News Article | April 6, 2016
Neuroscientists desperately need better tools to develop drugs for treating mental illness and neurodegenerative diseases. Experiments in lab animals more often than not produce drug candidates that ultimately fail in clinical trials. If a laboratory cell culture existed that accurately mimicked the intricate cellular networks that make up the cerebral cortex, it would provide a reality check and help answer the persistent question every drug developer wants to know: if this chemical works in mice that mimic some of the effects of schizophrenia or autism, will it work in humans? A team from Johns Hopkins Medical School and Nanjing University has just created a microcosm of the cerebral cortex that fits inside a lab dish. The “cortex in a dish” consists of an interwoven mesh of neurons that transmit electrical signals and other cells that damp down this activityThe cultured neurons may expand the toolkit needed to lift neurological and psychiatric drug development out of its present rut. Scientific American talked with Valina Dawson, co-director of the Institute for Cell Engineering at Johns Hopkins University School of Medicine, about a paper published April 6 in Science Translational Medicine on which she was the senior author. Scientific American: Describe what a 'cortex in a dish' is? Valina Dawson: The brain is divided into different structures and regions. The cerebral cortex is the largest part of the brain and manages higher brain functions such as thought and actions, language, sensory processing such as hearing and vision. One way to study how the neurons in the cortex function is to grow them in culture, “in a dish.” SA: Why have researchers wanted to create this laboratory model of the cortex? VD: Having human neurons in culture allows experimental investigations into signaling events at the chemical, protein and genetic levels that underlie normal and diseased actions in a manner that would not be possible or ethical in an intact human brain. Understanding how the neurons in the cortex work and what goes wrong when disease occurs will provide, we hope, new therapeutic opportunities to treat patients who suffer from brain injury and disease. SA: Why has it been so difficult to build this and how did you overcome hurdles along the way? VD: The ability to create human neurons from stem cells is relatively new. Established protocols to make different types of neurons from stem cells are limited to a few types of neurons out of the hundreds that exist in the human brain. Most of our understanding about the carefully controlled and elegantly choreographed events in the development of the cortex are from less complex organisms. Thus some of the key elements necessary to generate a protocol to produce the complex network of neuronal populations had to be guessed and identified by trial and error. SA:. What will scientists be able to do with your system? VD: We hope they will use this system to understand important mechanisms in cortical function and communication. We hope these cultures will also be useful in understanding how to provide neuroprotection against stroke and trauma and to investigate what goes wrong in diseases such as schizophrenia, autism and epilepsy. SA: Didn’t you show in the paper just published how this might work? Yes, in a model of stroke. Stroke is a common cause of death, disability and loss of quality of life world-wide but unfortunately there are few treatments to reduce the brain injury suffered. During a stroke there is a loss of blood flow to brain tissue. This can be mimicked in a culture dish by removing oxygen and glucose or by activating with the chemical NMDA a specific protein on the surface of neurons, an excitatory receptor. We have used our cortical culture system to study the cellular signaling events that occur and lead to neuronal cell death. Previously we found in rodent systems, that a member of a biochemical pathway, poly(ADP-Ribose) polymerase-1 (PARP-1) is pathologically activated and serves as a switch, directing the cell away from DNA repair towards cell death. Preventing PARP-1 activation protects neurons from ischemic cell death. For the first time we were able to determine using our cortical culture if human neurons respond in a similar manner, and they do! Inhibitors of PARP have been developed for the treatment of patients with certain types of cancer and some of these clinically useful drugs cross the blood brain barrier. Finding agents that can gain access to the brain has been a major hurdle in developing good treatments for neurologic disease and injury. Our studies raise the potential that these drugs could also be useful in the treatment of stroke. SA: What are the next steps? VD: Besides using these cultures to study how cortical neurons protect themselves from injury, we are also using these cultures to probe the molecular signaling events that underlie a form of autism with the hopes of finding a way to intervene. Additionally, other brain regions connect to the cortex, providing important information or receiving instructions from cortical regions. In the future, cultures could be established so that these connections could be studied at the cellular and sub-cellular level. One could also envision using sophisticated bioengineered scaffolds to permit the normal layering of the cortex so that interconnectivity between layers could be studied. In the science fiction future, perhaps cortical plugs would be developed that could be implanted into patients with stroke or trauma to replace the brain material that was damaged and lost.
Prabhakar N.R.,University of Chicago |
Prabhakar N.R.,Institute for Cell Engineering |
Prabhakar N.R.,Johns Hopkins University |
Semenza G.L.,University of Chicago |
And 2 more authors.
Physiological Reviews | Year: 2012
Hypoxia is a fundamental stimulus that impacts cells, tissues, organs, and physiological systems. The discovery of hypoxia-inducible factor-1 (HIF-1) and subsequent identification of other members of the HIF family of transcriptional activators has provided insight into the molecular underpinnings of oxygen homeostasis. This review focuses on the mechanisms of HIF activation and their roles in physiological and pathophysiological responses to hypoxia, with an emphasis on the cardiorespiratory systems. HIFs are heterodimers comprised of an O2-regulated HIF-1αor HIF-2αsubunit and a constitutively expressed HIF-1βsubunit. Induction of HIF activity under conditions of reduced O2 αvailability requires stabilization of HIF-1α and HIF-2α due to reduced prolyl hydroxylation, dimerization with HIF-1β, and interaction with coactivators due to decreased asparaginyl hydroxylation. Stimuli other than hypoxia, such as nitric oxide and reactive oxygen species, can also activate HIFs. HIF-1 and HIF-2αre essential for acute O2 sensing by the carotid body, and their coordinated transcriptional activation is critical for physiological adaptations to chronic hypoxia including erythropoiesis, vascularization, metabolic reprogramming, and ventilatory acclimatization. In contrast, intermittent hypoxia, which occurs in association with sleep-disordered breathing, results in an imbalance between HIF-1α and HIF-2α that causes oxidative stress, leading to cardiorespiratory pathology. © 2012 the American Physiological Society.
Christian K.M.,Institute for Cell Engineering |
Song H.,Institute for Cell Engineering |
Song H.,Johns Hopkins University |
Ming G.-L.,Institute for Cell Engineering |
Ming G.-L.,Johns Hopkins University
Annual Review of Neuroscience | Year: 2014
Adult neurogenesis, a developmental process of generating functionally integrated neurons, occurs throughout life in the hippocampus of the mammalian brain and showcases the highly plastic nature of the mature central nervous system. Significant progress has been made in recent years to decipher how adult neurogenesis contributes to brain functions. Here we review recent findings that inform our understanding of adult hippocampal neurogenesis processes and special properties of adult-born neurons. We further discuss potential roles of adult-born neurons at the circuitry and behavioral levels in cognitive and affective functions and how their dysfunction may contribute to various brain disorders. We end by considering a general model proposing that adult neurogenesis is not a cell-replacement mechanism, but instead maintains a plastic hippocampal neuronal circuit via the continuous addition of immature, new neurons with unique properties and structural plasticity of mature neurons induced by new-neuron integration. © Copyright ©2014 by Annual Reviews. All rights reserved.
Semenza G.L.,Institute for Cell Engineering |
Semenza G.L.,Johns Hopkins University
Arteriosclerosis, Thrombosis, and Vascular Biology | Year: 2010
Blood vessels function as conduits for the delivery of O2 and nutrients. Hypoxia-inducible factor 1 (HIF-1) mediates adaptive transcriptional responses to hypoxia/ischemia that include expression of angiogenic cytokines/growth factors by hypoxic cells and expression of cognate receptors for these ligands by vascular cells and their progenitors. Impairment of HIF-1-dependent responses to hypoxia is a major factor contributing to the impaired vascular responses to ischemia that are associated with aging and diabetes. © 2010 American Heart Association, Inc.
News Article | October 8, 2016
Strokes, injuries and even neurological diseases such as Alzheimer's, Parkinson's or Huntington's seem to follow the same chain of events, in spite of their distinct triggers. New research has found the end of the chain, responsible for the fatal step through carving up a cell's DNA. The conclusions of the research create new possibilities in the development of drugs aimed at treating and even preventing the process. The study, published on Oct. 7 in Science, documents the experiments conducted inside lab-grown cells, signed by Ted Dawson, director of the Institute for Cell Engineering at the Johns Hopkins University School of Medicine, and Valina Dawson, professor of neurology. Cell death occurs because of the toxic and/or stressful insults, and a scientific way to stop its activity would represent a huge step in the fight against neurodegenerative disorders, as well as any other form of cell injury. The shared mechanisms of these diseases share a programmed brain cell death the team named parthanatos, which is an enzyme involved in almost all forms of cellular injuries. Other scientific studies showed that a protein named mitochondrial apoptosis-inducing factor (AIF) sometimes leaves its normal place in the cellular mitochondria moving toward the nucleus, triggering the carving up of the genome housed in the nucleus. The process is responsible for cell death. However, AIF cannot cut DNA alone. The endonuclease G promotes DNA degradation cooperating with AIF but doesn't play an important role in the chromatinolysis that is PARP-dependend, therefore not being responsible for cell death. But Yingfei Wang, assistant professor at the University of Texas Southwestern Medical Center, tested which proteins interact most powerfully with the AIF by screening other human proteins. Using custom molecules known as small interfering RNAs, she tried to stop the formation of proteins in human cells grown in her laboratory to test the hypothesis of preventing cell death. Of the total number of proteins, the macrophage migration inhibitory factor (MIF) was the one found responsible for the interaction. Although MIF's capacity to affect DNA was only clearly linked to stroke. Disabling the MIF gene in mice reduced the number of strokes. The paper also mentioned that some chemical compounds blocking the action of MIF in the cells grown in the lab protected them from parthanatos. According to the scientists, they also plan on testing them on animals to maximize the efficiency. "We're interested in finding out whether MIF is also involved in Parkinson's, Alzheimer's and other neurodegenerative diseases," explained Dawson. Blocking the interaction of the two or the entire nuclease activity of MIF would open a major line of therapeutic opportunities for patients of various diseases. © 2017 Tech Times, All rights reserved. Do not reproduce without permission.
News Article | October 7, 2016
Despite their different triggers, the same molecular chain of events appears to be responsible for brain cell death from strokes, injuries and even such neurodegenerative diseases as Alzheimer's. Now, researchers at Johns Hopkins say they have pinpointed the protein at the end of that chain of events, one that delivers the fatal strike by carving up a cell's DNA. The find, they say, potentially opens up a new avenue for the development of drugs to prevent, stop or weaken the process. A report on the research appears in the Oct. 7 issue of the journal Science. The new experiments, conducted in laboratory-grown cells, build on earlier work by research partners Ted Dawson, M.D., Ph.D., now director of the Institute for Cell Engineering at the Johns Hopkins University School of Medicine, and Valina Dawson, Ph.D., professor of neurology. Their research groups found that despite their very different causes and symptoms, injury, stroke, Alzheimer's disease, Parkinson's disease and the rare, fatal genetic disorder Huntington's disease have a shared mechanism of a distinct form of "programmed" brain cell death they named parthanatos after the personification of death in Greek mythology and PARP, an enzyme involved in the process. "I can't overemphasize what an important form of cell death it is; it plays a role in almost all forms of cellular injury," Dawson says. His and Valina Dawson's research groups have spent years delineating each of the links in the parthanatos chain of events and the roles of the proteins involved. The current study, they say, has completed the chain. From previous studies, the researchers knew that when a protein called mitochondrial apoptosis-inducing factor, or AIF, leaves its usual place in the energy-producing mitochondria of the cell and moves to the nucleus, it sparks the carving up of the genome housed in the nucleus and leads to cell death. But AIF itself, they say, can't cut DNA. So then-postdoctoral fellow Yingfei Wang, Ph.D., now an assistant professor at the University of Texas Southwestern Medical Center, used a protein chip to screen thousands of human proteins to find those that interacted most strongly with AIF. Working with the 160 candidates she uncovered, she then used custom molecules called small interfering RNAs to stop each of those proteins' manufacture, one by one, in lab-grown human cells to see if doing so would prevent cell death. One of the 160 proteins, known as macrophage migration inhibitory factor (MIF), was a winner. "We found that AIF binds to MIF and carries it into the nucleus, where MIF chops up DNA," Dawson says. "We think that's the final execution step in parthanatos." The group reports that in work to be published, it also identified a few chemical compounds that block MIF's action in the lab-grown cells, protecting them from parthanatos. Dawson says they plan to test these in animals, and modify them to maximize their safety and effectiveness. He cautions that while parthanatos is known to cause cell death in many brain conditions, MIF's ability to chop up DNA has so far only been definitively linked with stroke -- when the MIF gene was disabled in mice, the damage caused by a stroke was dramatically reduced. "We're interested in finding out whether MIF is also involved in Parkinson's, Alzheimer's and other neurodegenerative diseases," he says. If so, and if an inhibitor of MIF proves successful in testing, it could have implications for treating many conditions, he says.
News Article | December 14, 2016
The investigators say the ability to reset the stem cells' developmental clock to an earlier stage offers new opportunities to successfully coax human stem cells into making any kind of cell on demand for use as transplants and in genetic disease modeling. Eventually, they may be used to create chimeric animals from which human organs could be harvested. Reporting on their work in the Nov. 29 Development, the researchers wrote that their so-called 3i cocktail, named for its three chemical inhibitors, produced stem cells with all the same features of classic mouse ESCs: they are easy to grow, manipulate and steer to differentiate into a variety of cell types—without the genetic instability that resulted from previous efforts to transform human stem cells. "These cells are exactly what we've been hoping for ever since the first human ESCs were derived," says Elias Zambidis, M.D., Ph.D., associate professor of oncology at the Johns Hopkins University School of Medicine and member of the Johns Hopkins Kimmel Cancer Center and Institute for Cell Engineering. When the first human ESCs were isolated in 1998, researchers working with these cells quickly noticed differences between them and those isolated nearly two decades earlier from mice. Mouse ESCs easily thrived in petri dishes, were able to generate nearly every cell or tissue type, could be genetically manipulated without much effort, and could make chimeras (organisms containing cells with at least two different sets of DNA). However, researchers found it far more difficult to coax conventional human ESCs into similarly performing these tasks. The human cells were more difficult to keep alive in laboratory cultures, could make only a limited selection of tissue types with far more work, and inserting or removing genes took substantially more effort. Researchers also had similar difficulties with other human stem cells derived from mature adult cells through a process called induced pluripotency. "For many years, people just kind of shrugged their shoulders and said, 'Humans and mice are different species. You can't expect their cells to behave the same,'" says Zambidis. Then, in 2007, researchers discovered a new type of primitive mouse stem cell known as an epiblast stem cell, derived from cells just a couple of days older than classic mouse ESCs. Rather than the facile features of their conventional mouse ESC counterparts, these epiblast stem cells behaved like the less malleable conventional human ESCs already in use. Suddenly, says Zambidis, many researchers began to suspect that human ESCs were more akin to these less pliable mouse epiblast stem cells and not to the more versatile mouse ESCs, and that an authentic human ESC with more useful features had yet to be discovered. Getting human ESCs to revert to the classical mouse ESC state, however, has proven difficult, Zambidis says. In 2015, several research groups published scientific findings suggesting that dosing conventional human ESCs with certain chemical mixtures could create this "ground state" similar to mouse ESCs, but subsequent research showed that these cells may have unstable genetic properties that may be related to the chemicals used to create them. In a new effort to create more malleable human ESCs, Zambidis and his colleagues dosed conventional ESCs with novel combinations of chemicals known to regulate well-studied developmental signaling pathways. Eventually, he says, they hit on a mixture of just three compounds that inhibit signaling pathways known to be pivotal when early cells mature into defined cell types. Two of the compounds in the 3i cocktail, inhibitors of the WNT and MEK/ERK signaling pathways, were previously used to help mouse embryonic stem cells maintain a primitive state. The third chemical that Zambidis and his colleagues added to the mixture is an anti-cancer agent called a tankyrase inhibitor. Zambidis and his team were able to "reset" a broad range of more than 25 human stem cell lines by using this new cocktail of three chemical inhibitors. They showed that these reset human ESCs expressed genes and proteins common only in the more malleable mouse ESCs, as well as in human preimplantation embryos, but not in conventional human ESCs. They also found these newly reverted human ESCs did not have the abnormal changes in their DNA that other methods induced. The new human ESCs differentiated into transplantable vascular and neural cell types at double or triple the frequencies of conventional human ESCs. Zambidis says he and his colleagues are now using this new class of human ESCs to develop blood, vascular and retinal tissues that are more suitable for transplantation than those previously derived from conventional human ESCs. Zambidis says this new class of human ESCs is more amenable to correction of genes known to be associated with diseases that include sickle cell disease and Parkinson's disease. The new ESCs could also create chimeras in which human organs can grow in animals and provide a potentially unlimited source of transplantable organs, he says. His team is now studying the cellular mechanisms behind this new 3i cocktail to better understand how it rewinds the clock in these cells. Explore further: Defining what it means to be a naive stem cell More information: Ludovic Zimmerlin et al. Tankyrase inhibition promotes a stable human naïve pluripotent state with improved functionality, Development (2016). DOI: 10.1242/dev.138982
News Article | April 23, 2016
The SpinΩ bioreactor used to create the conditions for mini-brains to grow.Image: John Hopkins Medicine Researchers from Johns Hopkins University have confirmed one of the most important ways that Zika causes microcephaly in the brains of infants via an innovative new technology, according to a study published Friday in Cell. By growing realistic brain organoids (groups of tissue meant to mimic actual organs) inside 3D printed bioreactors (vessels in which the brains are grown) the researchers confirmed that Zika infects specialized stem cells responsible for building the cerebral cortex, the outer layer of the brain. Zika then turns these cells into virus factories. The mini brains consisted of many different kinds of cells mixed together Xuyu Qian, the lead author of the paper and a PhD candidate at Johns Hopkins University told me. His team’s work showed that the virus prefers attacking neurological stem cells—the kind found in the cranium of developing fetuses—over other types. “In this paper we confirmed the preferential target of the virus,” Qian said. Their work also showed that the extent of the damage caused by Zika differs depending on when cells are infected. The "mini brains" survive for up to 100 days according to the study, allowing the researchers to examine what happens when a mother is infected very early and towards the middle of her pregnancy. Their work supported other clinical findings that suggest babies are most at risk during their first trimester, although there are still disastrous effects later in a mother’s pregnancy. A mini-brain infected with Zika virus. The virus is shown in green, vulnerable neural progenitor cells are shown in red, and neurons are shown in blue. The researchers grew the "brains" using human stem cells. A number of researchers in different fields have grown tiny organs for years, but the process is typically incredibly difficult due to the complexity of various organs and the high cost of the nutrients needed to cultivate human stem cells in the lab. In order to help overcome these hurdles, several high school students who spent a summer working in the Johns Hopkins University School of Medicine’s Institute for Cell Engineering came up with the idea for a smaller 3D printed bioreactor that would require less nutrients than the larger, commercially available options. After three years of testing different prototypes, the researchers were finally able to develop one that provided a suitable environment for the development of tiny brains. Dubbed SpinΩ, the paper’s authors included plans for it in their work, so that it could be recreated in other labs. “The design isn’t sophisticated, but it’s very intuitive,” Qian said. “This technology is not restricted to the Zika virus, it can be used to study all sorts of developmental disorders,” Qian told me. In the future, he hopes to commercialize the bioreactor that his team created in order to lower its price. “We hope it becomes a standard model for studying human development,” he said.
News Article | March 2, 2017
Researchers have identified a biochemical chain of events that allows some breast cancer cells to survive chemotherapy and restart tumor growth. The recurrence of cancer stem cells is tied to the drug resistance that develops in many breast tumors. Because of that resistance, the benefits of chemo are short-lived for many patients. Cancer recurrence after chemotherapy is frequently fatal. “Breast cancer stem cells pose a serious problem for therapy,” says lead investigator Gregg Semenza, professor of medicine and director of vascular biology at Johns Hopkins University’s Institute for Cell Engineering. “These are the cells that can break away from a tumor and metastasize,” Semenza says. “These are the cells you most want to kill with chemotherapy. Paradoxically, though, cancer stem cells are quite resistant to chemotherapy.” Earlier studies showed that resistance arises from the hardy nature of cancer stem cells, Semenza says. They often are found in the centers of tumors, where oxygen levels are low. Proteins known as hypoxia-inducible factors, or HIFs, turn on genes that help the cells survive in a low-oxygen environment. In the new study, Semenza and his Johns Hopkins colleagues analyzed human breast cancer cell lines grown in the laboratory after exposure to chemotherapy drugs like carboplatin, which stops tumor growth by damaging cancer cell DNA. The team found that the cancer cells that survived tended to have higher levels of a protein known as glutathione-S-transferase O1, or GSTO1. Experiments showed that HIFs controlled the production of GSTO1 in breast cancer cells when they were exposed to chemotherapy. If the researchers blocked HIF activity in these lab-grown cells, the cells did not produce GSTO1. After exposure to a chemotherapy drug, GSTO1 binds to a protein, RYR1, triggering the release of calcium. That causes a chain reaction that transforms ordinary breast cancer cells into cancer stem cells. To more directly assess the role of GSTO1 and RYR1 in the breast tumor response to chemotherapy, the researchers injected human breast cancer cells into the mammary gland of mice and then treated the mice with carboplatin after tumors had formed. In addition to using normal breast cancer cells in the experiments, the team also used cancer cells genetically engineered to lack either GSTO1 or RYR1. Loss of either GSTO1 or RYR1, the researchers report, decreased the number of cancer stem cells in the primary tumor, blocked metastasis of cancer cells from the primary tumor to the lungs, decreased the duration of chemotherapy required to induce remission, and increased the duration of time after chemotherapy was stopped that the mice remained tumor-free. Although the study showed that blocking the production of GSTO1 may improve the usefulness of chemotherapy drugs, GSTO1 is only one of many proteins produced under the control of HIFs in breast cancer cells after chemotherapy. Semenza and his team are working to develop HIF inhibitor drugs that they hope will make chemotherapy more effective. The Department of Defense and the American Cancer Society funded the work, which appears in the journal Cell Reports.
News Article | September 16, 2016
Working with animals, a team of scientists reports it has delivered stem cells to the brain with unprecedented precision by threading a catheter through an artery and infusing the cells under real-time MRI guidance. In a description of the work, published online Sept. 12 in the Journal of Cerebral Blood Flow and Metabolism, they express hope that the tests in anesthetized dogs and pigs are a step toward human trials of a technique to treat Parkinson’s disease, stroke, and other brain damaging disorders. “Although stem cell-based therapies seem very promising, we’ve seen many clinical trials fail. In our view, what’s needed are tools to precisely target and deliver stem cells to larger areas of the brain,” says Piotr Walczak, M.D., Ph.D., associate professor of radiology at the Johns Hopkins University School of Medicine’s Institute for Cell Engineering. The therapeutic promise of human stem cells is derived from their ability to develop into any kind of cell and, in theory, regenerate injured or diseased tissues ranging from the insulin-making islet cells of the pancreas that are lost in type 1 diabetes to the dopamine-producing brain cells that die off in Parkinson’s disease. Ten years ago, Shinya Yamanaka’s research group in Japan raised hopes further when it developed a technique for “resetting” mature cells, such as skin cells, to become so-called induced pluripotent stem cells. That gave researchers an alternative to embryonic stem cells that could allow the creation of therapeutic stem cells that matched the genetic makeup of each patient, greatly reducing the chances of cell rejection after they were infused or transplanted. But while induced pluripotent stem cells have enabled great strides forward in research, Walczak says they are not yet approved for any treatment, and barriers to success remain. In a bid to address once such barrier – how to get the cells exactly where needed and no place else – Walczak and his colleague Miroslaw Janowski, M.D., Ph.D., assistant professor of radiology, sought a way around strategies that require physicians to puncture patients’ skulls or inject them intravenously. The former, Walczak says, is not only unpleasant, but also only allows delivery of stem cells to one limited place in the brain. In contrast, injecting cells intravenously scatters the cells throughout the body, with few likely to land where they’re most needed, says Walczak.