells for current experiments and for possible
future therapeutic uses. However, like all dividing cells, it is probable that
cultured hESC undergo a low level of spontaneous mutation, which in some cases
could adversely affect their therapeutic potential. Maitra et al examined this
issue by comparing several parameters of genomic stability in 9 hESC lines that
were available as both early and late passage cells, ie, early and late passage
paired cell lines. Cells normally stop dividing when they reach high density in
culture, but they will start dividing again if diluted. Passage refers to this
dilution process; it is a crude measure of the number of times cells have
divided, ie, late passage cells have divided many more times than early passage
cells.
The authors used 3
assays to search for alterations of cellular DNA: nuclear DNA copy number,
mitochondrial DNA sequence, and gene promoter methylation. In the first case,
initial Affymetrix high-density array analysis of approximately 115 000 single
nucleotide polymorphisms (SNPs) distributed across the genome showed no
significant differences between the early and late passage hESC. However,
further analysis revealed copy number alterations in late but not early passage
cells from 4 of the 9 paired cell lines. The alterations ranged from large
genomic regions of amplification or deletions, such as amplification of the
entire chromosome 17q arm, to discrete changes such as a 2-Mb amplification
that included the MYC oncogene. These changes were verified by in situ
hybridization (FISH) or quantitative genomic PCR.
Next, they screened the mitochondrial genome, which is
often mutated in cancer, again using array technology. Sequence alterations
were detected in 2 of the late passage hESC cell lines that were not observed
in early passage cells from the same cell lines.
Promoter methylation, an epigenetic phenomenon observed in
almost all cancers, was assessed in a panel of 14 genes known to be
differentially methylated in cancer cells. Differential methylation of 3 genes
was detected in late passage cells. For one gene, RASSF1 – a putative tumor
suppressor gene, increased methylation was found in late but not early passage
cells from 7 of the 9 paired hESC lines.
In conclusion, the
authors suggest that most but not all hESC lines maintained in cell culture
acquire clonal DNA alterations over time. Many of these alterations are similar
to what has been observed in cancer, such as loss of tumor suppressor genes or
amplification of oncogenes. These alterations may provide a growth advantage
that allows the cells that harbor them to dominate late passages. The authors
acknowledge that much more work is needed to better define the nature of these
alterations and their functional consequences. However, they argue that their
findings underscore the need to periodically monitor hESC lines before they are
used in in vivo applications and that some late-passage hESC may be
unusable for therapeutic purposes due to genomic alterations over time.
Maitra
A, Arking DE, Shivapurkar N, et al. Genomic alterations in cultured human
embryonic stem cells. Nat Genet. 37:1099 - 1103.
Editor’s Comment: It is clear that hESC have
great potential in regenerative medicine. However, this paper illustrates that
the field is still relatively young with many troublesome issues, such as
long-term genomic fidelity, must be resolved before it can be applied
clinically.
William A. Horton, MD
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Current GGH - Vol. 24 No. 1
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