Researchers at the Salk Institute for Biological Studies in La Jolla, California, have successfully generated a new type of iPS (induced pluripotent stem) cells from the keratinocytes that are attached to a single human hair. The procedure represents a doubling of the speed and a 100-fold improvement in the overall efficiency of generating iPS cells when compared to the original methods that were initially used in early experiments, in which only one out of every 10,000 cells was successfully reprogrammed into an iPS cell. According to Dr. Juan Carlos Belmonte, who led the study, “Having a very efficient and practical way of generating patient-specific stem cells, which, unlike human embryonic stem cells, wouldn’t be rejected by the patient’s immune system after transplantation, brings us a step closer to the clinical application of stem cell therapy.”

After a single human hair is plucked from the scalp, the keratinocytes are reprogrammed into “keratinocyte-derived iPS cells”, or KiPS cells, to distinguish them from ordinary iPS cells which are derived from fibroblasts in the skin. Keratinocytes, which develop from the basal layer of the epidermis to form the uppermost layer of skin, produce the protein keratin which is the primary constituent not only of hair and skin but also of nails. According to Dr. Trond Aasen, a postdoctoral fellow at the Center of Regenerative Medicine in Barcelona who collaborated on the research, “We plucked a single hair from a co-worker’s scalp and cultured the keratinocytes, which are found in the outer root sheet area”, and which were then reprogrammed into KiPS cells. As Dr. Belmonte explains, “We checked a whole rainbow of cells and found keratinocytes to be the easiest to reprogram. It is still not clear exactly why that is, and knowing it will be very important for the technology to develop fully.”

The results of the research offer a fast and relatively simplified way to mass-produce cells which resemble embryonic stem cells in their pluripotency, yet which are obtained from an easily accessible source with noninvasive methods.

Although the topic was at one time front and center in the debates among U.S. presidential candidates, stem cells have receded somewhat from the public consciousness in recent months, due in part to breakthroughs with iPS (induced pluripotent stem) cells, which avoid the need for embryos and which thereby sidestep the related ethical controversies, and also due in part to the exigencies of more urgent matters such as the recent global financial crisis.

Nevertheless, stem cells remain a subject of intense interest and fierce argument among the general public if not among the presidential candidates, although the topic did resurface briefly during the final presidential debate. While both candidates endorse the idea of relaxing restrictions that currently exist on embryonic stem cell funding, subtle differences separate their policies. A brief summarization of such policies and of the most immediate historical background is provided herein.

As every stem cell scientist clearly remembers, in excrutiating detail, August 9 of 2001 was the day that President George W. Bush banned the use of federal funds for research on embryonic stem cell lines created after that date. He did not ban the use of federal funds for research on embryonic stem cell lines that already existed prior to that date, nor, contrary to widespread misperception, did he ban the use of private funds or state funds for embryonic stem cell research. Indeed, embryonic stem cell research is flourishing in laboratories across the entire U.S., precisely because the restrictions imposed by President Bush had the reverse effect of galvanizing private investors and scientists who were determined to pursue embryonic stem cell research even without federal funding. Harvard University is an excellent example, where the Harvard Stem Cell Institute is heavily involved in embryonic stem cell research and yet it is entirely privately funded. The state of California is another example, where the approval of Proposition 71 allowed for the allocation of $6 billion worth of state money – $3 billion in principal created through bonds, and $3 billion in interest, to be repaid over 30 years – for the funding of embryonic stem cell research, and which thereby also established the California Institute for Regenerative Medicine. It may be noted that Proposition 71 passed by a narrow margin of only 59.1% of voters, and was strongly opposed by many individuals and groups including the organization known as “Doctors, Patients and Taxpayers for Fiscal Responsibility”. Nevertheless, having been voted into state law by a narrow majority, Proposition 71 and the embryonic stem cell research that it finances are fully legal and in full accord with President Bush’s restrictions on embryonic stem cell research, which apply neither to private funding nor to funding by state tax or bond dollars. Contrary to common belief, therefore, President Bush’s restrictions on embryonic stem cell research – which consisted of a specific ban only on the use of federal tax dollars for embryonic stem cell research conducted on embryonic stem cell lines created after August 9th of 2001 – did not preclude embryonic stem cell research in the U.S. but in fact had the exact opposite effect by igniting a number of very strong and active privately and state funded embryonic stem cell institutes and projects.

Fast-forwarding several years, the U.S. now awaits a change of administration and with it a probable change in embryonic stem cell policy, although the exact nature of such changes in policy has not yet been explicity defined. Contrary to the GOP platform, John McCain – along with 57 other, mostly Democratic, senators – signed a letter to President Bush in 2004 requesting that restrictions on the federal funding of embryonic stem cell research be relaxed. Similarly, in 2006 and 2007, both Senators John McCain and Barack Obama voted for a bill to expand the federal funding of embryonic stem cell research. Both Senator Obama and his running mate, Senator Joe Biden, support a complete overturn of President Bush’s restrictions on embryonic stem cell research, while Senator McCain’s running mate, Governor Sarah Palin, adheres more closely to the official GOP platform by opposing embryonic stem cell research. Although some people question whether or not Senator McCain’s position on the issue may have shifted, or will shift, as a result of his running mate’s views, McCain spokesman Brian Rogers asserts that, if elected president, McCain would “absolutely” support embryonic stem cell research. Rogers adds, “He bucked his party and the Bush administration in supporting stem cell research, including embryonic stem cell research, and will continue to do so.” Previously, however, Senator McCain has stated that, “Recent scientific breakthroughs raise the hope that one day this debate will be rendered academic”, in a direct reference to recent breakthroughs with iPS (induced pluripotent stem) cells. Precisely because of such statements made by Senator McCain, a number of people, such as Father Tadeusz Pacholczyk, education director for the National Catholic Bioethics Center, which opposes embryonic stem cell research, have said that “a certain level of ambiguity” may still be found in McCain’s stance. In other words, the full extent to which McCain might “relax” or “expand” the restrictions imposed by President Bush are not entirely clear.

Nevertheless, the day when embryonic stem cell scientists and their laboratories throughout the U.S. might be competing for N.I.H. (National Institutes of Health) grants may not be very far away, but voters will have to wait until after November 4th to learn the exact details of such a prospect.

In 2007, a team of researchers in Madison, Wisconsin, led by the renowned stem cell pioneer Dr. James Thomson, announced their success in creating iPS (induced pluripotent stem) cells from the mature, non-stem cell, somatic cells of adult human skin. Such iPS cells exhibit the same pluripotency as that of embryonic stem cells, but no embryo is involved in the process. Such an announcement was a “shot heard ’round the world”, since it seemed to solve the ethical controversies that surround embryonic stem cells, by avoiding embryonic stem cells altogether. Now, the methods for conducting the iPS cell procedure are being replicated in hundreds of new laboratories across the nation and around the world.

In total, 812 new laboratories have sprung up in dozens of countries in recent months, the exclusive focus of which is iPS cells, according to the Massachusetts-based company Addgene, which is a repository for the laboratory supplies. According to Dr. Thomson, “People are jumping in very rapidly, much more rapidly than they did ten years ago,” in reference to his own, groundbreaking discovery that he made a decade ago.

Dr. James Thomson is the first person ever to have isolated an embryonic stem cell, first from a rhesus monkey in 1995, and then from a human in 1998. Human embryonic stem cell science in general, therefore, which is exactly one decade old this year, owes its very existence to Dr. Thomson, who invented the embryonic stem cell techniques that are now used throughout the world, and who is widely regarded as “the founder of the field”. With his success last year in reprogramming mature, adult human skin cells into iPS cells, Dr. Thomson once again pioneered a new direction in regenerative medicine which has very quickly become a booming industry.

Even Dr. Thomson, however, has expressed his reservations regarding the applicability of embryonic stem cells and of iPS cells to clinical therapies, at least in the foreseeable future. Many technical hurdles still remain, and any hope of being able to use embryonic stem cells or iPS cells for the actual treatment of human patients is generally considered to be decades away. According to Dr. Thomson, “It’s certainly going to happen, but it’s going to be hard, and people are not prepared for how hard it’s likely to be.”

Indeed, the only medical application of embryonic stem cells that is not still decades away is in the field of drug screening. In other words, instead of testing newly developed pharmaceuticals on live people, the new drugs could be tested on human tissue, derived from embryonic stem cells, in the laboratory. As Dr. Thomson explains, “It simply means that for the very first time we have access to the human body in the lab. And for drug screening and drug discovery, that’s going to make a huge difference. When you use one of those drugs you won’t know that human embryonic stem cells or iPS cells were involved. It won’t make the front pages at all.”

Yet one, critically important problem still remains unsolved. As pluripotent cells, iPS cells do, by definition, cause the formation of teratomas, which is a very particular type of tumor. Like embryonic stem cells, non-embryonic iPS cells are prized for their pluripotency, which is formally defined as a capacity to differentiate into the tissues of all 3 germ layers – the ectoderm, the mesoderm and the endoderm – as specifically found in teratomas. To date, scientists have not yet been able to guarantee that such cells will not also cause teratomas in the patients whose diseases they are intended to treat. The creation of iPS cells may have solved the ethical problems that are inextricably intertwined with embryonic stem cells, since embryos are not needed at all for iPS cells, but iPS cells have not solved the technical, medical and health problems that are an inherent part of all pluripotent cells, especially the very high risk of teratoma formation. By sharp contrast, adult stem cells do not pose such risks since adult stem cells cannot form teratomas since adult stem cells are not pluripotent.

Additionally, the exclusive use of embryonic stem cells for drug screening brings us back once again into the very same ethical dilemma as before, since the destruction of human embryos is required even for this type of drug screening. Meanwhile, however, approximately 70 new iPS cell laboratories are springing up around the world every month.

Led by Dr. Thomas Skutella, a team of researchers at the University of Tubingen in Germany has reported the creation of “germline stem cells” from the sperm-producing testicular tissue of 22 men, via a culturing process which is similar to that of culturing human embryonic stem cells. Following the standard, global test for pluripotency, the scientists then injected the new “germline stem cells” into immune compromised mice, where the newly created cells formed teratomas and hence are now celebrated for their similarity to embryonic stem cells.

According to Dr. Gerd Hasenfuss of the University of Gottingen in Germany, who has published similar studies involving the transformation of mouse testicular cells into pluripotent cells, “My summary is, it is a nice paper, they made big progress in getting to a pluripotent cell, and they show pluripotency with a teratoma experiment. But I think we are not completely there where we want to be, namely, at pluripotency absolutely comparable to that of embryonic stem cells.” In particular, although the “germline stem cells” were shown to differentiate into most cell types of the body, they did not differentiate into cardiomyocytes, from which heart tissue is formed.

Nevertheless, Dr. Skutella and his colleagues have demonstrated that the transformation and reprogramming of the cells may be accomplished through growth factors in the culture medium rather than through retroviral and oncogenic vectors, as were originally used in the first experiments conducted with iPS (induced pluripotent stem) cells. According to Dr. Dirk de Rooij, professor emeritus of endocrinology at the Utrech University in the Netherlands, “In comparison to the mouse studies that have been done, there is a big leap forward in efficiency with which these people get these germline stem cells.”

Similarly, as Dr. Peter Donovan of the University of California at Irvine explains, “The ability to make a pluripotent stem cell from an individual without the ethical and immunological problems associated with human embryonic stem cells is a big deal.” He adds the following caveat, however: “I suspect people would be much more willing to give up a piece of skin to make an iPS cell than to have a testicular biopsy to give rise to an adult germline stem cell.”

On the eve of the World Stem Cell Summit in Madison, Wisconsin, Dr. James Thomson offers realistic and sobering words. Interjecting a healthy dose of reality into the unrestrained hyperbole that typically characterizes embryonic stem cell discussions, Dr. Thomson speaks with a tone of authority as well as guarded optimism.

When Dr. Thomson was featured on the cover of Time Magazine in August of 2001, the headline next to his picture read, “The Man Who Brought You Stem Cells”. Indeed, as the world’s first person ever to isolate an embryonic stem cell, first from a rhesus monkey in 1995 and then from a human in 1998, Dr. Thomson is often acknowledged by his peers as “the founder of the field”. Once again leading a major breakthrough when his team announced their success with the development of human iPS (induced pluripotent stem) cells in 2007, Dr. Thomson is revered in stem cell laboratories throughout the world, wherein his name is to stem cell scientists what Einstein’s, Newton’s, and Galileo’s names are to physicists. It may, therefore, come as somewhat of a surprise to many people to discover that Dr. Thomson is considerably more reserved in his enthusiasm for embryonic stem cells and iPS cells than are most advocates.

When asked to predict a timeline for the development of actual therapies which might result from either embryonic stem cells or iPS cells, Dr. Thomson cautiously elaborated upon the technical difficulties that remain and which must still be resolved before such achievements could be realized. Although he said that such therapies might possibly be developed within the next decade, he added that there are still many scientific hurdles to overcome and the public should be prepared for setbacks and disappointment. According to Dr. Thomson, “It’s one thing to make a tissue in culture. It’s another thing to get it into the body and re-establish function. We need to roll up our sleeves and do a great deal of work here, but it’s not going to happen overnight.”

It is well known that embryonic stem cells are inherently problematic, which is why they have never advanced beyond the laboratory stage. Such problems typically include immune rejection, biological contamination, genetic mutation, lack of controllability during differentiation, and the natural tendency of embryonic stem cells to form a particular type of tumor known as a teratoma, among other problems. Additionally, embryonic stem cells are highly controversial for the ethical dilemmas that are inextricably tied to the destruction of embryos, which is required for the derivation of embryonic stem cells. While iPS cells have solved the ethical controversies, to some extent, by avoiding the need for embryonic stem cells entirely, iPS cells are still pluripotent and therefore still do, by definition, cause the formation of teratomas. Clearly, any medical therapy which is given to a human patient should not be worse than the disease or injury that it is meant to treat, which is why anything that causes teratomas cannot be administered as a “therapy” to people until the underlying cellular mechanisms are fully understood and controlled. Such a level of scientific understanding, of such genetically and biochemically complex matters, could take another decade to achieve, if not longer. Meanwhile, exactly how a clinical therapy could be developed from a tumor-causing, pluripotent cell, even if the cell is of non-embryonic origin, such as an iPS cell, is a topic that is not without its own far-reaching ethical controversies.

An embryologist by training, Dr. Thomson is the first to admit that his primary contribution to the world is not so much in the medical profession, nor in the realm of clinical therapies, as it is in the basic science of embryology and developmental biology. Nevertheless, the world turns to him for authoritative direction in medicine and in the treatment of human disease, and for guidance in navigating this brave new world which he played a major role in creating. The public would therefore be wise to heed his prudent and cautionary restraint.

The biotech company BioTime, Inc., and its subsidiary Embryome Sciences, Inc., have recently acquired multiple licenses for stem cell technology patents and their applications, which are now scheduled to be the topics of tomorrow’s conference call. Included within BioTime’s portfolio of patent licenses are proprietary methods for the production of induced pluripotent stem (iPS) cells as well as a number of embryonic stem cell technologies that include the rapid isolation and differentiation of highly purified novel embryonic progenitor cells, with the ultimate goal being the development and commercialization of related products.

BioTime is well known for its development of various surgical technologies that are used in emergency trauma and which include methods of organ preservation, blood plasma expanders, and blood replacement in hypothermic and hypovolemic surgery, among other applications. Through its wholly owned subsidiary Embryome Sciences, Inc., Biotime has recently entered the field of regenerative medicine, in which embryonic stem cell technology is its primary focus.

Among their proprietary technologies thus far, Embryome Sciences utilizes several patents that allow for “vector-free iPS technology”, which is the ability to generate iPS cells without the use of dangerous “vectors”, which include viral vectors such as retroviruses, lentiviruses and adenoviruses, as well as (cancer causing) oncogenes, such as the c-Myc oncogene which has been routinely used in the production of iPS cells and which was found to cause cancer in 20% of the chimeric mice that were utilized in early iPS cell experiements. Embryome Sciences can boast proprietary vector-free technology which avoids such problems, by utilizing iPS transcription factors to transform collateral cells into a cytoplasm that reprograms the patient’s own cells, rather than creating the iPS cells directly by reprogramming adult cells with viral and oncogenic vectors. However, as pluripotent cells, and regardless of how they are produced, iPS cells still do, by definition, cause the formation of teratomas, which are a very specific type of tumor and this unsolved problem remains one of the obstacles yet to be overcome in bringing iPS cells into the realm of clinical therapy. Nevertheless, Embryome Sciences is working to overcome these problems and health risks that are inherent to pluripotent stem cells, whether of embryonic or nonembryonic origin, and Embryome Sciences is hoping to collaborate with other companies on the development and commercialization of related therapies.

Such plans will no doubt be very eagerly addressed in tomorrow’s highly anticipated conference call.

The successful reprogramming of mature, non-stem cell, somatic cells to a more primitive state in which they behave with the same pluripotency as embryonic stem cells, was a major scientific breakthrough that has offered great hope in the field of regenerative medicine. Known as iPS (induced pluripotent stem) cells, these newly formed cells also avoid the ethical dilemmas surrounding embryonic stem cells, since no embryos are required for the generation of iPS cells. However, the conversion of regular somatic cells into iPS cells has been an extremely inefficient process, in addition to dangerous, since the production of these cells involves the use of retroviruses, lentiviruses, adenoviruses and (cancer causing) oncogenes, none of which are allowable for clinical use, and all of which are specifically prohibited by the FDA (Food and Drug Administration) and are grounds for disqualification from the FDA approval process of medical therapies. Furthermore, the precise molecular, genetic and biochemical mechanisms of cellular reprogramming that are at work in the production of iPS cells are still not yet fully understood.

Two independently published papers, however, now signify further progress in the field. A team led by Dr. Konrad Hochedlinger of the Harvard Stem Cell Institute, and another team led by Dr. Rudolf Jaenisch of the Massachusetts Institute of Technology and the Whitehead Institute, have both published findings which corroborate each other’s work.

Dr. Hochedlinger’s team was able to generate iPS cells with a pluripotency similar to that of embryonic stem cells, not by using the viral and oncogenic vectors that have been previously used, but instead by using the drug doxycycline for reprogramming of the cells. Perhaps of greatest importance, however, was the discovery that after the “primary” iPS cells were allowed to differentiate into mature cells, the researchers then re-exposed the cells to doxycycline a second time, which induced the production of a “secondary” group of iPS cells that was generated even more quickly and efficienty than the “primary” group that was produced after the first exposure.

Dr. Jaenish and his team also addressed the idea of generating a secondary round of iPS cells, in a separate paper wherein they describe experiments in which they were able to derive secondary iPS cells by using doxycycline-inducible transgenes. According to Dr. Jaenisch, “The drug-inducible system we describe represents a novel, predictable, and highly reproducible platform to study the kinetics of iPS cell generation. Furthermore, the genetic homogeneity of secondary cells makes chemical and genetic screening approaches to enhance reprogramming efficiency or to replace any of the original reprogramming factors feasible.” Dr. Hochedlinger adds, “The secondary system will enable chemical and genetic screening efforts to identify key molecular constituents of reprogramming, as well as important obstacles in this process, and will ultimately lend itself as a powerful tool in the development and optimization methods to produce human iPS cells.”

Both research teams confirmed that generation of the secondary group of iPS cells is faster than the generation of the primary group, although the precise time that is involved depends upon the types of skin cells that are used. Human keratinocytes, for example, were found to take approximately 10 days for production, whereas fibroblasts required several weeks. According to Dr. Hochedlinger, “The fast kinetics of reprogramming observed for keratinocytes suggests that these cells would be useful for development and optimization of methods to reprogram cells by transient delivery of factors.”

One problem still remains, however, which is the ability of all iPS cells to cause teratomas, which is a specific type of tumor. By formal definition, any cell which is “pluripotent” is capable of forming a teratoma, and if a cell cannot form a teratoma then it is recognized as not being pluripotent. Exactly how pluripotency in a cell may be turned on or off, like a switch, or controlled to the extent that anyone can guarantee, with 100% certainty, that the cell will not cause tumors when administered to human patients, remains to be seen.

Meanwhile, the ability to produce a “secondary” round of iPS cells, more quickly and efficiently than the first round, via a second exposure to doxycycline, represents a significant and important discovery, as scientists advance one step further along the path of elucidating the complex cellular mechanisms that are at work in reprogramming and differentiation.