A Safer Way to Reprogram Cells

Science, March 26, 2009

Dr. James Thomson and his colleagues at the University of Wisconsin at Madison are once again in the news as they announce in their latest publication in the journal Science that they have created human iPS (induced pluripotent stem) cells without the use of some of the dangerous reprogramming methods by which such cells were created in the past.

First derived from the skin of mice, then from the skin of humans, and most recently from the blood of humans, iPS cells are generated from ordinary adult somatic (non-stem cell) cells and therefore circumvent the ethical controversies that surround embryonic stem cell research, since iPS cells are not derived from embryos. Ethics and politics aside, however, iPS cells still have a long way to go before they can be incorporated into clinical therapies, since the laboratory methods by which iPS cells are derived pose too many medical dangers for any potential patient. Not only are (cancer-causing) oncogenes used as cellular reprogramming agents, but dangerous retroviruses have also typically been used as delivery agents that carry the reprogramming genes into the cell. Now, however, scientists may have found an alternative, at least to the retroviral delivery methods.

In a paper entitled "Human Induced Pluripotent Stem Cells Free of Vector and Transgene Sequences", Dr. Thomson and his colleagues describe how they used "non-integrating episomal vectors" instead of retroviruses to derive human iPS cells. As they further explain, "Human iPS cell derivation previously required vectors that integrate into the genome, which can create mutations and limit the utility of the cells in both research and clinical applications." Such integrating "vectors" have typically consisted of retroviruses or their lentiviral subset, which have been used as vehicles of gene delivery since the 1970s. The principle exploits the highly evolved molecular capability of viruses by which they infect cells, and through which transportation of their own viral genome into the host cell occurs. Also known as transduction, the process is a commonly used tool throughout molecular biology and genetics, especially in the field of gene therapy. The disadvantage, of course, is that the use of a live virus is not without risks, and while such risks may be modified to some extent for laboratory research, the risks can never be fully eliminated which therefore disqualifies such methods for use in any human clinical therapy.

Episomes, also known as plasmids, which are usually double-stranded circular molecules and can either exist independently of, or integrated with, the chromosome, can also be used as "vectors" to deliver genes inside of cells. In contrast to retroviruses and lentiviruses, the non-integrating episomal vectors that Dr. Thomson and his colleagues chose, which consist of "circles of DNA" plasmid vectors, will eventually vanish from the host cells over time, leaving no trace of their presence or activity other than the delivery of the desired genes into the cells.

As Dr. Thomson explains, "That means they [the iPS cells] are less likely to form tumors, less likely to destroy the function of some important gene." According to Dr. Jeremy Berg, director of the National Institute of General Medical Sciences at the National Institutes of Health, "What Dr. Thomson has done for the first time in human iPS cells is create methods which don't involve inserting DNA into the host genome at all - using plasmids which go into the cells but never get incorporated into the DNA."

In their study, Dr. Thomson and his colleagues used a number of genes which included OCT4, SOX2, NANOG, LIN28, c-Myc and KLF4, all of which are transcription factors involved in cellular self-renewal and differentiation except for LIN28 which is a marker of undifferentiated human embryonic stem cells, and c-Myc which is a gene involved in protein-coding, a mutated version of which is known to be oncogenic (in other words, it can cause cancer) and in fact the entire Myc family of genes are known as proto-oncogenes and are implicated in some types of cancer when mutated or overexpressed. As the authors noted, transgene linkage and the addition of KLF4 and c-Myc improved reprogramming efficiency; nevertheless, the continued use of the c-Myc oncogene still poses a risk of cancer and therefore the overall method is still in need of further "refinement" before it can be translated into a human clinical therapy.

For the construction of expression vectors, the authors describe how "Transgenes were cloned into a modified lentiviral vector or the oriP/EBNA1-based pCEP episomal vector (Invitrogen) for reprogramming", and human fibroblasts were thereby transduced. Additionally the authors elucidate the mechanisms by which combinations of episomal plasmids were co-transfected into fibroblasts via nucleofection for reprogramming with oriP/EBNA1-based episomal vectors. As Dr. Thomson and his colleagues further describe, "After removal of the episome, iPS cells completely free of vector and transgene sequences are derived that are similar to human embryonic stem cells in proliferative and developmental potential." Indeed, the new iPS cells also resemble human embryonic stem cells in their ability to form teratomas (tumors), and in fact one section of the paper which addresses this fact is specifically entitled "Teratoma formation", in which the authors confirm that the episomal-vector-generated human iPS cells were injected into the hind limb muscles of 6-week-old immunocompromised SCID-beige mice, from which, "After five to ten weeks, teratomas were dissected and fixed in 4% paraformaldehyde. Samples were embedded in paraffin and processed with hematoxylin and eosin staining at the Histology Lab at the School of Veterinary Medicine, University of Wisconsin-Madison, WI."

Technically speaking, iPS cells are not stem cells but, as the name implies, they are cells which have been artifically "induced" to exhibit pluripotency. As such, iPS cells resemble embryonic stem cells in their ability to differentiate, at least hypothetically, into all 220 types of tissue that exist throughout the human body - although in actuality nobody has ever differentiated any type of cell into all the various types of human body tissue. Nevertheless, despite the fact that iPS cells can be created without the need for embryos, iPS cells are still plagued by many of the problems that are inherent in embryonic stem cells, not the least of which is the risk of teratoma (tumor) formation, which is characteristic of all pluripotent cells since teratoma formation is part of the official scientific definition of pluripotency. Exactly how the strong, natural tendency toward teratoma formation could be "turned off" in any pluripotent cell with any certainty remains merely one of many scientific problems associated with pluripotent cells that have yet to be resolved. By sharp contrast, adult stem cells do not pose such problems since adult stem cells are not pluripotent and therefore are not able to form teratomas. Not surprisingly, therefore, adult stem cells are already being used in clinics around the world as therapies for a wide variety of diseases and injuries, whereas any type of pluripotent cell - whether of embryonic or iPS origin - will require at least another decade of research, if not more, before it could be available in the form of a clinical therapy.

Precisely for such reasons, Dr. Thomson is among the first to point out that the most immediate uses for iPS cells will not be as clinical therapies but instead will most likely be in the field of drug testing and development, which previously had been conducted on laboratory animals, not on humans nor on human tissue. As the authors state at the very beginning of their paper, iPS cell technology "has applications in basic biology, drug development, and transplantation", but the words "clinical therapy" are conspicuous by their absence since such words do not appear in the short list of applications of iPS cells, at least not for the immediate future. Until their multiple inherent medical dangers, only one of which is the formation of teratomas, can somehow be eliminated, iPS cells are not, and will not be, ready for the clinic. Even though Dr. Thomson and his colleagues have solved the problem posed by the previous use of retroviral vectors, their continued use of the c-Myc oncogene, which "improved reprogramming efficiency", still poses a risk of cancer and therefore a safer substitute for this oncogene must be found before iPS cells can be translated into a human clinical therapy.

As the authors conclude, "These results demonstrate that reprogramming human somatic cells does not require genomic integration or the continued presence of exogenous reprogramming factors, and removes one obstacle to the clinical application of human iPS cells."

Even though "one obstacle" has been removed, several more still remain.

The paper was first published online by Science Express, which provides the electronic publication of selected papers from the journal Science in advance of print. Both Science and Science Express are publications of the American Association for the Advancement of Science (AAAS), which was founded in 1848 and serves approximately 10 million individuals worldwide through 262 affiliated societies and scientific academies. According to their website, the journal Science "has the largest paid circulation of any peer-reviewed general science journal in the world, with an estimated total readership of one million."