Cells Reprogrammed With Single Gene

Nature, August 28, 2009

Researchers at the Max Planck Institute for Molecular Biomedicine in Munster, Germany have simplified the method for generating iPS (induced pluripotent stem) cells from ordinary cells. Specifically, they have reduced the number of required reprogramming genes from four to one. The success of the new procedure, however, is heavily dependent upon the types of cells that are chosen to be reprogrammed.

Led by Dr. Hans Scholer, the team of scientists created iPS cells by reprogramming ordinary somatic (non-stem-cell) neural cells that were derived from aborted human fetal tissue. Clearly, the practicality of applying such a procedure to actual clinical therapies is quite low, due to the fact that brain biopsies are not easily obtainable from living subjects, and most countries have laws restricing the use of fetal and embryonic tissue. At least hypothetically, therefore, Dr. Scholer and his colleagues have proposed that neural cells derived from dental pulp might ultimately yield clinical applications.

Nevertheless, the choice of neural cells was critical to the success of the new procedure, since neural cells already express three of the four factors required for reprogramming into iPS cells, namely, Sox2, KLF4, and c-myc. Only the fourth factor, Oct4, is not naturally expressed and is therefore still required for administration to the cells. Although Oct4 was delivered via the conventional use of viral vectors, it was done without genetic integration, thereby yielding a final iPS cell which does not pose as many dangers and risks to potential patients as do those iPS cells that were reprogrammed from 4 genes.

The ultimate point of generating iPS cells is to create patient-specific stem cell lines which might then be utilized for the development of patient-specific therapies for the treatment of diseases that are unique to the individual patient. Of course, the creation of patient-specific stem cell lines has already been available with adult stem cells, and patient-specific therapies already exist from autologous (in which the donor and recipient are the same person) adult stem cells. Furthermore, with the "immune privileged", "universal donor" adult stem cells such as mesenchymal stem cells, patient-specific therapies are unnecessary since even allogeneic (in which the donor and recipient are not the same person) therapies have already been developed from adult stem cells. Adult stem cells, of course, are not nearly as "sexy" as embryonic stem cells nor even iPS cells, and therapies which already exist do not hold nearly the same irresistible fascination and "mystique" as do potential therapies which have not even been developed yet. Consequently, most of the media focus is on embryonic or iPS cells, not adult stem cells.

Last year, Dr. Scholer and his colleagues had succeeded in reducing the required number of reprogramming genes from 4 to 2. Prior to this latest achievement in which only one reprogramming gene is required, previous animal studies had indicated that the origin of the tissue plays an important role in determining differentiatability. In particular, cells derived from the stomach and the liver were found in mouse studies to be the most easily reprogrammable into iPS cells, but attaining stomach and liver biopsies from living human patients is also not always practical.

The 4 genes that have been conventionally used in the past to create iPS cells are precisely the same agents that render the iPS cells ineligible for clinical use, since the genes - one of which is an oncogene (which causes cancer) - introduce serious medical risks to the cells. Among other properties, the transcription factor Sox2 (sex-determining region Y) is essential for the undifferentiated self-renewal of embryonic stem cells, as is the protein and transcription factor Oct-4 (Octamer-4), a delicate balance of which is necessary for determining whether pluripotent cells differentiate or remain undifferentiated. A member of the Kruppel-like family of transcription factors, KLF4 is also simultaneously a gene and an antibody which plays a key role in cell proliferation and has been extensively studied for its role in cancer. Perhaps of greatest concern, however, is the proto-oncogene cMyc, mutations and overexpression of which have been implicated in many types of cancers. That fact that neural cells naturally express all of these factors except OCT4 is no doubt a topic of widespread research interest with applications in oncology and other fields beyond the immediate realm of neurology.

As Dr. Boris Greber, a member of Scholer's team, explains, "Remarkably, it turns out that three of these four essential factors are already expressed in human neural stem cells, although not in skin cells, so we only needed to add one factor, OCT4. Ideally, we will be able to find a chemical that does the same job of expressing the factor without the need for a gene." Earlier this year, in fact, scientists in California announced the successful creation of iPS cells from mouse fibroblasts that were reprogrammed with a "cocktail" of proteins instead of the ordinary four genes, although this process is overall much less efficient. As Dr. Greber explains, "Without stable intervention using viruses, the frequency of reprogramming goes down and you have to wait a long time. We don't have the perfect method yet." Nevertheless, as Dr. Greber further adds, in addition to being easier to reprogram, cells from neural tissue are also less prone to mutations than are cells from the skin.

Induced pluripotent stem (iPS) cells first burst into the news in 2006 when Dr. Shinya Yamanaka of Kyoto University in Japan announced the creation of these cells from ordinary mouse fibroblasts, which was succeeded the following year by the creation of iPS cells from human fibroblasts. Since then, the procedure has been reproduced numerous times by scientists around the world, a number of whom have contributed significant improvements to the process. Nevertheless, it is still not yet known whether or not these artifically produced cells will perform in vivo in a manner similar to that of naturally occuring cells. Since none of the iPS cells that have been created thus far are safe enough for clinical use, the actual therapeutic efficacy of these cells as a medical treatment remains unknown.