Posts Tagged ‘Developmental biology’

Colon cancer and organoids

Larry H. Bernstein, MD, FCAP, Curator





Guts and Glory

An open mind and collaborative spirit have taken Hans Clevers on a journey from medicine to developmental biology, gastroenterology, cancer, and stem cells.

By Anna Azvolinsky

Ihave had to talk a lot about my science recently and it’s made me think about how science works,” says Hans Clevers. “Scientists are trained to think science is driven by hypotheses, but for [my lab], hypothesis-driven research has never worked. Instead, it has been about trying to be as open-minded as possible—which is not natural for our brains,” adds the Utrecht University molecular genetics professor. “The human mind is such that it tries to prove it’s right, so pursuing a hypothesis can result in disaster. My advice to my own team and others is to not preformulate an answer to a scientific question, but just observe and never be afraid of the unknown. What has worked well for us is to keep an open mind and do the experiments. And find a collaborator if it is outside our niche.”

“One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory.”

Clevers entered medical school at Utrecht University in The Netherlands in 1978 while simultaneously pursuing a master’s degree in biology. Drawn to working with people in the clinic, Clevers had a training position in pediatrics lined up after medical school, but then mentors persuaded him to spend an additional year converting the master’s degree to a PhD in immunology. “At the end of that year, looking back, I got more satisfaction from the research than from seeing patients.” Clevers also had an aptitude for benchwork, publishing four papers from his PhD year. “They were all projects I had made up myself. The department didn’t do the kind of research I was doing,” he says. “Now that I look back, it’s surprising that an inexperienced PhD student could come up with a project and publish independently.”

Clevers studied T- and B-cell signaling; he set up assays to visualize calcium ion flux and demonstrated that the ions act as messengers to activate human B cells, signaling through antibodies on the cell surface. “As soon as the experiment worked, I got T cells from the lab next door and did the same experiment. That was my strategy: as soon as something worked, I would apply it elsewhere and didn’t stop just because I was a B-cell biologist and not a T-cell biologist. What I learned then, that I have continued to benefit from, is that a lot of scientists tend to adhere to a niche. They cling to these niches and are not that flexible. You think scientists are, but really most are not.”

Here, Clevers talks about promoting a collaborative spirit in research, the art of doing a pilot experiment, and growing miniature organs in a dish.

Clevers Creates

Re-search? Clevers was born in Eindhoven, in the south of The Netherlands. The town was headquarters to Philips Electronics, where his father worked as a businessman, and his mother took care of Clevers and his three brothers. Clevers did well in school but his passion was sports, especially tennis and field hockey, “a big thing in Holland.” Then in 1975, at age 18, he moved to Utrecht University, where he entered an intensive, biology-focused program. “I knew I wanted to be a biology researcher since I was young. In Dutch, the word for research is ‘onderzoek’ and I knew the English word ‘research’ and had wondered why there was the ‘re’ in the word, because I wanted to search but I didn’t want to do re-search—to find what someone else had already found.”

Opportunity to travel. “I was very disappointed in my biology studies, which were old-fashioned and descriptive,” says Clevers. He thought medicine might be more interesting and enrolled in medical school while still pursuing a master’s degree in biology at Utrecht. For the master’s, Clevers had to do three rotations. He spent a year at the International Laboratory for Research on Animal Diseases (ILRAD) in Nairobi, Kenya, and six months in Bethesda, Maryland, at the National Institutes of Health. “Holland is really small, so everyone travels.” Clevers saw those two rotations more as travel explorations. In Nairobi, he went on safaris and explored the country in Land Rovers borrowed from the institute. While in Maryland in 1980, Clevers—with the consent of his advisor, who thought it was a good idea for him to get a feel for the U.S.—flew to Portland, Oregon, and drove back to Boston with a musician friend along the Canadian border. He met the fiancé of political activist and academic Angela Davis in New York City and even stayed in their empty apartment there.

Life and lab lessons. Back in Holland, Clevers joined Rudolf Eugène Ballieux’s lab at Utrecht University to pursue his PhD, for which he studied immune cell signaling. “I didn’t learn much science from him, but I learned that you always have to create trust and to trust people around you. This became a major theme in my own lab. We don’t distrust journals or reviewers or collaborators. We trust everyone and we share. There will be people who take advantage, but there have only been a few of those. So I learned from Ballieux to give everyone maximum trust and then change this strategy only if they fail that trust. We collaborate easily because we give out everything and we also easily get reagents and tools that we may need. It’s been valuable to me in my career. And it is fun!”

Clevers Concentrates

On a mission. “Once I decided to become a scientist, I knew I needed to train seriously. Up to that point, I was totally self-trained.” From an extensive reading of the immunology literature, Clevers became interested in how T cells recognize antigens, and headed off to spend a postdoc studying the problem in Cox Terhorst’s lab at Dana-Farber Cancer Institute in Boston. “Immunology was young, but it was very exciting and there was a lot to discover. I became a professional scientist there and experienced how tough science is.” In 1988, Clevers cloned and characterized the gene for a component of the T-cell receptor (TCR) called CD3-epsilon, which binds antigen and activates intracellular signaling pathways.

On the fast track in Holland. Clevers returned to Utrecht University in 1989 as a professor of immunology. Within one month of setting up his lab, he had two graduate students and a technician, and the lab had cloned the first T cell–specific transcription factor, which they called TCF-1, in human T cells. When his former thesis advisor retired, Clevers was asked, at age 33, to become head of the immunology department. While the appointment was high-risk for him and for the department, Clevers says, he was chosen because he was good at multitasking and because he got along well with everyone.

Problem-solving strategy. “My strategy in research has always been opportunistic. One thing I have learned is that hypothesis-driven research tends not to be productive when you are in an unknown territory. I think there is an art to doing pilot experiments. So we have always just set up systems in which something happens and then you try and try things until a pattern appears and maybe you formulate a small hypothesis. But as soon as it turns out not to be exactly right, you abandon it. It’s a very open-minded type of research where you question whether what you are seeing is a real phenomenon without spending a year on doing all of the proper controls.”

Trial and error. Clevers’s lab found that while TCF-1 bound to DNA, it did not alter gene expression, despite the researchers’ tinkering with promoter and enhancer assays. “For about five years this was a problem. My first PhD students were leaving and they thought the whole TCF project was a failure,” says Clevers. His lab meanwhile cloned TCF homologs from several model organisms and made many reagents including antibodies against these homologs. To try to figure out the function of TCF-1, the lab performed a two-hybrid screen and identified components of the Wnt signaling pathway as binding partners of TCF-1. “We started to read about Wnt and realized that you study Wnt not in T cells but in frogs and flies, so we rapidly transformed into a developmental biology lab. We showed that we held the key for a major issue in developmental biology, the final protein in the Wnt cascade: TCF-1 binds b-catenin when b-catenin becomes available and activates transcription.” In 1996, Clevers published the mechanism of how the TCF-1 homolog in Xenopus embryos, called XTcf-3, is integrated into the Wnt signaling pathway.

Clevers Catapults


3DCrypt building and colon cancer.

Clevers next collaborated with Bert Vogelstein’s lab at Johns Hopkins, linking TCF to Wnt signaling in colon cancer. In colon cancer cell lines with mutated forms of the tumor suppressor gene APC, the APC protein can’t rein in b-catenin, which accumulates in the cytoplasm, forms a complex with TCF-4 (later renamed TCF7L2) in the nucleus, and caninitiate colon cancer by changing gene expression. Then, the lab showed that Wnt signaling is necessary for self-renewal of adult stem cells, as mice missing TCF-4 do not have intestinal crypts, the site in the gut where stem cells reside. “This was the first time Wnt was shown to play a role in adults, not just during development, and to be crucial for adult stem cell maintenance,” says Clevers. “Then, when I started thinking about studying the gut, I realized it was by far the best way to study stem cells. And I also realized that almost no one in the world was studying the healthy gut. Almost everyone who researched the gut was studying a disease.” The main advantages of the murine model are rapid cell turnover and the presence of millions of stereotypic crypts throughout the entire intestine.

Against the grain. In 2007, Nick Barker, a senior scientist in the Clevers lab, identified the Wnt target gene Lgr5 as a unique marker of adult stem cells in several epithelial organs, including the intestine, hair follicle, and stomach. In the intestine, the gene codes for a plasma membrane protein on crypt stem cells that enable the intestinal epithelium to self-renew, but can also give rise to adenomas of the gut. Upon making mice with adult stem cell populations tagged with a fluorescent Lgr5-binding marker, the lab helped to overturn assumptions that “stem cells are rare, impossible to find, quiescent, and divide asymmetrically.”

On to organoids. Once the lab could identify adult stem cells within the crypts of the gut, postdoc Toshiro Sato discovered that a single stem cell, in the presence of Matrigel and just three growth factors, could generate a miniature crypt structure—what is now called an organoid. “Toshi is very Japanese and doesn’t always talk much,” says Clevers. “One day I had asked him, while he was at the microscope, if the gut stem cells were growing, and he said, ‘Yes.’ Then I looked under the microscope and saw the beautiful structures and said, ‘Why didn’t you tell me?’ and he said, ‘You didn’t ask.’ For three months he had been growing them!” The lab has since also grown mini-pancreases, -livers, -stomachs, and many other mini-organs.

Tumor Organoids. Clevers showed that organoids can be grown from diseased patients’ samples, a technique that could be used in the future to screen drugs. The lab is also building biobanks of organoidsderived from tumor samples and adjacent normal tissue, which could be especially useful for monitoring responses to chemotherapies. “It’s a similar approach to getting a bacterium cultured to identify which antibiotic to take. The most basic goal is not to give a toxic chemotherapy to a patient who will not respond anyway,” says Clevers. “Tumor organoids grow slower than healthy organoids, which seems counterintuitive, but with cancer cells, often they try to divide and often things go wrong because they don’t have normal numbers of chromosomes and [have] lots of mutations. So, I am not yet convinced that this approach will work for every patient. Sometimes, the tumor organoids may just grow too slowly.”

Selective memory. “When I received the Breakthrough Prize in 2013, I invited everyone who has ever worked with me to Amsterdam, about 100 people, and the lab organized a symposium where many of the researchers gave an account of what they had done in the lab,” says Clevers. “In my experience, my lab has been a straight line from cloning TCF-1 to where we are now. But when you hear them talk it was ‘Hans told me to try this and stop this’ and ‘Half of our knockout mice were never published,’ and I realized that the lab is an endless list of failures,” Clevers recalls. “The one thing we did well is that we would start something and, as soon as it didn’t look very good, we would stop it and try something else. And the few times when we seemed to hit gold, I would regroup my entire lab. We just tried a lot of things, and the 10 percent of what worked, those are the things I remember.”

Greatest Hits

  • Cloned the first T cell–specific transcription factor, TCF-1, and identified homologous genes in model organisms including the fruit fly, frog, and worm
  • Found that transcriptional activation by the abundant β-catenin/TCF-4 [TCF7L2] complex drives cancer initiation in colon cells missing the tumor suppressor protein APC
  • First to extend the role of Wnt signaling from developmental biology to adult stem cells by showing that the two Wnt pathway transcription factors, TCF-1 and TCF-4, are necessary for maintaining the stem cell compartments in the thymus and in the crypt structures of the small intestine, respectively
  • Identified Lgr5 as an adult stem cell marker of many epithelial stem cells including those of the colon, small intestine, hair follicle, and stomach, and found that Lgr5-expressing crypt cells in the small intestine divide constantly and symmetrically, disproving the common belief that stem cell division is asymmetrical and uncommon
  • Established a three-dimensional, stable model, the “organoid,” grown from adult stem cells, to study diseased patients’ tissues from the gut, stomach, liver, and prostate
 Regenerative Medicine Comes of Age   
“Anti-Aging Medicine” Sounds Vaguely Disreputable, So Serious Scientists Prefer to Speak of “Regenerative Medicine”
  • Induced pluripotent stem cells (iPSCs) and genome-editing techniques have facilitated manipulation of living organisms in innumerable ways at the cellular and genetic levels, respectively, and will underpin many aspects of regenerative medicine as it continues to evolve.

    An attitudinal change is also occurring. Experts in regenerative medicine have increasingly begun to embrace the view that comprehensively repairing the damage of aging is a practical and feasible goal.

    A notable proponent of this view is Aubrey de Grey, Ph.D., a biomedical gerontologist who has pioneered an regenerative medicine approach called Strategies for Engineered Negligible Senescence (SENS). He works to “develop, promote, and ensure widespread access to regenerative medicine solutions to the disabilities and diseases of aging” as CSO and co-founder of the SENS Research Foundation. He is also the editor-in-chief of Rejuvenation Research, published by Mary Ann Liebert.

    Dr. de Grey points out that stem cell treatments for age-related conditions such as Parkinson’s are already in clinical trials, and immune therapies to remove molecular waste products in the extracellular space, such as amyloid in Alzheimer’s, have succeeded in such trials. Recently, there has been progress in animal models in removing toxic cells that the body is failing to kill. The most encouraging work is in cancer immunotherapy, which is rapidly advancing after decades in the doldrums.

    Many damage-repair strategies are at an  early stage of research. Although these strategies look promising, they are handicapped by a lack of funding. If that does not change soon, the scientific community is at risk of failing to capitalize on the relevant technological advances.

    Regenerative medicine has moved beyond boutique applications. In degenerative disease, cells lose their function or suffer elimination because they harbor genetic defects. iPSC therapies have the potential to be curative, replacing the defective cells and eliminating symptoms in their entirety. One of the biggest hurdles to commercialization of iPSC therapies is manufacturing.

  • Building Stem Cell Factories

    Cellular Dynamics International (CDI) has been developing clinically compatible induced pluripotent stem cells (iPSCs) and iPSC-derived human retinal pigment epithelial (RPE) cells. CDI’s MyCell Retinal Pigment Epithelial Cells are part of a possible therapy for macular degeneration. They can be grown on bioengineered, nanofibrous scaffolds, and then the RPE cell–enriched scaffolds can be transplanted into patients’ eyes. In this pseudo-colored image, RPE cells are shown growing over the nanofibers. Each cell has thousands of “tongue” and “rod” protrusions that could naturally support rod and cone cells in the eye.

    “Now that an infrastructure is being developed to make unlimited cells for the tools business, new opportunities are being created. These cells can be employed in a therapeutic context, and they can be used to understand the efficacy and safety of drugs,” asserts Chris Parker, executive vice president and CBO, Cellular Dynamics International (CDI). “CDI has the capability to make a lot of cells from a single iPSC line that represents one person (a capability termed scale-up) as well as the capability to do it in parallel for multiple individuals (a capability termed scale-out).”

    Minimally manipulated adult stem cells have progressed relatively quickly to the clinic. In this scenario, cells are taken out of the body, expanded unchanged, then reintroduced. More preclinical rigor applies to potential iPSC therapy. In this case, hematopoietic blood cells are used to make stem cells, which are manufactured into the cell type of interest before reintroduction. Preclinical tests must demonstrate that iPSC-derived cells perform as intended, are safe, and possess little or no off-target activity.

    For example, CDI developed a Parkinsonian model in which iPSC-derived dopaminergic neurons were introduced to primates. The model showed engraftment and enervation, and it appeared to be free of proliferative stem cells.

    • “You will see iPSCs first used in clinical trials as a surrogate to understand efficacy and safety,” notes Mr. Parker. “In an ongoing drug-repurposing trial with GlaxoSmithKline and Harvard University, iPSC-derived motor neurons will be produced from patients with amyotrophic lateral sclerosis and tested in parallel with the drug.” CDI has three cell-therapy programs in their commercialization pipeline focusing on macular degeneration, Parkinson’s disease, and postmyocardial infarction.

    • Keeping an Eye on Aging Eyes

      The California Project to Cure Blindness is evaluating a stem cell–based treatment strategy for age-related macular degeneration. The strategy involves growing retinal pigment epithelium (RPE) cells on a biostable, synthetic scaffold, then implanting the RPE cell–enriched scaffold to replace RPE cells that are dying or dysfunctional. One of the project’s directors, Dennis Clegg, Ph.D., a researcher at the University of California, Santa Barbara, provided this image, which shows stem cell–derived RPE cells. Cell borders are green, and nuclei are red.

      The eye has multiple advantages over other organ systems for regenerative medicine. Advanced surgical methods can access the back of the eye, noninvasive imaging methods can follow the transplanted cells, good outcome parameters exist, and relatively few cells are needed.

      These advantages have attracted many groups to tackle ocular disease, in particular age-related macular degeneration, the leading cause of blindness in the elderly in the United States. Most cases of age-related macular degeneration are thought to be due to the death or dysfunction of cells in the retinal pigment epithelium (RPE). RPE cells are crucial support cells for the rods, cones, and photoreceptors. When RPE cells stop working or die, the photoreceptors die and a vision deficit results.

      A regenerated and restored RPE might prevent the irreversible loss of photoreceptors, possibly via the the transplantation of functionally polarized RPE monolayers derived from human embryonic stem cells. This approach is being explored by the California Project to Cure Blindness, a collaborative effort involving the University of Southern California (USC), the University of California, Santa Barbara (UCSB), the California Institute of Technology, City of Hope, and Regenerative Patch Technologies.

      The project, which is funded by the California Institute of Regenerative Medicine (CIRM), started in 2010, and an IND was filed early 2015. Clinical trial recruitment has begun.

      One of the project’s leaders is Dennis Clegg, Ph.D., Wilcox Family Chair in BioMedicine, UCSB. His laboratory developed the protocol to turn undifferentiated H9 embryonic stem cells into a homogenous population of RPE cells.

      “These are not easy experiments,” remarks Dr. Clegg. “Figuring out the biology and how to make the cell of interest is a challenge that everyone in regenerative medicine faces. About 100,000 RPE cells will be grown as a sheet on a 3 × 5 mm biostable, synthetic scaffold, and then implanted in the patients to replace the cells that are dying or dysfunctional. The idea is to preserve the photoreceptors and to halt disease progression.”

      Moving therapies such as this RPE treatment from concept to clinic is a huge team effort and requires various kinds of expertise. Besides benefitting from Dr. Clegg’s contribution, the RPE project incorporates the work of Mark Humayun, M.D., Ph.D., co-director of the USC Eye Institute and director of the USC Institute for Biomedical Therapeutics and recipient of the National Medal of Technology and Innovation, and David Hinton, Ph.D., a researcher at USC who has studied how actvated RPE cells can alter the local retinal microenvironment.


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Fat Cells Reprogrammed to Make Insulin

Curator: Larry H. Bernstein, MD, FCAP


A New Use for Love Handles, Insulin-Producing Beta Cells


Scientists at the Swiss Federal Institute of Technology (ETH) in Zurich have found an exciting new use for the cells that reside in the undesirable flabby tissue—creating pancreatic beta cells. The ETH researchers extracted stem cells from a 50-year-old test subject’s fatty tissue and reprogrammed them into mature, insulin-producing beta cells.

The findings from this study were published recently in Nature Communications in an article entitled “A Programmable Synthetic Lineage-Control Network That Differentiates Human IPSCs into Glucose-Sensitive Insulin-Secreting Beta-Like Cells.”

The investigators added a highly complex synthetic network of genes to the stem cells to recreate precisely the key growth factors involved in this maturation process. Central to the process were the growth factors Ngn3, Pdx1, and MafA; the researchers found that concentrations of these factors change during the differentiation process.

For instance, MafA is not present at the start of maturation. Only on day 4, in the final maturation step, does it appear, its concentration rising steeply and then remaining at a high level. The changes in the concentrations of Ngn3 and Pdx1, however, are very complex: while the concentration of Ngn3 rises and then falls again, the level of Pdx1 rises at the beginning and toward the end of maturation.

Senior study author Martin Fussenegger, Ph.D., professor of biotechnology and bioengineering at ETH Zurich’s department of biosystems science and engineering stressed that it was essential to reproduce these natural processes as closely as possible to produce functioning beta cells, stating that “the timing and the quantities of these growth factors are extremely important.”

The ETH researchers believe that their work is a real breakthrough, in that a synthetic gene network has been used successfully to achieve genetic reprogramming that delivers beta cells. Until now, scientists have controlled such stem cell differentiation processes by adding various chemicals and proteins exogenously.

“It’s not only really hard to add just the right quantities of these components at just the right time, but it’s also inefficient and impossible to scale up,” Dr. Fussenegger noted.

While the beta cells not only looked very similar to their natural counterparts—containing dark spots known as granules that store insulin—the artificial beta cells also functioned in a very similar manner. However, the researchers admit that more work needs to be done to increase the insulin output.

“At the present time, the quantities of insulin they secrete are not as great as with natural beta cells,” Dr. Fussenegger stated. Yet, the key point is that the researchers have for the first time succeeded in reproducing the entire natural process chain, from stem cell to differentiated beta cell.

In future, the ETH scientists’ novel technique might make it possible to implant new functional beta cells in diabetes sufferers that are made from their adipose tissue. While beta cells have been transplanted in the past, this has always required subsequent suppression of the recipient’s immune system—as with any transplant of donor organs or tissue.

“With our beta cells, there would likely be no need for this action since we can make them using endogenous cell material taken from the patient’s own body,” Dr. Fussenegger said. “This is why our work is of such interest in the treatment of diabetes.”

A programmable synthetic lineage-control network that differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells

Pratik SaxenaBoon Chin HengPeng BaiMarc FolcherHenryk Zulewski & Martin Fussenegger
Nature Communications7,Article number:11247

Synthetic biology has advanced the design of standardized transcription control devices that programme cellular behaviour. By coupling synthetic signalling cascade- and transcription factor-based gene switches with reverse and differential sensitivity to the licensed food additive vanillic acid, we designed a synthetic lineage-control network combining vanillic acid-triggered mutually exclusive expression switches for the transcription factors Ngn3 (neurogenin 3; OFF-ON-OFF) and Pdx1 (pancreatic and duodenal homeobox 1; ON-OFF-ON) with the concomitant induction of MafA (V-maf musculoaponeurotic fibrosarcoma oncogene homologue A; OFF-ON). This designer network consisting of different network topologies orchestrating the timely control of transgenic and genomic Ngn3, Pdx1 and MafA variants is able to programme human induced pluripotent stem cells (hIPSCs)-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells, whose glucose-stimulated insulin-release dynamics are comparable to human pancreatic islets. Synthetic lineage-control networks may provide the missing link to genetically programme somatic cells into autologous cell phenotypes for regenerative medicine.

Cell-fate decisions during development are regulated by various mechanisms, including morphogen gradients, regulated activation and silencing of key transcription factors, microRNAs, epigenetic modification and lateral inhibition. The latter implies that the decision of one cell to adopt a specific phenotype is associated with the inhibition of neighbouring cells to enter the same developmental path. In mammals, insights into the role of key transcription factors that control development of highly specialized organs like the pancreas were derived from experiments in mice, especially various genetically modified animals1, 2, 3, 4. Normal development of the pancreas requires the activation of pancreatic duodenal homeobox protein (Pdx1) in pre-patterned cells of the endoderm. Inactivating mutations of Pdx1 are associated with pancreas agenesis in mouse and humans5, 6. A similar cell fate decision occurs later with the activation of Ngn3 that is required for the development of all endocrine cells in the pancreas7. Absence of Ngn3 is associated with the loss of pancreatic endocrine cells, whereas the activation of Ngn3 not only allows the differentiation of endocrine cells but also induces lateral inhibition of neighbouring cells—via Delta-Notch pathway—to enter the same pancreatic endocrine cell fate8. This Ngn3-mediated cell-switch occurs at a specific time point and for a short period of time in mice9. Thereafter, it is silenced and becomes almost undetectable in postnatal pancreatic islets. Conversely, Pdx1-positive Ngn3-positive cells reduce Pdx1 expression, as Ngn3-positive cells are Pdx1 negative10. They re-express Pdx1, however, as they go on their path towards glucose-sensitive insulin-secreting cells with parallel induction of MafA that is required for proper differentiation and maturation of pancreatic beta cells11. Data supporting these expression dynamics are derived from mice experiments1, 11, 12. A synthetic gene-switch governing cell fate decision in human induced pluripotent stem cells (hIPSCs) could facilitate the differentiation of glucose-sensitive insulin-secreting cells.

In recent years, synthetic biology has significantly advanced the rational design of synthetic gene networks that can interface with host metabolism, correct physiological disturbances13 and provide treatment strategies for a variety of metabolic disorders, including gouty arthritis14, obesity15 and type-2 diabetes16. Currently, synthetic biology principles may provide the componentry and gene network topologies for the assembly of synthetic lineage-control networks that can programme cell-fate decisions and provide targeted differentiation of stem cells into terminally differentiated somatic cells. Synthetic lineage-control networks may therefore provide the missing link between human pluripotent stem cells17 and their true impact on regenerative medicine18, 19, 20. The use of autologous stem cells in regenerative medicine holds great promise for curing many diseases, including type-1 diabetes mellitus (T1DM), which is characterized by the autoimmune destruction of insulin-producing pancreatic beta cells, thus making patients dependent on exogenous insulin to control their blood glucose21, 22. Although insulin therapy has changed the prospects and survival of T1DM patients, these patients still suffer from diabetic complications arising from the lack of physiological insulin secretion and excessive glucose levels23. The replacement of the pancreatic beta cells either by pancreas transplantation or by transplantation of pancreatic islets has been shown to normalize blood glucose and even improve existing complications of diabetes24. However, insulin independence 5 years after islet transplantation can only be achieved in up to 55% of the patients even when using the latest generation of immune suppression strategies25, 26. Transplantation of human islets or the entire pancreas has allowed T1DM patients to become somewhat insulin independent, which provides a proof-of-concept for beta-cell replacement therapies27, 28. However, because of the shortage of donor pancreases and islets, as well as the significant risk associated with transplantation and life-long immunosuppression, the rational differentiation of stem cells into functional beta-cells remains an attractive alternative29, 30. Nevertheless, a definitive cure for T1DM should address both the beta-cell deficit and the autoimmune response to cells that express insulin. Any beta-cell mimetic should be able to store large amounts of insulin and secrete it on demand, as in response to glucose stimulation29, 31. The most effective protocols for the in vitro generation of bonafide insulin-secreting beta-like cells that are suitable for transplantation have been the result of sophisticated trial-and-error studies elaborating timely addition of complex growth factor and small-molecule compound cocktails to human pancreatic progenitor cells32, 33, 34. The differentiation of pancreatic progenitor cells to beta-like cells is the most challenging part as current protocols provide inconsistent results and limited success in programming pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells35, 36, 37. One of the reasons for these observations could be the heterogeneity in endocrine differentiation and maturation towards a beta cell phenotype. Here we show that a synthetic lineage-control network programming the dynamic expression of the transcription factors Ngn3, Pdx1 and MafA enables the differentiation of hIPSC-derived pancreatic progenitor cells to glucose-sensitive insulin-secreting beta-like cells (Supplementary Fig. 1).


Vanillic acid-programmable positive band-pass filter

The differentiation pathway from pancreatic progenitor cells to glucose-sensitive insulin-secreting pancreatic beta-cells combines the transient mutually exclusive expression switches of Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) with the concomitant induction of MafA (OFF-ON) expression10,11. Since independent control of the pancreatic transcription factors Ngn3, Pdx1 and MafA by different antibiotic transgene control systems responsive to tetracycline, erythromycin and pristinamycin did not result in the desired differential control dynamics (Supplementary Fig. 2), we have designed a vanillic acid-programmable synthetic lineage-control network that programmes hIPSC-derived pancreatic progenitor cells to specifically differentiate into glucose-sensitive insulin-secreting beta-like cells in a seamless and self-sufficient manner. The timely coordination of mutually exclusive Ngn3 and Pdx1 expression with MafA induction requires the trigger-controlled execution of a complex genetic programme that orchestrates two overlapping antagonistic band-pass filter expression profiles (OFF-ON-OFF and ON-OFF-ON), a positive band-pass filter for Ngn3 (OFF-ON-OFF) and a negative band-pass filter, also known as band-stop filter, for Pdx1 (ON-OFF-ON), the ramp-up expression phase of which is linked to a graded induction of MafA (OFF-ON).

The core of the synthetic lineage-control network consists of two transgene control devices that are sensitive to the food component and licensed food additive vanillic acid. These devices are a synthetic vanillic acid-inducible (ON-type) signalling cascade that is gradually induced by increasing the vanillic acid concentration and a vanillic acid-repressible (OFF-type) gene switch that is repressed in a vanillic acid dose-dependent manner (Fig. 1a,b). The designer cascade consists of the vanillic acid-sensitive mammalian olfactory receptor MOR9-1, which sequentially activates the G protein Sα (GSα) and adenylyl cyclase to produce a cyclic AMP (cAMP) second messenger surge38 that is rewired via the cAMP-responsive protein kinase A-mediated phospho-activation of the cAMP-response element-binding protein 1 (CREB1) to the induction of synthetic promoters (PCRE) containing CREB1-specific cAMP response elements (CRE; Fig. 1a). The co-transfection of pCI-MOR9-1 (PhCMV-MOR9-1-pASV40) and pCK53 (PCRE-SEAP-pASV40) into human mesenchymal stem cells (hMSC-TERT) confirmed the vanillic acid-adjustable secreted alkaline phosphatase (SEAP) induction of the designer cascade (>10nM vanillic acid; Fig. 1a). The vanillic acid-repressible gene switch consists of the vanillic acid-dependent transactivator (VanA1), which binds and activates vanillic acid-responsive promoters (for example, P1VanO2) at low and medium vanillic acid levels (<2μM). At high vanillic acid concentrations (>2μM), VanA1 dissociates from P1VanO2, which results in the dose-dependent repression of transgene expression39 (Fig. 1b). The co-transfection of pMG250 (PSV40-VanA1-pASV40) and pMG252 (P1VanO2-SEAP-pASV40) into hMSC-TERT corroborated the fine-tuning of the vanillic acid-repressible SEAP expression (Fig. 1b).

Figure 1: Design of a vanillic acid-responsive positive band-pass filter providing an OFF-ON-OFF expression profile.

Design of a vanillic acid-responsive positive band-pass filter providing an OFF-ON-OFF expression profile.

a) Vanillic acid-inducible transgene expression. The constitutively expressed vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers G protein (Gs)-mediated activation of the membrane-bound adenylyl cyclase (AC) that converts ATP into cyclic AMP (cAMP). The resulting intracellular cAMP surge activates PKA (protein kinase A), whose catalytic subunits translocate into the nucleus to phosphorylate cAMP response element-binding protein 1 (CREB1). Activated CREB1 binds to synthetic promoters (PCRE) containing cAMP-response elements (CRE) and induces PCRE-driven expression of human placental secreted alkaline phosphatase (SEAP; pCK53, PCRE-SEAP-pA). Co-transfection of pCI-MOR9-1 and pCK53 into human mesenchymal stem cells (hMSC-TERT) grown for 48h in the presence of increasing vanillic acid concentrations results in a dose-inducible SEAP expression profile. (b) Vanillic acid-repressible transgene expression. The constitutively expressed, vanillic acid-dependent transactivator VanA1(pMG250, PSV40-VanA1-pA, VanA1, VanR-VP16) binds and activates the chimeric promoter P1VanO2 (pMG252, P1VanO2-SEAP-pA) in the absence of vanillic acid. In the presence of increasing vanillic acid concentrations, VanA1 is released from P1VanO2, and transgene expression is shut down. Co-transfection of pMG250 and pMG252 into hMSC-TERT grown for 48h in the presence of increasing vanillic acid concentrations results in a dose-repressible SEAP expression profile. (c) Positive band-pass expression filter. Serial interconnection of the synthetic vanillic acid-inducible signalling cascade (a) with the vanillic acid-repressible transcription factor-based gene switch (b) by PCRE-mediated expression of VanA1 (pSP1, PCRE-VanA1-pA) results in a two-level feed-forward cascade. Owing to the opposing responsiveness and differential sensitivity to vanillic acid, this synthetic gene network programmes SEAP expression with a positive band-pass filter profile (OFF-ON-OFF) as vanillic acid levels are increased. Medium vanillic acid levels activate MOR9-1, which induces PCRE-driven VanA1 expression. VanA1remains active and triggers P1VanO2-mediated SEAP expression in feed-forward manner, which increases to maximum levels. At high vanillic acid concentrations, MOR9-1 maintains PCRE-driven VanA1 expression, but the transactivator dissociates from P1VanO2, which shuts SEAP expression down. Co-transfection of pCI-MOR9-1, pSP1 and pMG252 into hMSC-TERT grown for 48h in the presence of increasing vanillic acid concentrations programmes SEAP expression with a positive band-pass profile (OFF-ON-OFF). Data are the means±s.d. of triplicate experiments (n=9).

The opposing responsiveness and differential sensitivity of the control devices to vanillic acid are essential to programme band-pass filter expression profiles. Upon daisy-chaining the designer cascade (pCI-MOR9-1; PhCMV-MOR9-1-pASV40; pSP1, PCRE-VanA1-pASV40) and the gene switch (pSP1, PCRE-VanA1-pASV40; pMG252, P1VanO2-SEAP-pASV40) in the same cell, the network executes a band-pass filter SEAP expression profile when exposed to increasing concentrations of vanillic acid (Fig. 1c). Medium vanillic acid levels (10nM to 2μM) activate MOR9-1, which induces PCRE-driven VanA1 expression. VanA1 remains active within this concentration range and, in a feed-forward amplifier manner, triggers P1VanO2-mediated SEAP expression, which gradually increases to maximum levels (Fig. 1c). At high vanillic acid concentrations (2μM to 400μM), MOR9-1 maintains PCRE-driven VanA1 expression, but the transactivator is inactivated and dissociates from P1VanO2, which results in the gradual shutdown of SEAP expression (Fig. 1c).

Vanillic acid-programmable lineage-control network

For the design of the vanillic acid-programmable synthetic lineage-control network, constitutive MOR9-1 expression and PCRE-driven VanA1 expression were combined with pSP12 (pASV40-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pASV40) for endocrine specification and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40) for maturation of developing beta-cells (Fig. 2a,b). ThepSP12-encoded expression unit enables the VanA1-controlled induction of the optimized bidirectional vanillic acid-responsive promoter (P3VanO2) that drives expression of a codon-modified Ngn3cm, the nucleic acid sequence of which is distinct from its genomic counterpart (Ngn3g) to allow for quantitative reverse transcription–PCR (qRT–PCR)-based discrimination. In the opposite direction, P3VanO2 transcribes miR30Pdx1g-shRNA, which exclusively targets genomicPdx1 (Pdx1g) transcripts for RNA interference-based destruction and is linked to the production of a blue-to-red medium fluorescent timer40 (mFT) for precise visualization of the unit’s expression dynamics in situ. pSP17 contains a dicistronic expression unit in which the modified high-tightness and lower-sensitivity PCREm promoter (see below) drives co-cistronic expression of Pdx1cm andMafAcm, which are codon-modified versions producing native transcription factors that specifically differ from their genomic counterparts (Pdx1g, MafAg) in their nucleic acid sequence. After individual validation of the vanillic acid-controlled expression and functionality of all network components (Supplementary Figs 2–9), the lineage-control network was ready to be transfected into hIPSC-derived pancreatic progenitor cells. These cells are characterized by high expression of Pdx1g and Nkx6.1 levels and the absence of Ngn3g and MafAg production32, 33, 34 (day 0:Supplementary Figs 10–16).


Figure 2: Synthetic lineage-control network programming differential expression dynamics of pancreatic transcription factors.

Synthetic lineage-control network programming differential expression dynamics of pancreatic transcription factors.

(a) Schematic of the synthetic lineage-control network. The constitutively expressed, vanillic acid-sensitive olfactory G protein-coupled receptor MOR9-1 (pCI-MOR9-1; PhCMV-MOR9-1-pA) senses extracellular vanillic acid levels and triggers a synthetic signalling cascade, inducing PCRE-driven expression of the transcription factor VanA1 (pSP1, PCRE-VanA1-pA). At medium vanillic acid concentrations (purple arrows), VanA1 binds and activates the bidirectional vanillic acid-responsive promoter P3VanO2 (pSP12, pA-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pA), which drives the induction of codon-modified Neurogenin 3 (Ngn3cm) as well as the coexpression of both the blue-to-red medium fluorescent timer (mFT) for precise visualization of the unit’s expression dynamics and miR30pdx1g-shRNA (a small hairpin RNA programming the exclusive destruction of genomic pancreatic and duodenal homeobox 1 (Pdx1g) transcripts). Consequently, Ngn3cm levels switch from low to high (OFF-to-ON), and Pdx1g levels toggle from high to low (ON-to-OFF). In addition, Ngn3cm triggers the transcription of Ngn3g from its genomic promoter, which initiates a positive-feedback loop. At high vanillic acid levels (orange arrows), VanA1 is inactivated, and both Ngn3cm and miR30pdx1g-shRNA are shut down. At the same time, the MOR9-1-driven signalling cascade induces the modified high-tightness and lower-sensitivity PCREm promoter that drives the co-cistronic expression of the codon-modified variants of Pdx1 (Pdx1cm) and V-maf musculoaponeurotic fibrosarcoma oncogene homologue A (MafAcm; pSP17, PCREm-Pdx1cm-2A-MafAcm-pA). Consequently, Pdx1cm and MafAcm become fully induced. As Pdx1cm expression ramps up, it initiates a positive-feedback loop by inducing the genomic counterparts Pdx1g and MafAg. Importantly, Pdx1cm levels are not affected by miR30Pdx1g-shRNA because the latter is specific for genomic Pdx1g transcripts and because the positive feedback loop-mediated amplification of Pdx1gexpression becomes active only after the shutdown of miR30Pdx1g-shRNA. Overall, the synthetic lineage-control network provides vanillic acid-programmable, transient, mutually exclusive expression switches for Ngn3 (OFF-ON-OFF) and Pdx1 (ON-OFF-ON) as well as the concomitant induction of MafA (OFF-ON) expression, which can be followed in real time (Supplementary Movies 1 and 2). (b) Schematic illustrating the individual differentiation steps from human IPSCs towards beta-like cells. The colours match the cell phenotypes reached during the individual differentiation stages programmed by the lineage-control network shown in a.

Following the co-transfection of pCI-MOR9-1 (PhCMV-MOR9-1-pASV40), pSP1 (PCRE-VanA1-pASV40), pSP12 (pASV40-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pASV40) and pSP17(PCREm-Pdx1cm-2A-MafAcm-pASV40) into hIPSC-derived pancreatic progenitor cells, the synthetic lineage-control network should override random endogenous differentiation activities and execute the pancreatic beta-cell-specific differentiation programme in a vanillic acid remote-controlled manner. To confirm that the lineage-control network operates as programmed, we cultivated network-containing and pEGFP-N1-transfected (negative-control) cells for 4 days at medium (2μM) and then 7 days at high (400μM) vanillic acid concentrations and profiled the differential expression dynamics of all of the network components and their genomic counterparts as well as the interrelated transcription factors and hormones in both whole populations and individual cells at days 0, 4, 11 and 14 (Figs 2 and 3 and Supplementary Figs 11–17).


Figure 3: Dynamics of the lineage-control network.

Dynamics of the lineage-control network.

(a,b) Quantitative RT–PCR-based expression profiling of the pancreatic transcription factors Ngn3cm/g, Pdx1cm/g and MafAcm/g in hIPSC-derived pancreatic progenitor cells containing the synthetic lineage-control network at days 4 and 11. Data are the means±s.d. of triplicate experiments (n=9). (cg) Immunocytochemistry of pancreatic transcription factors Ngn3cm/g, Pdx1cm/g and MafAcm/g in hIPSC-derived pancreatic progenitor cells containing the synthetic lineage-control network at days 4 and 11. hIPSC-derived pancreatic progenitor cells were co-transfected with the lineage-control vectors pCI-MOR9-1 (PhCMV-MOR9-1-pA), pSP1 (PCRE-VanA1-pA), pSP12 (pA-Ngn3cm←P3VanO2right arrowmFT-miR30Pdx1g-shRNA-pA) and pSP17 (PCREm-Pdx1cm-2A-MafAcm) and immunocytochemically stained for (c) VanA1 and Pdx1 (day 4), (d) VanA1 and Ngn3 (day 4), (e) VanA1 and Pdx1 (day 11), (f) MafA and Pdx1 (day 11) as well as (g) VanA1 and insulin (C-peptide) (day 11). The cells staining positive for VanA1 are containing the lineage-control network. DAPI, 4′,6-diamidino-2-phenylindole. Scale bar, 100μm.


Multicellular organisms, including humans, consist of a highly structured assembly of a multitude of specialized cell phenotypes that originate from the same zygote and have traversed a preprogrammed multifactorial developmental plan that orchestrates sequential differentiation steps with high precision in space and time19, 51. Because of the complexity of terminally differentiated cells, the function of damaged tissues can for most medical indications only be restored via the transplantation of donor material, which is in chronically short supply52.

Despite significant progress in regenerative medicine and the availability of stem cells, the design of protocols that replicate natural differentiation programmes and provide fully functional cell mimetics remains challenging29, 53. For example, efforts to generate beta-cells from human embryonic stem cells (hESCs) have led to reliable protocols involving the sequential administration of growth factors (activin A, bone morphogenetic protein 4 (BMP-4), basic fibroblast growth factor (bFGF), FGF-10, Noggin, vascular endothelial growth factor (VEGF) and Wnt3A) and small-molecule compounds (cyclopamine, forskolin, indolactam V, IDE1, IDE2, nicotinamide, retinoic acid, SB−431542 and γ-secretase inhibitor) that modulate differentiation-specific signalling pathways31, 54, 55. In vitro differentiation of hESC-derived pancreatic progenitor cells into beta-like cells is more challenging and has been achieved recently by a complex media formulation with chemicals and growth factors32, 33, 34.

hIPSCs have become a promising alternative to hESCs; however, their use remains restricted in many countries56. Most hIPSCs used for directed differentiation studies were derived from a juvenescent cell source that is expected to show a higher degree of differentiation potential compared with older donors that typically have a higher need for medical interventions37, 57, 58. We previously succeeded in producing mRNA-reprogrammed hIPSCs from adipose tissue-derived mesenchymal stem cells of a 50-year-old donor, demonstrating that the reprogramming of cells from a donor of advanced age is possible in principle59.

Recent studies applying similar hESC-based differentiation protocols to hIPSCs have produced cells that release insulin in response to high glucose32, 33, 34. This observation suggests that functional beta-like cells can eventually be derived from hIPSCs32, 33. In our hands, the growth-factor/chemical-based technique for differentiating human IPSCs resulted in beta-like cells with poor glucose responsiveness. Recent studies have revealed significant variability in the lineage specification propensity of different hIPSC lines35, 60 and substantial differences in the expression profiles of key transcription factors in hIPSC-derived beta-like cells33. Therefore, the growth-factor/chemical-based protocols may require further optimization and need to be customized for specific hIPSC lines35. Synthetic lineage-control networks providing precise dynamic control of transcription factor expression may overcome the challenges associated with the programming of beta-like cells from different hIPSC lines.

Rather than exposing hIPSCs to a refined compound cocktail that triggers the desired differentiation in a fraction of the stem cell population, we chose to design a synthetic lineage-control network to enable single input-programmable differentiation of hIPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells. In contrast with the use of growth-factor/chemical-based cocktails, synthetic lineage-control networks are expected to (i) be more economical because of in situ production of the required transcription factors, (ii) enable simultaneous control of ectopic and chromosomally encoded transcription factor variants, (iii) tap into endogenous pathways and not be limited to cell-surface input, (iv) display improved reversibility that is not dependent on the removal of exogenous growth factors via culture media replacement, (v) provide lateral inhibition, thereby reducing the random differentiation of neighbouring cells and (vi) enable trigger-programmable and (vii) precise differential transcription factor expression switches.

The synthetic lineage-control network that precisely replicates the endogenous relative expression dynamics of the transcription factors Pdx-1, Ngn3 and MafA required the design of a new network topology that interconnects a synthetic signalling cascade and a gene switch with differential and opposing sensitivity to the food additive vanillic acid. This differentiation device provides different band-pass filter, time-delay and feed-forward amplifier topologies that interface with endogenous positive-feedback loops to orchestrate the timely expression and repression of heterologous and chromosomally encoded Ngn3, Pdx1 and MafA variants. The temporary nature of the engineering intervention, which consists of transient transfection of the genetic lineage-control components in the absence of any selection, is expected to avoid stable modification of host chromosomes and alleviate potential safety concerns. In addition, the resulting beta-cell mass could be encapsulated inside vascularized microcontainers28, a proven containment strategy in prototypic cell-based therapies currently being tested in animal models of prominent human diseases14, 15, 16, 61, 62 as well as in human clinical trials28.

The hIPSC-derived beta-like cells resulting from this trigger-induced synthetic lineage-control network exhibited glucose-stimulated insulin-release dynamics and capacity matching the human physiological range and transcriptional profiling, flow cytometric analysis and electron microscopy corroborated the lineage-controlled stem cells reached a mature beta-cell phenotype. In principle, the combination of hIPSCs derived from the adipose tissue of a 50-year-old donor59 with a synthetic lineage-control network programming glucose-sensitive insulin-secreting beta-like cells closes the design cycle of regenerative medicine63. However, hIPSCs that are derived from T1DM patients, differentiated into beta-like cells and transplanted back into the donor would still be targeted by the immune system, as demonstrated in the transplantation of segmental pancreatic grafts from identical twins64. Therefore, any beta-cell-replacement therapy will require complementary modulation of the immune system either via drugs30, 65, engineering or cell-based approaches66, 67 or packaging inside vascularizing, semi-permeable immunoprotective microcontainers28.

Capitalizing on the design principles of synthetic biology, we have successfully constructed and validated a synthetic lineage-control network that replicates the differential expression dynamics of critical transcription factors and mimicks the native differentiation pathway to programme hIPSC-derived pancreatic progenitor cells into glucose-sensitive insulin-secreting beta-like cells that compare with human pancreatic islets at a high level. The design of input-triggered synthetic lineage-control networks that execute a preprogrammed sequential differentiation agenda coordinating the timely induction and repression of multiple genes could provide a new impetus for the advancement of developmental biology and regenerative medicine.

Other related articles published in this Open Access Online Scientific Journal include the following:

Adipocyte Derived Stroma Cells: Their Usage in Regenerative Medicine and Reprogramming into Pancreatic Beta-Like Cells

Curator: Evelina Cohn, Ph.D.

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Cell-cell communication in brain

Larry H. Bernstein, MD, FCAP, Curator



Adjustable Brain Cells

Neighboring neurons can manipulate astrocytes.

By Ruth Williams | February 18, 2016

Neurons in the adult mouse brain can shape the features and physiologies of nearby astroglial cells, according to a study published today (February 18) in Science. Researchers at McGill University in Montreal and their colleagues have identified a molecular signal called sonic hedgehog (Shh), secreted by neurons, that acts as the agent of change.

“What’s very exciting about the paper is this notion that a cell’s fate might be determined—after it has already established its morphology and location in the brain—based on interactions with its neighbors,” said neurologist Ed Ruthazer of the Montreal Neurological Institute at McGill who was not involved in the research. “And the conversion is not superficial,” he added, “it really does seem to fundamentally reorganize the transcriptome of the cell.”

Astroglia are non-neuronal cells in the central nervous system that generally support and modulate neuronal function. The mammalian brain has an assortment of astrocytes, which perform a variety of specialized functions. This diversity was largely thought to be established during embryogenesis and early postnatal development, said Keith Murai of McGill who led the new research. “But after that,” he said, “the properties of these cells were thought to be solidified . . . for the rest of their lives.”

Murai and his colleagues had a different view, however. “Some of these [astrocytes] are so specialized around certain neural circuits that it was hard to imagine that all of the properties of these cells could be determined by that point [in development],” he said. After all, the neural circuitry itself isn’t fully formed until much later.

To investigate whether astrocyte identity might continue to be shaped beyond the perinatal period, Murai’s team searched for gene products in adult neurons and astrocytes that might govern continuing development. To simplify matters, the researchers focused on the cerebellar cortex, where just two types of astrocyte exist—Bergmann glial cells (BGs), which encapsulate the impulse-receiving regions of Purkinji cell neurons (PCs), and velate astrocytes (VAs), which surround granule cell neurons (GCs). Their searches revealed many candidate factors, said Murai, but one pathway kept coming up: Shh signaling.

Shh is a developmental morphogen known to have many important roles in the developing embryo, including the specification of cells in the brain, explained Murai. “People thought that the pathway was shut down and eliminated from the brain after it developed,” he said, “but as it turns out, this pathway is very potent even in the adult brain.”

The team found that the Shh protein itself was produced by PC neurons in the cerebellum, and that Shh receptors were abundantly expressed in BG, but not VA cells.

Furthermore, BGs required Shh signals from PCs to maintain their identities. When transgenic techniques were used to switch off either Shh production in PCs or Shh signaling in BGs in adult mouse brains, the BG cells adopted a transcription profile similar to that of VAs. If Shh signaling in VAs was given a boost on the other hand, these cells became more like BGs.

We could “almost interconvert one type of astrocyte into another based upon the level of Shh signaling,” said Murai. And it wasn’t just a handful of factors, he added: “We’re talking about hundreds of genes that are either being turned on or turned off in response to this pathway.”

The team also found evidence that astrocytes in other brain regions were influenced by Shh manipulations, and that these cells’ electrophysiologies were altered as a result.

“The key message is that astrocytes’ molecular fate is not hardwired,” said cell biologist Cagla Eroglu of Duke University in Durham, North Carolina, who did not participate in the study. The shapes of these cells appear to be less malleable, however. While Shh signaling influenced astrocyte expression profiles and electrical behaviors, the cells’ morphologies remained largely unchanged.

The finding that astrocyte identity is considerably more plastic than previously thought is “exciting and interesting,” added Cagla, “but it remains to be seen what the exact function of this will be in terms of an animal’s behavior or its ability to learn.”

W.T. Farmer et al., “Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling,” Science, 351:849-54, 2016.



Neurons diversify astrocytes in the adult brain through sonic hedgehog signaling

Glial cell properties dictated by neurons

Neurons in the brain coexist with astrocytes, a type of glial cell, which help support many functions of their neighboring nerve cells. Farmer et al. now show that the support goes both ways (see the Perspective by Stevens and Muthukumar). They explored the influence of neurons on two specialized types of astrocytes in the mouse cerebellar cortex. The neurons produced the morphogen known as Sonic Hedgehog. Hedgehog signaling adjusted distinctive gene expression within the two astrocyte cell types. Thus, mature neurons appear to promote and maintain specific properties of associated astrocytes.

Science, this issue p. 849; see also p. 813


Astrocytes are specialized and heterogeneous cells that contribute to central nervous system function and homeostasis. However, the mechanisms that create and maintain differences among astrocytes and allow them to fulfill particular physiological roles remain poorly defined. We reveal that neurons actively determine the features of astrocytes in the healthy adult brain and define a role for neuron-derived sonic hedgehog (Shh) in regulating the molecular and functional profile of astrocytes. Thus, the molecular and physiological program of astrocytes is not hardwired during development but, rather, depends on cues from neurons that drive and sustain their specialized properties.


Sonic hedgehog regulates discrete populations of astrocytes in the adult mouse forebrain.

Astrocytes are an essential component of the CNS, and recent evidence points to an increasing diversity of their functions. Identifying molecular pathways that mediate distinct astrocyte functions, is key to understanding how the nervous system operates in the intact and pathological states. In this study, we demonstrate that the Hedgehog (Hh) pathway, well known for its roles in the developing CNS, is active in astrocytes of the mature mouse forebrain in vivo. Using multiple genetic approaches, we show that regionally distinct subsets of astrocytes receive Hh signaling, indicating a molecular diversity between specific astrocyte populations. Furthermore, we identified neurons as a source of Sonic hedgehog (Shh) in the adult forebrain, suggesting that Shh signaling is involved in neuron-astrocyte communication. Attenuation of Shh signaling in postnatal astrocytes by targeted removal of Smoothened, an obligate Shh coreceptor, resulted in upregulation of GFAP and cellular hypertrophy specifically in astrocyte populations regulated by Shh signaling. Collectively, our findings demonstrate a role for neuron-derived Shh in regulating specific populations of differentiated astrocytes.
Emerging evidence indicates that astrocytes play active and diverse roles in the central nervous system (CNS). Astrocytes actively regulate cerebral blood flow (Takano et al., 2006), and respond to sensory stimuli in both the visual and somatosensory cortices (Wang et al., 2006; Schummers et al., 2008). During development, astrocytes play key roles in regulating synapse formation and function (Ullian et al., 2001), and promote maturation of dendritic spines (Nishida and Okabe, 2007). In addition, bidirectional communication between astrocytes and neurons is an important element of synaptic transmission (Zhang et al., 2003; Araque, 2008). Although it is becoming increasingly clear that astrocytes actively contribute to normal CNS function, the underlying molecular mechanisms that mediate the functional properties of astrocytes remain poorly understood.
Several lines of evidence suggest that Shh signaling could play a role in astrocyte development and/or function. In the developing optic nerve, Shh mediates proliferation of astrocyte precursors (Wallace and Raff, 1999), and application of Shh agonists to early postnatal cortical astrocyte cultures upregulates Shh target genes (Atkinson et al., 2009). In addition, the transcription factors Gli1, 2, and 3, essential components of the Shh signaling pathway, are enriched in postnatal and adult astrocyte cultures (Cahoy et al., 2008). Moreover, a recent study suggests that reactive astrocytes produce and respond to Shh following cortical freeze injury (Amankulor et al., 2009). However, it remains unknown whether astrocytes in the mature CNS participate in Shh signaling in vivo, under normal physiological conditions. Transduction of Hh signaling is mediated by the Gli family of transcription factors, Gli1, 2, and 3. Binding of Shh to the transmembrane receptor Patched (Ptc) relieves constitutive inhibition of a second transmembrane receptor, Smoothened (Smo), thereby initiating a cascade of events leading to induction of Gli1 transcription (Fuccillo et al., 2006). Gli1 transcription in the embryo is dependent on Hh signaling through Gli2 and Gli3 (Bai et al., 2004). Thus, the presence of Gli1 can be used as an indicator of cells actively responding to high levels of Hh signaling (Bai et al., 2002). In this study, we show that regionally distinct populations of mature, differentiated astrocytes are the primary cell type responding to Shh in the adult forebrain. We further identified neurons as a source of Shh, suggesting a novel role for Shh signaling in neuronastrocyte communication. Finally, we demonstrate that conditional deletion of Smo in postnatal astrocytes results in a mild astrogliosis in the cortex, suggesting that Shh is an important regulator of specific astrocyte populations.
Gli1 is expressed in many non-proliferating cells in the adult forebrain In order to examine the distribution and identity of Hh-responding cells in the adult forebrain, we used Gli1nlacZ/+ mice in which nuclear lacZ is expressed from the Gli1 locus (Bai et al., 2002). In the developing neural tube, Gli1 is expressed predominantly in ventral populations of proliferating neuronal and oligodendrocyte precursors (Platt et al., 1997; Jessell, 2000). In contrast, we found that Gli1-expressing cells were distributed in the adult forebrain as far dorsally as the cortex, where they were localized primarily to layers 3, 4, and 5 (Fig. 1A). Dense populations of Gli1-expressing cells were also found in multiple basal forebrain nuclei, including the septum (data not shown) and globus pallidus, as well as in the thalamus, and hypothalamus (Fig. 1B–D). These results are consistent with a previous report that Ptc and Smo mRNA can be detected in the globus pallidus, thalamus and hypothalamus of the adult rat (Traiffort et al., 1999). However, whereas low levels of Ptc transcript were detected only in the piriform cortex (Traiffort et al., 1999), our findings indicate that cells in the entorhinal, motor and somatosensory cortex express Gli1, suggesting a more widespread cortical distribution of Hh-responding cells. Shh signaling plays a critical role in regulating adult neural stem and progenitor cells in the adult forebrain (Han et al., 2008). Although constitutive neurogenesis does not occur outside the subependymal zone (SEZ) and subgranular zone (SGZ), glial progenitors proliferate throughout the parenchyma of the adult CNS (Nishiyama et al., 2002; Dawson et al., 2003). To examine whether Gli1-expressing cells outside the neurogenic regions correspond to glial progenitors, we used a second line of mice in which an inducible Cre recombinase (CreERT2) is expressed from the Gli1 locus (Gli1CreER/+; Ahn and Joyner, 2005). When combined with the Rosa26loxP-STOP-loxP-lacZ reporter allele (R26lacZ; Soriano, 1999), cells expressing CreERT2 from the Gli1 locus express lacZ following tamoxifen administration. LacZ expression is permanent and heritable, and because CreER is active for only ~36 hours (Nakamura et al., 2006), expression of the βGal reporter protein becomes a permanent marker of cells that were expressing Gli1 at the time of tamoxifen administration. Moreover, because βGal expression is cytoplasmic, it is possible to examine the morphology of marked cells.
The absence of an apparent expansion of marked cells between 1 and 6 months posttamoxifen suggested that Gli1-expressing cells are not proliferating. Consistent with this, analysis of double staining for BrdU and βGal in the cortex showed that βGal-expressing cells were not double labeled with BrdU at all time points examined (Fig. 2G). As expected, double labeled cells were readily observed in the SEZ (Fig. 2H), corresponding to adult neural stem and progenitor cells (Ahn and Joyner, 2005). However, since BrdU labeled cells were only rarely observed in the cortex, we could not rule out the possibility that some Gli1- expressing cells divided, but escaped detection due to insufficient BrdU. We therefore used a more extensive BrdU labeling protocol that has been shown to label glial progenitor cells throughout the adult forebrain and spinal cord (Horner et al., 2000). Adult Gli1nlacZ/+ animals were given 50mg/kg BrdU for 12 days, and examined 3 days after the last BrdU injection. Despite a greater number of BrdU-labeled cells than in the previous experiments, none of the cortical BrdU-positive cells co-expressed βGal (n=148 cells, Supp. Fig. 1). Glial progenitors in the adult forebrain and spinal cord express the proteoglycan, NG2 (Horner et al., 2000). Analysis of βGal and NG2 double labeling showed that none of the βGalexpressing cells corresponded to NG2-positive glial progenitor cells (Supp. Fig. 1). The apparent increase in βGal-expressing cells between day 8 and 1 month post-tamoxifen in Gli1CreER;R26lacZ forebrains therefore cannot be due to proliferation of glial progenitors, but instead must be due to accumulation of βGal protein within cells. Taken together, these data suggest that, with the exception of adult neural stem and progenitor cells, the vast majority of Gli1-expressing cells in the adult forebrain are terminally differentiated. Moreover, the regional distribution of cells expressing Gli1 indicates that Hh signaling occurs in discrete cell populations throughout the dorsal/ventral and anterior/posterior axes.
Regionally distinct populations of astrocytes express Gli1
We next examined the cell types that express Gli1 in the adult forebrain. We primarily analyzed Gli1CreER;R26lacZ animals because the cytoplasmic localization of βGal permitted morphological analysis. In addition, since our previous experiments indicated that reporter expression stabilizes by 3 months post-tamoxifen, we restricted our analysis to adult Gli1CreER;R26lacZ animals given tamoxifen 3 months earlier. Throughout the cortex, Gli1- expressing cells exhibited small cell bodies with an elaborate branching morphology (Fig. 1I and 3B). The processes were highly ramified and very fine, creating a bushy appearance, consistent with the morphology of protoplasmic astrocytes. In addition, some labeled cells extended processes to nearby blood vessels (Fig. 3B), further suggesting that these cells correspond to astrocytes.
Interestingly, although the majority of Gli1-positive cells are astrocytes, not all astrocytes express Gli1. In addition, the proportion of astrocytes that express Gli1 differs between specific forebrain regions. In the cortex of tamoxifen-treated Gli1CreER;R26lacZ mice, only 24% of the S100β-positive cells were marked with βGal (n=1932 cells), whereas 56% (n=1233 cells) and 80% (n=1489 cells) of S100β-expressing cells in the globus pallidus and hypothalamus respectively, were βGal-positive (Fig. 4). In contrast, the caudate putamen and area CA1 of the hippocampus exhibit few βGal-positive cells (Fig. 1), indicating that the vast majority of astrocytes in these regions do not express Gli1. Similarly, white matter astrocytes do not express Gli1, as indicated by the lack of βGal staining in the corpus callosum, anterior commissure, and fimbria (Fig. 1F–J).
Gli1 is a sensitive readout of Shh in the adult forebrain
Although transcriptional activation of Gli1 has been shown to be a reliable readout of Hh signaling in the developing CNS (Lee et al., 1997), it is possible that signaling pathways other than Hh might activate Gli1 transcription in the adult. In order to address whether Hh signaling is responsible for Gli1 expression in adult forebrain astrocytes, we took several approaches.



Astrocytes in Gli1-expressing regions of the forebrain show signs of mild reactive gliosis following postnatal interruption of Shh signaling
During development, Shh plays a critical role in specification of neuronal and oligodendrocyte precursors (Jessell, 2000). Shh has also been implicated in regulating proliferation of astrocytes in the developing optic nerve (Wallace and Raff, 1999; Dakubo et al., 2008).

Upregulation of GFAP and hypertrophy are hallmarks of reactive astrogliosis, which occurs in response to injury or disease (Eng and Ghirnikar, 1994). Reactive astrocytes have also been shown to undergo increased proliferation and synthesis of nestin and vimentin (Sofroniew, 2009). However BrdU labeling showed no change in proliferation between mutants and controls (data not shown), and staining for nestin and vimentin showed no change in expression of these intermediate filaments (data not shown). Taken together, these data show that the cellular response to interrupting Shh signaling includes key characteristics of reactive astrogliosis and indicates that Shh signaling plays an important role in mediating intracellular properties of specific astrocyte populations.


In this study, we show that high level Shh signaling in the adult CNS occurs in regionally distinct populations of mature, differentiated astrocytes. Our data demonstrate that neurons are a source of Shh, suggesting a novel signaling pathway involved in direct neuronastrocyte communication. Furthermore, we provide evidence that Shh signaling is required to maintain normal cellular functions in specific astrocyte populations. Taken together, our data are the first to demonstrate a critical role for Shh signaling in neuron-astrocyte communication in vivo, in the adult CNS.


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Astrocytes that express Gli1 are differentially distributed between specific forebrain regions

(A–C) Double labeling immunohistochemistry for βGal (red) and S100β (green) in the cortex (A), globus pallidus (B), and hypothalamus (C) of adult Gli1CreER/+;R26lacZ animals given tamoxifen 3 months earlier. Counterstained with DAPI (blue). Scale bar, 25μm. (D) Quantitative analysis of the proportion of S100β-positive astrocytes within the cortex, globus pallidus, and hypothalamus that express Gli1. (mean ± SEM, n300 cells/region/animal, from 3 animals, *p < 0.05, **p < 0.01, one-way, repeated measures ANOVA and Tukey’s post-test.)


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Neurogenesis in the embryonic and adult brain: same regulators, different roles

Front. Cell. Neurosci., 27 November 2014 |

Neurogenesis persists in adult mammals in specific brain areas, known as neurogenic niches. Adult neurogenesis is highly dynamic and is modulated by multiple physiological stimuli and pathological states. There is a strong interest in understanding how this process is regulated, particularly since active neuronal production has been demonstrated in both the hippocampus and the subventricular zone (SVZ) of adult humans. The molecular mechanisms that control neurogenesis have been extensively studied during embryonic development. Therefore, we have a broad knowledge of the intrinsic factors and extracellular signaling pathways driving proliferation and differentiation of embryonic neural precursors. Many of these factors also play important roles during adult neurogenesis, but essential differences exist in the biological responses of neural precursors in the embryonic and adult contexts. Because adult neural stem cells (NSCs) are normally found in a quiescent state, regulatory pathways can affect adult neurogenesis in ways that have no clear counterpart during embryogenesis. BMP signaling, for instance, regulates NSC behavior both during embryonic and adult neurogenesis. However, this pathway maintains stem cell proliferation in the embryo, while it promotes quiescence to prevent stem cell exhaustion in the adult brain. In this review, we will compare and contrast the functions of transcription factors (TFs) and other regulatory molecules in the embryonic brain and in adult neurogenic regions of the adult brain in the mouse, with a special focus on the hippocampal niche and on the regulation of the balance between quiescence and activation of adult NSCs in this region.

Neural stem cells (NSCs) in the embryonic and early postnatal murine brain generate neurons and glia, including astrocytes and oligodendrocytes. The transition of proliferative and multipotent NSCs to fully differentiated neurons and glia is called neurogenesis and gliogenesis, respectively. Neurons are generated from early embryonic development until early postnatal stages, with only a few neurogenic zones remaining active in the adult (Götz and Huttner, 2005; Ming and Song, 2011;Paridaen and Huttner, 2014). In contrast, gliogenesis starts during late embryogenesis and continues in postnatal stages, with low but widespread production of both astrocytes and oligodendrocytes also occurring throughout the adult brain (Rowitch and Kriegstein, 2010; Gallo and Deneen, 2014; Guérout et al., 2014). The main neurogenic regions in the adult murine brain are the subependymal zone of the lateral ventricles, also called ventricular-subventricular Zone (V-SVZ) and the subgranular zone (SGZ) of the dentate gyrus (DG) in the hippocampus (Altman and Das, 1965; Doetsch et al., 1999; Ming and Song, 2011;Fuentealba et al., 2012). Both of these neurogenic regions have been shown to also be active in the adult human brain, with the V-SVZ thought to contribute new neurons to the striatum (whereas it produces neurons migrating to the olfactory bulb in mice) and the SGZ contributing neurons to the DG (Eriksson et al., 1998; Spalding et al., 2013; Ernst et al., 2014). The addition of new neurons to the complex circuitry of the adult brain is the focus of intensive research, which is uncovering crucial functions for the newly generated neurons in memory and behavior (Deng et al., 2010). In particular, the integration of adult-born granule cells to the hippocampus circuitry confers an extra degree of plasticity that is crucial for the acquisition of certain types of contextual memory (Jessberger et al., 2009; Sahay et al., 2011). Although adult neurogenesis is an ancient trait, with widespread neurogenesis occurring, for instance, in 16 different adult brain areas of zebrafish, the appearance of the DG as a structural and functional unit seems exclusive to mammals (Treves et al., 2008; Grandel and Brand, 2013). This fact, amongst others, has prompted the idea that hippocampal neurogenesis might be a newly evolved trait in some species, including humans, aimed to enhance adaptation to a continuously changing environment (Kempermann, 2012).

Significant advances have been made in our understanding of the regulation of mouse adult hippocampal neurogenesis in the last few years. Thus, our focus for the rest of the review will be on the mouse model of neurogenesis. The coordinated action of multiple signals acting on embryonic NSCs gives rise to the vast diversity of neuronal and glial populations that populate the mature brain. Embryonic neurogenesis is, thus, tightly linked to cell fate specification. In adult neurogenic regions, however, stem cells are tightly restricted to the generation of one (granule neurons of the DG) or a few types of neurons (granule neurons and periglomerular neurons in the V-SVZ) (Zhao et al., 2008; Ming and Song, 2011). Therefore signals and factors that specify subtype identities during development can control more subtle aspects of adult stem cell behavior.

In recent years, it has become evident that, at the single cell level, stem cells in the embryonic and the adult brain are not as versatile as previously thought. Instead of their classically attributed multipotency, they appear to be already committed to the generation of specific types of neural cells (Taverna et al., 2014). The causes and functions of the emerging heterogeneity of adult NSCs are among the most exciting questions remaining to be addressed in the field (DeCarolis et al., 2013; Encinas et al., 2013; Giachino et al., 2014b). In the case of the murine V-SVZ, different populations of adult NSCs, also called type-B cells, co-exist and give rise to distinct types of periglomerular cells and granule cells in the olfactory bulb. Different adult NSCs are characterized by the differential expression of specific transcription factors (TFs), including Nkx2.1, Pax6, Gsx2 and Nkx6.2, which also pattern the different domains of the embryonic telencephalon (Merkle et al., 2007; Brill et al., 2008;López-Juárez et al., 2013; Merkle et al., 2014). The distinct adult NSC populations are located in different regions along the V-SVZ and their distinct properties are acquired during development (Obernier et al., 2014). Despite the spatial separation of these stem cell populations, all their progeny follow the same long migratory path, the rostral migratory stream (RMS), towards their final destination in the olfactory bulb. In the hippocampus, adult NSCs, also called type-I cells or radial glial-like cells, generate exclusively granule neurons in the DG. The migration of granule neurons is very limited, as they settle, differentiate and integrate into the hippocampal circuitry in the granule cell layer (GCL) located just above the NSC from which they originated in the SGZ. While they appear uniform, adult NSCs in the DG respond to diverse and complex signals, raising the possibility that they are functionally heterogeneous.

Despite their many differences, adult NSCs in the two adult neurogenic niches share several key characteristics. Neural stem cells in both V-SVZ and SGZ, like radial glial stem cells in the embryo, express the molecules GFAP, Nestin and Sox2 and they directly contact blood vessels. Both NSC populations share a restricted potential, as just discussed, with each generating a unique neuronal subtype and one type of glia: in the V-SVZ they generate neurons and oligodendrocytes, while in the SGZ they generate neurons and astrocytes. Perhaps the two characteristics that distinguish adult NSCs most clearly from their embryonic counterparts are the acquisition of quiescence and their situation in a complex and stable cellular niche. While one of the main features of embryonic NSCs is their high proliferative rate, the opposite is true for adult NSCs, which remain for long periods out of the cell cycle, in G0. This is a characteristic that adult NSCs share with many stem cells in other mature tissues and one that is crucial to maintain tissue homeostasis and avoid stem cell exhaustion (Orford and Scadden, 2008;Simons and Clevers, 2011). The existence of adult neurogenic niches (complex cellular microenvironments surrounding adult NSCs) is also a characteristic shared with other tissues (Fuchs et al., 2004; Kuang et al., 2008; Mirzadeh et al., 2008; Ming and Song, 2011; Fuentealba et al., 2012; Goldstein and Horsley, 2012). The niche is comprised of diverse cell types and structures, such as astrocytes, neurons, axon projections and blood vessels, and one of its main functions is to create an appropriate environment that keeps the majority of stem cells quiescent and undifferentiated (Morrison and Spradling, 2008). The niche also provides a great variety of signals that modulate the behavior of adult stem cells and adjust the production of new cells to the needs of the tissue (Fuchs et al., 2004; Blank et al., 2008; Faigle and Song, 2013).

Embryonic and Adult Origin of Granule Cells

From a developmental point of view, the generation of the DG is unique. While the V-SVZ is seen as a continuation of the embryonic ventricular zone (VZ) of the telencephalon, the formation of the DG involves the generation of a dedicated progenitor cell source away from the VZ and in close proximity to the pial surface. This additional proliferative zone remains active during postnatal stages and eventually becomes the SGZ, which is the site of adult hippocampal neurogenesis (Figure 1;Bayer, 1980a,b; Altman and Bayer, 1990; Pleasure et al., 2000; Khalaf-Nazzal and Francis, 2013; Sugiyama et al., 2013).

Development of the mouse hippocampus. Schematic representation of the dorsal telencephalon at different embryonic (E) stages and at birth (P0). The indicated area in each picture corresponds to the hippocampal region and is magnified on its right handside (blue squares). (A) At E12.5 the presumptive DNE is located between the HNE and the CH, which produces Cajal-Retzius cells (orange), shown lining the pial side of the cortex. (B) At E14.5 dentate precursors of the primary matrix (dark blue circles) are located in the VZ, and precursor cells start to migrate towards the pial side of the cortex forming the secondary matrix. In the VZ of the HNE, radial glial precursors (depicted in dark blue and triangular body shape) will give rise to hippocampal neurons. (C) At E17.5 the hippocampal fissure is formed and dentate precursor cells migrate to and accumulate there, forming the tertiary matrix (light blue). Cajal-Retzius cells are also present and follow the hippocampal fissure. At this stage the glial scaffold (not shown) extends from the CH to the hippocampal fissure and pial surface, directing the migration of dentate precursor cells. From the HNE, hippocampal neurons (red triangles) are born and migrate along radial glial cells towards their location in the hippocampal fields (CA1 and CA3 are shown). (D) At birth the blades of the DG start to form. Granule neurons in the DG (red triangles) appear first in the upper blade, below the hippocampal fissure. The continuous migration of Cajal-Retzius cells reaches the pial side and promotes the formation of the lower blade of the DG. Precursor cells in the primary and secondary matrix will soon disappear, but cells in the tertiary matrix continue actively dividing and producing granule neurons through postnatal DG development. HNE, hippocampal neuroepithelium; DNE, dentate neuroepithelium; CH, cortical hem; VZ, ventricular zone; 1ry, primary matrix; 2ry, secondary matrix; 3ry, tertiary matrix; DG, dentate gyrus; D, dorsal; M, medial; V, ventral; L, lateral.

Regulation of Adult Neurogenesis

The late maturation of the hippocampus, which spans late embryonic and early postnatal stages, means that the process of DG formation and the appearance of NSCs with adult characteristics are overlapping processes. It can therefore be difficult to distinguish between developmental and adult cues regulating hippocampal neurogenesis. However, several physiological and pathological situations, such as physical exercise, task learning, an enriched environment and seizures, have been shown to stimulate neurogenesis specifically in the adult DG (Rolando and Taylor, 2014). Although no direct link has been clearly established between those external stimuli and signaling pathways, numerous extracellular signaling molecules, including Bone Morphogenetic Proteins (BMPs), Notch, GABA, WNT, insulin growth factors (IGFs) and SHH, have been shown to regulate the rate of neurogenesis in the adult DG (Ming and Song, 2011; Faigle and Song, 2013). However, due to limitations of in vivo studies, little is known about the mechanisms by which these signals exert their effects. In the adult DG, NSCs generate granule cells via a well characterized cell lineage that includes a succession of transit amplifying or intermediate progenitor cells (IPCs), characterized by rapid divisions and the expression of a series of neurogenic TFs (Figure 2; Hsieh, 2012). Extrinsic stimuli can affect the proliferation and survival of NSCs but also of IPCs (typeIIa and typeIIb) or differentiating neuroblasts (typeIII) further along the lineage (Figure 2). The selective death of IPCs, for instance, is a major mechanism of regulation of neurogenesis in the DG, with as many as two thirds of these cells being actively eliminated by microglia (Sierra et al., 2010, 2014). Therefore, in order to understand the effects of signaling pathways and intrinsic factors on neurogenesis, it is crucial to determine the stages in the adult neurogenic lineage at which they act, and the cellular processes they regulate. In fact, one of the main difficulties faced by the adult neurogenesis field concerns the scarcity of markers for adult NSCs, which are often shared by other cell types (for instance, GFAP marks subpopulations of astrocytes and Nestin is expressed by early intermediate progenitors). This problem is only more evident in the case of distinguishing quiescent from activated adult NSCs, in which case there is an absolute lack of specific markers apart from the use of cell cycle genes. This issue has been partly addressed in a recent report in which an unbiased approach was used to identify genes differentially expressed by activated and quiescent adult NSCs isolated from the V-SVZ (Codega et al., 2014). This work demonstrates that the quiescent state is a much more complex state than simply the lack of proliferation markers, as the list of differentially expressed genes is enriched in genes related to very diverse cellular processes, such as lipids metabolism, signaling or adhesion. This quiescence signature is shared by adult quiescent stem cells from other organs, such as the blood, muscle or intestine (Cheung and Rando, 2013; Codega et al., 2014). It is thus likely that many of the general characteristics of quiescent stem cells will be shared between DG and SVZ, although no studies on the expression profile of adult DG NSCs have been performed to date.

Ageing of the brain is marked by a major decrease in the number of new neurons generated in the DG. This decrease has been attributed both to a reduction of the NSC pool and to an increased state of quiescence of the remaining stem cells (Lugert et al., 2010; Encinas et al., 2011; Jaskelioff et al., 2011; Seib et al., 2013). The possibility to increase neurogenesis in ageing mice by activating the quiescent stem cell pool is currently the focus of intensive research. In this regard, it was recently shown that systemic factors from young animals can re-activate neurogenesis in aged mice (Katsimpardi et al., 2014). However, disruption of quiescence signals can lead to a short-lived increase in neurogenesis, followed by a sharp decrease caused by a loss of quiescent NSCs (Ehm et al., 2010; Mira et al., 2010; Song et al., 2012). Assessing precisely how factors and signals affect stem cell behavior will be vital to understand their long-term effects on adult neurogenesis. Lineage tracing and particularly clonal analysis of NSCs in the DG have begun to provide evidence of the great diversity of responses of adult NSCs to stimuli, which can affect both their proliferation and differentiation potentials (Bonaguidi et al., 2011; Dranovsky et al., 2011; Song et al., 2012).

Notch Signaling

The functions of Notch signaling during embryonic brain development have been extensively reviewed elsewhere (Kageyama et al., 2008; Imayoshi and Kageyama, 2011). During development of the hippocampus, Notch does not seem to be involved in neural precursor specification or differentiation, but rather in broader decisions, including the regulation of neural lineage commitment, the tempo of neuronal and glial generation and the maintenance of stem cells. Notch receptors and ligands are broadly expressed during all stages of development of the hippocampus (Pleasure et al., 2000). Loss of the essential Notch signaling component RBPJk in the developing brain results in proliferation defects and premature differentiation of embryonic NSCs (Imayoshi et al., 2010). Similarly, loss of RBPJk or of the Notch ligand Jagged1 during hippocampal development leads to defects in proliferation and stem cell maintenance, although the formation of the DG is not prevented (Breunig et al., 2007; Lavado and Oliver, 2014). Therefore, the main function of the Notch pathway in embryonic NSCs is to maintain their proliferative and undifferentiated state.

Other Signaling Pathways: SHH, IGF and Neurotransmitters

We will briefly discuss here the roles of other key signaling pathways for which specific roles in hippocampal development or in adult neurogenesis have not been reported.

Sonic Hedgehog signaling has crucial roles in early patterning and cell fate specification in the embryonic brain. Recently, NSCs in the adult DG have been shown to originate from SHH-responsive progenitors in the ventral hippocampus (Li et al., 2013). Sonic Hedgehog signaling has been implicated in the proliferation and maintenance of both DG and V-SVZ adult NSCs (Machold et al., 2003; Álvarez-Buylla and Ihrie, 2014). Although the sources of SHH that regulate V-SVZ and SGZ neurogenesis have not been clearly identified yet, tracing the activity of SHH by the expression of the SHH-inducible geneGli1 in Gli1nLacZ mice has shown that NSCs in both adult neurogenic regions as well as a fraction of mature astrocytes express the beta galactosidase reporter protein and therefore receive SHH signals (Ahn and Joyner, 2005; Garcia et al., 2010;Ihrie et al., 2011; Petrova et al., 2013). Removal of SHH signaling from V-SVZ stem cells by deletion of the receptor Smoothened has revealed that SHH is necessary for the proliferation and long term maintenance of the stem cells, as well as the subtype specification of the neurons they generate (Palma et al., 2005; Balordi and Fishell, 2007; Kim et al., 2007; Ihrie et al., 2011; Petrova et al., 2013; Merkle et al., 2014). In adult DG stem cells, conditional disruption of primary cilia, which are required for SHH signaling, decreases the production of IPCs, supporting a role for SHH in NSC divisions in the DG as well (Breunig et al., 2008; Amador-Arjona et al., 2011). However, a more direct investigation of the role of SHH in adult DG neurogenesis has not yet been performed.

The Transition from Postnatal to Adult Neurogenesis: NFIX, Tlx, CcnD2 and Ascl1

Granule neurons in the adult DG are exclusively generated by NSCs located in the SGZ. During embryonic and postnatal development, in contrast, neurons are generated by a heterogeneous population of precursor cells in the dentate matrices (Figures 1, 2). The exact time at which the switch from embryonic to adult modes of neurogenesis occurs in the DG is still not well defined. Several independent pieces of evidence suggest that this happens around the second week of life in mice. At postnatal day 14 (P14), the blades of the DG are already formed and the source of new neurons in the DG becomes restricted to the tertiary matrix, which gradually becomes the SGZ (Pleasure et al., 2000; Sugiyama et al., 2013). At the same time, the first presumptive GFAP- and Nestin-positive NSCs adopt their characteristic location, with the nucleus residing in the SGZ and the basal process extending through the GCL (Li and Pleasure, 2005; Martynoga et al., 2013).


Adult neurogenic niches can be conceptualized as remnants of embryonic signaling centers (i.e., the septum/antihem giving rise to the V-SVZ and the CH generating the SGZ): they are the source of instructive signals that determine the fate of neighboring stem cells. However, in contrast with stem cells in the developing brain that must cope with a continuously changing environment, adult stem cells are surrounded by a relatively stable niche. The V-SVZ and the SGZ niches share many common features. However, while the cellular and molecular composition of the V-SVZ niche has been relatively well investigated, we lack a similar level of understanding of the SGZ niche. Further studies of the signals and cellular interactions that control NSC behavior in the DG will be required before we can appreciate the similarities and divergences in the regulation and function of stem cells in the two adult neurogenic niches (Figure 3).

Niche regulation of mouse adult stem cells in the dentate gyrus. (A) Representation of a neural stem cell (blue) in the adult subgranular zone of the dentate gyrus and some of its interactions with the niche. Granule neurons (yellow), interneurons (red), intermediate precursors (green) and astrocytes (purple) are shown providing quiescence cues, while blood vessels and astrocytes are shown providing activation cues. (B) How quiescence and activation signals are interpreted by adult stem cells is still not known. Here we show several intracellular factors that have been linked to the quiescent (left, Hes5, p. 57, FoxO3 and REST) or active (right, Tlx, Ascl1 and CcnD2) state of stem cells in the adult DG. We also show other factors expressed in NSCs with no clear function in the switch from quiescence to activation (Sox2, Pax6, GFAP and GLAST) in the central part of the schematized cell.

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Neural stem cells aging

Larry H. Bernstein, MD, FCAP, Curator



Aging Stem Cells Provide Clues into Tumor Development

With the enzyme Eyeless knocked out, cells over-proliferate (shown in green) in a fruit fly’s larval brain during reactivated Notch signaling. [University of Oregon]

Investigators at the University of Oregon (UO) have recently uncovered molecular events experienced by stem cells as they age, which could provide new avenues toward the discovery of novel therapies for cancer and neurological disorders. The researchers noticed that these changes arise in Drosophila during the development of the central nervous and at that time a specific protein is expressed, blocking tumor formation.

The UO researchers were focused on the larval stage of fruit fly development, as this is when stem cells generate most of the neurons that form the adult’s brain. During this process the stem cells are rapidly dividing in order to populate the central nervous system of the fly, and they rely heavily on the Notch signaling pathway—a developmentally important signal transduction pathway that has also been linked to cancer.

In previous studies, scientists have described scenarios where Notch signaling ran efficiently and stem cells produce neurons that populate the adult central nervous system. However, with too much Notch, stem cells lose control and over-proliferate—forming large tumors. In humans, adult T-cell leukemia is tied to overactive Notch signaling.

“Stem cells have a really tough job because they have to divide to make the millions of neurons in our brain,” explained Howard Hughes Medical Institute Investigator and senior author Chris Doe, Ph.D., professor of biology at the UO. “If they don’t divide enough, it results in microcephaly or other small brain diseases, but if they divide too much, they make tumors. They have to stay right on that boundary of dividing to make neurons but not dividing excessively and forming a tumor. It’s really walking a tightrope.”

The findings from this study were published recently in Current Biology through an article entitled “Aging Neural Progenitors Lose Competence to Respond to Mitogenic Notch Signaling.”

In the current study, the scientists discovered that if they waited for stem cells to divide a few times and age a bit, they quit responding to Notch. Moreover, the stem cells could not be pushed by high doses of Notch signaling to form tumors.

As the UO researchers looked closer, they uncovered a host of age-related molecular changes. As the stem cells get older and around the same time they begin to resist tumor formation, the stem cells begin expressing a transcription factor protein, known as Eyeless in Drosophila and Pax6 in humans. Its presence blocks Notch signaling.

Dr. Doe and his team described the genetic knockout of Eyeless in these stem cells, which led Notch signaling to overwhelm the precise growth balance and form tumors within the fruit flies.

“If we can identify the stem cells that are relied upon during development, maybe we could find a way to use them later to recreate conditions that might be therapeutic,” noted Dylan Farnsworth, a doctoral candidate at the UO. “If you do it incorrectly, you risk over-proliferation and the development of masses—and cancer.”

“This paper shows that Eyeless is important for winding down the lifespan of the stem cells that are giving rise to the adult brain,” Dr. Doe added. “It’s a stop signal that says it is time to cease responding to Notch signals.”

The UO researchers were excited by their findings and believe that with more extensive research, their system could provide a roadmap for fine-tuning the timing of stem cell-based therapies to restart healthy activity in adult stem cells.


Aging Neural Progenitors Lose Competence to Respond to Mitogenic Notch Signaling

Dylan R. Farnsworth, Omer Ali Bayraktar, and Chris Q. Doe
Cell 7 Dec 2015; 25(23):3058–3068  DOI:


  • Aging INPs lose competence to respond to constitutively active Notch signaling
  • The late temporal factor Eyeless blocks Notch-induced target gene expression
  • Eyeless blocks Notch-induced INP tumor formation


Drosophila neural stem cells (neuroblasts) are a powerful model system for investigating stem cell self-renewal, specification of temporal identity, and progressive restriction in competence. Notch signaling is a conserved cue that is an important determinant of cell fate in many contexts across animal development; for example, mammalian T cell differentiation in the thymus and neuroblast specification in Drosophila are both regulated by Notch signaling. However, Notch also functions as a mitogen, and constitutive Notch signaling potentiates T cell leukemia as well as Drosophila neuroblast tumors. While the role of Notch signaling has been studied in these and other cell types, it remains unclear how stem cells and progenitors change competence to respond to Notch over time. Notch is required in type II neuroblasts for normal development of their transit amplifying progeny, intermediate neural progenitors (INPs). Here, we find that aging INPs lose competence to respond to constitutively active Notch signaling. Moreover, we show that reducing the levels of the old INP temporal transcription factor Eyeless/Pax6 allows Notch signaling to promote the de-differentiation of INP progeny into ectopic INPs, thereby creating a proliferative mass of ectopic progenitors in the brain. These findings provide a new system for studying progenitor competence and identify a novel role for the conserved transcription factor Eyeless/Pax6 in blocking Notch signaling during development.

Supplemental Information

Figure S1, related to Figure 1. Notchintra is nuclear and at qualitatively similar levels when expressed in young or old INP lineages. (A-C’) R9D11-gal4, R16B06-gal and OK107-gal4 driving UAS-Notchintra result in efficient Notchintra protein expression, as visualized by antibody staining. Yellow dashed outlines show INP lineages in central brain labeled by each driver. Scale bar = 10 µm

Figure S2, related to Figure 3. R16B06-gal4 labels old INPs in third instar larval brains. (A) Expression pattern of R16B06-gal4 in both brain lobes of third instar larva. R9D11-gal4 marks young INPs and is shown for comparison. (B-B’’) High magnification images show Dpn+ INPs distal to their parental Type II NB (white dashed line) are labeled by R16B06-gal4. Arrow indicates direction of age progression in lineage. (C-C’’’) Distal INPs (yellow dashed line) labeled by R16B06-gal4 express the old INP specific transcription factor Eyeless (Ey) but not the young INP specific transcription factor Dichaete (D). Images are a single, one micron plane. All panels show third instar larvae; scale bar = 10 µm

Figure S3, related to Figure 7. Notchintra signaling can induce expression of the Notch response element reporter (NRE-PGR) in old INPs but not in GMCs. (A) In wild type, the NRE reporter is expressed at high levels in Type II neuroblasts (arrowhead) and shows progressively weaker levels in the progeny. There are low levels in old INPs (dashed yellow outline; identified by Ey expression), but is not detectable in Prospero (Pros)+ GMCs (arrow). (B) Expression of Notchintra in the old INPs and their progeny using R16B06-gal4 results in elevated expression of the NRE reporter (dashed yellow outline; compare to level in adjacent cells); only Dpn+ INPs show elevated levels of the reporter, Prospero (Pros)+ GMCs show no detectable expression. (C-C’’’) The Notch target E(spl)mγ is expressed in both young and old INPs. (C) Young INPs expressing Dichaete (small white circles) and (C’) old INPs expressing Eyeless (small dashed yellow circles) are positive for Deadpan and the Notch target E(spl)mγ- GFP fusion reporter. Asterisk marks Type II NB, arrow indicates direction of lineage from young to old. All panels show third instar larvae; scale bar = 10 µm.

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Heroes in Medical Research: Developing Models for Cancer Research

Author, Curator: Stephen J. Williams, Ph.D.


The current rapid progress in cancer research would have never come about if not for the dedication of past researchers who had developed many of the scientific tools we use today. In this issue of Heroes in Medical Research I would like to give tribute to the researchers who had developed the some of the in-vivo and in-vitro models which are critical for cancer research.


The Animal Modelers in Cancer Research

Helen Dean King, Ph.D. (1869-1955)

Helen Dean King

Helen Dean King, Ph.D. from; photo Courtesy of the Wistar Institute Archive Collection, Philadelphia, PA



The work of Dr. Helen Dean King on rat inbreeding led to development of strains of laboratory animals. Dr. King taught at Bryn Mawr College, then worked at University of Pennsylvania and the Wistar Institute under famed geneticist Thomas Hunt Morgan, researching if inbreeding would produce harmful genetic traits.   At University of Pennsylvania she examined environmental and genetic factors on gender determination.





Important papers include [1-6]as well as the following contributions:

“Studies in Inbreeding”, “Life Processes in Gray Norway Rats During Fourteen Years in Captivity”, doctoral thesis on embryologic development in toads (1899)


Milestones include:


1909    started albino rat breeding and bred 20 female and male from same litter (King colony) to 25

successive generations (inbreeding did not cause harmful traits)


1919     started to domesticate the wild Norwegian rats that ran thru Philadelphia (six pairs Norway rats

thru 28 generations)

A good reference for definitions of rat inbreeding versus line generation including a history of Dr. King’s work can be found at the site: Munificent Mischief Rattery and a brief history here.[7] In addition, Dr. King had investigated using rat strains as a possible recipient for tumor cells. The work was an important advent to the use of immunodeficient models for cancer research.


As shown below Philadelphia became a hotbed for research into embryology, development, genetics, and animal model development.


Beatrice Mintz, Ph.D.

(Beatrice Minz, Ph.D.; photo credit Fox Chase Cancer Center, Mintz

Dr. Mintz, an embryologist and cancer researcher from Fox Chase Cancer Center in Philadelphia, PA, contributed some of the most seminal discoveries leading to our current understanding of genetics, embryo development, cellular differentiation, and oncogenesis, especially melanoma, while pioneering techniques which allowed the development of genetically modified mice.

If you get the privilege of hearing her talk, take advantage of it. Dr. Mintz is one of those brilliant scientists who have the ability to look at a clinical problem from the viewpoint of a basic biological question and, at the same time, has the ability to approach the well-thought out questions with equally well thought out experimental design. For example, Dr. Mintz asked if a cell’s developmental fate was affected by location in the embryo. This led to her work by showing teratocarcinoma tumor cells in the developing embryo could revert to a more normal phenotype, essentially proving two important concepts in development and tumor biology:

  1. The existence of pluripotent stem cells
  2. That tumor cells are affected by their environment (which led to future concepts of the importance of tumor microenvironment on tumor growth

Other seminal discoveries included:

  • Development of the first mouse chimeras using novel cell fusion techniques
  • With Rudolf Jaenisch in 1974, showed integration of viral DNA from SV40, could be integrated into the DNA of developing mice and persist into adulthood somatic cells, the first transgenesis in mice which led ultimately to:
  • Development of the first genetically modified mouse model of human melanoma in 1993

Her current work, seen on the faculty webpage here, is developing mice with predisposition to melanoma to uncover risk factors associated with the early development of melanoma.

In keeping with the Philadelphia tradition another major mouse model which became seminal to cancer drug discovery was co-developed in the same city, same institute and described in the next section.

It is interesting to note that the first cloning of an animal, a frog, had taken place at the Institute for Cancer Research, later becoming Fox Chase Cancer Center, which was performed by Drs. Robert Briggs and Thomas J. King and reported in the 152 PNAS paper Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs.[8]


 The Immunodeficient Animal as a Model System for Cancer Research – Dr. Mel Bosma, Ph.D.



Melvin J. Bosma, Ph.D.; photo credit Fox Chase Cancer Center

In the summer of 1980 at Fox Chase Cancer Center, Dr. Melvin J. Bosma and his co-researcher wife Gayle discovered mice with deficiencies in common circulating antibodies and since, these mice were littermates, realized they had found a genetic defect which rendered the mice immunodeficient (upon further investigation these mice were unable to produce mature B and T cells). These mice were the first scid (severe combined immunodeficiency) colony. The scid phenotype was later found to be a result of a spontaneous mutation in the enzyme Prkdc {protein kinase, DNA activated, catalytic polypeptide} involved in DNA repair, and ultimately led to a defect in V(D)J recombination of immunoglobulins.

The emergence of this scid mouse was not only crucial for AIDS research but was another turning point in cancer research , as researchers now had a robust in-vivo recipient for human tumor cells. The orthotopic xenograft of human tumor cells now allowed for studies on genetic and microenvironmental factors affecting tumorigenicity, as well as providing a model for chemotherapeutic drug development (see Suggitt for review and references)[9]. A discussion of the pros and cons of the xenograft system for cancer drug discovery would be too voluminous for this post and would warrant a full review by itself. But before the advent of such scid mouse systems researchers relied on spontaneous and syngeneic mouse tumor models such as the B16 mouse melanoma and Lewis lung tumor model.

Other scid systems have been developed such as in the dog, horse, and pig. Please see the following post on this site The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research. The athymic (nude) mouse (nu/nu) also is a popular immunodeficient mouse model used for cancer research

Two other in-vivo tumor models: Patient Derived Xenografts (PDX) and Genetically Engineered Mouse models (GEM) deserve their own separate discussion however the success of these new models can be attributed to the hard work of the aforementioned investigators. Therefore I will post separately and curate PDX and GEM models of cancer and highlight some new models which are having great impact on cancer drug development.



1.         Loeb L, King HD: Transplantation and Individuality Differential in Strains of Inbred Rats. The American journal of pathology 1927, 3(2):143-167.

2.         Lewis MR, Aptekman PM, King HD: Retarding action of adrenal gland on growth of sarcoma grafts in rats. J Immunol 1949, 61(4):315-319.

3.         Aptekman PM, Lewis MR, King HD: Tumor-immunity induced in rats by subcutaneous injection of tumor extract. J Immunol 1949, 63(4):435-440.

4.         Lewis MR, Aptekman PM, King HD: Inactivation of malignant tissue in tumor-immune rats. J Immunol 1949, 61(4):321-326.

5.         Lewis MR, King HD, et al.: Further studies on oncolysis and tumor immunity in rats. J Immunol 1948, 60(4):517-528.

6.         Aptekman PM, Lewis MR, King HD: A method of producing in inbred albino rats a high percentage of immunity from tumors native in their strain. J Immunol 1946, 52:77-86.

7.         Ogilvie MB: Inbreeding, eugenics, and Helen Dean King (1869-1955). Journal of the history of biology 2007, 40(3):467-507.

8.         Briggs R, King TJ: Transplantation of Living Nuclei From Blastula Cells into Enucleated Frogs’ Eggs. Proceedings of the National Academy of Sciences of the United States of America 1952, 38(5):455-463.

9.         Suggitt M, Bibby MC: 50 years of preclinical anticancer drug screening: empirical to target-driven approaches. Clinical cancer research : an official journal of the American Association for Cancer Research 2005, 11(3):971-981.


Other posts on this site about Cancer, Animal Models of Disease, and other articles in this series include:

The SCID Pig: How Pigs are becoming a Great Alternate Model for Cancer Research

A Synthesis of the Beauty and Complexity of How We View Cancer

Guidelines for the welfare and use of animals in cancer research

Importance of Funding Replication Studies: NIH on Credibility of Basic Biomedical Studies

FDA Guidelines For Developmental and Reproductive Toxicology (DART) Studies for Small Molecules

Report on the Fall Mid-Atlantic Society of Toxicology Meeting “Reproductive Toxicology of Biologics: Challenges and Considerations:

What`s new in pancreatic cancer research and treatment?

Heroes in Medical Research: Dr. Carmine Paul Bianchi Pharmacologist, Leader, and Mentor

Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer

Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension

Reuben Shaw, Ph.D., a geneticist and researcher at the Salk Institute: Metabolism Influences Cancer


Read Full Post »



The immune response mechanism is the holy grail of the human defense system for health.   IDO, indolamine 2, 3-dioxygenase, is a key gene for homeostasis of immune responses and producing an enzyme catabolizing the first rate-limiting step in tryptophan degradation metabolism. The hemostasis of immune system is complicated.  In this review, the  properties of IDO such as basic molecular genetics, biochemistry and genesis will be discussed.

IDO belongs to globin gene family to carry oxygen and heme.  The main function and genesis of IDO comes from the immune responses during host-microbial invasion and choice between tolerance and immunegenity.  In human there are three kinds of IDOs, which are IDO1, IDO2, and TDO, with distinguished mechanisms and expression profiles. , IDO mechanism includes three distinguished pathways: enzymatic acts through IFNgamma, non-enzymatic acts through TGFbeta-IFNalpha/IFNbeta and moonlighting acts through AhR/Kyn.

The well understood functional genomics and mechanisms is important to translate basic science for clinical interventions of human health needs. In conclusion, overall purpose is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.

The first part of the review concerns the basic science information gained overall several years that lay the foundation where translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.

Table of Contents:

  • Abstract

1         Introduction: IDO gene encodes a heme enzyme

2        Location, location, location

3        Molecular genetics

4        Types of IDO:

4.1       IDO1,

4.2       IDO2,

4.3       IDO-like proteins

5        Working mechanisms of IDO

6        Infection Diseases and IDO

7. Conclusion

  1. 1.     Indoleamine 2, 3-dioxygenase (IDO) gene encodes a heme enzyme

IDO is a key homeostatic regulator and confined in immune system mechanism for the balance between tolerance and immunity.  This gene encodes indoleamine 2, 3-dioxygenase (IDO) – a heme enzyme (EC= that catalyzes the first rate-limiting step in tryptophan catabolism to N-formyl-kynurenine and acts on multiple tryptophan substrates including D-tryptophan, L-tryptophan, 5-hydroxy-tryptophan, tryptamine, and serotonin.

The basic genetic information describes indoleamine 2, 3-dioxygenase 1 (IDO1, IDO, INDO) as an enzyme located at Chromosome 8p12-p11 (5; 6) that active at the first step of the Tryptophan catabolism.    The cloned gene structure showed that IDO contains 10 exons ad 9 introns (7; 8) producing 9 transcripts.

After alternative splicing only five of the transcripts encode a protein but the other four does not make protein products, three of transcripts retain intron and one of them create a nonsense code (7).  Based on IDO related studies 15 phenotypes of IDO is identified, of which, twelve in cancer tumor models of lung, kidney, endometrium, intestine, two in nervous system, and one HGMD- deletion.

  1. 2.     Location, Location and Location

The specific cellular location of IDO is in cytosol, smooth muscle contractile fibers and stereocilium bundle. The expression specificity shows that IDO is present very widely in all cell types but there is an elevation of expression in placenta, pancreas, pancreas islets, including dendritic cells (DCs) according to gene atlas of transcriptome (9).  Expression of IDO is common in antigen presenting cells (APCs), monocytes (MO), macrophages (MQs), DCs, T-cells, and some B-cells. IDO present in APCs (10; 11), due to magnitude of role play hierarchy and level of expression DCs are the better choice but including MOs during establishment of three DC cell subset, CD14+CD25+, CD14++CD25+ and CD14+CD25++ may increase the longevity and efficacy of the interventions.

IDO is strictly regulated and confined to immune system with diverse functions based on either positive or negative stimulations. The positive stimulations are T cell tolerance induction, apoptotic process, and chronic inflammatory response, type 2 immune response, interleukin-12 production (12).  The negative stimulations are interleukin-10 production, activated T cell proliferation, T cell apoptotic process.  Furthermore, there are more functions allocating fetus during female pregnancy; changing behavior, responding to lipopolysaccharide or multicellular organismal response to stress possible due to degradation of tryptophan, kynurenic acid biosynthetic process, cellular nitrogen compound metabolic process, small molecule metabolic process, producing kynurenine process (13; 14; 15).

IDO plays a role in a variety of pathophysiological processes such as antimicrobial and antitumor defense, neuropathology, immunoregulation, and antioxidant activity (16; 17; 18; 19).


 3.     Molecular Genetics of IDO:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3' untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database. (reference:

A: Structure of human IDO2 gene and transcripts. Complete coding region is 1260 bps encoding a 420 aa polypeptide. Alternate splice isoforms lacking the exons indicated are noted. Hatch boxes represent a frameshift in the coding region to an alternate reading frame leading to termination. Black boxes represent 3′ untranslated regions. Nucleotide numbers, intron sizes, and positioning are based on IDO sequence files NW_923907.1 and GI:89028628 in the Genbank database.

Molecular genetics data from earlier findings based on reporter assay results showed that IDO promoter is regulated by ISRE-like elements and GAS-sequence at -1126 and -1083 region (20).  Two cis-acting elements are ISRE1 (interferon sequence response element 1) and interferon sequence response element 2 (ISRE2).

Analyses of site directed and deletion mutation with transfected cells demonstrated that introduction of point mutations at these elements decreases the IDO expression. Removing ISRE1 decreases the effects of IFNgamma induction 50 fold and deleting ISRE1 at -1126 reduced by 25 fold (3). Introducing point mutations in conserved t residues at -1124 and -1122 (from T to C or G) in ISRE consensus sequence NAGtttCA/tntttNCC of IFNa/b inducible gene ISG4 eliminates the promoter activity by 24 fold (21).

ISRE2 have two boxes, X box (-114/1104) and Y Box 9-144/-135), which are essential part of the IFNgamma response region of major histocompatibility complex class II promoters (22; 23).  When these were removed from ISRE2 or introducing point mutations at two A residues of ISRE2 at -111 showed a sharp decrease after IFNgamma treatment by 4 fold (3).

The lack of responses related to truncated or deleted IRF-1 interactions whereas IRF-2, Jak2 and STAT91 levels were similar in the cells, HEPg2 and ME180 (3). Furthermore, 748 bp deleted between these elements did not affect the IDO expression, thus the distance between ISRE1 and ISRE2 elements have no function or influence on IDO (3; 24)

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

B: Amino acid alignment of IDO and IDO2. Amino acids determined by mutagenesis and the crystal structure of IDO that are critical for catalytic activity are positioned below the human IDO sequence. Two commonly occurring SNPs identified in the coding region of human IDO2 are shown above the sequence which alter a critical amino acid (R248W) or introduce a premature termination codon (Y359stop).

4.     There are three types of IDO in human genome:

IDO was originally discovered in 1967 in rabbit intestine (25). Later, in 1990 the human IDO gene is cloned and sequenced (7).  However, its importance and relevance in immunology was not created until prevention of allocation of fetal rejection and founding expression in wide range of human cancers (26; 27).

There are three types of IDO, pro-IDO like, IDO1, and IDO2.  In addition, another enzyme called TDO, tryptophan 2, 3, dehydrogenase solely degrade L-Trp at first-rate limiting mechanism in liver and brain.

4.1.  IDO1:

IDO1 mechanism is the target for immunotherapy applications. The initial discovery of IDO in human physiology is protection of pregnancy (1) since lack of IDO results in premature recurrent abortion (28; 26; 29).   The initial rate-limiting step of tryptophan metabolism is catalyzed by either IDO or tryptophan 2, 3-dioxygenase (TDO).

Structural studies of IDO versus TDO presenting active site environments, conserved Arg 117 and Tyr113, found both in TDO and IDO for the Tyr-Glu motif, but His55 in TDO replaced by Ser167b in IDO (30; 2). As a result, they are regulated with different mechanisms (1; 2) (30).  The short-lived TDO, about 2h, responds to level of tryptophan and its expression regulated by glucorticoids (31; 32).  Thus, it is a useful target for regulation and induced by tryptophan so that increasing tryptophan induces NAD biosynthesis. Whereas, IDO is not activated by the level of Trp presence but inflammatory agents with its interferon stimulated response elements (ISRE1 and ISRE2) in its (33; 34; 35; 36; 3; 10) promoter.

TDO promoter contains glucorticoid response elements (37; 38) and regulated by glucocorticoids and other available amino acids for gluconeogenesis. This is how IDO binds to only immune response cells and TDO relates to NAD biosynthesis mechanisms. Furthermore, TDO is express solely in liver and brain (36).  NAD synthesis (39) showed increased IDO ubiquitous and TDO in liver and causing NAD level increase in rat with neuronal degeneration (40; 41).  NAM has protective function in beta-cells could be used to cure Type1 diabetes (40; 42; 43). In addition, knowledge on NADH/NAD, Kyn/Trp or Trp/Kyn ratios as well as Th1/Th2, CD4/CD8 or Th17/Threg are equally important (44; 40).

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (

Active site of IDO–PI complex. (A) Stereoview of the residues around the heme of IDO viewed from the side of heme plane. The proximal ligand H346 is H-bonded to wa1. The 6-propionate of the heme contacts with wa2 and R343 Nε. The wa2 is H-bonded to wa1, L388 O, and 6-propionate. Mutations of F226, F227, and R231 do not lose the substrate affinity but produce the inactive enzyme. Two CHES molecules are bound in the distal pocket. The cyclohexan ring of CHES-1 (green) contacts with F226 and R231. The 7-propionate of the heme interacts with the amino group of CHES-1 and side chain of Ser-263. The mutational analyses for these distal residues are shown in Table 1. (B) Top view of A by a rotation of 90°. The proximal residues are omitted. (

4.2. IDO2:

The third type of IDO, called IDO2 exists in lower vertebrates like chicken, fish and frogs (45) and in human with differential expression properties. The expression of IDO2 is only in DCs, unlike IDO1 expresses on both tumors and DCs in human tissues.  Yet, in lower invertebrates IDO2 is not inhibited by general inhibitor of IDO, D-1-methyl-tryptophan (1MT) (46).   Recently, two structurally unusual natural inhibitors of IDO molecules, EXIGUAMINES A and B, are synthesized (47).  LIP mechanism cannot be switch back to activation after its induction in IDO2 (46).

Crucial cancer progression can continue with production of IL6, IL10 and TGF-beta1 to help invasion and metastasis.  Inclusion of two common SNPs affects the function of IDO2 in certain populations.  SNP1 reduces 90% of IDO2 catalytic activity in 50% of European and Asian descent and SNP2 produce premature protein through inclusion of stop-codon in 25% of African descent lack functional IDO2 (Uniport).

4.3. IDO-like proteins: The Origin of IDO:

Knowing the evolutionary steps will helps us to identify how we can manage the regulator function to protect human health in cancer, immune disorders, diabetes, and infectious diseases.

Bacterial IDO has two types of IDOs that are group I and group II IDO (48).  These are the earliest version of the IDO, pro-IDO like, proteins with a quite complicated function.  Each microorganism recognized by a specific set of receptors, called Toll-Like Receptors (TLR), to activate the IDO-like protein expression based on the origin of the bacteria or virus (49; 35).   Thus, the genesis of human IDO originates from gene duplication of these early bacterial versions of IDO-like proteins after their invasion interactions with human host.  IDO1 only exists in mammals and fungi.

Fungi also has three types of IDO; IDOa, IDO beta, and IDO gamma (50) with different properties than human IDOs, perhaps multiple IDO is necessary for the world’s decomposers.

All globins, haemoglobins and myoglobins are destined to evolve from a common ancestor, which  is only 14-16kDa (51) length. Binding of a heme and being oxygen carrier are central to the enzyme mechanism of this family.  Globins are classified under three distinct origins; a universal globin, a compact globin, and IDO-like globin (52) IDO like globin widely distributed among gastropodic mollusks (53; 51).  The indoleamine 2, 3-dioxygenase 1–like “myoglobin” (Myb) was discovered in 1989 in the buccal mass of the abalone Sulculus diversicolor (54).

The conserved region between Myb and IDO-like Myb existed for at least 600 million years (53) Even though the splice junction of seven introns was kept intact, the overall homolog region between Myb and IDO is only about 35%.

No significant evolutionary relationship is found between them after their amino acid sequence of each exon is compared to usual globin sequences. This led the hint that molluscan IDO-like protein must have other functions besides carrying oxygen, like myoglobin.   Alignment of S. cerevisiae cDNA, mollusk and vertebrate IDO–like globins show the key regions for controlling IDO or myoglobin function (55). These data suggest that there is an alternative pathways of myoglobin evolution.  In addition, understanding the diversity of globin may help to design better protocols for interventions of diseases.

Mechanisms of IDO:

The dichotomy of IDO mechanism lead the discovery that IDO is more than an enzyme as a versatile regulator of innate and adaptive immune responses in DCs (66; 67; 68). Meantime IDO also involve with Th2 response and B cell mediated autoimmunity showing that it has three paths, short term (acute) based on enzymatic actions, long term (chronic) based on non-enzymatic role, and moonlighting relies of downstream metabolites of tryptophan metabolism (69; 70).

IFNgamma produced by DC, MQ, NK, NKT, CD4+ T cells and CD8+ T cells, after stimulation with IL12 and IL8.  Inflammatory cytokine(s) expressed by DCs produce IFNgamma to stimulate IDO’s enzymatic reactions in acute response.  Then, TDO in liver and tryptophan catabolites act through Aryl hydrocarbon receptor induction for prevention of T cell proliferation. This mechanism is common among IDO, IDO2 (expresses in brain and liver) and TDO expresses in liver) provide an acute response for an innate immunity (30). When the pDCs are stimulated with IFNgamma, activation of IDO is go through Jak, STAT signaling pathway to degrade Trp to Kyn causing Trp depletion. The starvation of tryptophan in microenvironment inhibits generation of T cells by un-read t-RNAs and induce apoptosis through myc pathway.  In sum, lack of tryptophan halts T cell proliferation and put the T cells in apoptosis at S1 phase of cell division (71; 62).

The intermediary enzymes, functioning during Tryptophan degradation in Kynurenine (Kyn) pathway like kynurenine 3-hydroxylase and kynureninase, are also induced after stimulation with liposaccaride and proinflammatory cytokines (72). They exhibit their function in homeostasis through aryl-hydrocarbon receptor (AhR) induction by kynurenine as an endogenous signal (73; 74).  The endogenous tumor-promoting ligand of AhR are usually activated by environmental stress or xenobiotic toxic chemicals in several cellular processes like tumorigenesis, inflammation, transformation, and embryogenesis (Opitz ET. Al, 2011).

Human tumor cells constitutively produce TDO also contributes to production of Kyn as an endogenous ligand of the AhR (75; 27).  Degradation of tryptophan by IDO1/2 in tumors and tumor-draining lymph nodes occur. As a result, there are animal studies and Phase I/II clinical trials to inhibit the IDO1/2 to prevent cancer and poor prognosis (NewLink Genetics Corp. NCT00739609, 2007).

 IDO mechanism for immune response

Systemic inflammation (like in sepsis, cerebral malaria and brain tumor) creates hypotension and IDO expression has the central role on vascular tone control (63).  Moreover, inflammation activates the endothelial coagulation activation system causing coagulopathies on patients.  This reaction is namely endothelial cell activation of IDO by IFNgamma inducing Trp to Kyn conversion. After infection with malaria the blood vessel tone has decreases, inflammation induce IDO expression in endothelial cells producing Kyn causing decreased trp, lower arterial relaxation, and develop hypotension (Wang, Y. et. al 2010).  Furthermore, existing hypotension in knock out Ido mice point out a secondary mechanism driven by Kyn as an endogenous ligand to activate non-canonical NfKB pathway (63).

Another study also hints this “back –up” mechanism by a significant outcome with a differential response in pDCs against IMT treatment.  Unlike IFN gamma conditioned pDC blocks T cell proliferation and apoptosis, methyl tryptophan fails to inhibit IDO activity for activating naïve T cells to make Tregs at TGF-b1 conditioned pDCs (77; 78).

 Indoleamine-Pyrrole 2,3,-Dioxygenase; IDO dioxygenase; Indeolamine-2,3

The second role of the IDO relies on non-enzymatic action as being a signal molecule. Yet, IDO2 and TDO are devoid of this function. This role mainly for maintenance of microenvironment condition. DCs response to TGFbeta-1 exposure starts the kinase Fyn induce phosphorylation of IDO-associated immunoreceptor tyrosine–based inhibitory motifs (ITIMs) for propagation of the downstream signals involving non-canonical (anti-inflammatory) NF-kB pathway for a long term response. When the pDCs are conditioned with TGF-beta1 the signaling (68; 77; 78) Phospho Inositol Kinase3 (PIK-3)-dependent and Smad independent pathways (79; 80; 81; 82; 83) induce Fyn-dependent phosphorylation of IDO ITIMs.  A prototypic ITIM has the I/V/L/SxYxxL/V/F sequence (84), where x in place of an amino acid and Y is phosphorylation sites of tyrosines (85; 86).

Smad independent pathway stimulates SHP and PIK3 induce both SHP and IDO phosphorylation. Then, formed SHP-IDO complex can induce non-canonical (non-inflammatory) NF-kB pathway (64; 79; 80; 82) by phosphorylation of kinase IKKa to induce nuclear translocation of p52-Relb towards their targets.  Furthermore, the SHP-IDO complex also may inhibit IRAK1 (68). SHP-IDO complex activates genes through Nf-KB for production of Ido1 and Tgfb1 genes and secretion of IFNalpha/IFNbeta.  IFNa/IFNb establishes a second short positive feedback loop towards p52-RelB for continuous gene expression of IDO, TGFb1, IFNa and IFNb (87; 68).  However, SHP-IDO inhibited IRAK1 also activates p52-RelB.  Nf-KB induction at three path, one main and two positive feedback loops, is also critical.  Finally, based on TGF-beta1 induction (76) cellular differentiation occurs to stimulate naïve CD4+ T cell differentiation to regulatory T cells (Tregs).  In sum, TGF-b1 and IFNalpha/IFNbeta stimulate pDCs to keep inducing naïve T cells for generation of Treg cells at various stages, initiate, maintain, differentiate, infect, amplify, during long-term immune responses (67; 66).

Moonlighting function of Kyn/AhR is an adaptation mechanism after the catalytic (enzymatic) role of IDO depletes tryptophan and produce high concentration of Kyn induce Treg and Tr1 cell expansion leading Tregs to use TGFbeta for maintaining this environment (67; 76). In this role, Kyn pathway has positive-feedback-loop function to induce IDO expression.

In T cells, tryptophan starvation induces Gcn2-dependent stress signaling pathway, which initiates uncharged Trp-tRNA binding onto ribosomes. Elevated GCN2 expression stimulates elF2alfa phosphorylation to stop translation initiation (88). Therefore, most genes downregulated and LIP, an alternatively initiated isoform of the b/ZIP transcription factor NF-IL6/CEBP-beta (89).

This mechanism happens in tumor cells based on Prendergast group observations. As a result, not only IDO1 propagates itself while producing IFNalpha/IFNbeta, but also demonstrates homeostasis choosing between immunegenity by production of TH17or tolerance by Tregs. This mechanism acts like a see-saw. Yet, tolerance also can be broken by IL6 induction so reversal mechanism by SOC-3 dependent proteosomal degradation of the enzyme (90).  All proper responses require functional peripheral DCs to generate mature DCs for T cells to avoid autoimmunity (91).

Niacin (vitamin B3) is the final product of tryptophan catabolism and first molecule at Nicotinomic acid (NDA) Biosynthesis.  The function of IDO in tryptophan and NDA metabolism has a great importance to develop new clinical applications (40; 42; 41).  NAD+, biosynthesis and tryptophan metabolisms regulate several steps that can be utilize pharmacologically for reformation of healthy physiology (40).

IDO for protection in Microbial Infection with Toll-like Receptors

The mechanism of microbial response and infectious tolerance are complex and the origination of IDO based on duplication of microbial IDO (49).  During microbial responses, Toll-like receptors (TLRs) play a role to differentiate and determine the microbial structures as a ligand to initiate production of cytokines and pro-inflammatory agents to activate specific T helper cells (92; 93; 94; 95). Uniqueness of TLR comes from four major characteristics of each individual TLR by ligand specificity, signal transduction pathways, expression profiles and cellular localization (96). Thus, TLRs are important part of the immune response signaling mechanism to initiate and design adoptive responses from innate (naïve) immune system to defend the host.

TLRs are expressed cell type specific patterns and present themselves on APCs (DCs, MQs, monocytes) with a rich expression levels (96; 97; 98; 99; 93; 100; 101; 102; 87). Induction signals originate from microbial stimuli for the genesis of mature immune response cells.  Co-stimulation mechanisms stimulate immature DCs to travel from lymphoid organs to blood stream for proliferation of specific T cells (96).  After the induction of iDCs by microbial stimuli, they produce proinflammatory cytokines such as TNF and IL-12, which can activate differentiation of T cells into T helper cell, type one (Th1) cells. (103).

Utilizing specific TLR stimulation to link between innate and acquired responses can be possible through simple recognition of pathogen-associated molecular patterns (PAMPs) or co-stimulation of PAMPs with other TLR or non-TLR receptors, or even better with proinflammatory cytokines.   Some examples of ligand- TLR specificity shown in Table1, which are bacterial lipopeptides, Pam3Cys through TLR2 (92; 104; 105).  Double stranded (ds) RNAs through TLR3 (106; 107), Lipopolysaccharide (LPS) through TLR4, bacterial flagellin through TLR5 (108; 109), single stranded RNAs through TLR7/8 (97; 98), synthetic anti-viral compounds imiquinod through TLR 7 and resiquimod through TLR8, unmethylated CpG DNA motifs through TLR9 (Krieg, 2000).

IDO action

Then, the specificity is established by correct pairing of a TLR with its proinflammatory cytokines, so that these permutations influence creation and maintenance of cell differentiation. For example, leading the T cell response toward a preferred Th1 or Th2 response possible if the cytokines TLR-2 mediated signals induce a Th2 profile when combined with IL-2 but TLR4 mediated signals lean towards Th1 if it is combined with IL-10 or Il-12, (110; 111)  (112).

TLR ligand TLR Reference
Lipopolysaccharide, LPS TLR4 (96).  (112).
Lipopeptides, Pam3Cys TLR2 (92; 104; 105)
Double stranded (ds) RNAs TLR3 (106; 107)
Bacterial flagellin TLR5 (108; 109)
Single stranded RNAs TLR7/8 (97; 98)
Unmethylated CpG DNA motifs TLR9 (Krieg, 2000)
Synthetic anti-viral compounds imiquinod and resiquimod TLR7 and TLR8 (Lee J, 2003)

Furthermore, if the DCs are stimulated with IL-6, DCs relieve the suppression of effector T cells by regulatory T cells (113).

The modification of IDO+ monocytes manage towards specific subset of T cell activation with specific TLRs are significantly important (94).

The type of cell with correct TLR and stimuli improves or decreases the effectiveness of stimuli. Induction of IDO in monocytes by synthetic viral RNAs (isRNA) and CMV was possible, but not in monocyte derived DCs or TLR2 ligand lipopeptide Pam3Cys since single- stranded RNA ligands target TLR7/8 in monocytes derive DCs only (Lee J, 2003).  These data show that TLRs has ligand specificity, signal transduction pathways, expression profiles and cellular localization so design of experiments should follow these rules.


Overall our purpose of this information is to find a method to manipulate IDO to correct/fix/modulate immune responses for clinical applications.  This first part of the review concerns the basic science information gained overall several years that lay the foundation that translational research scientist should familiar to develop a new technology for clinic. The first connection of IDO and human health came from a very natural event that is protection of pregnancy in human. The focus of the translational medicine is treatment of cancer or prevention/delay cancer by stem cell based Dendritic Cell Vaccine (DCvax) development.


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Zebrafish—Susceptible to Cancer

Reporter: Larry H Bernstein, MD, FCAP



Models of Transparency

Researchers are taking advantage of small, transparent zebrafish embryos and larvae—and a special strain of see-through adults—to understand the development and spread of cancer.
By Joan K. Heath, David Langenau, Kirsten C. Sadler, and Richard White | April 1, 2013
LOOKING INSIDE DISEASE: The wild-type zebrafish larva on the left is stained for the two neuronal proteins (green) and membrane-trafficking proteins expressed near synapses (blue). On the right, the neurons of a transgenic zebrafish larva produce the dementia-associated Tau protein (red), a disease-specific form of which is stained in blue. Tubulin is stained in green.
From frogs to dogs and people, cancer wreaks havoc across the animal kingdom—and fish are no exception. Coral trout, for example, develop melanoma from overexposure to sun, just as humans do. Rainbow trout develop liver cancer in response to environmental toxins. And zebrafish—small, striped fish indigenous to the rivers of India and a widely used model organism—are susceptible to both malignant and benign tumors of the brain, nervous system, blood, liver, pancreas, skin, muscle, and intestine.
Importantly, tumors that arise in the same organs in humans and fish look and behave alike, and the cancers often share common genetic underpinnings. As a result, most researchers believe that the basic mechanisms underlying tumor formation are conserved across species, allowing them to study the formation, expansion, and spread of tumors in animal models with the hope of eventually finding new insights into cancer in people.
Zebrafish are an increasingly popular choice among cancer biologists. Between 1995 and 2012, there was a 10-fold increase in the number of yearly PubMed citations of cancer studies in the species, with more than 200 research papers published last year.  Although dwarfed by cancer studies using human tissue and mouse models, the optical transparency of zebrafish embryos and larvae—and now, adult fish of a recently created strain—allows researchers to track tumors in a way that is not possible in other vertebrate models. Furthermore, their small size—embryos are small enough to be reared in 96-well plates—make them a more practical laboratory system than other cancer models. Indeed, researchers are now using these fish to identify druggable oncogenic drivers of specific tumor types, to tease apart the complex network of cancer genes that cooperate in tumor formation and progression, to probe the interplay between the genes that govern embryonic development and those that cause cancer, and to uncover how tumors metastasize and kill their host. The zebrafish model offers a major opportunity to discover important pathways underlying cancer and to identify novel therapies in high-throughput drug screens in a way that mice never could.

The zebrafish toolbox

Zebrafish (Danio rerio) have fast made their way from pet stores and home aquaria into research laboratories worldwide. Their weekly matings produce 100 to 200 embryos that rapidly and synchronously march through embryonic development, so that within 5 days of fertilization, they are mature, feeding larvae. Zebrafish are small and inexpensive to maintain in high numbers, facilitating large-scale experimentation and cheap in vivo drug screens. Famously, the fish are transparent during early larval stages, allowing investigators to directly observe internal development and making the fish a favorite of developmental biologists since the 1960s. But in recent years, the utility of zebrafish has been proven beyond developmental fields, and they are now being found in more and more laboratories studying behavior, diabetes, heart disease, regeneration, stem cell biology—and cancer.
Critically, zebrafish can be used to identify the important pathways and processes that cause cancer in people. Common organ systems and cell types are shared between human and zebrafish, and whether induced by transgenesis or carcinogens, cancers arising from the blood (leukemia and lymphoma), pigmented cells of the skin (melanoma), and the cells that line the bile ducts (cholangiocarcinoma) have microscopic features that are essentially indistinguishable between humans and zebrafish.
The zebrafish model offers a major opportunity to discover important pathways underlying cancer and to identify novel therapies in high-throughput drug screens, in a way that mice never could.
Comparing gene-expression profiles of tumors across various species provides a powerful mechanism for identifying genes that likely represent core functions of cancer. For example, microarray gene-expression analyses have compared the gene signatures of fish hepatocellular carcinoma to that of human liver, gastric, prostate, and lung tumors. Remarkably, this analysis revealed that fish and human liver tumors are more similar to each other than either tumor type is to human tumors derived from different tissues. Moreover, comparative studies can often be used to pinpoint pathways that are active in human disease. This is illustrated by work on a zebrafish model of rhabdomyosarcoma (RMS), a cancer of skeletal muscle, which revealed a gene signature that is also commonly found in human RMS, highlighting the importance of the RAS signaling pathway in the genesis of human RMS.1

A window into cancer

In 2003, the laboratory of A. Thomas Look at Children’s Hospital Boston was the first to realize the long-held dream of following the behavior of cancer cells as they initiate tumor growth and invade structures within live animals. Specifically, the researchers engineered leukemia-afflicted T cells to express green fluorescent protein (GFP) and visualized cancer onset within the zebrafish thymus.2 Moreover, these GFP-positive tumor cells were transplantable into recipient fish, a hallmark of the malignant cell type. Following up on this work, several researchers have now begun transplanting fluorescently labeled human cancer cells into zebrafish larvae to visualize tumor growth and spread in a manner not achievable in more common mouse xenograft models.
Capitalizing on the lack of an acquired immune system during larval stages and the ability to rear zebrafish at temperatures that mimic the human core temperature, Stefania Nicoli of the University of Brescia in Italy and colleagues implanted human cancer cells expressing high levels of vascular endothelial growth factor (VEGF) into zebrafish larvae with GFP-labeled blood vessels.3 VEGF is a factor commonly produced by growing cancers and is responsible for coaxing blood vessels to invade the developing tumor. Nicoli’s work allowed the direct visualization of vasculature remodeling and new vessel formation—and showed that it could be blocked by the addition of VEGF-inhibitory drugs to the water in which the larvae lived. Using similar approaches, many laboratories have successfully engrafted human cancer cells from a range of tumor types into zebrafish embryos.
Researchers have also used zebrafish to visualize the role of tumor heterogeneity within cancer over time. Work from one of our labs (David Langenau’s) has utilized a model of RAS-induced RMS to fluorescently label tumor cells based on differentiation status. This allowed the team to watch the never-before-visualized birth of cancer—the acquisition of invasive properties by normal muscle stem cells and the breakdown of normal muscle architecture, clearing the way for continued tumor expansion. The researchers also characterized two molecularly distinct cell populations, one that is responsible for tumor growth and another that drives cancer spread or metastasis. (See photos below—Imaging Blood Cancers; Solid Tumor Development.)
SEE-THROUGH SUBJECTS: Zebrafish embryos (bottom right), shown here at 28 hours, are naturally transparent, as are zebrafish larvae (bottom left) until around 3 weeks of age. A new strain of zebrafish, called casper, maintains its transparency into adulthood (top right), allowing researchers to observe cancer formation in adult fish. A wall of tanks (top left) at the Zebrafish Resource Center, Karlsruhe Institute of Technology.
T-cell leukemia and RMS are pediatric diseases, favoring cancer development in early larval stages of these zebrafish models. However, cancer is predominantly a disease that affects adults, and zebrafish lose their transparency at around 3 weeks of age. To visualize tumor formation in older zebrafish, one of us (Richard White) and Len Zon of Children’s Hospital Boston have developed a strain of zebrafish called casper, which lacks pigment and is optically clear into adulthood.4 (See photo here.) Using these animals, investigators have implanted pigmented melanomas and witnessed local spread and metastasis over time. First described in 2008, casper is now the zebrafish strain of choice for imaging studies in the field.
Zebrafish have truly proven to be ideal organisms for visualizing cancer; there is no other animal system that allows researchers to literally watch tumors grow and spread. Because we can follow cells as they escape from the primary tumor, migrate, and form metastases in a variety of organs, zebrafish provide an unsurpassed model to describe the distinct steps of cancer progression, and researchers using this model are already contributing much to our understanding of cancer.

 Screening for drugs

In terms of drug discovery, zebrafish have emerged as the only vertebrate organism amenable to high-throughput and high-content chemical screening in vivo. The small size of freshly hatched zebrafish embryos means that up to 20 embryos can be dispensed into the individual wells of a 96-well plate, thereby providing a platform for the robotic delivery and testing of hundreds or thousands of compounds in living animals. Because of the parallels between embryonic development and cancer, compounds producing changes in the growth or proliferation of developing organs may also be relevant to cancer. However, to more directly search for small molecules capable of putting the brakes on cancerous growth, researchers are turning to several established tumor-prone zebrafish lines.
One prominent success from such endeavors is the identification of a drug to treat melanoma. Like human melanoma skin lesions, zebrafish melanomas exhibit a gene-expression signature characteristic of the embryonic neural crest, a multipotent group of stem cells that give rise to dozens of cell types, including pigmented skin cells called melanocytes. The genes in this signature, including sox10 and mitf, were hypothesized to be important for melanoma growth, so in 2011 White and Zon performed an in vivo screen to identify small molecules that suppressed the expression of these neural crest genes in developing embryos.5 After screening 2,000 molecules, they identified leflunomide, an approved treatment for rheumatoid arthritis. Importantly, leflunomide was then found to inhibit the growth of both zebrafish and human melanoma xenografts in vivo, and the drug moved from discovery to Phase 1/2 trials in only 4 years, demonstrating just how quickly discoveries in zebrafish can have clinical impact. Similar efforts to discover novel modulators of leukemia growth have recently been reported to work in both fish and humans, suggesting that this approach will be broadly applicable to a wide range of solid and liquid tumors.
Probing the cancer genome
The genesis of cancer generally depends on the inactivation of one or more tumor suppressor genes in conjunction with signaling from oncogenes. Indeed, rapid advances in sequencing technologies and efforts such as The Cancer Genome Atlas (TCGA) have revealed surprisingly few “driver” mutations capable of causing cancer alone. Instead, TCGA and other sequencing studies have identified vast genetic heterogeneity both across and within tumor types, with mutations extending well beyond genes likely to represent classical oncogenes or tumor suppressor genes. How these mutations influence tumor growth remains a major unanswered question in cancer biology.
IMAGING BLOOD CANCERS: Developing T lymphocytes in the thymus of a transgenic zebrafish (top right) express green fluorescent protein (GFP). A transgenic zebrafish (bottom right) that coexpresses the Myc oncogene with GFP shows signs of prominent leukemia, which has spread well beyond the boundaries of the thymus (T).
Enter zebrafish, and a range of high-throughput reverse-genetic techniques for cancer gene discovery. By transiently overexpressing each of 30 candidate genes in zebrafish larvae, for example, Craig Ceol and Yariv Houvras in Zon’s group identified a single cooperating oncogene, SETDB1, as a new player in melanoma.6 In this study, the researchers created and analyzed more than 3,000 transgenic animals. Because they used a transposon-based transgenic approach that leads to high-level, uniform expression, they could directly assess how the injected genes affected tumor onset without going through the lengthy process of germline transgenics, a major bottleneck in mouse genetics. This rapid screening approach is a prime example of how the mountains of data generated by TCGA can be quickly assessed for biological function, and zebrafish are the only in vivo whole-animal vertebrate system that enables researchers to rapidly sift through these data to understand which mutations drive cancer.
Other new approaches are advancing loss-of-function analyses. Until recently, such studies relied on TiLLING (targeting induced local lesions in genomes), in which a chemical carcinogen, ethylnitrosourea (ENU), introduces point mutations throughout the genome and high-throughput methods then look for mutations in genes of interest. This method yielded several valuable strains of tumor-prone zebrafish harboring clinically relevant mutations in the well-known tumor-suppressor genes p53, apc, and pten, and these have been pivotal to the development of multiple zebrafish cancer models. However, the unbiased nature of ENU mutagenesis makes TiLLING a labor-intensive and impractical business in most laboratory settings. Instead, precision editing of the genome has emerged as the method of choice for the systematic creation of knockout and mutant animals. Specifically, homology-based editing, using TALENs (transcription activator–like effector nucleases) and, more recently, CRISPR (clustered regularly interspaced short palindromic repeats)/Cas systems, has revolutionized the field.7,8 Using relatively simple procedures, virtually any gene can now be mutated in zebrafish, allowing for very large-scale, in vivo assessments of novel cancer genes and the analysis of interacting mutations—one of the greatest challenges facing the cancer field in the coming decade.
SOLID TUMOR DEVELOPMENT: A transgenic zebrafish (bottom left) with fluorescent-labeled RAS-induced rhabdomyosacoma. Green fluorescent protein is expressed in the tumor-propagating cells, which drive continued tumor growth. Red fluorescent protein is localized to the nucleus and expressed in myoblast-like cells, while blue fluorescent protein is confined to terminally differentiated cancer cells that express myosin (magnified image, bottom right). Live-cell imaging permits dynamic visualization of the birth of cancer and the functional consequences of tumor cell heterogeneity within established tumors.
To complement approaches that directly inactivate genes within the genome, strategies to achieve interference RNA-mediated gene silencing in zebrafish have come of age as well. Expression of short hairpin RNAs, for example, have produced stable and tissue-specific knockdown in cancer-related genes such as chordin and wnt5b.9,10 Because of the ease of manipulating the genome as well as the large number of well-characterized zebrafish gene promoters, such strategies immediately afford the opportunity to knockdown known gene functions in a tissue-specific fashion, and it is likely that temporal control will be readily achievable as well.
In all, combining the descriptive data from TCGA with zebrafish transgenesis, high-throughput overexpression and knockout techniques, and unbiased genetic screens offers an unprecedented opportunity to functionally probe the cancer genome.

The future of the field

Given its power for imaging, transplantation, small-molecule screens, and high-throughput transgenesis, the zebrafish model should become a major platform for deeply interrogating cancer biology in vivo over the next decade. One major area where zebrafish are particularly valuable is in teasing apart the extreme complexity of cancer. Because combinations of genetic pathways can be assessed simultaneously, potentially dozens of genomic alterations found in human cancer could be tested for their effects in the fish, allowing us to sort biologically meaningful alterations from neutral ones. These techniques will also allow us to understand how numerous small changes, which on their own have little phenotypic effect, can combine to cause cancer.
The success of the field will depend upon improved funding for zebrafish cancer research, however. Currently, only a small fraction of National Institutes of Health RO1 grants for cancer research are awarded for zebrafish studies, with the vast majority going to work in mice, humans, and human cells. Consortium efforts analogous to the Mouse Model Consortium will be necessary to develop more faithful zebrafish models of human cancer, which can then be used as the basis for further screens. Whereas mouse models of cancer have delivered great insights into the biological mechanisms underlying human malignancy, we view zebrafish models as a springboard for the rapid launch of unbiased genetic and chemical screens.
With any cancer model, bridging the gap between the animals and human patients is the ultimate proof of its utility. For the zebrafish, this can occur not only through bringing drugs to the clinic, but also in the development of novel biomarkers and early detection methods. The next 10 years will be an exciting time, and we have great confidence that the zebrafish will contribute major discoveries to the treatment of human cancers.
Joan K. Heath is an associate professor in the ACRF Chemical Biology Division at the Walter and Eliza Hall Institute of Medical Research and the Department of Medical Biology at the University of  Melbourne, Australia, where her laboratory is studying the genetic regulation of  intestinal organogenesis and colorectal cancer.
David Langenau, an assistant professor of pathology at Harvard Medical School, studies the mechanisms that drive pediatric cancer relapse within the Molecular Pathology Unit and the Cancer Center at Massachusetts General Hospital.
Kirsten C. Sadler is an assistant professor in the Division of Liver Diseases/Department of Medicine and in Developmental and Regenerative Biology at the Icahn School of Medicine at Mount Sinai in New York City, where she studies the mechanisms of liver development, regeneration, and cancer.
Richard White is an assistant professor at the Memorial Sloan-Kettering Cancer Center and Weill Cornell Medical College in New York City. His laboratory studies the evolutionary mechanisms by which tumors develop the capacity for metastasis.


D.M. Langenau et al., “Effects of RAS on the genesis of embryonal rhabdomyosarcoma,” Genes Dev, 21:1382-95, 2007.
D.M. Langenau et al., “Myc-induced T cell leukemia in transgenic zebrafish,” Science, 299:887-90, 2003.
S. Nicoli et al., “Mammalian tumor xenografts induce neovascularization in zebrafish embryos,” Cancer Res, 67:2927-31, 2007.
R.M. White et al., “Transparent adult zebrafish as a tool for in vivo transplantation analysis,” Cell Stem Cell, 2:183-89, 2008.
R.M. White et al., “DHODH modulates transcriptional elongation in the neural crest and melanoma,” Nature, 471:518-22, 2011.
V.M. Bedell et al., “In vivo genome editing using a high-efficiency TALEN system,” Nature, 491:114-18, 2012.
W.Y. Hwang et al., “Efficient genome editing in zebrafish using a CRISPR-Cas system,” Nat Biotechnol, doi: 10.1038/nbt.2501, 2013.
M. Dong et al., “Heritable and lineage-specific gene knockdown in zebrafish embryo,” PLOS ONE, 4:e6125, 2009.
G. De Rienzo et al., “Efficient shRNA-mediated inhibition of gene expression in zebrafish,” Zebrafish, 9:97-107, 2012.
zebrafish, model organisms, cancer therapetics, cancer research, cancer genomics, cancer gene expression, cancer and animal models
Danio rerio, better known as the zebrafish

Danio rerio, better known as the zebrafish (Photo credit: Wikipedia)

Zebrafish embryo development

Zebrafish embryo development (Photo credit: Carl Zeiss Microscopy)

English: zebrafish (Danio rerio) ovary oocyte ...

English: zebrafish (Danio rerio) ovary oocyte maturation (Photo credit: Wikipedia)

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