Stem Cells and Their Fat Neighbors

We recently published a PLOS ONE paper (Mitochondrial respiration regulates adipogenic differentiation of human mesenchymal stem cells) in which we studied how the metabolism of an adult stem cell can influence its ability to differentiate. Human bone marrow mesenchymal stem cells (also known as marrow stromal cells, marrow progenitor cells or MSCs) can be converted into fat (adipocytes), cartilage (chondrocytes) or bone (osteoblasts). The work performed by Yanmin Zhang and Glenn Marsboom in my lab showed that MSCs undergo a major metabolic shift towards increased mitochondrial oxidation when they become fat cells and that suppressing mitochondrial respiration can prevent their differentiation. The metabolic state of the adult stem cells is therefore not only an indicator of their “stemness”, it can be used to either promote or suppress their differentiation.

 

Dr. Peter Toth, one of the co-authors on the paper, helped us acquire some really beautiful images of the cells that I would like to share with the readers of the blog. The image below shows undifferentiated adult human bone marrow mesenchymal stem cells (MSCs) that were exposed to an adipogenic differentiation medium, i.e a combination of factors which induces the formation of fat cells (adipocytes). However, as with many stem cell differentiation protocols, not all stem cells turned into fat cells. The cells on the right have a typical fat-like structure in which cells are full of round lipid droplets. The neighboring cells on the left are MSCs that have not (yet?) become fat cells. We stained the cells with the fluorescent mitochondrial dye JC-1. Depolarized mitochondria appear green and hyperpolarized mitochondria red. As you can see, the cells on the left have a much higher mitochondrial membrane potential (significant amount of red among the green mitochondria) than their fat neighbors on the right (mostly green mitochondria, all of them located between lipid droplets). By capturing both cell types next to each other, we could show an illustrative example of how entwined metabolism and stem cell differentiation are. The morphology and metabolic state of neighboring cells in this image were quite different, despite the fact that all cells were subjected to the same cocktail of differentiation factors. The blue-appearing dye is DAPI and stains nuclei of cells so one can tell the cells apart. Each cell in this image has one blue nucleus.

 

 

The image was published with a PLoS ONE CC-BY license. Feel free to use it as an example of adult stem cell differentiation or how mitochondrial morphology and function can vary between stem cell and its differentiated progeny, as long as you attribute the original PLoS One paper. The image in the paper also has a scale bar and asterisks/arrows pointing out the specific cells.

 

ResearchBlogging.org

Zhang Y, Marsboom G, Toth PT, & Rehman J (2013). Mitochondrial respiration regulates adipogenic differentiation of human mesenchymal stem cells. PLoS ONE 8(10): e77077;  PMID: 24204740; DOI: 10.1371/journal.pone.0077077

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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

 

ResearchBlogging.org
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

Bone Marrow Cell Infusions Do NOT Improve Cardiac Function After Heart Attack

For over a decade, cardiologists have been conducting trials in patients using cells extracted from the bone marrow and infusing them into the blood vessels of the heart in patients who have suffered a heart attack. This type of a procedure is not without risks. It involves multiple invasive procedures in patients who are already quite ill, because they are experiencing a major heart attack:

1) Patients with a major heart attack (also referred to as ST-elevation Myocardial Infarction or STEMI) usually undergo an immediate angiogram of the heart to treat the blockage that is causing the heart attack by impeding the blood flow. This is the standard of care for heart attack patients in the developed world.

2) Patients enrolled in an experimental cell therapy trial are then brought back for a second procedure during which bone marrow is extracted with a needle under local anesthesia.

3) The research patients then undergo another angiogram of the heart using a catheter which allows for the infusion of bone marrow cells into the heart.

The hope is that the stem cells contained within the bone marrow are able to help regenerate the heart, either by turning into heart cells (cardiomyocytes), blood vessel cells (endothelial cells) or releasing factors that protect the heart and prevent the formation of a large scar. Unfortunately, there is very limited scientific evidence that bone marrow stem cells can actually turn into functional heart cells. The trials that have been conducted so far have yielded mixed results – some show that infusing the bone marrow cells indeed improves heart function, others show that patients who just receive the standard therapy with cell infusions do just as well. Most of the trials have been quite small – often studying only 10-50 patients.

The SWISS-AMI cell therapy trial, published online on April 17, 2013 in the world’s leading cardiovascular research journal Circulation, addressed this question in a randomized, controlled trial, which enrolled 200 patients who had suffered a major heart attack. The published paper is entitled “Intracoronary Injection of Bone Marrow Derived Mononuclear Cells, Early or Late after Acute Myocardial Infarction: Effects on Global Left Ventricular Function” and was conducted in Switzerland.

The researchers assigned the patients to three groups: a) Standard heart attack treatment, b) Standard heart attack treatment and infusion of bone marrow cells 5-7 days after the heart attack or c) Standard heart attack treatment and infusion of bone marrow cells 3-4 weeks after the heart attack. They assessed heart function four months later using cardiac magnetic resonance imaging, one of the best tools available to determine heart function. The results were rather disappointing: Neither of the two cell treatment groups showed any improvement in their cardiac function.

This trial had some important limitations: Even though this study enrolled 200 patients and was thus larger than most other cell therapy trials for heart attack patients, it is still a rather small study when compared to other cardiovascular studies, which routinely enroll thousands of patients. Furthermore, this study only assessed heart function after four months and it is possible that if they had waited longer, they might have seen some benefit of the cell therapy. Despite these limitations, the trial will dampen the general enthusiasm for injecting bone marrow cells into heart attack patients.

Is this study a set-back for cardiac stem cell treatments? Not really. As the authors reveal in their data analysis, most of the cells contained in the bone marrow preparation that they used for the infusion were plain old white blood cells and NOT stem cells. Actually, only 1% of the infused cells were hematopoietic stem cells (stem cells that give rise to blood cells) and there was an undisclosed percentage of other stem cell types (such as mesenchymal stem cells) contained in the infused bone marrow extract. As I point out in the accompanying editorial “Bone Marrow Tinctures for Cardiovascular Disease: Lost in Translation“, using such a mixture of poorly defined cells is ill-suited to promote cardiac regeneration or repair. Therefore, this important study is not a set-back for cardiac stem cell therapy, but a well-deserved setback for injections of undefined cells, most of which are not true stem cells!

Even if the majority of infused cells had been stem cells, there is no guarantee that merely infusing them into the heart would necessarily result in the formation of new heart tissue. Regenerating heart tissue from adult stem cells requires priming or directing stem cells towards becoming heart cells and ensuring that the cells can attach and integrate into the heart, not just infusing or injecting them into the heart.

It is commendable that the journal published this negative study, because too many treatments are being marketed as “stem cell therapies” without clarifying whether the injected cells are truly efficacious. Hopefully, the results of this trial will lead to more caution when rushing to perform “stem cell treatments” in patients without carefully defining the scientific characteristics and therapeutic potential of the cells that are being used.

 

Link to the original paper:  “Intracoronary Injection of Bone Marrow Derived Mononuclear Cells, Early or Late after Acute Myocardial Infarction: Effects on Global Left Ventricular Function

Link to the editorial: “Bone Marrow Tinctures for Cardiovascular Disease: Lost in Translation

Image credit: Surgeon extracting bone marrow from a patient (Public Domain image via Wikimedia)

ResearchBlogging.org
Surder, D., Manka, R., Lo Cicero, V., Moccetti, T., Rufibach, K., Soncin, S., Turchetto, L., Radrizzani, M., Astori, G., Schwitter, J., Erne, P., Zuber, M., Auf der Maur, C., Jamshidi, P., Gaemperli, O., Windecker, S., Moschovitis, A., Wahl, A., Buhler, I., Wyss, C., Kozerke, S., Landmesser, U., Luscher, T., & Corti, R. (2013). Intracoronary Injection of Bone Marrow Derived Mononuclear Cells, Early or Late after Acute Myocardial Infarction: Effects on Global Left Ventricular Function Four months results of the SWISS-AMI trial Circulation DOI: 10.1161/CIRCULATIONAHA.112.001035
ResearchBlogging.org
Rehman, J. (2013). Bone Marrow Tinctures for Cardiovascular Disease: Lost in Translation Circulation DOI: 10.1161/CIRCULATIONAHA.113.002775

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.

ResearchBlogging.org
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

To Branch, Or Not To Branch – Plant Hormones Help Turn A Stem Into A Bush

When we hear the expression “stem cells”, we tend to think of cells from animals or patients that are used to treat diseases or promote regeneration. However, stem cells are also present in plants. The growing tips of plants are called meristems and they are reservoirs of plant stem cells. A meristem is formed at the base of each leaf and can remain dormant as a small bud or be activated and give rise to a whole new branch. Gardeners know that pruning leaves can activate the buds and help transform a single stem plant into a multi-branched bush, but the exact mechanisms that govern branch formation are not fully understood.

 

The recent paper “Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane” published in PLOS Biology by Naoki Shinohara and colleagues has uncovered an important novel pathway that regulates the formation of branches in plants. The researchers based their work on an existing model which states that the plant growth hormone auxin is a central regulator of branch formation. Auxin levels are highest in activated buds because this is where auxin is produced. Auxin then flows to the roots, where auxin levels are low (“auxin sinks“). The removal of auxin from the activated bud allows for further auxin production and thus creates a continuous auxin flow pattern. This is thought to establish a positive feedback loop for the activated bud, which then ultimately results in the formation of branch emanating from the activated bud. This model is called the “auxin transport canalization” and is explained in an excellent accompanying article “Transforming a Stem into a Bush” by Amy Coombs, also published in PLOS Biology.

Once an activated bud initiates the positive auxin feedback loop, it also becomes necessary to inhibit the branch formation from other buds. If all the buds in a plant started making branches at once, the plant’s resources would probably become depleted very quickly, possibly resulting in the chaotic formation of too many suboptimal branches. There is a clearly a need for a system that allows some activated buds to go on to make branches, while putting the brake on other buds so that they bide their time. The details of such a fine-tuned balance of selected activation and inhibition have been a bit of a mystery, but the work by Shinohara and colleagues is a major step forward in unraveling this puzzle.

The researchers show that the plant hormone strigolactone removes the auxin export protein PIN1 from the cell surface and increases its degradation. Therefore, a plant without strigolactone would have more PIN1, sustain greater auxin flux and thus increase branching. Genetically engineered plants that do not produce strigolactone did indeed show more branch formation. When the researchers added back synthetic strigolactone (called GR24), they were able to suppress the excessive branch formation. However, the researchers also obtained a somewhat counter-intuitive result: When they gave GR24 to plants with defective auxin transport, low doses of GR24 actually helped branch formation and only higher doses suppressed branch formation. The problem with these results is that the synthetic strigolactone also severely impacted the general growth of the plants (not just branch formation) in the auxin transport mutants, and it is difficult to interpret whether the subtle differences between low and high doses were just generalized effects due to reduced overall plant health or whether they were truly related to aberrant branching.

The oddly opposite results obtained with low dose and high dose GR24 treatment are probably going to raise some controversy, and as Amy Coombs pointed out, not all scientists agree with the auxin transport canalization theory of branch formation in plants. This is not the first study to propose an interaction between strigolactone and auxin as regulators of plant branch formation, but it is one of the most comprehensive papers in this area. It includes a mathematical model of the interaction between these two regulators, tests the model with experiments and identifies a novel cellular mechanism for how strigolactone reduces PIN1. These results do suggest that plants have a very finely-tuned system involving at least two hormones, auxin and strigolactone, that act together to promote branch formation in some buds, while suppressing bud formation in others. As a stem cell biologist who works with mammalian stem cells, I am quite intrigued by this fascinating interplay between activating and suppressing hormones in plants that permit a self-organized branch formation. In mammals, we still do not fully understand how during development, some embryonic stem cells commit to one lineage and form organs such as a heart, while also preventing other stem cells in the developing embryo to form a second or third heart in other areas. It is quite likely that developing mammalian embryonic stem cells also depend on positive feedback loops and inhibitory systems, similar to what the researchers found in the plants. Many major discoveries in cell biology and molecular biology are first made in plants and we then discover similar principles of regulation in animals and humans.

 

Image credit: Panel from Figure 5 of the PLOS Biology (2013) paper by Shinohara N., et al, Green indicates the PIN protein and magenta shows the autofluorescence of chloroplasts

 

ResearchBlogging.org

Shinohara, N., Taylor, C., & Leyser, O. (2013). Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane PLoS Biology, 11 (1) DOI: 10.1371/journal.pbio.1001474

Radical Tails: Antioxidants Can Prevent Regeneration

Amphibians such as frogs or salamanders have a remarkable ability to regenerate amputated limbs and tails. The regenerative process involves the formation of endogenous pluripotent stem cells, which then expand and differentiate into the tissue types that give rise to the regenerated body part. The complex interplay of the cell types and signals involved in this regenerative response to the injury are not fully known and there is considerable interest in identifying all the necessary steps. The ultimate hope is that by identifying the specific mechanisms of injury response and regeneration, one might be able to activate similar repair processes in humans, who lack the extraordinary regenerative capacity of amphibians.

The recent paper “Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration” by Nick Love and colleagues published online in the journal Nature Cell Biology on January 13, 2013 elegantly demonstrates that reactive oxygen species (ROS), also known as oxygen radicals or oxidants, play a critical role in the regeneration of amphibian tails. Using a rather elegant approach, the researchers generated Xenopus tadpoles with a genetically integrated sensor of the oxidant-sensitive protein HyPerYFP that emits fluorescence upon contact with ROS, and is thought to be rather specific for the oxidant H2O2, more commonly known as hydrogen peroxide. This allowed them to study the hydrogen peroxide levels in all cells of the live tadpole, while it was responding to an injury. They found that within 6 hours after the tail amputation, the residual tail tissue was flooded with high levels of the hydrogen peroxide and that as the tail started growing back, the regenerative edge of the growing tail continued to show high levels of this oxidant.

After excluding the possible confounding phenomenon that the increase in ROS was merely a bystander effect of increases in inflammatory cells, the researchers then performed a pivotal set of experiments in which they used anti-oxidants to see if these would impact the tail regeneration. The researchers first utilized pharmacological inhibitors that reduce the production of oxidants as well as the therapeutic antioxidant MCI-186 (its trade-name is Edaravone and is marketed for use in patients in Japan). These pharmacological agents were all very effective in terms of lowering the hydrogen peroxide levels in the regenerating tail, but they also significantly impaired the regeneration itself. In another intriguing set of experiments, the researchers treated the tadpoles with these agents immediately after the injury and then withdrew them after three days, to see if the regeneration would set in after their removal. Interestingly, when the tails were exposed to agents that prevented the generation of the oxidants, the regenerative program remained blocked even when they were removed. On the other hand, the antioxidant scavenger that soaks up oxidants being produced did not permit regeneration while it was present, but regeneration resumed after the antioxidant was removed.

The researchers also performed complementary genetic experiments in which they reduced oxidant revels by suppressing the enzymes that produce oxidants. The results all point to an important conclusion: There is a burst of oxidants that are released after injury and that are necessary to initiate the regenerative program. The exact molecular targets of the oxidant hydrogen peroxide that enable regeneration remain unknown, but some of the data in the paper points to the Wnt protein pathway as a potential oxidant-sensitive regenerative signal in the tadpole tail.

One has to bear in mind that this work was performed in tadpoles and may not be necessarily fully applicable to the human setting, but Wnt is a key regulator of stem cell renewal, differentiation and regeneration in human tissues. This does suggest that there may be some key similarities between the tadpole regeneration pathways and those found in humans. Despite the shared Wnt signals in tadpoles and humans, building a bridge from this work in Xenopus tadpoles to research and therapeutic applications in humans will be quite challenging. After all, the elegance of this study lies in the genetically integrated oxidant sensor that allows live tracking of oxidants as well as the fact that tadpoles can regenerate whole limbs and tails. Current tools do not permit real-time tracking of human oxidant levels in tissues and humans can usually only regenerate very small amounts of tissue, such as superficial skin injury.

Nevertheless, this work is an important milestone in understanding the role of oxidants as promoters of regeneration and it is very likely that at least some similar pro-regenerative role of oxidants may also be present in human tissues. One of the most important take home messages of this work is that we need get rid of the common “oxidants are bad guys and antioxidants are good guys” myth. Oxidants can be harmful in some context, but they can also serve as important regenerative signals. Indiscriminate use of antioxidants can actually impair these important endogenous signals. Instead of consuming large quantities of non-specific antioxidants, we need to use antioxidants in a very targeted, context-specific and perhaps time-limited manner so that they only prevent oxidative damage without affecting beneficial oxidant signaling.

 

Image credit: Image of a Xenopus hybrid from Figure S1 in Narbonne P, Simpson D, Gurdon J (2011). “Deficient Induction Response in a Xenopus Nucleocytoplasmic Hybrid“. PLOS Biology. 

ResearchBlogging.org

Love, N., Chen, Y., Ishibashi, S., Kritsiligkou, P., Lea, R., Koh, Y., Gallop, J., Dorey, K., & Amaya, E. (2013). Amputation-induced reactive oxygen species are required for successful Xenopus tadpole tail regeneration Nature Cell Biology DOI: 10.1038/ncb2659