Restoring Vision with Stem Cells

Researchers have succeed in producing photoreceptors from human embryonic stem cells

Tuesday, 06 October 2015

Age-related macular degeneration (AMRD) could be treated by transplanting photoreceptors produced by the directed differentiation of stem cells, thanks to findings published today by Professor Gilbert Bernier of the University of Montreal and its affiliated Maisonneuve-Rosemont Hospital. ARMD is a common eye problem caused by the loss of cones. Bernier's team has developed a highly effective in vitro technique for producing light sensitive retina cells from human embryonic stem cells.

"Our method has the capacity to differentiate 80% of the stem cells into pure cones," Professor Gilbert explained.

"Within 45 days, the cones that we allowed to grow towards confluence spontaneously formed organised retinal tissue that was 150 microns thick. This has never been achieved before."

In order to verify the technique, Bernier injected clusters of retinal cells into the eyes of healthy mice. The transplanted photoreceptors migrated naturally within the retina of their host.

Age-related macular degeneration could be

treated by transplanting photoreceptors

produced by the directed differentiation of stem

cells, thanks to findings published today by

Professor Gilbert Bernier of the University of

Montreal and its affiliated Maisonneuve-

Rosemont Hospital. This image illustrates the 3D

reconstruction of the tissue that was produced in

vitro and labelled with antibodies against

photoreceptor-specific proteins. Credit: G.

Bernier, Université de Montréal.

"Cone transplant represents a therapeutic solution for retinal pathologies caused by the degeneration of photoreceptor cells," Bernier explained.

"To date, it has been difficult to obtain great quantities of human cones."

His discovery offers a way to overcome this problem, offering hope that treatments may be developed for currently non-curable degenerative diseases, like Stargardt disease and ARMD.

"Researchers have been trying to achieve this kind of trial for years," he said.

"Thanks to our simple and effective approach, any laboratory in the world will now be able to create masses of photoreceptors. Even if there's a long way to go before launching clinical trials, this means, in theory, that will be eventually be able to treat countless patients."

The findings are particularly significant in the light of improving life expectancies and the associated increase in cases of ARMD. ARMD is in fact the greatest cause of blindness amongst people over the age of 50 and affects millions of people worldwide. And as we age, it is more and more difficult to avoid - amongst people over 80, this accelerated aging of the retina affects nearly one in four. People with ARMD gradually lose their perception of colours and details to the point that they can no longer read, write, watch television or even recognize a face.

ARMD is due to the degeneration of the macula, which is the central part of the retina that enables the majority of eyesight. This degeneration is caused by the destruction of the cones and cells in the retinal pigment epithelium (RPE), a tissue that is responsible for the reparation of the visual cells in the retina and for the elimination of cells that are too worn out. However, there is only so much reparation that can be done as we are born with a fixed number of cones. They therefore cannot naturally be replaced. Moreover, as we age, the RPE's maintenance is less and less effective - waste accumulates, forming deposits.

"Differentiating RPE cells is quite easy. But in order to undertake a complete therapy, we need neuronal tissue that links all RPE cells to the cones. That is much more complex to develop," Bernier explains, noting nonetheless that he believes his research team is up to the challenge.

Bernier has been interested in the genes that code and enable the induction of the retina during embryonic development since completing his PhD in Molecular Biology in 1997.

"During my post-doc at the Max-Planck Institute in Germany, I developed the idea that there was a natural molecule that must exist and be capable of forcing embryonic stem cells into becoming cones," he said.

Indeed, bioinformatics analysis led him to predict the existence of a mysterious protein: COCO, a "recombinational" human molecule that is normally expressed within photoreceptors during their development.

In 2001, he launched his laboratory at Maisonneuve-Rosemont Hospital and immediately isolated the molecule. But it took several years of research to demystify the molecular pathways involved in the photoreceptors development mechanism. His latest research shows that in order to create cones, COCO can systematically block all the signalling pathways leading to the differentiation of the other retinal cells in the eye. It's by uncovering this molecular process that Bernier was able to produce photoreceptors. More specifically, he has produced S-cones, which are photoreceptor prototypes that are found in the most primitive organisms.

Beyond the clinical applications, Professor Bernier's findings could enable the modelling of human retinal degenerative diseases through the use of induced pluripotent stem cells, offering the possibility of directly testing potential avenues for therapy on the patient's own tissues.

Source: University of Montréal

Contact: William Raillant-Clark

Reference:

Differentiation of human embryonic stem cells into cone photoreceptors through simultaneous inhibition of BMP, TGFβ and Wnt signaling

Shufeng Zhou, Anthony Flamier, Mohamed Abdouh, Nicolas Tétreault, Andrea Barabino, Shashi Wadhwa and Gilbert Bernier

Development, 42, 3294-3306, October 1, 2015, doi: 10.1242/dev.125385

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Vitamin C Boosts the Reprogramming of Adult Cells into Stem Cells

Vitamin C Boosts the Reprogramming of Adult Cells into Stem Cells Monday, 04 January 2010 Famous for its antioxidant properties and role in tissue repair, vitamin C is touted as beneficial for illnesses ranging from the common cold to cancer and perhaps even for slowing the aging process. Now, a study published online on December 24th by Cell Press in the journal Cell Stem Cell uncovers an unexpected new role for this natural compound: facilitating the generation of embryonic-like stem cells from adult cells. Over the past few years, we have learned that adult cells can be reprogrammed into cells with characteristics similar to embryonic stem cells by turning on a select set of genes. Although the reprogrammed cells, called induced pluripotent stem cells (iPSCs), have tremendous potential for regenerative medicine, the conversion is extremely inefficient. "The low efficiency of the reprogramming process has hampered progress with this technology and is indicative of how little we understand it. Further, this process is most challenging in human cells, raising a significant barrier for producing iPSCs and serious concerns about the quality of the cells that are generated," explains senior study author Dr. Duanqing Pei from the South China Institute for Stem Cell Biology and Regenerative Medicine at the Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences. Dr. Pei and colleagues measured the production of reactive oxygen species or ROS during reprogramming and discovered a potential link between high ROS and low reprogramming efficiency. They became particularly interested in antioxidants, hypothesizing that they might suppress ROS and cell senescence, which seems to be a major roadblock for the generation of iPSCs. The researchers found that adding vitamin C, an essential nutrient that is abundant in citrus fruits, enhanced iPSC generation from both mouse and human cells. Vitamin C accelerated gene expression changes and promoted a more efficient transition to the fully reprogrammed state. Somewhat to their surprise, they found that other antioxidants do not have the same effect, but vitamin C does seem to act at least in part through slowing cell senescence. "Our results highlight a simple way to improve iPSC generation and provide additional insight into the mechanistic basis of reprogramming," concludes Dr. Pei. "It is also of interest that a vitamin with long-suspected anti-aging effects has such a potent influence on reprogramming, which can be considered a reversal of the aging process at the cellular level. It is likely that our work may stimulate further research in this area as well." ......... ZenMaster

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Cellular Damage in Huntington's Disease

New clues emerge from study Monday, 17 November 2008 "Huntington's disease presents an ideal vantage point to study neurodegenerative disease, because we know the misfolded protein that's responsible," says Martin Duennwald, formerly a postdoctoral researcher in the lab of Whitehead Institute for Biomedical Research member Susan Lindquist. "But we don't understand how this protein causes cellular damage and death for the neurons that are affected." In a study published in Genes & Development online on November 17, however, Duennwald and Lindquist report the discovery of a mechanism driven by the misfolded proteins that could be one early trigger for cell death. In the U.S., about 1 in 20,000 people suffers from Huntington's. Better understanding of the cellular toxicity may allow new therapies for this fatal and incurable disorder. "This is a diabolical disease, because the misfolded protein interacts with and probably traps many other proteins in the cell," notes Lindquist, who is also a Howard Hughes Medical Institute investigator and a professor of biology at Massachusetts Institute of Technology. Scientists have long known that a single mutated gene that creates proteins with abnormally long repeats of the amino acid glutamine (“Q”) drives Huntington’s. In certain neurons, these "polyQ-expanded" proteins misfold and clump together, damaging and eventually killing the cells. But the steps that kick off the process of cell damage and death have remained a mystery, remarks Duennwald, now a principal scientist at Boston Biomedical Research Institute in Watertown, Mass. In the study, Duennwald first examined what makes polyQ-expanded proteins toxic in yeast. He then performed similar experiments in two kinds of mammalian cells — rat cells that model neurons and mouse striated cells (from the part of the brain most afflicted in Huntington's). He found that cells generated with polyQ-expanded fragments quickly showed problems with proteins that had been marked for degradation in the endoplasmic reticulum (ER, a cell component that folds and finalizes proteins). Such proteins were not expelled for tagging and degradation in the cytosol, the intracellular fluid, outside the ER. "With no garbage disposal, all of a sudden the ER is flooded with proteins that need to be degraded," he says. This breakdown in protein quality control may lead toward cell damage and death. "We were quite surprised because the ER didn't seem to have any connection with the misfolded proteins in the cytosol," Duennwald adds. "This study tells us to investigate cellular pathways beyond the usual suspects." He went on to uncover the basis for this breakdown: The polyQ-expanded fragments glom onto the key VCP/Npl4/Ufd1 protein complex that aids in the transport and degradation of the proteins that flunk quality control in the ER. When Duennwald genetically modified cells to over-express two crucial proteins in the protein complex, the toxic effect dropped. Additionally, his experiments showed that polyQ-expanded proteins avoid a main method by which cells deal with misfolded proteins. Generally, a class of proteins called "chaperone" or "heat shock" proteins move in and either help the misfolded proteins assume their normal shape or help to get rid of them. "Amazingly, polyQ-expanded proteins don't elicit the heat shock response, and that might contribute to their toxicity," Duennwald says. Such findings may help in eventually treating the disease. The research suggests that activating the cell's protein quality control mechanisms may provide novel and effective strategies for combating Huntington's and other illnesses driven by polyQ-expanded proteins. Reference: Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity Martin L. Duennwald and Susan Lindquist Genes & Development, December 2008 ......... ZenMaster

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The Genetics of the White Horse Unravelled

The Genetics of the White Horse Unravelled Sunday, 20 July 2008 The white horse is an icon for dignity which has had a huge impact on human culture across the world. An international team led by researchers at Uppsala University has now identified the mutation causing this spectacular trait and show that white horses carry an identical mutation that can be traced back to a common ancestor that lived thousands of years ago. The study is interesting for medical research since this mutation also enhance the risk for melanoma. The paper is published on July 20 on the website of Nature Genetics. Exactly when the mutation for ‘Greying with age’ was appearing is not known, but it was first described in western literature 2.500 years ago by the Greek historian Herodotus, telling that the Persian King Xerxes had a whole stable with Holy White Horses. The great majority of white horses carry the dominant mutation ‘Greying with age’. A Grey horse is born coloured (black, brown or chestnut) but the greying process starts already during its first year and they are normally completely white by six to eight years of age but the skin remains pigmented. Thus, the process resembles greying in humans but the process is ultrafast in these horses. The research presented now demonstrates that all Grey horses carry exactly the same mutation which must have been inherited from a common ancestor that lived thousands of years ago. “It is a fascinating thought that once upon a time a horse was born that turned grey and subsequently white and the people that observed it were so fascinated by its spectacular appearance that they used the horse for breeding so that the mutation could be transmitted from generation to generation,” says Leif Andersson, who led the study, and professor in functional genomic at the Department of Medical Biochemistry and Microbiology (IMBIM), Uppsala Biomedical Centre, at Uppsala University. Today about one horse in ten carries the mutation for Greying with age. It is obvious that humans across the world have greatly valued these white horses as documented by the rich collection of stories and paintings featuring white horses. In the paper the white horse as an icon for dignity is illustrated by reproducing a painting from the late 17th century of the Swedish king Karl XI on his white horse Brilliant. “The discovery was quite unexpected, since the mutation for ‘Greying with age’ was found in a DNA segment never before associated with colour pigmentation,” says Leif Andersson.

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Lipizzaner horses.

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DNA from more than 900 horses was analysed, several of them from the white Lipizzaner horses from the Spanish Riding School in Vienna. In all white horses, but not in any black, brown or chestnut coloured horse, the exact same DNA change was found, a large segment of DNA duplication. This mutation does not change any protein coding gene, but regulate how active two other genes will be. The Grey horse is also very interesting from a medical point of view since the mutation also predisposes for development of melanoma. About 75% of Grey horses older than 15 years of age have a benign form of melanoma that in some cases develops into a malignant melanoma. Thus, the study reported today has also given new insight in a molecular pathway that may lead to tumour development. “We propose that the Grey mutation stimulates growth of melanocytes and that this leads to a premature loss of the melanocyte stem cells needed for hair pigmentation whereas the mutation promotes an expansion of some of the melanocytes causing skin pigmentation,” says Leif Andersson. Domestic animals constitute extraordinary models for evolution of biological diversity as already recognized by Charles Darwin. The white horse is a beautiful illustration of the importance of regulatory mutations as a major underlying mechanism for phenotypic diversity within and between species. The Grey mutation does not change any protein structure but it affects the genetic regulation of two genes. The researchers found that the white horses carry an extra copy of a DNA segment located in one of these genes. “It is very likely that regulatory mutations like the one we found in these white horses constitute the dominating class of mutations explaining differences between breeds of domestic animals as well as between species like humans and chimpanzee,” concludes Leif Andersson. Referens: A cis-acting regulatory mutation causes premature hair greying and susceptibility to melanoma in the horse Gerli Rosengren Pielberg, Anna Golovko, Elisabeth Sundström, Ino Curik, Johan Lennartsson, Monika H Seltenhammer, Thomas Druml, Matthew Binns, Carolyn Fitzsimmons, Gabriella Lindgren, Kaj Sandberg, Roswitha Baumung, Monika Vetterlein, Sara Strömberg, Manfred Grabherr, Claire Wade, Kerstin Lindblad-Toh, Fredrik Pontén, Carl-Henrik Heldin, Johann Sölkner & Leif Andersson Nature Genetics Published online: 20 July 2008, doi:10.1038/ng.185 ......... ZenMaster

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Stem Cell Researchers Give Old Muscle New Vigour

Stem Cell Researchers Give Old Muscle New Vigour Tuesday, 17 June 2008 Old muscle got a shot of youthful vigour in a stem cell experiment by bioengineers at the University of California, Berkeley, setting the path for research on new treatments for age-related degenerative conditions such as muscle atrophy or Alzheimer's and Parkinson's diseases. In a new study to be published June 15 in an advanced online issue of the journal Nature, researchers identified two key regulatory pathways that control how well adult stem cells repair and replace damaged tissue. They then tweaked how those stem cells reacted to those biochemical signals to revive the ability of muscle tissue in old mice to repair itself nearly as well as the muscle in the mice's much younger counterparts. Irina Conboy, an assistant professor of bioengineering and an investigator at the Berkeley Stem Cell Center and at the California Institute for Quantitative Biosciences (QB3), led the research team conducting this study.

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Shown above is muscle tissue from a young mouse. On the right side are healthy, new cells created to replace damaged tissue. This ability to regenerate new cells diminishes with age. Credit: Photo courtesy of Morgan Carlson and Irina Conboy, UC Berkeley

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Because the findings relate to adult stem cells that reside in existing tissue, this approach to rejuvenating degenerating muscle eliminates the ethical and medical complications associated with transplanting tissues grown from embryonic stem cells. "We are one step closer to having a point of intervention where we can rejuvenate the body's own stem cells so we don't have to suffer from some of the debilitating diseases associated with aging," said the study's lead author, Morgan Carlson, a recent Ph.D. graduate of Conboy's lab. The researchers focused on the interplay of two competing molecular pathways that control the stem cells, which sit next to the mature, differentiated cells that make up our working body parts. When the mature cells are damaged or wear out, the stem cells are called into action to begin the process of rebuilding. "We don't realize it, but as we grow our bodies are constantly being remodelled," said Conboy. "We are constantly falling apart, but we don't notice it much when we're young because we're always being restored. As we age, our stem cells are prevented, through chemical signals, from doing their jobs."

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Shown here is muscle tissue from an old mouse. After UC Berkeley researchers manipulated the biochemical response of adult stem cells in the old tissue, the muscle was able to repair itself from damage almost as well as muscle from young mice. Credit: Photo courtesy of Morgan Carlson and Irina Conboy, UC Berkeley

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The good news, the researchers said, is that the stem cells in old tissue are still ready and able to perform their regenerative function if they receive the appropriate chemical signals. Studies have shown that when old tissue is placed in an environment of young blood, the stem cells behave as if they are young again. "Conversely, we have found in a study published last year that even young stem cells rapidly age when placed among blood and tissue from old mice," said Carlson, who will stay on at UC Berkeley to expand his work on stem cell engineering either as a QB3 fellow or a postdoctoral researcher.

Adult stem cells have a receptor called Notch that, when activated, tells them that it is time to grow and divide, the researchers said. But stem cells also have a receptor for the protein TGF-beta that sets off a chain reaction activating the molecule pSmad3 and ultimately producing cyclin-dependent kinase (CDK) inhibitors, which regulate the cell's ability to divide. "Interestingly, activated Notch competes with activated pSmad3 for binding to the regulatory regions of the same CDK inhibitors in the stem cell," said Conboy. "We found that Notch is capable of physically kicking off pSmad3 from the promoters for the CDK inhibitors within the stem cell's nucleus, which tells us that a precise manipulation of the balance of these pathways would allow the ability to control stem cell responses." Notch and TGF-beta are well known in molecular biology, but Conboy's lab is the first to connect them to the process of aging, and the first to show that they act in opposition to each other within the nucleus of the adult stem cell.

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As muscle tissue ages, it loses its ability to adequately repair itself from damage. Instead of creating healthy, new cells to replace damaged ones, the old muscle tissue is left with fibroblasts and scar tissue, as shown here. Credit: Photo courtesy of Morgan Carlson and Irina Conboy, UC Berkeley.

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Aging and the inevitable march towards death are, in part, due to the progressive decline of Notch and the increased levels of TGF-beta, producing a one-two punch to the stem cell's capacity to effectively rebuild the body, the researchers said. "What we discovered is the interplay between two pathways - one an aging pathway, and the other a youthful pathway," said Conboy. But what would happen if researchers blocked the adult stem cells in old tissues from reacting to those TGF-beta signals? The researchers put that question to the test in a living organism by comparing the muscle regeneration capacity of old, 2-year-old mice, comparable in age to a 75- to 80-year-old human, with that of 2-month-old mice, similar in age to a 20- to 25-year-old human. For a group of the old mice, the researchers disabled the "aging pathway" that tells stem cells to stop dividing by using an established method of RNA interference that reduced levels of pSmad3. The researchers then examined the muscle of the different groups of mice one to five days after injury to compare how well the tissue repaired itself. As expected, the researchers found that muscle tissue in the young mice easily replaced damaged cells with new, healthy cells. In contrast, the areas of damaged muscle in the control group of old mice were characterized by fibroblasts and scar tissue. However, muscles in the old mice whose stem cell "aging pathway" had been dampened showed levels of cellular regeneration that were comparable to their much younger peers, and that were 3 to 4 times greater than those of the group of "untreated" old mice. The researchers cautioned that shutting down the TGF-beta/pSmad3 pathway altogether by turning off the gene that controls it could lead to many health problems. The ability to suppress cell division is critical in controlling the development of tumours, for instance. "When we are young, there is an optimal balance between Notch and TGF-beta," said Conboy. "We need to find out what the levels of these chemicals are in the young so we can calibrate the system when we're older. If we can do that, we could rejuvenate tissue repair for a very long time." The researchers also warn against interpreting this research as the cure-all for aging. "We're not at a point where we're ready to inject ourselves with TGF-beta antibodies and call it a day," said Carlson. "There are multiple mechanisms involved in how our body functions. We know that TGF-beta is involved in one aspect of aging, but we don't know where it fits in the global scheme of aging." In addition to their work on adult stem cells, Carlson and Conboy have also discovered that human embryonic stem cells can actually neutralize the effects of aging. Conboy received funding last year from the California Institute for Regenerative Medicine (CIRM) to pursue this line of research. Reference: Imbalance between pSmad3 and Notch induces CDK inhibitors in old muscle stem cells Morgan E. Carlson, Michael Hsu & Irina M. Conboy Nature advance online publication 15 June 2008, doi:10.1038/nature07034 ......... ZenMaster

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Ovaries and Testes Age in Unique Ways

Ovaries and Testes Age in Unique Ways Tuesday, 03 June 2008 Aging leads to large changes in gene activity in the ovaries of mice, but only limited changes in testes, according to research published in the open-access journal, BMC Biology. A lifespan-extending calorie-restricted diet reversed some of the aging effects – but, unlike the widespread changes observed in somatic organs, it had an impact only in a small number of gonad-specific genes. As well as tackling one of the key questions of ageing – by exploring if reproductive organs age in the same way as other body organs – this research is important in the light of the trend for some women in developed countries to put off childbearing until later in life. A research team led by Minoru Ko, MD, PhD, from the National Institute on Aging, Baltimore, USA used whole-genome DNA microarrays to study the effects of age, sex and diet on the global gene expression in mouse ovaries and testes. They found that reproductive organs age in a different way to other body tissues and, furthermore, that ovaries age in a different way from testes. Age-related changes in gene expression occurred in gonads – as they are known to in other body tissues – but these changes tended to be in different classes of genes. Only two of the six categories of genes previously associated with aging in muscle, kidney and brain were associated with aging in the ovary; none were associated with aging in the testis. The changes seen in ovaries could be influenced by changes in the tissue composition of ovaries as female’s age and ovulation ceases. The researchers also found that calorie restriction in females reduced the expression of genes involved in metabolism and follicle growth, which seems to be consistent with a popular view that the calorie restriction causes a shift in energy use away from reproduction towards general body maintenance and repair. However, male mice on the same diet did not appear to sacrifice reproductive function, suggesting an evolutionary difference between males and females when coping with a food shortage. Reference: Effects of aging and calorie restriction on the global gene expression profiles of testis and ovary Alexei A Sharov, Geppino Falco, Yulan Piao, Suresh Poosala, Kevin G Becker, Alan B Zonderman, Dan L Longo, David Schlessinger and Minoru SH Ko BMC Biology 2008, 6:24, doi:10.1186/1741-7007-6-24 ......... ZenMaster

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A molecular map for aging

A molecular map for aging in mice and humans Wednesday, 28 November 2007 Researchers at the National Institute of Aging and Stanford University have used gene arrays to identify genes whose activity changes with age in 16 different mouse tissues. The study, published November 30 in PLoS Genetics, uses a newly available database called AGEMAP to document the process of aging in mice at the molecular level. The work describes how aging affects different tissues in mice, and ultimately could help explain why lifespan is limited to just two years in mice. As an organism ages, most tissues change their structure (for example, muscle tissues become weaker and have slow twitch rather than fast twitch fibres), and all tissues are subject to cellular damage that accumulates with age. Both changes in tissues and cellular damage lead to changes in gene expression, and thus probing which genes change expression in old age can lead to insights about the process of aging itself. Previous studies have studied gene expression changes during aging in just one tissue. The new work stands out because it is much larger and more complete, including aging data for 16 different tissues and containing over 5.5 million expression measurements. One noteworthy result is that some tissues (such as the thymus, eyes and lung) show large changes in which genes are active in old age whereas other tissues (such as liver and cerebrum) show little or none, suggesting that different tissues may degenerate to different degrees in old mice. Another insight is that there are three distinct patterns of aging, and that tissues can be grouped according to which aging pathway they take. This result indicates that there are three different clocks for aging that may or may not change synchronously, and that an old animal may be a mixture of tissues affected by each of the different aging clocks. Finally, the report compares aging in mice to aging in humans. Several aging pathways were found to be the same, and these could be interesting because they are relevant to human aging and can also be scientifically studied in mice. Reference: AGEMAP: A gene expression database for aging in mice. PLoS Genet 3(11): e201. ......... ZenMaster

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