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Apr 26, 2010

Silencing of Genes May Be Responsible for Murine iPSCs’ Limitations

  • An important cluster of genes is inactivated in mouse induced pluripotent stem cells (iPSCs), according to investigators from the Massachusetts General Hospital Center for Regenerative Medicine (MGH-CRM) and the Harvard Stem Cell Institute (HSCI). “We found that a segment of chromosome 12 containing genes important for fetal development was abnormally shut off in most iPSCs,” says Konrad Hochedlinger, Ph.D., of the MGH-CRM and HSCI who led the study.

    “These findings indicate we need to keep improving the way we produce iPSCs and suggest the need for new reprogramming strategies,” Dr. Hochedlinger states. His paper, called “Aberrant silencing of imprinted genes on chromosome 12qF1 in mouse induced pluripotent stem cells,” appeared online April 25 in Nature.

    Several molecular differences have been observed between iPSCs and embryonic stem cells (ESCs), particularly in the epigenetic processes that control which genes are expressed. Additionally, procedures that are able to generate live animals from the embryonic stem cells of mice are much less successful with iPSCs.

    Previous studies have compared iPSCs generated through the use of viruses to embryonic stem cells from unrelated animals, according to the MGH/HSCI team. To reduce the chance that the different sources of the cells were responsible for observed molecular differences, they prepared two genetically matched cell lines. After generating mice from embryonic stem cells, they used a technique that does not use viruses to prepare lines of iPSCs from several types of cells taken from those animals. They then compared the iPSCs with the original, genetically identical embryonic stem cells.

    The most stringent assay of cells' developmental potential showed that two tested lines of embryonic stem cells were able to generate live mice as successfully as in previous studies but no animals could be generated from genetically matched iPSCs, the researchers report. Closely comparing RNA transcription profiles of several matched cell lines revealed significantly reduced transcription of two genes in the iPSCs. Both genes are part of a gene cluster on chromosome 12 that is normally maternally imprinted.

    Examination of more than 60 iPSC lines developed from several types of cells revealed that this gene cluster was silenced in the vast majority of cell lines. While the gene-silenced iPSCs were able to generate many types of mouse tissues, their overall developmental potential was limited.

    In an assay that produces chimeric animals that incorporate cells from two different stem cells, mice produced from gene-silenced cells had very few tissues that originated from the iPSCs. Notably, treatment of an iPSC that had the genes silenced with a histone deacetylase inhibitor reactivated the locus and rescued its ability to support full-term development of all iPSC mice.

    In the few iPSC lines where the gene cluster was normally activated, the most rigorous developmental assay showed that those iPSCs were as successful in producing live animals as embryonic stem cells have been. The authors believe this is the first report of animals being produced entirely from adult-derived iPSCs.

    “The activation status of this imprinted cluster allowed us to prospectively identify iPSCs that have the full developmental potential of embryonic stem cells,” says Matthias Stadtfeld, Ph.D., of the MGH-CRM and HSCI, a co-lead author of the report. “Identifying pluripotent cells of the highest quality is crucial to the development of therapeutic applications so we can ensure that any transplanted cells function as well as normal cells. It's going to be important to see whether iPSCs derived from human patients have similar differences in gene expression and if they can be as good as embryonic stem cells, which continue to be the gold standard in giving rise to the 220 functional cell types in the human body.”

     


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