Growing Skepticism about the Stem Cell Acid Trip

In January 2014, the two papers “Stimulus-triggered fate conversion of somatic cells into pluripotency” and “Bidirectional developmental potential in reprogrammed cells with acquired pluripotency” published in the journal Nature by Haruko Obokata and colleagues took the world of stem cell research by surprise.

Since Shinya Yamanaka’s landmark discovery that adult skin cells could be reprogrammed into embryonic-like induced pluripotent stem cells (iPSCs) by introducing selected embryonic genes into adult cells, laboratories all over the world have been using modifications of the “Yamanaka method” to create their own stem cell lines. The original Yamanaka method published in 2006 used a virus which integrated into the genome of the adult cell to introduce the necessary genes. Any introduction of genetic material into a cell carries the risk of causing genetic aberrancies that could lead to complications, especially if the newly generated stem cells are intended for therapeutic usage in patients.

Researchers have therefore tried to modify the “Yamanaka method” and reduce the risk of genetic aberrations by either using genetic tools to remove the introduced genes once the cells are fully reprogrammed to a stem cell state, introducing genes without non-integrating viruses or by using complex cocktails of chemicals and growth factors in order to generate stem cells without the introduction of any genes into the adult cells.

The papers by Obokata and colleagues at the RIKEN center in Kobe, Japan use a far more simple method to reprogram adult cells. Instead of introducing foreign genes, they suggest that one can expose adult mouse cells to a severe stress such as an acidic solution. The cells which survive acid-dipping adventure (25 minutes in a solution with pH 5.7) activate their endogenous dormant embryonic genes by an unknown mechanism. The researchers then show that these activated cells take on properties of embryonic stem cells or iPSCs if they are maintained in a stem cell culture medium and treated with the necessary growth factors. Once the cells reach the stem cell state, they can then be converted into cells of any desired tissue, both in a culture dish as well as in a developing mouse embryo. Many of the experiments in the papers were performed by starting out with adult mouse lymphocytes, but the researchers also found that mouse skin fibroblasts and other cells could also be successfully converted into an embryonic-like state using the acid stress.

My first reaction was incredulity. How could such a simple and yet noxious stress such as exposing cells to acid be sufficient to initiate a complex “stemness” program? Research labs have spent years fine-tuning the introduction of the embryonic genes, trying to figure out the optimal combination of genes and timing of when the genes are essential during the reprogramming process. These two papers propose that the whole business of introducing stem cell genes into adult cells was unnecessary – All You Need Is Acid.

 

This sounds too good to be true. The recent history in stem cell research has taught us that we need to be skeptical. Some of the most widely cited stem cell papers cannot be replicated. This problem is not unique to stem cell research, because other biomedical research areas such as cancer biology are also struggling with issues of replicability, but the high scientific impact of burgeoning stem cell research has forced its replicability issues into the limelight. Nowadays, whenever stem cell researchers hear about a ground-breaking new stem cell discovery, they often tend to respond with some degree of skepticism until multiple independent laboratories can confirm the results.

My second reaction was that I really liked the idea. Maybe we had never tried something as straightforward as an acid stress because we were too narrow-minded, always looking for complex ways to create stem cells instead of trying simple approaches. The stress-induction of stem cell behavior may also represent a regenerative mechanism that has been conserved by evolution. When our amphibian cousins regenerate limbs following an injury, adult tissue cells are also reprogrammed to a premature state by the stress of the injury before they start building a new limb.

The idea of stress-induced reprogramming of adult cells to an embryonic-like state also has a powerful poetic appeal, which inspired me to write the following haiku:

 

The old warrior

plunges into an acid lake

to emerge reborn.

 

(Read more about science-related haikus here)

Just because the idea of acid-induced reprogramming is so attractive does not mean that it is scientifically accurate or replicable.

A number of concerns about potential scientific misconduct in the context of the two papers have been raised and it appears that the RIKEN center is investigating these concerns. Specifically, anonymous bloggers have pointed out irregularities in the figures of the papers and that some of the images may be duplicated. We will have to wait for the results of the investigation, but even if image errors or duplications are found, this does not necessarily mean that this was intentional misconduct or fraud. Assembling manuscripts with so many images is no easy task and unintentional errors do occur. These errors are probably far more common than we think. High profile papers undergo much more scrutiny than the average peer-reviewed paper, and this is probably why we tend to uncover them more readily in such papers. For example, image duplication errors were discovered in the 2013 Cell paper on human cloning, but many researchers agreed that the errors in the 2013 Cell paper were likely due to sloppiness during the assembly of the submitted manuscript and did not constitute intentional fraud.

Irrespective of the investigation into the irregularities of figures in the two Nature papers, the key question that stem cell researchers have to now address is whether the core findings of the Obokata papers are replicable. Can adult cells – lymphocytes, skin fibroblasts or other cells – be converted into embryonic-like stem cells by an acid stress? If yes, then this will make stem cell generation far easier and it will open up a whole new field of inquiry, leading to many new exciting questions. Do human cells also respond to acid stress in the same manner as the mouse cells? How does acid stress reprogram the adult cells? Is there an acid-stress signal that directly acts on stem cell transcription factors or does the stress merely activate global epigenetic switches? Are other stressors equally effective? Does this kind of reprogramming occur in our bodies in response to an injury such as low oxygen or inflammation because these kinds of injuries can transiently create an acidic environment in our tissues?

Researchers all around the world are currently attempting to test the effect of acid exposure on the activation of stem cell genes. Paul Knoepfler’s stem cell blog is currently soliciting input from researchers trying to replicate the work. Paul makes it very clear that this is an informal exchange of ideas so that researchers can learn from each other on a “real-time” basis. It is an opportunity to find out about how colleagues are progressing without having to wait for 6-12 months for the next big stem cell meeting or the publication of a paper confirming or denying the replication of acid-induced reprogramming. Posting one’s summary of results on a blog is not as rigorous as publishing a peer-reviewed paper with all the necessary methodological details, but it can at least provide some clues as to whether some or all of the results in the controversial Obokata papers can be replicated.

If the preliminary findings of multiple labs posted on the blog indicate that lymphocytes or skin cells begin to activate their stem cell gene signature after acid stress, then we at least know that this is a project which merits further investigation and researchers will be more willing to invest valuable time and resources to conduct additional replication experiments. On the other hand, if nearly all the researchers post negative results on the blog, then it is probably not a good investment of resources to spend the next year or so trying to replicate the results.

It does not hurt to have one’s paradigms or ideas challenged by new scientific papers as long as we realize that paradigm-challenging papers need to be replicated. The Nature papers must have undergone rigorous peer review before their publication, but scientific peer review does not involve checking replicability of the results. Peer reviewers focus on assessing the internal logic, experimental design, novelty, significance and validity of the conclusions based on the presented data. The crucial step of replicability testing occurs in the post-publication phase. The post-publication exchange of results on scientific blogs by independent research labs is an opportunity to crowd-source replicability testing and thus accelerate the scientific authentication process. Irrespective of whether or not the attempts to replicate acid-induced reprogramming succeed, the willingness of the stem cell community to engage in a dialogue using scientific blogs and evaluate replicability is an important step forward.

 

Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, Yamato M, & Vacanti CA (2014). Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature, 505 (7485), 641-7 PMID: 24476887

Replicability of High-Impact Papers in Stem Cell Research

I recently used the Web of Science database to generate a list of the most highly cited papers in stem cell research. As of July 2013, the search for original research articles which use the key word “stem cells” resulted in the following list of the ten most widely cited papers to date:

Human ES cell colony – Nuclei labeled in blue, Mitochondria labeled in green- Rehman lab.1. Pittenger M et al. (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143-147

Citations: 8,157

2.  Thomson JA et al. (1998) Embryonic stem cell lines derived from human blastocysts. Science 282(5391):1145-1147

Citations: 5,565

3. Takahashi K and Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126(4): 663-676

Citations: 5,034

4. Takahashi K et al. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131(5):861-872

Citations: 4,061

5. Donehower LA et al  (1992) Mice deficient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356(6366): 215-221

Citations: 3,279

6. Al-Hajj M et al (2003) Prospective identification of tumorigenic breast cancer cells. Proceedings of the National Academy of Sciences 100(7): 3983-3988

Citations: 3,183

 7. Yu J et al (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science 318(5858): 1917-1920

Citations: 3,086

 8. Jiang YH et al (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418(6893):41-49

Citations: 2,983

9. Orlic D et al (2001) Bone marrow cells regenerate infarcted myocardium. Nature 410 (6829):701-705

Citations: 2,961

10. Lu J et al (2005) MicroRNA expression profiles classify human cancers. Nature 435(7043): 834-838

Citations: 2,917

 

Three of the articles (Donehower et al, Al-Hajj et al and Lu et al) in this “top ten list” do not focus on stem cells but are actually cancer research papers. They were probably identified by the search because the authors may have made comparisons to stem cells or used stem cells as tools.The remaining seven articles are indeed widely known in the stem cell field.

The Science paper by Pittenger and colleagues in 1999 provided a very comprehensive description of mesenchymal stem cells (MSCs), a type of adult stem cell which is found in the bone marrow alongside hematopoietic stem cells (HSCs). Despite the fact that MSCs and HSCs are both adult stem cells in the bone marrow, they have very different functions. HSCs give rise to circulating blood cells, whereas MSCs primarily form bone, fat and cartilage as was nicely demonstrated by Pittenger and colleagues.

The article by Thomson and colleagues was published in 1998 in the journal Science described the derivation of human embryonic stem cells (ESCs) and revolutionized the field of stem cell research. While adult stem cells have a very limited capacity in terms of lineages they can turn into, ESCs are derived from the early blastocyst stage of embryonic development (within the first 1-2 weeks following fertilization) and thus retain the capacity to turn into a very wide range of tissues, such as neurons, heart cells, blood vessel cells or liver cells. This paper not only identified the methods for isolating human ESCs, but also how to keep them in culture and expand them as undifferentiated stem cells.

The Cell paper by Takahashi and Yamanaka in 2006 represented another major advancement in the field of stem cell biology, because it showed for the first time that a mouse adult skin cell (fibroblast) could be reprogrammed and converted into a truly pluripotent stem cell (an induced pluripotent stem cell or iPSC) which exhibited all the major characteristics of an embryonic stem cell (ESC). It was as if the adult skin cell was traveling back in time, erasing its identity of having been a skin cell and returning to primordial, embryonic-like stem cell. Only one year later, Dr. Yamanaka’s group was able to demonstrate the same phenomena for adult human skin cells in the 2007 Cell paper (Takahashi et al), and in the same year a different group independently confirmed that adult human cells could be reprogrammed to the iPSC state (Science paper by Yu et al in 2007). The generation of iPSCs described in these three papers is probably the most remarkable discovery in stem cell biology during the past decade. It is no wonder that each of these three papers have been cited several thousand times even though they were published only six or seven years ago, and that Dr. Yamanaka was awarded the 2012 Nobel prize for this pioneering work.

All five of the above-mentioned stem cell papers have one thing in common: the results have been repeated and confirmed by numerous independent laboratories all over the world. However, this does not necessarily hold true for the other two highly cited stem cell papers on this list.

The 2002 Nature paper by Jiang and colleagues from Dr. Verfaillie’s laboratory at the University of Minnesota proposed that the bone marrow contained a rather special subset of adult MSCs which had a much broader differentiation potential than had been previously recognized. While adult MSCs were thought to primarily turn into bone, cartilage or fat when given the appropriate cues, this rare new cell type – referred to as MAPCs (multipotent adult progenitor cells) – appeared to differentiate into a much broader range of tissues. The paper even showed data from an experiment in which these adult mouse bone marrow stem cells were combined with embryonic cells and gave rise to a chimeric mouse. i.e. a mouse in which the tissues were in part derived from standard embryonic cells and in part from the newly discovered adult MAPCs. Such chimerism suggested that the MAPCs were embryonic-like, contributing to the formation of all the tissues in the mice. At the time of its publication, this paper was met with great enthusiasm because it proved that the adult body contained embryonic-like cells, hidden away in the bone marrow, and that these MAPCs could be used to regenerate ailing organs and tissues without having to use ethically problematic human embryonic stem cells.

There was just one major catch. Many laboratories around the world tried to replicate the results and were unable to identify the MAPCs, and even when they found cells that were MAPCs, they were unable to confirm the embryonic-like nature of the cells. In a remarkable example of investigative journalism, the science journalists Peter Aldhous and Eugenie Reich identified multiple irregularities in the publications involving MAPCs and documented the inability of researchers to replicate the findings by publishing the results of their investigation in the New Scientist (PDF).

The second high profile stem cell paper which was also plagued by an inability to replicate the results was the 2001 Nature paper by Orlic and colleagues. In this paper from Dr. Anversa’s laboratory, the authors suggested that adult hematopoietic (blood-forming) stem cells from the bone marrow could regenerate an infarcted heart by becoming heart cells (cardiomyocytes). It was a rather bold claim, because simply injecting these blood-forming stem cells into the heart seemed to be sufficient to redirect their fate. Instead of giving rise to red and white blood cells, these bone marrow cells were generating functional heart cells. If this were the case, then every patient could be potentially treated with their own bone marrow and grow back damaged heart tissue after a heart attack. Unfortunately, it was too good to be true. Two leading stem cell laboratories partnered up to confirm the results, but even after years of experiments, they were unable to find any evidence of adult bone marrow stem cells converting into functional heart cells. They published their findings three years later, also in the journal Nature:

Murry CE et al (2004) Haematopoietic stem cells do not transdifferentiate into cardiac myocytes in myocardial infarcts. Nature 428(6983): 664-668

Citations: 1,150

Interestingly, the original paper which had made the claim that bone marrow cells can become functional heart cells has been cited nearly 3,000 times, whereas the refutation by Murry and colleagues, published in the same high-profile journal has been cited only 1,150 times. The vast majority of the nearly 3,000 citations of the 2001 paper by Orlic and colleagues occurred after it had been refuted in 2004! The 2001 Orlic et al paper has even been used to justify clinical trials in which bone marrow was obtained from heart attack patients and injected into their hearts. As expected after the refutation by Murry and colleagues, the success of these clinical trials was rather limited One of the largest bone marrow infusion trials in heart attack patients was recently published, showing no success of the therapy.

These claims of the two papers (Orlic et al and Jiang et al) were quite innovative and exciting, and they were also published in a high-profile, peer-reviewed journal, just like the other five stem cell papers. The crucial difference was the fact that their findings could not be replicated by other laboratories. Despite their lack of replicability, both papers had an enormous impact on the field of stem cell research. Senior scientists, postdocs and graduate students may have devoted a substantial amount of time and resources to developing projects that built on the findings of these two papers, only to find out that they could not be replicated. If there is a lesson to be learned, it is that we need to be rather cautious in terms of our enthusiasm for new claims in stem cell biology until they have been appropriately confirmed by other researchers. Furthermore, we need to streamline the replicability testing process so that we do not have to wait years before we find out that one of the most highly prized discoveries cannot be independently confirmed.

 

Update 7/24/2013: Peter Aldhous reminded me that the superb job of investigative journalism into the question of MAPCs was performed in partnership with the science writer Eugenie Reich, the author of a book on scientific fraud. I have updated the blog post to reflect this.

Cellular Alchemy: Converting Fibroblasts Into Heart Cells

Medieval alchemists devoted their lives to the pursuit of the infamous Philosopher’s Stone, an elusive substance that was thought to convert base metals into valuable gold. Needless to say, nobody ever discovered the Philosopher’s Stone. Well, perhaps some alchemist did get lucky but was wise enough to keep the discovery secret. Instead of publishing the discovery and receiving the Nobel Prize for Alchemy, the lucky alchemist probably just walked around in junkyards, surreptitiously collected scraps of metal and brought them to home to create a Scrooge-McDuck-style money bin.  Today, we view the Philosopher’s Stone as just a myth that occasionally resurfaces in the titles of popular fantasy novels, but cell biologists have discovered their own version of the Philosopher’s Stone: The conversion of fibroblast cells into precious heart cells (cardiomyocytes) or brain cells (neurons).

 

Fibroblasts are an abundant cell type, found in many organs such as the heart, liver and the skin. One of their main functions is to repair wounds and form scars in this process. They are fairly easy to grow or to expand, both in the body as well as in a culture dish. The easy access to large quantities of fibroblasts makes them analogous to the “base metals” of the alchemist. Adult cardiomyocytes, on the other hand, are not able to grow, which is why a heart attack which causes death of cardiomyocytes can be so devastating. There is a tiny fraction of regenerative stem-cell like cells in the heart that are activated after a heart attack and regenerate some cardiomyocytes, but most of the damaged and dying heart cells are replaced by a scar – formed by the fibroblasts in the heart. This scar keeps the heart intact so that the wall of the heart does not rupture, but it is unable to contract or beat, thus weakening the overall pump function of the heart. In a large heart attack, a substantial portion of cardiomycoytes are replaced with scar tissue, which can result in heart failure and heart failure.

A few years back, a research group at the Gladstone Institute of Cardiovascular Disease (University of California, San Francisco) headed by Deepak Srivastava pioneered a very interesting new approach to rescuing heart function after a heart attack.  In a 2010 paper published in the journal Cell, the researchers were able to show that plain-old fibroblasts from the heart or from the tail of a mouse could be converted into beating cardiomyocytes! The key to this cellular alchemy was the introduction of three genes – Gata4, Mef2C and Tbx5 also known as the GMT cocktail– into the fibroblasts. These genes encode for developmental cardiac transcription factors, i.e. proteins that regulate the expression of genes which direct the formation of heart cells. The basic idea was that by introducing these regulatory factors, they would act as switches that turn on the whole heart gene program machinery. Unlike the approach of the Nobel Prize laureate Shinya Yamanaka, who had developed a method to generate stem cells (induced pluripotent stem cells or iPSCs) from fibroblasts, Srivastava’s group bypassed the whole stem cell generation process and directly created heart cells from fibroblasts. In a follow-up paper published in the journal Nature in 2012, the Srivastava group took this research to the next level by introducing the GMT cocktail directly into the heart of mice and showing that this substantially improved heart function after a heart attack. Instead of merely forming scars, the fibroblasts in the heart were being converted into functional, beating heart cells – cellular alchemy with great promise for new cardiovascular therapies.

As exciting as these discoveries were, many researchers remained skeptical because the cardiac stem cell field has so often seen paradigm-shifting discoveries appear on the horizon, only to later on find out that they cannot be replicated by other laboratories. Fortunately, Eric Olson’s group at the University of Texas, Southwestern Medical Center also published a paper in Nature in 2012, independently confirming that cardiac fibroblasts could indeed be converted into cardiomyocytes. They added on a fourth factor to the GMT cocktail because it appeared to increase the success of conversion. Olson’s group was also able to confirm Srivastava’s finding that directly treating the mouse hearts with these genes helped convert cardiac fibroblasts into heart cells. They also noticed an interesting oddity. Their success of creating heart cells from fibroblasts in the living mouse was far better than what they would have expected from their experiments in a dish. They attributed this to the special cardiac environment and the presence of other cells in the heart that may have helped the fibroblasts convert to beating heart cells. However, another group of scientists attempted to replicate the findings of the 2010 Cell paper and found that their success rate was far lower than that of the Srivastava group. In the paper entitled “Inefficient Reprogramming of Fibroblasts into Cardiomyocytes Using Gata4, Mef2c, and Tbx5” published in the journal Circulation Research in 2012, Chen and colleagues found that very few fibroblasts could be converted into cardiomyocytes and that the electrical properties of the newly generated heart cells did not match up to those of adult heart cells. One of the key differences between this Circulation Research paper and the 2010 paper of the Srivastava group was that Chen and colleagues used fibroblasts from older mice, whereas the Srivastava group had used fibroblasts from newly born mice. Arguably, the use of older cells by Chen and colleagues might be a closer approximation to the cells one would use in patients. Most patients with heart attacks are older than 40 years and not newborns.

These studies were all performed on mouse fibroblasts being converted into heart cells, but they did not address the question whether human fibroblasts would behave the same way. A recent paper in the Proceedings of the National Academy of Sciences by Eric Olson’s laboratory (published online before print on March 4, 2013 by Nam and colleagues) has now attempted to answer this question. Their findings confirm that human fibroblasts can also be converted into beating heart cells, however the group of genes required to coax the fibroblasts into converting is slightly different and also requires the introduction of microRNAs – tiny RNA molecules that can also regulate the expression of a whole group of genes. Their paper also points out an important caveat.  The generated heart-like cells were not uniform and showed a broad range of function, with only some of the spontaneously contracting and with an electrical activity pattern that was not the same as in adult heart cells.

Where does this whole body of work leave us? One major finding seems to be fairly solid. Fibroblasts can be converted into beating heart cells. The efficiency of conversion and the quality of the generated heart cells – from mouse or human fibroblasts – still needs to be optimized. Even though the idea of cellular alchemy sounds fascinating, there are many additional obstacles that need to be overcome before such therapies could ever be tested in humans. The method to introduce these genes into the fibroblasts used viruses which permanently integrate into the DNA of the fibroblast and could cause genetic anomalies in the fibroblasts. It is unlikely that such viruses could be used in patients. The fact that the generated heart cells show heterogeneity in their electrical activity could become a major problem for patients because patches of newly generated heart cells in one portion of the heart might be beating at a different rate of rhythm than other patches. Such electrical dyssynchony can cause life threatening heart rhythm problems, which means that the electrical properties of the generated cells need to be carefully understood and standardized. We also know little about the long-term survival of these converted cells in the heart and whether the converted cells maintain their heart-cell-like activity for months or years. The idea of directly converting fibroblasts by introducing the genes into the heart instead of first obtaining the fibroblasts, then converting them in a dish and lastly implanting the converted cells back into the heart sounds very convenient. But this convenience comes at a price. It requires human gene therapy which has its own risks and it is very difficult to control the cell conversion process in an intact heart of a patient. On the other hand, if cells are converted in a dish, one can easily test and discard the suboptimal cells and only implant the most mature or functional heart cells.

This process of cellular alchemy is still in its infancy. It is one of the most exciting new areas in the field of regenerative medicine, because it shows how plastic cells are. Hopefully, as more and more labs begin to investigate the direct reprogramming of cells, we will be able to address the obstacles and challenges posed by this emerging field.

 

Image credit: Painting in 1771 by Joseph Wright of Derby – The Alchymist, In Search of the Philosopher’s Stone via Wikimedia Commons

 

Nam, Y., Song, K., Luo, X., Daniel, E., Lambeth, K., West, K., Hill, J., DiMaio, J., Baker, L., Bassel-Duby, R., & Olson, E. (2013). Reprogramming of human fibroblasts toward a cardiac fate Proceedings of the National Academy of Sciences, 110 (14), 5588-5593 DOI: 10.1073/pnas.1301019110

Stemming the Flow: Using Stem Cells To Treat Urinary Bladder Dysfunction

Neurogenic bladder is a disorder which occurs in spinal cord diseases such as spina bifida and is characterized by an inability of the nervous system to properly control the urinary bladder and the muscle tissue contained in the bladder wall. This can lead to spasms and a build-up of pressure in the bladder, often resulting in urinary incontinence. Children with spina bifida and neurogenic bladder often feel urges to urinate after drinking comparatively small amounts of liquid and they can also involuntarily leak urine. This is a source of a lot of emotional stress, especially in social settings such as when they are around friends or in school. If untreated, the long-standing and frequent pressure build-up in the bladder can have even more devastating effects such as infections or kidney damage.

Current treatments for neurogenic bladder involve surgeries which reconstruct and increase the size of the bladder by using tissue patches obtained from the bowel of the patient. Since such a gastrointestinal patch is derived from the patient’s own body, it is less likely to elicit an immune response and these intestinal tissue patches tend to be strong enough to withstand the pressures in the bladder. Unfortunately, the incompatibility of intestinal tissue and bladder tissue can lead to long-term complications, such as urinary tract infections, formation of urinary tract stones and in some rare cases even cancers. For this reason, researchers have been searching for newer safer patches which resemble the actual bladder wall.

 

A team of researchers at Northwestern University recently published a study which used stem cells of children with spina bifida to generate tissue patches that could be used for bladder surgery. In the paper “Cotransplantation with specific populations of spina bifida bone marrow stem/progenitor cells enhances urinary bladder regeneration” published in the Proceedings of the National Academy of Sciences (online publication on February 19, 2013), Arun Sharma and colleagues isolated two types of cells from the bone marrow of children with spina bifida: Mesenchymal stem cells (MSCs) and CD34+ cells (stem and progenitor cells which usually give rise to blood cells). They then coated a special polymer scaffold called POC with the cells and implanted this newly created patch into a rat bladder after performing a bladder augmentation surgery, similar to what is performed in patients with spina bifida. They then assessed the survival and formation of human muscle tissue on the implanted patch. When both human cell types (MSCs and CD34+) were combined, more than half of the implanted patch was covered with muscle tissue, four weeks after the implantation. If they only used CD34+ cells, they found that only a quarter of the patch was covered with muscle tissue. What is even more remarkable is that in addition to the newly formed muscle tissue, the implanted patch also showed evidence of some peripheral nerve growth and of blood vessel formation, both of which are found in healthy, normal bladder walls. These findings suggest that a patient’s own bone marrow stem cells can be used to help construct a tissue patch which could be used for bladder augmentation surgeries. The observation of some nerve growth in the implanted patch is also an exciting finding. One could conceivably try to re-connect the reconstructed bladder tissue with the main nervous system, but its success would largely depend on the severity of the neurologic disease.

One has to keep in mind that there are some key limitations to this study. The authors of the paper believe that the newly formed muscle tissue on the implanted patches was all derived from the patients’ bone marrow stem cells. However, there were no experiments performed to convincingly demonstrate this. The authors report that in previous studies, merely implanting the empty POC scaffold without any human stem cells resulted in 20% coverage with muscle tissue. This suggests that a big chunk of the newly formed muscle tissue is actually derived from the host rat and not from human stem cells. The authors also did not compare the effectiveness of this newly formed stem cell patch to the currently used intestinal patches, and there is no assessment of whether the newly formed muscle tissue on the reconstructed bladder is less prone to spasms and involuntary contractions. Lastly, all the in vivo testing of the tissue patches was performed in rats without neurogenic bladder and it is possible that the highly successful formation of muscle tissue may have been diminished if the animals had a neurologic disease.

A second study published in PLOS One took a different approach. In “Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells” (online publication February 7, 2013), Debra Franck and colleagues describe how they coated a scaffold consisting of silk threads with extracellular matrix proteins such as fibronectin. Instead of using bone marrow stem cells, they converted induced pluripotent stem cells into the smooth muscle cells that are typically found inside the bladder wall and placed these newly differentiated cells on the silk scaffold. The induced pluripotent stem cells (iPSCs) used by Franck and colleagues can be generated from a patient’s own skin cells which reduces the risk of being rejected by a patient’s immune system. The advantage of this approach is that it starts out with a pure and truly pluripotent stem cell population, which is easier to direct and control than bone marrow stem cells. There are also a few important limitations to this second study. Franck and colleagues used mouse pluripotent stem cells and it is not clear that their approach would necessarily work with human pluripotent stem cells. They also did not test the function of these differentiated cells on the silk scaffold to check if they actually behaved like true bladder wall smooth muscle cells. Unlike the first study, Franck and colleagues did not evaluate the newly created patch in an animal model.

Both studies are purely experimental and much additional work is needed before they can be tested in humans, but both show promising new approaches to help improve bladder dysfunction. It is heartening to see that researchers are developing new cell-based therapies to help children and adults who suffer from neurogenic bladder. The results from these two experimental studies are still too preliminary to predict whether cell-based therapies can be successfully used in patients, but they represent important first steps.

 

Image credit: Taken from Franck D, Gil ES, Adam RM, Kaplan DL, Chung YG, et al. (2013) Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells. PLoS ONE 8(2): e56237. doi:10.1371/journal.pone.0056237- Figure 6 B: Differentiated mouse induced pluripotent stem cells cultured on fibronectin-coated silk matrices show protein markers typically found in bladder smooth muscle cells.

Franck, D., Gil, E., Adam, R., Kaplan, D., Chung, Y., Estrada, C., & Mauney, J. (2013). Evaluation of Silk Biomaterials in Combination with Extracellular Matrix Coatings for Bladder Tissue Engineering with Primary and Pluripotent Cells PLoS ONE, 8 (2) DOI: 10.1371/journal.pone.0056237

Immune Cells Can Remember Past Lives

The generation of induced pluripotent stem cells (iPSCs) is one of the most fascinating discoveries in the history of stem cell biology. John Gurdon and Shinya Yamanaka received the 2012 Nobel Prize for showing that adult cells could be induced to become embryonic-like stem cells (iPSCs). Many stem cell laboratories now routinely convert skin cells or blood cells from an adult patient into iPSCs. The stem cell properties of the generated iPSCs then allow researchers to convert them into a desired cell type, such as heart cells (cardiomyocytes) or brain cells (neurons), which can then be used for cell-based therapies or for the screening of novel drugs. The initial conversion of adult cells to iPSCs is referred to as “reprogramming” and is thought to represent a form of rejuvenation, because the adult cell appears to lose its adult cell identity and reverts to an immature embryonic-like state. However, we know surprisingly little about the specific mechanisms that allow adult cells to become embryonic-like. For example, how does a blood immune cell such as a lymphocyte lose its lymphocyte characteristics during the reprogramming process? Does the lymphocyte that is converted into an immature iPSC state “remember” that it used to be a lymphocyte? If yes, does this memory affect what types of cells the newly generated iPSCs can be converted into, i.e. are iPSCs derived from lymphocytes very different from iPSCs that are derived from skin cells?

There have been a number of recent studies that have tried to address the question of the “memory” in iPSCs, but two recent papers published in the January 3, 2013 issue of the journal Cell Stem Cell provide some of the most compelling proofs of an iPSC “memory” and also show that this “memory” could be used for therapeutic purposes. In the paper “Regeneration of Human Tumor Antigen-Specific T Cells from iPSCs Derived from Mature CD8+ T Cells“, Vizcardo and colleagues studied the reprogramming of T-lymphocytes derived from the tumor of a melanoma patient. Mature T-lymphocytes are immune cells that can recognize specific targets, depending on what antigen they have been exposed to. The tumor infiltrating cells used by Vizcardo and colleagues have been previously shown to recognize the melanoma tumor antigen MART-1. The researchers were able to successfully generate iPSCs from the T-lymphocytes, and they then converted the iPSCs back to T-lymphocytes. What they found was that the newly generated T-lymphocytes expressed a receptor that was specific for the MART tumor antigen. Even though the newly generated T-lymphocytes had not been exposed to the tumor, they had retained their capacity to respond to the melanoma antigen. The most likely explanation for this is that the generated iPSCs “remembered” their previous exposure to the tumor in their past lives as T-lymphocytes before they had been converted to embryonic-like iPSCs and then “reborn” as new T-lymphocytes. The iPSC reprogramming apparently did not wipe out their “memory”.

This finding has important therapeutic implications. One key problem that the immune system faces when fighting a malignant tumor is that the demand for immune cells outpaces their availability. The new study suggests that one can take activated immune cells from a cancer patient, convert them to the iPSC state, differentiate them back into rejuvenated immune cells, expand them and inject them back into the patient. The expanded and rejuvenated immune cells would retain their prior anti-tumor memory, be primed to fight the tumor and thus significantly augment the ability of the immune system to slow down the tumor growth.

The paper by Vizcardo and colleagues did not actually show the rejuvenation and anti-tumor efficacy of the iPSC-derived T-lymphocytes and this needs to be addressed in future studies. However, the paper “Generation of Rejuvenated Antigen-Specific T Cells by Reprogramming to Pluripotency and Redifferentiation” by Nishimura and colleagues in the same issue of Cell Stem Cell, did address the rejuvenation question, albeit in a slightly different context. This group of researchers obtained T-lymphocytes from a patient with HIV, then generated iPSC and re-differentiated the iPSCs back into T-lymphocytes. Similar to what Vizcardo and colleagues had observed, Nishimura and colleagues found that their iPSC derived T-lymphocytes retained an immunological memory against HIV antigens. Importantly, the newly derived T-lymphocytes were highly proliferative and had longer telomeres. The telomeres are chunks of DNA that become shorter as cells age, so the lengthening of telomeres and the high growth rate of the iPSC derived T-lymphocytes were both indicators that the iPSC reprogramming process had made the cells younger while also retaining their “memory” or ability to respond to HIV.

Further studies are now needed to test whether adding the rejuvenated cells back into the body does actually help prevent tumor growth and can treat HIV infections. There is also a need to ensure that the cells are safe and the rejuvenation process itself did not cause any harmful genetic changes. Long telomeres have been associated with the formation of tumors and one has to make sure that the iPSC-derived lymphocytes do not become malignant. These two studies represent an exciting new development in iPSC research. They not only clearly document that iPSCs retain a memory of the original adult cell type they are derived from but they also show that this memory can be put to good use. This is especially true for immune cells, because retaining an immunological memory allows rejuvenated iPSC-derived immune cells to resume the fight against a tumor or a virus.

 

Image credit: “Surface of HIV infected macrophage” by Sriram Subramaniam at the National Cancer Institute (NCI) via National Institutes of Health Image Bank

Inspired By Snake Venom

When I remember the 80s, I think of Nena’s 99 Luftballons, Duran Duran’s Wild Boys and ….snake venom. Back in those days, I used to be a typical high school science nerd. My science nerdiness interfered with my ability to socialize with non-nerds and it was characterized by an unnecessary desire to read science books and articles that I did not really understand, just so that I could show off with some fancy science terminology. I did not have much of an audience to impress, because my class-mates usually ignored me. My high school biology teacher, Herr Sperr, was the only one who had the patience to listen to me. One of the science books that I purchased was called “Gehirn und Nervensystem” (i.e. “Brain and Nervous System”), published by Spektrum der Wissenschaft, the German publisher of Scientific American. It was a collection of Scientific American articles in the field of neuroscience that had been translated into German. I was thumbing through it, looking for some new neurobiology idea or expression that I could use to impress Herr Sperr. While browsing the book, I came across the article “Der Nervenwachstumsfaktor” (originally published in Scientific American as “The Nerve-Growth Factor” in 1979) by Rita Levi-Montalcini and Pietro Calissano.

My curiosity was piqued by this article, because I did not realize that nerves had “growth factors” and because one of the authors, Rita Levi-Montalcini, had just won the Nobel Prize in the preceding year. I started reading the article and loved it, reading it over and over again. I liked the article so much, that I did not even try to show off about it and kept the newly discovered inspiration to myself. There are many reasons why I loved the article and I will just mention two of them:

1. Scientific discovery is an exciting journey, starting and ending with unanswered questions

Levi-Montalcini and Calissano started off by describing the state of knowledge and the unanswered questions in the field of developmental neurobiology and neuronal differentiation in the 1940s, when Levi-Montalcini was about to launch her career as a scientist. They commented on how the simple yet brilliant idea to test whether tumors could influence the growth of nerves sparked a whole new field of investigation. They narrated a beautiful story of scientific discovery, from postulating a “nerve growth factor” to actually isolating and sequencing it. Despite all the advances that Levi-Montalcini and her colleagues had made, the article ended with a new mystery, that the role of the nerve growth factor may be much bigger than all the researchers suspected. The nerve growth factor was able to act on cells that were not neurons and it was unclear why this was the case. By hinting at these yet to be defined roles, the article made it clear that so much more work was necessary and I felt that an invitation was being extended to the readers to participate in the future discovery.

2. Scientific tools can harbor surprises and important clues

The article mentioned one important coincidence that helped shape the progress of discovering the sequence of the nerve growth factor. To assess whether the putative nerve growth factor contained nucleic acids, Levi-Montalcini and her colleagues exposed the “soup” that was inducing the growth of nerves to snake venom. The rationale was that snake venom (by the way, the German expression “Schlangengift” sounds even more impressive than the English “snake venom”) would degrade nucleic acids and if the growth enhancing properties disappeared, it would mean that the nerve growth inducing factor contained nucleic acids. It turned out that the snake venom unexpectedly magnified the nerve growth enhancing effects, because the venom contained large quantities of the nerve growth factor itself. This unexpected finding made it much easier for the researchers to sequence the nerve growth factor, because the snake venom now provided access to a large source of the nerve growth factor and it resulted in a new mystery: Why would snake venom contain a nerve growth factor?

In the subsequent decades, as I embarked on my own career as a scientist, I often thought about this article that I read back in high school. It inspired me to become a cell biologist and many of the projects in my laboratory today focus on the effects of growth factors on blood vessels and stem cells. The article also made me think about the importance of continuously re-evaluating the tools that we use. Sometimes our tools are not as neutral or straight-forward as we think, and this lesson is just as valid today as it was half a century ago. For example, a recent paper in Cell found that the virus used for reprogramming adult cells into stem cells is not merely a tool that allows entry of the reprogramming factors, as was previously thought. The virus tool can actually activate the stem cell reprogramming itself, reminiscent of how the “snake venom” tool was able to induce nerve growth.

Rita Levi-Montalcini was one of the world’s greatest biologists and passed away on December 30, 2012. In addition to her outstanding scientific work, she was also a shining example of an activist scientist with a conscience, who fought for education and research. I never had the opportunity to meet her in person, but I was inspired by her work and I will always see her as a role model.

Image credit: Cover of the book “Gehirn und Nervensystem” by Spektrum der Wissenschaft

Somatic Mosaicism: Genetic Differences Between Individual Cells

The cells in the body of a healthy person all have the same DNA, right? Not really! It has been known for quite some time now that there are genetic differences between cells within one person. The expression to describe these between-cell differences is “somatic mosaicism“, because cells can represent a mosaic of genetic profiles, even within a single organ. During embryonic development, all cells are derived from one fertilized egg and ought to be genetically identical. However, during every cell division errors and differences during DNA replication can occur and this can lead to genetic differences between cells. This process not only occurs during embryonic development but continues after birth.

As we age, our cells are exposed to numerous factors such as radiation, chemicals or other stressors which can causes genetic alterations, ranging from single nucleotide mutations to duplications and deletions of large chunks of DNA. Some mutations are known to cause cancer by making a single cell grow rapidly, but not all mutations lead to cancer. Many spontaneous mutations can either result in the death of a cell or do not even impact its function in any significant manner. DNA copy number variations (CNVs) is an expression used to describe a variable copy number of larger DNA segments of one kilobase (kb). Most recent studies on CNVs have compared CNVs between people, i.e. how many CNVs does person A have when compared to person B. It turns out that there may be quite a bit of genetic diversity between people that had previously been overlooked.

A new paper published in the journal Nature takes this one step further. It not only shows that there are significant CNVs between people, but even within a single person. In the study “Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells“, Alexej Abyzov and colleagues found significant CNVs in induced pluripotent stem cells (iPSCs) that they had generated from the adult skin cells of human subjects. Importantly, most of these CNVs were not the result of reprogramming adult skin cells to the stem cell state. They were already present in the skin fibroblasts obtained from the human subjects. Most analyses of CNVs are performed on whole tissues or biopsies, but not on single cells, which is why so little is known about between cell CNV differences. However, when iPSCs are generated from skin fibroblast cells, they are often derived from a single cell. This enables the evaluation of genetic diversity between cells.

Abyzov and colleagues estimate that 30% of adult skin fibroblasts carry large CNVs. This estimate is based on a very small number of fibroblast samples. It is not clear whether other cells such as neurons or heart cells also have similar CNVs and whether the 30% estimate would hold up in a larger sample. Their work leads to the intriguing question: What percentage of neighboring cells in a single heart, brain or kidney are actually genetically identical? Cell types, such as heart cells or adult neurons cannot be clonally expanded so it may be difficult to determine the genetic diversity within a heart or a brain using the methods employed by Abyzov and colleagues.

What are the implications of this work? On a practical level, this study suggests that it may be important to derive multiple iPSC clones from a subject’s or patient’s skin cells, if one wants to use the iPSCs for disease modeling. This will help control for the genetic diversity that exists among the skin cells. However, a much more profound implication of this work is that we have to think about between-cell diversity within a single organ. We need to develop better tools for how to analyze genetic diversity between individual cells, and more importantly, we have to understand how this genetic diversity impacts health and disease.

Image Credit: Wikimedia / Alexander Mosaic (Public Domain)

Abyzov A, Mariani J, Palejev D, Zhang Y, Haney MS, Tomasini L, Ferrandino AF, Rosenberg Belmaker LA, Szekely A, Wilson M, Kocabas A, Calixto NE, Grigorenko EL, Huttner A, Chawarska K, Weissman S, Urban AE, Gerstein M, & Vaccarino FM (2012). Somatic copy number mosaicism in human skin revealed by induced pluripotent stem cells. Nature, 492 (7429), 438-42 PMID: 23160490

Recent Study Raises Questions About Using Adult Stem Cells for Chronic Heart Disease

A recent clinical study (POSEIDON Randomized Trial) investigated the effects of transplanting bone marrow derived adult stem cells into patients with known heart disease. The results were presented at the 2012 American Heart Association (AHA) meeting in Los Angeles and also published in the article “Comparison of Allogeneic vs Autologous Bone Marrow–Derived Mesenchymal Stem Cells Delivered by Transendocardial Injection in Patients With Ischemic Cardiomyopathy: The POSEIDON Randomized Trial“. The article by Dr. Joshua Hare and colleagues appeared in the online edition of the Journal of the American Medical Association on November 6, 2012.

The primary goal of the study was to compare whether adult stem cells from other donors (allogeneic cells) are just as safe as the stem cells derived from the patients’ own bone marrow (autologous cells). Thirty patients with a prior heart attack and reduced cardiac function received either allogeneic or autologous cells. The injected cells were mesenchymal stem cells (MSCs), an adult stem cell type that resides within the bone marrow and primarily gives rise to bone, fat or cartilage tissue. MSCs are quite distinct from hematopoietic stem cells (HSCs) which are also present in the bone marrow but give rise to blood cells. In the POSEIDON study, patients underwent a cardiac catheterization and the MSCs were directly injected into the heart muscle. Various measurements of safety and cardiac function were performed before and up to one year after the cell injection.

The good news is that in terms of safety, there was no significant difference when either autologous or allogeneic MSCs were used. Within the first month after the cell injection, only one patient in each group was hospitalized for what may have been a major treatment related side effect. In the long-run, the number of adverse events was very similar in both groups. The implication of this finding is potentially significant. It suggests that one can use off-the-shelf adult stem cells from a healthy donor to treat a patient with heart disease. This is much more practical than having to isolate the bone marrow from a patient and wait for 4-8 weeks to expand his or her own bone marrow stem cells.

The disappointing news from this study is that one year following the stem cell injection, there was minimal improvement in the cardiac function of the patients. The ejection fraction of the heart is an indicator of how well the heart contracts and the normal range for healthy patients is roughly 55-60%. In the current study, patients who received allogeneic cells started out with an average ejection fraction of 27.9% and the value increased to 29.5% one year after the cell injection. The patients who received autologous cells had a mean ejection fraction of 26.2% prior to the cell transplantation and a mean ejection fraction of 28.5% one year after the stem cell therapy. In both groups, the improvement was minimal and not statistically significant. A different measure of the functional capacity of the heart is the assessment of the peak oxygen consumption. This measurement correlates well with the survival of a patient and is also used to help decide if a patient needs a heart transplant. There was no significant change in the peak oxygen consumption in either of the two groups of patients, one year after the treatment. Some other measures did indicate a minor improvement, such as the reduction of the heart attack scar size in both patient groups but this was apparently not enough to improve the ejection fraction or oxygen consumption.

One of the key issues in interpreting the results is the fact that there was no placebo control group. The enrollment in a research study and the cell injection procedure itself could have contributed to minor non-specific or placebo benefits that were unrelated to the stem cell treatments. One odd finding was that the patient sub-group which showed a statistically significant improvement in ejection fraction was the group which received the least stem cells. If the observed minor benefits were indeed the result of the injected cells turning into cardiac cells, one would expect that more cells would lead to greater functional improvement. The efficacy of the lowest number of cells points to non-specific effects from the cell injection or to an unknown mechanism by which the injected cells activate cardiac repair without necessarily becoming cardiac cells themselves.

The results of this study highlight some key problems with current attempts to use adult stem cells in cardiovascular patients. Many studies have shown that adult stem cells have a very limited differentiation potential and that they do not really turn into beating, functional heart cells. Especially in patients with established, long-standing heart disease, the utility of adult stem cells may be very limited. The damage that the heart of these patients has suffered is probably so severe that they need stem cells which can truly regenerate the heart. Examples of such regenerative stem cells are embryonic stem cells or induced pluripotent stem cells which have a very broad differentiation potential. Cardiac stem cells, which exist in very low numbers within the heart itself, are also able to become functional heart cells. Each of these three cell types is challenging to use in patients, which is why many current studies have resorted to using the more convenient adult bone marrow stem cells.

Human embryonic stem cells can develop into functional heart cells, but there have been numerous ethical and regulatory concerns about using them. Induced pluripotent stem cells (iPSCs) appear to have the capacity to become functional heart cells, similar to what has been observed for human embryonic stem cells. However, iPSCs were only discovered six years ago and we still have a lot to learn more about how they work. Lastly, cardiac stem cells are very promising but isolating them from the heart requires an additional biopsy procedure which can also carry some risks for the patients. Hopefully, the fact that adult bone marrow stem cells showed only minimal benefits in the POSEIDON study will encourage researchers to use these alternate stem cells (even if they are challenging to use) instead of adult bone marrow stem cells for future studies in patients with chronic heart disease.

One factor that makes it difficult to interpret the POSEIDON trial is the lack of a placebo control group. This is a major problem for many stem cell studies, because it is not easy to ethically justify a placebo group for invasive procedures such as a stem cell implantation. The placebo patients would also have to receive a cardiac catheterization and injections into the heart tissue, but instead of stem cells, the injections would just contain a cell-free liquid solution. Scientifically, such a placebo control group is necessary to determine whether the stem cells are effective, but this scientific need has to be weighed against the ethics of a “placebo” heart catheterization. Even if one were to ethically justify a “placebo” heart catheterization, it may not be easy to recruit volunteer patients for the study if they knew that they had a significant chance of receiving “empty” injections into their heart muscle.

There is one ongoing study which is very similar in design to the POSEIDON trial and it does contain a placebo group: The TAC-HFT trial. The results of this trial are not yet available, but they may have a major impact on whether or not bone marrow stem cells have a clinical future. If the TAC-HFT trial shows that the bone marrow stem cell treatment for patients with chronic heart disease has no benefits or only minor benefits when compared to the placebo group, it will become increasingly difficult to justify the use of these cells in heart patients.

In summary, the POSEIDON trial has shown that treating chronic heart disease patients with bone marrow derived stem cells is not yet ready for prime time. Bone marrow cells from strangers may be just as safe as one’s own cells, but if bone marrow stem cells are not very effective for treating chronic heart disease, than it may just be a moot point.

 

Image credit: Wikipedia

The Importance of Being Embryonic

Human ESC colony – Wikimedia

There are three broad categories of human stem cells: 1) adult stem cells, 2) embryonic stem cells (ESCs) and 3) induced pluripotent stem cells (iPSCs). Adult stem cells can be found in selected adult tissues, such as the hematopoietic stem cells in the bone marrow which give rise to a variety of blood cells on a daily basis in an adult. Such adult stem cells are quite rare and, when compared to ESCs, somewhat limited in the type of cells they can generate. Hematopoietic stem cells, for example, routinely produce leukocytes (white blood cells) and erythrocytes (red blood cells), but most researchers agree that they cannot give rise heart muscle cells. On the other hand, human ESCs are pluripotent, which refers to the fact that they can differentiate into nearly all cell types, from neurons to insulin-producing pancreatic cells or even heart muscle cells.

Human ESCs are usually derived from human eggs that were created in an in vitro fertilization clinic but never implanted in a woman. Such clinics often generate far more fertilized human eggs than they actually implant, because it is difficult to predict how many implantation attempts are necessary before a successful pregnancy can be achieved. The “back-up” eggs remain in a freezer at the in vitro fertilization clinic and the donors can then decide whether they want these eggs to be used for the generation of human ESCs, which can be used for either research or ESC-based therapies. The informed consent of the donors is critical and needs to be documented before the ethics committees at the research institutions permit their usage. In spite of these regulations, some religious groups in the US have voiced concerns about using the ESCs, because they feel that even though the donated fertilized egg was never implanted in a woman, it could have been implanted and that its fertilized state already indicates a degree of personhood that requires protection. When the fertilized egg is cultured in a lab and ESCs are derived from it, the fertilized egg is invariably destroyed and from a certain religious perspective, this constitutes a destruction of a human life. Due to concerns about the ethics of using human ESCs, multiple US-based Christian groups have championed the use of adult stem cells to help repair injured tissues and organs. However, since adult stem cells are very rare and limited in their differentiation potential, most stem cell biologists do not see adult stem cells as a suitable alternative to ESCs.

A landmark paper published by Shinya Yamanaka’s group in 2007 provided a new perspective in the gridlock between demands of Christian groups to ban human ESC research and the desire of stem cell biologists to use human ESCs for regenerative medicine.  Yamanaka and his colleagues were able to show that human adult skin fibroblasts could be converted into embryonic-like stem cells (induced pluripotent stem cells or iPSCs). The iPSCs were not adult stem cells with, but actually exhibited the broad differentiation capacity that was previously only seen in human ESCs. From an ethical perspective, iPSCs seemed like a perfect solution since they could be generated without the destruction of any fertilized eggs. Shinya Yamanaka and John Gurdon, whose earlier work had set the stage for Yamanaka’s discovery, received the 2012 Nobel Prize for these exciting findings. Yamanaka’s work was not only lauded by fellow scientists, but also by religious groups, who felt that his work abolished the need for human ESCs. What these religious organizations did not understand was that human ESC research provided the foundation for Yamanaka’s research. All the factors used to reprogram adult skin cells into iPSCs were derived from a careful analysis of ESCs and the culture of human iPSCs was only made possible after the culture of human ESCs had been established in the late 1990s. To this day, the comparison of human ESCs and iPSCs is a topic of active investigation. In many ways, iPSC research is still – pardon the pun – in its embryonic stage. We are still in the process of understanding how an adult cell can be reprogrammed into an iPSC and whether the reprogramming process leaves any kind of marks or blemishes that would affect the generated iPSC.

To understand the biology and nature of iPSCs, researchers routinely use them side-by-side with human ESCs, which still serve as the “gold-standard” for a pluripotent stem cell. At a symposium of the International Society of Stem Cell Research (ISSCR) in San Francisco on August 24, 2012, Yamanaka showed the results of a new study in which he compared the gene expression profiles of 49 different human iPSC lines and 10 different human ESC lines. The comparison revealed that the majority of iPSC lines are indistinguishable from human ESCs, but that there is a minority of iPSC cell lines that behave very differently from human ESCs. Other stem cell researchers have also shown both similarities and differences between ESCs and iPSCs, and definitive conclusions about whether human ESCs and iPSCs are equally suitable for regenerating human tissues and organs cannot yet be drawn.

These new studies remind us that human ESC research is still a very active area of investigation and that in the years to come, research on both ESCs and iPSCs is needed. This was also emphasized in a recent statement by the ISSCR:

 

Yamanaka’s recent and exciting advances demonstrate that it is possible to reprogram cells in adult human tissues into cells that very closely resemble, but may not be identical to, ES cells. Along with recent progress on redirecting cell fate to enhance tissue repair, these experiments have captured the imagination of the scientific community worldwide. While many scientists are very optimistic about the future of this new research, some people in political circles have incorrectly interpreted this enthusiasm as a verdict that research on human ES cells is no longer necessary. This conclusion is not yet scientifically justified.

At present, and in the foreseeable future, there is a strong scientific and medical consensus that continued research on all types of stem cells is critical to developing research strategies that will ultimately provide new therapies. Supporting all forms of stem cell research is in the best long-term interests of a broad spectrum of patients with debilitating diseases and injuries. In fact, predictions about what might or might not be possible cannot substitute for careful and rigorous research to discover what strategy will provide the most successful therapeutic intervention for a given disease or condition. The basic tools for these discoveries include human ES cells, which remain the benchmark for assessing pluripotency and the ability of cells to develop into all the different cell types of the body.

In the wake of the announcement of the Nobel Prize, the ISSCR (whose current president is Shinya Yamanaka) wanted to pre-empt any attempts to dismiss the importance of human ESC research, which remains a cornerstone of stem cell biology and regenerative medicine. I applaud the ISSCR for this pro-active approach. Taking ethical concerns into account is important, but one also needs to make sure that scientific discoveries are not misused to put forward political or religious agendas. In the next years or decades, we may indeed discover that iPSCs can completely replace human ESCs. On the other hand, we may discover that iPSCs and ESCs will play distinct and complementary roles in the future of regenerative medicine. We will not know the answer to the question until we conduct the research and keep an open mind when we assess the results. The nascent biology of iPSCs and ESCs is a journey into the unknown and this is what makes it such an exciting area of research.