Improving the efficiency of obtaining iPSCs: proteins – separately, viruses – separately
Unexpected relationship between inflammation and the cell's ability to "reprogram"
Dmitry Dzhagarov, "Biomolecule"
Researchers from Stanford University have unexpectedly discovered an effective way to increase the efficiency of reprogramming somatic cells into stem cells. It turned out that in addition to the inducer proteins read by the cell from the viral carrier vector, the presence of the virus itself is necessary, which activates one of the receptors of the innate immune system.
In a paper that won the Nobel Prize in Physiology or Medicine in 2012, Japanese researcher Shinya Yamanaka demonstrated that the cells of an adult organism can be returned to a state similar to an embryonic cell. For such a radical rejuvenation of the cell, hyperactivation of only four genes (Oct4, Klf4, Sox2 and c-Myc) delivered to the cell using retroviruses is required.
It would seem that the "spring of eternal youth and health" has been found. Yes, it was not there. Although using this method it is possible to rejuvenate even the cells of centenarians, clinicians are in no hurry to use this discovery, since the introduction of foreign DNA into the genome with the help of a virus may well lead to genetic disorders, up to malignant transformation. To overcome this disadvantage, according to a number of researchers, the use of reprogramming is not genes, but simply proteins that penetrate into the nucleus, which these genes encode.
However, attempts to use proteins instead of genes were unsuccessful. The effectiveness of reprogramming with the help of protein factors penetrating into the nucleus turned out to be negligible. Scientists wondered: "Why do proteins act several orders of magnitude less efficiently than "viral" gene delivery?" Maybe the virus somehow helps reprogramming? But how? The answer was obtained in experiments where, together with reprogramming proteins, an extraneous virus was used that did not carry the genes necessary for reprogramming. It turned out that the activation of the innate immune system is a necessary condition for reprogramming cells and obtaining induced pluripotent stem cells (iPSCs).
An article published by a group of scientists led by Kenneth Zaret on November 22 in the journal Cell (Facilitators and Impediments of the Pluripotency Reprogramming Factors' Initial Engagement with the Genome) describes other obstacles to reprogramming somatic cells in iPSCs, and also shows how to overcome them in order to significantly increase the efficiency and speed of IPSC generation. These researchers found a large region of the genome that resisted the binding of reprogramming factors for 48 hours, but was eventually activated, which was required for the formation of induced pluripotent cells. These "non-amenable" sequences are usually chemically marked by a modification of histone H3 by methyl groups (called H3K9me3). It turned out that if you block the enzymes that create such labels, you can significantly speed up the reprogramming process.
Further studies have shown that the virus causes an inflammatory reaction and initiates activation in the cell of the "customs officer" receptor 3 (toll-like receptor 3), abbreviated TLR3. (By the way, the Nobel Prize was also awarded for the discovery and study of toll-like receptors. And they got their name not from the English word toll – "duty", "tariff" – but from the German Toll – "crazy", "insane".)
Indeed, the "shutdown" of the TLR3 gene using RNA interference reduces the effectiveness of the viral reprogramming method, significantly reducing the number of iPSCs at the output. Stimulation of TLR3, caused by a synthetic analogue of double-stranded RNA instead of a virus, caused an increase in the effectiveness of reprogramming. It turned out that TLR3 activation leads to epigenetic changes in certain chromatin regions, making them available for protein reprogramming factors (Oct4, Klf4, Sox2 and c-Myc), as well as for epigenome "modifiers" (such as, for example, beta-catenin). This facilitates the induction of genes involved in reprogramming and causes the formation of induced pluripotent stem cells.
In general, this discovery may help to develop new methods for obtaining iPSCs using reprogramming proteins penetrating into the cell – a technique that is possibly safer, and therefore more acceptable for use in the clinic.
Written based on the materials of the works:
- Lee et al., Activation of Innate Immunity Is Required for Efficient Nuclear Reprogramming. Cell 151, 547-558
- O’Neill, "Transflammation": When Innate Immunity Meets Induced Pluripotency. Cell 151, 471-473
Portal "Eternal youth" http://vechnayamolodost.ru22.11.2012