Protein Induction of Pluripotent Stem Cells Made More Efficient

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.


Stanford study finds Induced pluripotent stem cells match embryonic stem cells in modeling human disease

Investigators from Stanford University School of Medicine have shown that induced Pluripotent Stem cells (iPSCs), which are made from adult cells through genetic engineering techniques, are a possible alternative to human embryonic stem cells when it comes to modeling those defects caused by a particular genetic condition. The example used in this study was Marfan syndrome, and in this study, iPSCs modeled the disease as well as embryonic stem cells (ESCs). Thus, iPSCs could be used to examine the molecular aspects of Marfan on a personalized basis. Embryonic stem cells, on the other hand, can’t do this because their genetic contents are those of the donated embryo are not the same as the patient’s.

Marfan syndrome is an inherited connective-tissue disorder that occurs in one in 10,000 to one in 20,000 individuals. It results from a large number of defects in one gene called “fibrillarin.” People with Marfan syndrome tend to be very tall and thin, and also tend to suffer from osteopenia, or poor bone mineralization. Medical experts have speculated that Abraham Lincoln, for example, suffered from this disorder. Marfan can also profoundly affect the eyes and cardiovascular system.

This proof-of-principle study, with regards to the utility of iPSCs also has more universal significance; it advances the credibility of using iPSCs to model a broad range of human diseases. iPSCs, unlike ESCs, are easily obtained from virtually anyone and possess a genetic background identical to the patient from which they were derived. Moreover, they carry none of the ethical controversy associated with the necessity of destroying embryos.

“Our in vitro findings strongly point to the underlying mechanisms that may explain the clinical manifestations of Marfan syndrome,” said Michael Longaker, MD, professor of surgery and senior author of the study, which will be published online Dec. 12 in Proceedings of the National Academy of Sciences. Longaker is the Dean P. and Louise Mitchell Professor in the School of Medicine and co-director of the school’s Institute for Stem Cell Biology and Regenerative Medicine. The study’s first author is Natalina Quarto, PhD, a senior research scientist in Longaker’s laboratory.

In this study, both iPSCs and ESCs, and embryonic stem cells that carried a mutation that causes Marfan syndrome showed impaired ability to form bone, and all too readily formed cartilage. These aberrations mirror the most prominent clinical manifestation of the disease.

iPSCs were discovered in 2006, and are derived from fully differentiated tissues such as the skin. However, they harbor the same capacity as embryonic stem cells; namely to differentiate into all the tissues of the body, and replicate for indefinite periods in a cell culture dish. Because iPSCs offer an ethically uncomplicated alternative to ESCs, IPSCs have fueled the hope that they can replace ESCs in scientists’ efforts to analyze, in a dish, those cellular defects ultimately responsible for diseases ranging from diabetes to Parkinson’s and even such complex conditions as cardiovascular disease and autism.

One hope for iPSCs is to be able to differentiate them in a dish into tissues of interest and then study these cells and their characteristics. This would help scientists better understand diseases in a patient-specific way, which would be impossible to do with ESCs unless ESCs were made from donated human eggs that were modified by cloning procedures. Cloning human embryos to the blastocyst stage has yet to occur, which makes this option technically impossible at the present time.

While scientists want to us iPSCs to develop therapeutic applications for regenerative medicine. This strategy, however, is technically more difficult, since scientists will have to develop the capacity first to repair genetic defects within cells before they can be used for regenerative medicine. iPSCs in theory might be a better bet because they are derived from patients’ own cells and, therefore, are less likely to provoke graft rejection than similar tissues produced using a donor embryo’s ESCs.

Unfortunately, several studies have reported subtle differences between iPSCs and ESCs, and these differences imply that the two cell types may not be equivalent. Stem cell experts have wondered whether these differences may render iPSCs inadequate substitutes for ESCs in modeling disease states, but this Stanford study suggests otherwise.