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Genomics Reveals Molecular Changes Behind Cellular Reprogramming

NEW YORK (GenomeWeb News) – Using a battery of genomic techniques, a group of American researchers have started characterizing the molecular changes underlying direct reprogramming — the process of returning differentiated adult cells to a pluripotent, stem cell-like state.
The researchers investigated mouse cells at various stages of reprogramming using everything from gene expression profiling to chromatin mapping and methylation analyses. By comparing successfully reprogrammed cells, known as induced pluripotent stem cells, or iPS cells, with cells that failed or stalled in the process, the team identified molecular signatures of pluripotency — as well as molecular stumbling blocks on the way to pluripotency. Their work appeared online today in Nature.
“We needed to understand what is happening while cells reprogram,” senior author Alex Meissner, a researcher affiliated with the Broad Institute and Harvard University’s department of stem cell and regenerative biology, told GenomeWeb Daily News today. 
The ability to reprogram adult cells is still relatively new. Less than two years ago, Japanese researchers demonstrated that they could reverse cellular differentiation in mouse fibroblast cells by expressing four transcription factors: Oct4, Sox2, Klf4, and c-Myc. The factors “kickstart” the stem cell program, Meissner explained. This returns cells to a pluripotent state, meaning they can become any type of cell in the body, and gives them the ability to renew themselves — another characteristic stem cell feature.
Since then, several studies have shown that tinkering with the same transcription factors can reprogram human cells, too. The approach holds promise for creating patient-specific iPS cells that could be used for regenerative medicine — bypassing controversial techniques such as nuclear transfer and human embryonic stem cell harvesting.
Still, the reprogramming process remains time-consuming and temperamental. And, it hinges on infecting somatic cells with genetically engineered viruses, an approach that’s less than ideal for regenerative medicine.
“Certainly, I don’t think anybody intends to use the current strategy — using viruses — in any patient,” Meissner said.
But if researchers can get a better handle on how reprogramming works, they may eventually be able to achieve the same end while circumventing the viral delivery system. “A clearer understanding of the process would enable development of safer and more efficient reprogramming strategies, and might shed light on fundamental questions concerning the establishment of cellular identity,” Meissner and his colleagues wrote.
To begin doing this, the team characterized mouse fibroblast and B lymphocyte cells at different stages of reprogramming based on their gene expression profiles, chromatin states, and/or epigenetic modifications.
Overall, the researchers noted that fully reprogrammed cells have gene expression profiles and epigenetic features that are much like those of embryonic stem cells. But the majority of cells stall en route to becoming iPS cells. Based on gene expression profiling, the researchers found that these cells switch on “fail-safe” mechanisms in response to reprogramming transcription factors, often arresting to prevent over-proliferation.
They also tend to ramp up certain differentiation genes, such as axon guidance factors and epidermal proteins, possibly in a separate response to the reprogramming transcription factors.
“Improving the low efficiency of the reprogramming process will require circumventing these [fail safe] mechanisms without disabling them permanently,” lead author Tarjei Mikkelsen, a graduate student at the Broad Institute, said in a statement.
When they looked specifically at three partially reprogrammed cell lines derived from mouse B lymphocyte cells, the researchers identified a mix of somatic and iPS cell features. While the partially reprogrammed cells looked essentially like stem cells, Meissner said, they could easily be distinguished based on their chromatin profile — assessed using ChIP-seq, chromatin immunoprecipitation coupled with Illumina sequencing.
Interestingly, Meissner said, the partially reprogrammed cells had gained the ability to self-renew and had lost their cell-type specific differentiation. But they still weren’t pluripotent, suggesting the two processes can be decoupled, Meissner noted.
Not surprisingly, the partially reprogrammed cells have unique gene expression patterns, re-expressing some stem cell genes and not others. Some cells also continued expressing lineage-specific transcription factors, preventing complete reprogramming.
Meissner and his colleagues reasoned that they may be able to nudge the process of reprogramming along using RNA interference to knock down certain transcription factors. Indeed, they showed that it is possible to improve the efficiency of reprogramming by suppressing lineage-specific transcription factors. Inhibiting DNA methyltransferase enzymes also improved the reprogramming efficiency.
In the future, Meissner said, the team plans to continue using genome-wide tools to further refine its understanding of iPS cells. Ultimately, they and others hope that by understanding direct reprogramming, it will be possible to create patient-specific iPS cells that can be used therapeutically. “We’re actually getting close to being able to do that,” Meissner said.

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