The work conducted by the Yamanaka lab led to the discovery that somatic cells can be reprogrammed into pluripotent cells (hiPSCs’s) by introducing four transcription factors (TFs): Oct4, Sox2, Klf4 and c-Myc—the infamous gene believed to be linked to cancer. Given the highly controversial role of c-Myc, additional studies were conducted to see if those factors could be replaced. It was found that only Oct4 and Sox2 are crucial to induce pluripotency, however, the efficiency suffered significantly. The exact mechanism by which this induction happens is still poorly understood today. It is believed that the expression of the two transcriptions factors among others—that are still being tested—leads to epigenetic modifications in the cell, from which little is known, resulting in inducing a pluripotent state within said cells. This process takes several weeks before yielding a very small percentage of pluripotent cells. This poses a serious problem, which needs to be resolved before using hiPSC’s in therapeutic applications.
Another difficulty, which will also have to be addressed, is the reprogramming technique itself. Indeed, the three most used reprogramming methods all have flaws that forbid them from being used in therapy.
The initial techniques used were done with lentiviral or retroviral transduction. They were and still are very efficient methods of inducing pluripotency, however, both vectors were found to integrate into the genome. This has for consequence to disrupt the gene at site of entry often leading to tumourigenesis. Furthermore, the expression of the TFs themselves must be regulated, as they are only needed for a set period of time, this leads to problems with differentiation. In order to respond to the difficulties engendered by this method, adenoviral vectors have been developed that do not integrate into the genome. However, the efficiency suffers and prolonged expression of the TFs remains a problem.
Given the difficulties encountered when using viral methods, non-viral methods were developed. Plasmid vectors containing the TFs’ complementary DNA’s were transfected into the cells and resulted for the most part with non-genomic integration. Because of contradictory results, another method was used: fusion of the TFs proteins to a membrane permeable peptide (constructs recombinant cell-penetrating reprogramming proteins). These peptides are broken down by the cell itself and nothing of the constructs remain inside the cells. It is labor intensive however as it requires multiple rounds of addition of recombinant proteins to ensure sufficient presence of the TFs proteins for long enough. A new method is showing great promises with microRNAs (miRNAs), the regulatory RNAs that repress the expression of a large set of target genes post transcriptionally. It was shown that reprogramming with miRNAs was faster and approximately 100-fold more efficient than with standard viral vectors. However, viral vectors are still used to deliver the miRNAs to the cell. There are ongoing works to overcome this problem with very promising results.
Once the reprogramming techniques perfected, methods to accurately measuring pluripotency must be in place in order to determine if the end product is achieved or not after reprogramming. This in itself is a difficulty as stemness must be clearly defined before being able to qualify an iPSC as having achieved an embryonic stem cell (ESC) like state: stemness is still ill understood and poorly quantified. For those reasons, molecular assays have been created to identify ESC-like condition as well as the epigenetic status of the hiPSCs based on the detection of mRNA associated with pluripotency and promoter activity. However, even after extensive testing it is still difficult to draw conclusive results about the success of the reprogramming technique as even if resemblance to ESCs is attained it might just be that in end, resemblance. Additionally, the efficiency of the reprogramming technique, percentage of viable hiPSC colony forming cells, is usually very low and is dependent on a combination of cell state and reprogramming strategy making the entire process that much more complicated.
Nonetheless, the application of hiPSCs in cell replacement therapy for neurodegenerative diseases including PD, Alzheimer’s Disease and ALS has been the subject of great interest given its potential to repair the damaged brain. And more importantly they can be obtained from the patient own somatic cells and thus bypass immune rejection issues, the ethical issues surrounding ESCs and the very invasive protocols to obtaining neural stem cells.
In the case of PD there were very positive outcome of several studies for developing cell therapies and therefore the use of hiPSCs is being explored extensively. Different labs are trying to optimize in vitro protocols to produce large amounts of functional dopaminergic cells. There are no clinical trials for PD as of yet, but animal studies have shown promising results. Indeed, transplantation performed with iPSC-derived dopaminergic neurons improved motor function in a rat model of PD, and even resulted in an improvement of PD signs, both functionally and behaviorally. There is one major issue however the survival rate of transplanted cells is often low. Another disease where the use of hiPSCs might be very useful is in vanishing white matter disorder (VWM), a progressive disease in which the white matter of the brain becomes increasingly abnormal and eventually virtually disappears. Indeed, the disease-causing mutations are known and, thus, genetic modification is possible using hiPSCs expanded in sufficient numbers in-vitro before transplantation. Of course the procedure still lacks safety and efficiency and no clinical trials or animal testing is underway as of today since there are no confirmed animal model of the disease yet.
In conclusion, even though hiPSCs show great promises in treating neurodegenerative diseases while avoiding the ethical concerns of ESCs and the problematic immune rejections, there are still many limitations preventing using hiPSCs in clinical settings. Furthermore, even if we overcome the known limitations, we must be aware of potential novel issues arising from using hiPSCs. Indeed, new ethical issues would have to be addressed, such as using patient-specific iPSCs derived cells without the consent of the donor, especially in the production of gametes for reproductive purposes, and, also, we would still need to confirm that differentiated hiPSCs would not trigger an immune response in the patient as well.