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Salk's 21st Century Revolution

Cell

This is a large example mosaic image of hESC (human embryonic stem cell)-derived neurons labeled in three colors taken on one of the laser-based microscopes in the biophotonics facility. Blue represents cell nuclei, green is a dendritic marker and red is a neuronal cell marker.
Sample provided by T.J. Eames (grad student, Gage Lab) and imaged by James Fitzpatrick, director of the Biophotonics Core.


Stem Cell Revolution

Born from a medical revolution half a century ago, the Salk Institute has spent the past couple of decades on the front lines of an even more ambitious one: the stem cell revolution.

So far, it has been a simmering revolution. Researchers are still busy analyzing and perfecting the tricky dynamics of safely reprogramming the body's ordinary cells into stem cells and clearing the many obstacles that stand between hopeful science and useful therapies. But such therapies, when perfected, won't merely improve medicine. They'll also simplify and expand it. And the pace of recent progress suggests that the culmination of these efforts may be only years, not decades, away.

The Salk Institute is one of the principal drivers propelling basic stem cell research toward discovery and drug therapies. Virtually since the origins of modern stem cell research in the 1980s, Salk scientists have been producing influential science in the field, whether they are conducting basic studies of stem cell biology or developing stem cell-based disease models and even prospective therapies. In the early 2000s, Salk officials and researchers helped push for passage of California's Proposition 71, which provided for $3 billion in stem cell research funding and facility construction over ten years. Now more than a dozen Salk laboratories, collaborating with other leading labs in the U.S. and abroad, are taking part in this critical biomedical endeavor. "Our involvement in stem cell research has been escalating dramatically, across all the main areas of investigation, from basic science to potential therapeutics," says Salk professor and stem cell pioneer Fred H. Gage.

The human impact


Cell

Salk's 21st Century Story

Stem cells are the cells from which all animals and plants begin. Their power lies in their versatility. The "pluripotent" stem cells of a developing embryo have the potential to develop into all the cell types in the body, from red blood cells to heart muscle cells to dopamine neurons in the brain. Even in a full-grown organism, certain organs and tissues contain "multipotent" stem cells that stand ready to generate new local cells to replace those cells lost to injury and aging.

The basic idea of stem cell medicine is to use the youthful, replenishing, self-renewing properties of stem cells to treat disease. Neural stem cells, for example—stem cells that make new nerve cells—might help repair spinal cord ruptures and free paralysis patients from their wheelchairs, or boost the brain's defenses against Alzheimer's and Parkinson's diseases. Cardiac stem cells, injected into a failing heart, could rebuild it in place, making a heart transplant unnecessary. Scientists also hope to use stem cells to grow—in the laboratory—youthful, healthy organs, such as livers and windpipes, that later can be transplanted into patients.

Dozens of institutions around the world are now working to turn these stem cell dreams into medical realities, and among them, the Salk Institute stands out for its high-impact work. "We have great core facilities, a highly collaborative environment, broad expertise and a good mix of basic and clinically oriented science," says Gage.

Adds Salk professor Juan Carlos Izpisúa Belmonte: "We still need to know many things at a fundamental level, to advance stem cell science to the clinic. To that end, it helps greatly to be surrounded by Salk colleagues who are among the world's best in basic areas of cell biology."

Seeing how they grow


One area of research focuses on the adult stem cells that help maintain and repair the organs and tissues where they reside. The activity of these stem cells tends to decline with age, so a major goal is to find drugs or other techniques that can boost this activity to treat or even prevent disease.

Neural stem cells have always been an inviting target because of their potential to help the brain ward off Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (Lou Gehrig's disease), Huntington's disease, multiple sclerosis and possibly even the ordinary cognitive decline of aging. Gage was the first scientist to identify adult neural stem cells in humans in the mid-1990s, and his lab at Salk is still heavily involved in studying how the ability of these stem cells to generate new brain cells—a process called neurogenesis—can be boosted. "One of the more encouraging things we've found is that even in very elderly mice, factors such as exercise and social enrichment can cause neural stem cells to proliferate and generate fully functional neurons," Gage says.

Stem cells are at work elsewhere in the body too, and again, they have limitations that stem cell science could one day overcome. Some amphibians and fish can regenerate their organs even in adulthood, but humans and other mammals lose this capacity in the womb or early infancy. Izpisúa Belmonte and his lab have shown that zebrafish regenerate heart muscle by activating a certain signaling pathway in heart muscle cells. "The signals make the heart muscle cells go a little bit backward in their development, in the direction of the stem cell state, so that they can proliferate again," Izpisúa Belmonte says. "We have found recently that in mammals, this process is switched off shortly after birth, which explains why they no longer have this regenerative capability."

Salk researcher Leanne Jones has used the fruit fly Drosophila as a model for studying the age-related decline in stem cells' ability to repair organs and tissues. Her work has shown that aging affects not only stem cells but also the stem cells' "niche"&8211;the other cells and proteins that make up stem cells' environment and that help regulate their normal functions. "To optimize the transplantation of stem cells in regenerative medicine, we may also need to consider rejuvenating the niche, especially in older patients," she says.

Reprogramming cells to the stem cell state


A decade ago, researchers thought that the only reliable way to get human stem cells that could be grown and studied in the lab was to take them from human embryos—typically the discarded pre-implantation embryos produced at fertility clinics. "We knew that human embryonic stem cells were potentially quite powerful, but there were ethical and technical issues that discouraged a lot of researchers from getting involved in that work," says Gage.

Even identifying adult stem cells in patient tissue samples was extremely difficult. Then in 2006 came a major breakthrough. Researchers reported that they had found a technique that would allow ordinary adult cells, such as skin cells, to be "reprogrammed" to a stem cell state by inserting four special genes into their DNA. The first demonstration was in mouse cells; then in 2007, researchers showed that the technique works in human cells too. "It meant that in principle anybody could have their own individualized stem cell therapy; it opened the floodgates of research," Gage says.

These "induced pluripotent stem cells," or iPS cells, are now the focus of most therapy-oriented stem cell research. One of the big challenges facing researchers is to understand how iPS cells differ from embryonic stem cells, and a major contribution to the understanding of those differences was reported by Salk scientists Joseph Ecker, Ronald Evans and colleagues in Nature last March. Their team found that the pattern of gene activity within iPS cells differs extensively from that in embryonic cells. "We need to understand what these differences would mean if you were to inject such iPS cells into people for therapies," says Ecker.

"Knowing these differences also gives us an opportunity to find drugs that can erase the changes and create a more effective cell," adds Evans. The researchers now are collaborating with other labs to see how these differences change depending on the techniques used to reprogram cells to the iPS state.

A mammary bud in a mouse at about 15 days of embryogenesis.
Image from Dannielle Engle in Geoff Wahl's lab.

Scientists normally reprogram adult tissue cells into iPS cells using an artificial virus that carries the four reprogramming genes into target cells and inserts the genes into the cells' DNA. But with this basic technique, the reprogramming genes may end up in a wide variety of places—and in some of these places, their insertion will promote cancer or otherwise harm the cell, which is a significant obstacle to the therapeutic use of iPS cells. Indeed, Izpisúa Belmonte and his lab, collaborating with the labs of Kun Zhang and Larry Goldstein at UC San Diego and the labs of Louise Laurent and Jeanne Loring at The Scripps Research Institute, have shown that iPS cells often contain unpredictable DNA abnormalities. Izpisúa Belmonte's laboratory and the laboratory of Salk professor Inder Verma are now developing a new technique that allows them to insert genes more safely, into precisely targeted locations in cellular DNA.

Another approach to making iPS cells safer is to start with cells that are already in the stem cell state. Salk researcher Ronald Evans and his team have developed a technique to make iPS cells from adult stem cells in people's fat deposits—the kind of deposits removed during liposuction, for example. Adult fat stem cells on their own can make only new fat cells, but when reprogrammed to the pluripotent iPS state, they can be directed to make virtually any other cell type. "Fat is a rich source of stem cells; we can efficiently turn them into iPS cells, and they also have a unique safety advantage," Evans says. Normally, cells that are reprogrammed to the iPS state have to be grown in the presence of special "feeder cells" that provide essential nutrients, but feeder cells typically come from other people or from mice and contain viruses and foreign proteins. "Exposure to this foreign material can create serious contamination problems, but fat cells don't require these foreign feeder cells, so they represent a real breakthrough," Evans says.

Therapies on the horizon


Cancer's stem cells


Studies of stem cells have been making a major impact on the scientific understanding of cancer. Researchers recently have learned, for example, that tumors typically contain their own stem cell-like cells that may survive chemo or radiation therapy and later seed the growth of new, more aggressive tumors. Several labs at Salk are now working in this area. Inder Verma recently reported that in the virtually incurable brain cancer known as glioma, some mature glial cells undergo an iPS-like transformation into a stem cell state. "That's why gliomas are so terrible," says Verma. "Even after a surgeon has operated, and 99.9 percent of cells are removed, tumor stem cells that have been left behind can make a whole new tumor."

Incipient cancer cells are often kept from proliferating by the activity of a protein called p53, known as a tumor suppressor and sometimes called the "Guardian of the Genome." Salk professor Geoffrey Wahl and his lab, in collaboration with Izpisúa Belmonte's lab, have shown that p53 also can obstruct the reprogramming events that produce iPS cells from ordinary mature cells. "When p53 is disabled, the reprogramming process happens much more efficiently," Wahl says.

Wahl's lab next asked whether cancer cells that contain inactivated p53—as many do— show signs of having reverted to a primitive, embryonic stem cell-like state. In collaboration with Arnold Levine's lab at Princeton, they studied p53-disabled breast cancer cells and found that they do indeed strongly resemble embryonic stem cells. More recently, the two groups extended this work by finding resemblances between these breast cancer cells and the normal stem cells that arise in the embryo and are destined to build the adult mammary gland. "These cancer cells become caricatures of normal developing cells by resurrecting gene activity patterns from their original developmental phase," Wahl says. "It's a phenomenon that could be very relevant to cancer treatment, drug resistance and metastasis."

Salk researcher Inder Verma was known as an expert on gene therapy research when he decided to begin studying stem cells a few years ago. The first successful gene therapy trials, for children born with severe immunodeficiencies, were carried out using viral vectors he designed. "With gene therapy, ideally you want to introduce a therapeutic gene that keeps working for the rest of the life of the patient. If you put a gene in a cell that lasts for only a few months, it doesn't really help. But in a stem cell it could work for the rest of the patient's life," he says.

A stem cell that comes from the patient, rather than from a donor, would be particularly useful, so when the first iPS techniques were reported, Verma decided to extend his work to stem cells. His main goal now is to use iPS cells to create therapeutic quantities of the adult bone marrow stem cells that replenish our blood and immune cells. This past May, in the journal Stem Cells, Verma and his lab reported reaching a significant milestone on the way to this goal: the highly efficient generation of human blood cells from iPS cells. The lessons learned from that study are now enabling them to zero in on the "recipe" for turning iPS cells into plentiful marrow stem cells. "We are quite close to being able to take ordinary cells from a patient, convert them to these marrow stem cells and give these marrow stem cells back to the patient, without worrying about immune rejection," says Verma.

That on its own would be therapeutic in many conditions—for example, in patients who need a bone marrow transplant. "Our ultimate goal is to use the marrow cells as vehicles for therapeutic genes, and then there is almost no limit to the medical applications," he adds.

Early gene therapy methods were risky because they used viruses to deliver new genes to unpredictable locations within the DNA of target cells. Researchers have needed new techniques that can deliver genes to precise locations and that can even cleanly replace bad genes with good ones. Izpisúa Belmonte's lab has made major progress in this area. Their technique (noted above) for inserting genes in a precisely targeted way can be used to repair the DNA of any cell, so that mutant, disease-causing genes are replaced with healthy versions of those genes. This past June, Izpisúa Belmonte's team reported a successful demonstration of their "gene-editing" technique in iPS cells derived from patients with Hutchinson-Gilford progeria syndrome, in which gene defects cause premature aging. "In principle, this technique could be applied to correct a wide variety of gene defects," Izpisúa Belmonte says.

Making stem cell-based models of disease


Even though stem cell therapies have not yet reached the clinic, stem cell science is having an impact on medicine. Researchers are using iPS techniques to take skin or other easily available cells from a patient, turn them into iPS cells, and then direct those iPS cells to mature into brain cells, heart cells or cells from whatever tissue is diseased in that patient. Researchers can then study these cells to gain insights into the disease process and even to test possible diseasemodifying drugs far more easily and quickly than they can in traditional animal models. They call these patient-derived cells "disease in a dish" models, and the number of papers describing them has been rising rapidly in the past few years.

One of the landmark achievements in stem cell-based disease modeling was reported by Gage and his lab last May in Nature. Taking cells from several schizophrenia patients, they converted the cells to iPS cells, then made them mature into neurons that grew so well that they formed spontaneous, brain-like networks in the lab dish. Using the iPS cell-derived neurons as a model of schizophrenia, Gage's team found that they had a host of abnormalities in gene expression as well as a reduced number of connections to each other. A standard antipsychotic drug, loxapine, reversed many of these abnormalities, thus demonstrating the potential of this iPS cell-based disease-modeling technique to help scientists test prospective drugs cheaply and easily. Gage and his lab now are improving their schizophrenia models and are working on autism-related and even Parkinson's disease models. "We're currently trying to get iPS cells to differentiate not just into basic neurons but into highly specific subtypes of neurons—for example, the midbrain dopamine neurons that are heavily affected in Parkinson's, to see if that gives us greater insight into the disease," Gage says.

Izpisúa Belmonte and his lab are active in this area too. They are using their new geneediting technique to repair the DNA in their iPS cell-based models of progeria. "In this way we can compare prematurely aging cells to normal cells to see in detail how they differ, and we can use these cells to screen drug compounds for their ability to correct these differences, and perhaps understand human aging," Izpisúa Belmonte says.

Looking forward


What stem cell medicine applications can we reasonably hope for in the next five years? "Certainly there will be drugs in clinical trials that have been discovered through the use of iPS-based cell models," says Gage.

Stem cell-based therapies are also likely to be in clinical testing, and Gage sees marrow stem cells as one of the first potential blockbuster applications. "There's already so much medical infrastructure in place for doing bone marrow transplants that this would be easy once we overcome the technical obstacles to making marrow stem cells safely," he says. "We're still in the early stages of developing these therapies. But it's all going to happen."

Salk's Stem Cell Core Facilty


Travis Berggren

W. Travis Berggren, director of the Salk Stem Cell Core.

At the core of Salk's stem cell research effort is, well, "The Core," a central facility for stem cell culturing and cold-storing and experimenting. It was set up in 2007 after Salk professor Verma led a team of Salk scientists to secure funding from the California Institute of Regenerative Medicine (CIRM). Initially it was justified as a dedicated space for research on human embryonic stem cells, which was at that time hampered by stiff federal funding restrictions even though they were the only stem cells that could be cultured in the lab. Techniques to make iPS cells were developed at about that time, and the facility's director, Travis Berggren, soon turned it into a broader center of excellence. "It's really a central resource to allow people here at Salk to get quickly up to speed in working with both embryonic and iPS cells," he says. "Growing these cells involves some significant technical challenges, and we're here so that no one at Salk has to start from scratch."

The core houses live and frozen stored cultures of both embryonic and iPS human cell lines, validates the chemical "reagents" that make up culture media, maintains the physical space and equipment where Salk researchers come to study the stem cells, trains the steady stream of new researchers who conduct stem cell experiments, and keeps abreast of the latest culturing techniques—which owe much to work done by Berggren at the University of Wisconsin, his last post before Salk. The success of the Salk stem cell core has led CIRM to renew Verma's original grant for the facility for another three years.

Leanne Jones, whose lab recently began studying human stem cells, is one of more than a dozen principal investigators at Salk whose work has been made easier by the core facility's equipment and training. "I'm first and foremost a fruit fly geneticist, and there's no way my lab could have made such a quick transition to working with human stem cells if we didn't have this facility," she says.

»1997 »1998 »1999 »2003
Gage lab reports that a socially "enriched" environment increases the generation of new neurons ("neurogenesis") in aged mice. Gage lab reports first evidence of neurogenesis in the brains of human subjects.

Gage lab reports that physical exercise boosts neurogenesis in mice and improves their cognitive abilities.

Verma lab reports the introduction of genes in human hematopoietic stem cells by lentiviral vectors that can reconstitute the repertoire of blood cells in the mouse.

Pfaff's lab describes details of how neural stem cells become motor neurons; the work suggests that stem cell therapies might help against neurodegenerative diseases such as ALS.

Izpisúa Belmonte lab describes a signaling pathway involved in heart regeneration.

»2004 »2005 »2006 »2007
Gage and Evans labs find key receptor that regulates neural stem cells. Gage lab finds key signaling molecule that regulates nerual stem cells.

Izpisúa Belmonte and Gage labs find factors that nudge embryonic cells toward muscle cell fate, or back to stem cell state.

Izpisúa Belmonte lab uses stem cell signaling factor to induce wing regeneration in fetal chicks, which normally cannot regenerate lost limbs.

Jones lab finds that stem cells' supportive environmental "niche" declines with age.
 

Stem Cell Research at Salk

Salk scientists have long been at the cutting edge of stem cell research. Supported in large part by Proposition 71, which provided $3 billion in funding for stem cell research through 2014 and set up the California Institute for Regenerative Medicine (CIRM), Salk researchers have made many pioneering discoveries.

 
»2008 »2009 »2010

Gage lab finds that switching off neurogenesis in adult mice impairs learning and memory.

Gage lab reports controlling neural stem cell fate within mouse brains.

Izpisúa Belmonte lab reports that iPS cells can be efficiently obtained from a single human hair.

Ecker lab maps the "epigenome" in human embryonic stem cells and skin cells by determining the patterns of gene-silencing methylation marks on their DNA.

Izpisúa Belmonte lab reports that human umbilical cord blood cells could be an ideal source of iPS cells.

Izpisúa Belmonte and Verma labs report the generation of disease-free hematopoietic progenitor cells from iPS cells derived from Fanconi anemia patients.

Wahl and Izpisúa Belmonte labs find that p53 tumor suppressor pathway inhibits iPS reprogramming.

Gage lab and lab of Salk alumnus Alysson Muotri at UCSD report iPS-derived cell model of Rett syndrome.

Gage lab finds hormonal signal that regulates neural stem cells and that may link exercise to neurogenesis.

Izpisúa Belmonte lab reports the mechanism by which zebrafish can regenerate lost heart tissue.

»2011

Izpisúa Belmonte lab reports successful gene-editing corrections of laminin mutations in patient-derived iPS cells.

Verma lab reports highly efficient production of hematopoetic progenitor cells from patient-derived iPS cells.

Ecker lab contributes to mapping of hydroxymethylation epigenetic marks in mouse embryonic stem cells.

Gage lab reports iPS-derived disease-in-a-dish model of schizophrenia and demonstrates its potential utility for drug testing.

Ecker and Evans labs report on major study of epigenetic differences between iPS and embryonic stem (ES) cells.

Evans lab reports that human fat stem cells can be used to generate iPS cells without need for a contaminating feeder layer.

Izpisúa Belmonte lab reports iPS-derived model of Hutchinson-Gilford progeria.

Izpisúa Belmonte lab demonstrates that the initiation of pluripotency in humans starts during preimplantation development, earlier than previously thought.

Izpisúa Belmonte lab successfully achieves complete meiosis from human iPS cells.

Izpisúa Belmonte lab, in collaboration with Kun Zhang and Larry Goldstein at UCSD and Louise Laurent and Jeanne Loring at Scripps, uncovers genomic and epigenomic alterations in human iPS cells.