Saturday, March 05, 2011

Stem Cells in Ophthalmology Update 5: Gene Defects Common in Induced Stem Cells

As the senior editor, John Gever, of MedPage Today reported, following the publication of three studies about induced pluripotent stem cells in the March 3rd, issue of Nature, “The road to regenerative medicine based on induced pluripotent stem cells (iPSCs) may have developed a giant pothole, with new studies showing that the cells are prone to several types of genetic defects.”

The three studies showed that the reprogramming process and subsequent culture of pluripotent stem cells in vitro can induce genetic and epigenetic abnormalities in these cells. The authors of the studies and the editorialist said that the results raise concerns over the implications of such aberrations for future applications of pluripotent stem cells.

Point mutations, copy number variations, and abnormal DNA methylation patterns all appear to crop up during generation of iPSCs. The frequency of such defects significantly exceeds what is normally found in human embryonic stem cells or in fibroblasts, the somatic cells from which iPSCs are usually derived.

"The studies raise concerns over the implications of such aberrations for future applications of iPSCs," wrote Martin F. Pera, PhD, of the University of Southern California in Los Angeles, in an accompanying editorial commentary. But he noted that it remains unknown whether the genetic "reprogramming" undertaken to generate iPSCs from fibroblasts is itself responsible for the genetic defects. Perhaps, Pera indicated, such defects "would be common to any experimental situation in which a cultured cell is subjected to strong selection and replication pressures in vitro."

Each of the three studies focused on a different type of genomic defect.

Kun Zhang, PhD, of the University of California San Diego, and colleagues from several other institutions looked at rates of point mutations in 22 iPSC lines and in the fibroblasts from which they were generated. The average number of mutations in protein-encoding genomic regions in each cell line was close to six. "Every single stem cell line we looked at had mutations. Based on our best knowledge, we expected to see 10 times fewer mutations than we actually observed," Zhang said.

All of the 22 iPSC lines had at least one "nonsilent" mutation that affected the resulting protein. One line showed 12 such alterations leading to mutated proteins. The mutations took different forms, the researchers reported, including splice variants and nonsense mutations.

In addition, they wrote, the abnormalities "were enriched in genes mutated or having causative effects in cancers."

Another of the Nature papers, by investigators from the Samuel Lunenfeld Research Institute in Toronto and elsewhere, examined gene copy-number variations that arise in iPSCs. Samer Hussein, PhD, of the Lunenfeld Institute, and colleagues found that these variations -- which included deletions as well as multiple copies -- were present in 37% of the iPSC lines they analyzed. Such variations were present in just 15% of fibroblasts and 25% of human embryonic stem cells (ESCs). The defects may be less significant in practical applications, however, relative to the point mutations. Hussein and colleagues reported that the highest rates of copy-number variations in iPSCs were seen in early-passage cells, whereas rates dropped with additional passages.

"Most of these novel CNVs rendered the affected cells at a selective disadvantage," the researchers explained. "Expansion of human iPSCs in culture selects rapidly against mutated cells, driving the lines towards a genetic state resembling human ESCs."

The third paper investigated what authors Joseph Ecker, PhD, of the Salk Institute for Biological Studies in La Jolla, Calif., and colleagues called "aberrant epigenomic reprogramming."

DNA methylation patterns help regulate gene expression and are variable during life as well as transmissible during reproduction. Ideally, causing an adult cell to revert to a stem-cell state would also involve reestablishing stem cell-like methylation patterns. Ecker and colleagues therefore generated whole-genome methylation profiles of five iPSC lines along with undifferentiated human ESCs, somatic cells, and differentiated iPSCs and ESCs.

They found that the reprogramming of methylation patterns in iPSCs was generally successful when looked at across the entire genome. "Overall, this process generates an iPSC methylome that, in general, is very similar to that of ESCs," they wrote. But in their base-by-base analysis, Ecker and colleagues discovered hundreds of differentially methylated regions compared with ESCs. Some of these were on the megabase scale, which apparently were "repeatedly resistant to reprogramming."

There were detectable differences in cell appearance or function as a result, the researchers noted. Moreover, whereas cells with copy-number variations failed to survive multiple passages, the abnormal methylation patterns did not appear to affect cell survival, as differentiated iPSCs retained the abnormal patterns.

That these aberrations "cannot be erased by passaging and are frequently transmitted through cellular differentiation has immediate consequences for the derivation and use of iPSCs," Ecker and colleagues warned.

In his "News and Views" commentary, Pera pointed out that these are not the first studies to warn of genomic irregularities in iPSCs. Two previous analyses, one published in 2010 and another earlier this year, also documented abnormal chromosome numbers and gene copy-number variations in the cells. These and the new Nature studies raise a number of questions about the future of iPSC research, Pera contended. Perhaps the most important, he wrote, "is the biological significance of the changes."

He indicated that missing or duplicated chromosomes would clearly disqualify cells from use in therapy, as would a high frequency of mutations in genes associated with cancer or known genetic disorders. "However, the many subchromosomal changes, copy-number variations, or point mutations that are not obviously associated with known disease-related genetic abnormalities pose challenges to interpretation," Pera argued.

He suggested that high-throughput functional genomics may be the best approach to resolving the problems -- which need to be addressed before iPSCs can become a basis for human disease treatments.

All three studies were supported by government and foundation grants. No commercial funding was reported.

The three studies are:

Gore A, et al, "Somatic coding mutations in human induced pluripotent stem cells" Nature 2011; 471: 63-67.

Hussein S, et al, "Copy number variation and selection during reprogramming to pluripotency"  Nature 2011; 471: 58-62.

Lister R, et al, "Hotspots of aberrant epigenomic reprogramming in human induced pluripotent stem cells" Nature 2011; 471: 68-73.

And the accompanying editorial:

Pera M, "The dark side of induced pluripotency" Nature 2011; 471: 46-47

MedPage Today, John Gever, Senior Editor
March 2, 2011

In addition, Healthzone Canada published a lengthy review of the Toronto study: “Toronto Scientists Report Roadblock in Stem Cell Field”; and a press release from the UC San Diego Health System“Mutations Found In Human Induced Pluripotent Stem Cells”, reported in depth on the study from the scientists and colleagues from the University of California, San Diego:

For more on “induced pluripotent stem cells “(iPSCs) and the three other types – "embryonic stem cells" (embryonic SCs, or human embryonic stem cells hESCs), "adult stem cells" (adult SCs) and "parthenogenetic stem cells" (hpSCs), please see my Primer on the Use of Stem Cells in Ophthalmology.


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