IPSC without the help of viruses and genes: simple, effective, safe
An effective model for obtaining human induced pluripotent
stem cells without DNA integration
LifeSciencesToday based on the materials of the University of California:
UC San Diego Researchers Develop Efficient Model for Generating Human iPSCsScientists at the University of California, San Diego School of Medicine have developed a simple and easily reproducible method for producing human induced pluripotent stem cells (iPSCs) based on RNA.
The results of their study are published in the journal Cell Stem Cell (Yoshioka et al., Efficient Generation of Human iPSCs by a Synthetic Self-Replicative RNA). The new approach opens up broad prospects for the use of iPSCs in scientific research and, possibly, in stem cell therapy.
American scientists have managed to develop a much more effective method than those based on DNA embedding. It avoids potential integration problems and obviously represents a safer and easier way to create iPSCs in terms of their future clinical use.
Obtaining human induced pluripotent stem cells opens up wide opportunities in the transition to the clinical use of regenerative medicine methods based on a particular patient's own stem cells. Pluripotency means that these cells have the ability to give rise to any type of cells in the body. Human iPSCs, as a rule, are artificially obtained from non-pluripotent differentiated cells, such as cells of the deep layers of the skin fibroblasts, representing connective tissue that forms the structural basis of many other tissues.
iPSCs retain the characteristics of natural pluripotent stem cells, known as embryonic stem cells. Since they develop from the patient's own cells, it was believed that their use for medicinal purposes would avoid any immunogenic reactions. However, depending on the methods of preparation, the use of induced pluripotent stem cells may be associated with a significant risk limiting their clinical use. For example, the use of viruses as a means of delivering genes to change the genome of differentiated cells can provoke their cancer degeneration.
Previously developed methods for obtaining iPSCs to avoid DNA integration are complex and inefficient in reproduction. Therefore, UCSD researchers focused their attention on an alternative approach based on self-replicating RNA. In this case, there is no need for embedding genes in DNA, while the method makes it possible to save and destroy RNAs in a controlled mode, which it is enough to introduce into the cell only once.
Obtaining human induced pluripotent stem cells opens up broad prospects in the development of treatment methods for a number of diseases, but without integrative DNA vectors it remains problematic. UCSD scientists have developed a simple and easily reproducible method for obtaining iPSCs based on RNA, which uses a single synthetic self-replicating RNA replicon (a replicative form of the Venezuelan equine encephalitis virus RNA, VEE-RF RNA) expressing four reprogramming factors at a consistently high level (OCT4, Klf4, SOX2 and either c-MYC or GLIS1) before regulated RNA degradation. A single transfection of VEE-RF RNA into newly formed or adult human fibroblasts leads to the effective generation of iPSCs with all the characteristics of stem cells, including cell surface markers, global gene expression profiles and the ability to differentiate in vivo into all three germ sheets. iPSCs created using RNA replicon are free of transgenes.
Artistic (from the UC Santa Barbara website) and schematic (from an article in Cell Stem Cell) image
a method for obtaining human induced pluripotent stem cells using RNA.Using Venezuelan equine encephalitis virus (Venezuelan equine encephalitis virus, VEE) with removed structural and preserved non-structural proteins, the scientists added four reprogramming factors (OCT4, KLF4, SOX2 and either c-MYC or GLIS1) to newly formed or adult human fibroblasts by performing a single transfection of the replicative form (replicative form, RF) Virus RNA.
"The result of this transfection was the effective generation of iPSCs with all the signs of stem cells," explains study leader Steven Dowdy, PhD, professor of the UCSD Department of Cellular and Molecular Medicine. "This method is highly reproducible, efficient, does not require integration – and it works."
The new method gives good results on both young and old human cells, and this is important, since induced pluripotent stem cells suitable for treating diseases or creating disease models should be obtained from cells of middle-aged or elderly people more prone to developing diseases that scientists are trying to treat. In addition, it makes it easy to replace reprogramming factors, adds Professor Dowdy.
The work was funded by the California Institute for Regenerative Medicine (California Institute for Regenerative Medicine) and the National Institutes of Health (National Institutes of Health) USA.
Portal "Eternal youth" http://vechnayamolodost.ru26.08.2013