Whereas embryonic stem cells generally pass all these tests, many iPSCs do not. Closer examination of the cells that fail has revealed that the viruses used to deliver the four key reprogramming genes into skin cells are often not properly shut off, and important genes in the cells’ original DNA are not properly turned on, resulting in cells that have lost their skin cell identity without gaining a pluripotent identity. These partially reprogrammed cells therefore do not qualify as authentic pluripotent cells.
Ongoing studies of iPSCs that do pass all the pluripotency tests are aimed at pinpointing the differences that distinguish a “good” from a “bad” iPSC. Thorsten Schlaeger, George Daley and their colleagues at Harvard University, for example, recently identified a pattern of gene activity in skin cells undergoing the lengthy (about three weeks) process of changing their identity to that of pluripotent cells. The fluorescent markers displayed by these cells during the transition distinguished them from cells in the same colony that would not ultimately become iPSCs, and so this marker pattern could be used as an early indicator of successful conversion.
Because scientists cannot ethically perform the most stringent pluripotency test by injecting human iPSCs into human embryos, it is absolutely critical to ensure that human iPSCs fulfill all other criteria of pluripotency. These include the complete silencing of the potentially harmful viruses employed to deliver the reprogramming genes. Yamanaka’s team members discovered, for example, that one third of the mice that they had generated by injecting iPSCs into developing mouse embryos later formed cancers as a consequence of residual retrovirus activity.
One of the main problems with using retroviruses as gene-delivery vehicles is that these kinds of viruses (HIV is one example) integrate themselves directly into the host cell’s DNA strand, becoming a part of its genome. This ability allows the added genes to reside permanently and remain active in the host cell, but depending on where the virus inserts itself, it can cause DNA damage that sparks cancerous changes in the cell. In efforts to produce safer iPSCs, therefore, many labs have developed methods that avoid permanent genetic manipulation of cells.
My research group has used a modified type of adenovirus, which normally causes the common cold in humans, to deliver the four reprogramming genes into mouse cells without integrating into the cellular genome. Adenoviruses persist inside the cells for only a short period—just long enough to convert them into iPSCs. When we injected the resulting pluripotent cells into mouse embryos, they readily became incorporated into the developing animals, which were all tumor-free as adults. This discovery, along with several alternative approaches to producing virus-free iPSCs, should eliminate a major roadblock to one day applying iPSCs directly in human therapies.
Ultimately, researchers hope to produce iPSCs without using any type of virus, but instead by simply exposing adult cells to a combination of drugs that mimic the effect of the reprogramming genes. Sheng Ding of the Scripps Research Institute, Douglas A. Melton of Harvard and others have already identified chemicals that can substitute for each of the four reprogramming genes in that each chemical activates a pathway of molecular interactions inside a cell that would be activated by the gene. When the four drugs have been tried together, however, they proved insufficient to make pluripotent cells. It may only be a matter of time, though, until researchers find the right cocktail and concentration of drugs to reprogram body cells into iPSCs without ever using viruses.
Because pluripotent cells are capable of generating any type of tissue in the body, the application that most captures the public imagination is the possibility of using iPSCs to produce replacement parts for cells and organs damaged by disease: neurons lost to Parkinson’s or a spinal cord injury, for instance, or cardiac tissue destroyed by a heart attack. The ability to convert adult cells from the intended recipient of such a transplant into pluripotent cells and then coax those cells into the desired tissue would mean the replacement part is perfectly matched, genetically and immunologically, with the recipient’s body. Moreover, easily accessible skin cells could be used to produce any kind of needed cell, including those in hard-to-reach organs and tissues, such as the brain or pancreas.