Clinicians and stem cells scientists have been hopeful but also quite cautious about the use of induced pluripotent stem cells iPSCs in human treatments. One of the primary concerns in the use of viral vectors that insert themselves into the genome of the cells they infect. Such insertions can create activating mutations or insertional inactivation mutations that can transform cells into tumors.
However, scientists at Stanford University School of Medicine have designed a safer way to make iPSCs that is also very efficient. This method is an extension of a protocol that has already been tried; treating the cells with recombinant proteins that can pass through the cell membrane and transform the cells into iPSCs without the use of viruses. Unfortunately, this protocol has proven to be rather inefficient relative to methods that use genetically engineered viruses.
The Stanford researchers discovered that viruses were not simply burrowing into cells to deposit genes. According to John Cooke, MD, PhD, professor of medicine and associate director of the Stanford Cardiovascular Institute and senior author of this work: “It had been thought that the virus served simply as a Trojan horse to deliver the genes into the cell. Now we know that the virus causes the cell to loosen its chromatin and make the DNA available for the changes necessary for it to revert to the pluripotent state.”
The derivation of iPSCs does not require the destruction of embryos. and therefore, offer an ethical alternative to embryonic stem cells (ESCs). Instead of using embryos, iPSCs are made from adult cells that have been genetically engineered to overexpress four different genes (Oct4, Sox2, Klf4 and c-Myc). These four genes are heavily expressed in ESCs and by transiently overexpressing them in adult cells, the adult cells revert to an ESC-like state.
The derivation of iPSCs from adult cells was discovered by Shinya Yamanaka and his colleagues, and Yamanaka won the Nobel Prize for this achievement.
The research of Cooke and his colleagues, however, provides an important clue as to how this reversion to the embryonic state occurs. Cooke noted, “We found that when a cell is exposed to a pathogen, it changes to adapt or defend itself against a challenge. Part of this innate immunity includes increasing access to its DNA, which is normally tightly packaged. This allows the cell to reach into its genetic toolbox and take out what it needs to survive.”
It is this loosening of the structure of DNA in adult cells that allows the pluripotency-inducing proteins to modify the expression pattern of the cell and transform it into an ESC-like cell.
This type of response to viral infections that causes the DNA of cells to loosen up has been termed “transflammation” by Cooke and his team. They think that this finding could easily simplify and increase the efficiency of iPSC derivation.
Cooke’s laboratory initially tried to increase the efficiency of cell-permeable proteins that can reprogram adult cells into iPSCs. These proteins can bind to their target sequences on DNA and can also enter the nucleus when they pass into the cell. Why were these proteins so inefficient when compared to viral-based techniques?
To answer this question, Cooke’s lab examined the gene expression patterns of cells treated with iPSC-inducing viruses or iPSC-transforming proteins. They discovered that the gene expression patterns differed extensively. This led Cooke to hypothesize the virus itself was causing some sort of change in the adult cells that was necessary for iPSC derivation.
To test this hypothesis, they repeated the experiment with recombinant proteins but also concomitantly treated the cells with an unrelated virus. This dramatically increased the rates of pluripotency transformation. The increased rate of transformation was also linked to a signaling pathway called the toll-like receptor-3 (TLR-3) pathway.
Toll-like receptors (TLRs) have been established to play an essential role in the activation of innate immunity by recognizing specific molecular patterns normally found on microbial components. Each TLR recognizes a different set of microbial-specific molecules, and TLR-3 binds to double-stranded RNA molecules. Therefore, these cells activate those pathways that are normally turned when they are infected by viruses.
According to Cooke, “These proteins are non-integrating, and so we don’t have to worry about any viral-induced damage to the host genome.” Cooke also pointed out that cell-permeable proteins can allow the researchers to exert greater amounts of control over the reprogramming process. This, essentially could speed the use of iPSCs in human therapies. Cooke continued: “Now that we understand that the cell assumes greater plasticity when challenged by a pathogen, we can theoretically use this information to further manipulate the cells to induce direct reprogramming.”
Therefore, to sum up, the elimination of TLR3 reduces the efficiency and yield of human iPSC generation, but if TLR3 is activated, it enhances human iPSC generation by cell permeant peptides. Also, TLR3 activation enables changes to the structure of DNA (epigenetic changes), and these changes promote an open chromatin state that makes iPSC generation much more efficient.