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Aug 1, 2013

New RNA-Based Method for Producing iPSCs

  • Researchers at the University of San Diego School of Medicine reported that they have developed a simple, easily reproducible RNA-based method of generating human induced pluripotent stem cells (iPSCs). Writing in the August 1 edition of Cell Stem Cell, the researchers described a method for producing these cells that they say dramatically improves upon existing DNA-based approaches to creating pluripotent stem cells for use in human stem cell studies, and eventually, cell therapies.

    To date, methods previously developed to generate integration-free iPSCs have not proven not easily and efficiently reproducible. Therefore, the UC San Diego researchers focused their approach on developing a self-replicating nonintegrating RNA-based method that could be retained and degraded in a controlled fashion, and that would only need to be introduced once into the cell.

    While terminally differentiated somatic cells such as fibroblasts can be reprogrammed to generate iPSCs, several drawbacks have complicated their clinical use. These include low efficiency of cell generation without genetic alterations, the potential for tumor formation in vivo, random integration of retroviral-based delivery vectors into the genome, and unregulated growth of the remaining cells that are partially reprogrammed and refractory to differentiation. Investigators say that the development of protein or RNA-based reprogramming strategies will help generate human iPSCs without permanent genetic alterations.

    The authors of the current paper describe a highly reproducible RNA-based iPSC generation approach using a single, synthetic self-replicating VEE-RF RNA replicon that expresses four reprogramming factors (OCT4, KLF4, SOX2, with c-MYC or GLIS1) at consistent high levels prior to regulated RNA degradation. A single VEE-RF RNA transfection into newborn or adult human fibroblasts resulted in efficient generation of iPSCs with all the hallmarks of stem cells, including cell surface markers, global gene expression profiles, and in vivo pluripotency, to differentiate into all three germ layers.

    The VEE-RF RNA-based approach has broad applicability, the authors said, for the generation of iPSCs for ultimate use in human stem cell therapies in regenerative medicine.

    "This resulted in efficient generation of iPSCs with all the hallmarks of stem cells," said principal investigator Steven Dowdy, Ph.D., professor in the UC San Diego Department of Cellular & Molecular Medicine. "The method is highly reproducible, efficient, nonintegrative—and it works.”

    Dowdy said that the method worked on both young and old human cells. He explained that this is important since in order to be used therapeutically in fighting disease or to create disease models for research iPSCs will need to be derived from the cells of middle-aged to old adults who are more prone to the diseases scientists are attempting to treat. In addition, reprogramming factors can be easily changed.

    The methods developed by the UC San Diego researchers dramatically improve upon existing DNA-based approaches, avoiding potential integration problems and providing what appears to be a safer and simpler method for future clinical applications. Their approach has broad applicability for the successful production of iPSCs for use in human stem cell studies and eventual cell therapies.

    This study, "Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA", was published in the August 1, 2013, issue of Cell Stem Cell (volume 13, issue 2).


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