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

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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

The Healing Power of Sweat Glands

Two kinds of sweat glands are present in the human body. Apocrine sweat glands are located in arm-pits or rectogenital areas and are responsible for “smelly” sweat. Eccrine sweat glands, on the other hand, are distributed all over the human body and produce a non-odorous sweat. The eccrine sweat glands are primarily found in humans and certain primates. They also exist in some other mammals, but are usually restricted to the footpads of non-primate mammals. There is some controversy about the actual purpose of eccrine sweat glands in humans. The functions traditionally ascribed to eccrine sweat glands include promoting grip, generating a protective acid mantle for the skin as well as regulation of temperature or the electrolyte balance.

The recent article “Eccrine Sweat Glands are Major Contributors to Reepithelialization of Human Wounds” published in the American Journal of Pathology by Laure Rittié and colleagues proposes a novel and very interesting function for eccrine sweat glands. In this study, CO2 laser treatment was used to create superficial wounds in human subjects, either on their palm or on their forearms. The researchers performed biopsies during the subsequent days to assess the wound healing process. They observed significant proliferation of cells at the bases of hair follicles (pilosebaceous units) as well as proliferation of cells within the eccrine sweat glands. The outgrowths of cells from these areas merged together to regenerate the skin layer. Wound healing (re-epithelialization) in the palms of hands was primarily driven by cell proliferation of sweat gland cells, because the palms do not contain hair follicles.

The findings suggest that wound healing and regeneration of damaged skin may be an important function of cells that reside within human eccrine sweat glands. The study did not quantify the exact contribution of the sweat glands to the wound healing process or compare it with the contributions of other cell types. It also did not address whether the sweat production itself regulates or facilitates the repair process. This would be an intriguing possibility, because we all know how our palms become sweaty when we are under stress. Is it possible that the eccrine sweat production is a way of preparing the body for potential wounds and the need for repair or regeneration? Is there a way to enhance the wound healing emanating from the eccrine sweat glands? These and other questions will need to be addressed in future studies.

In summary, the work by Rittié and colleagues presents an important new perspective on how sweat glands can participate in wound healing. It is also an important reminder of how some animal models of wound healing may have their limitations when their results are translated to the human setting. Most laboratory animals that are used for wound healing studies do not have eccrine sweat glands. Results derived from such animal wound healing studies may thus not be readily applicable to the human setting and should be interpreted with a grain of sweat (salt).

 

Image credit: Wikimedia / National Institutes of Health – Anatomy of Skin