Newswise — A newly discovered small molecule called IQ-1 plays a key role in preventing embryonic stem cells from differentiating into one or more specific cell types, allowing them to instead continue growing and dividing indefinitely, according to research performed by a team of scientists who have recently joined the stem-cell research efforts at the Keck School of Medicine of the University of Southern California. Their findings are being published today in an early online edition of the Proceedings of the National Academy of Sciences.

This discovery takes scientists another step closer to being able to grow embryonic stem cells without the “feeder layer” of mouse fibroblast cells that is essential for maintaining the pluripotency of embryonic stem cells, says the study’s primary investigator, Michael Kahn, Ph.D., who was recently named the first Provost’s Professor of Medicine and Pharmacy at USC. Such a layer is needed because it is currently the only proven method to provide the stem cells with the necessary chemical signals that prompt them to stay undifferentiated and to continue dividing over and over.

Still, growing human embryonic stem cells on a layer of mouse fibroblasts has never made much sense to the scientists forced to do just that. “Stem cells that grow on feeders are contaminated with mouse glycoproteins markers,” Kahn says.

“If you use them into humans, you’d potentially have a horrible immune response.”

And so, in order to take any eventual stem cell-based treatments from the laboratory to the clinic, there needs to be a way to keep the cells growing and dividing without the use of mouse fibroblasts. The discovery of IQ-1, says Kahn, is a significant step in that direction.

What IQ-1 does, Kahn explains, is to block one arm of a cell-signaling pathway called the Wnt pathway, while enhancing the signal coming from the other arm of the Wnt pathway. The Wnt pathway is known to have dichotomous effects on stem cells i.e. both proliferative and differentiative. More specifically, IQ-1 blocks the coactivator p300 from interacting with the protein ß-catenin; this prevents the stem cells from being ‘told’ to differentiate into a more specific cell type. At the same time, IQ-1 enhances the interaction between the coactivator CBP and ß-catenin, which signals the cells to keep dividing and to remain as fully potent stem cells.

“This way, you can essentially maintain the stem cell’s growth and potency for as long as you want,” Kahn says.

The studies of IQ-1 and its effects reported in the newly published PNAS paper were performed at the University of Washington in Seattle by Kahn and his colleagues (along with collaborators from the Asahi Kasei Corporation in Shizuoka, Japan) using mouse embryonic stem cells, but Kahn notes that subsequent pilot studies using human embryonic stem cells, in collaboration with Dr. Qilong Ying at the Center for Stem Cell and Regenerative Medicine at the Keck School of Medicine, have confirmed that IQ-1 plays a similar role in that system as well.

“If we can create a totally chemically defined system for growing human embryonic stem cells without any risk of contamination, it would make life much easier for scientists than it is at the moment,” says Kahn. “And that’s our goal.”

“Kahn’s study provides us with striking new insights into the molecular regulatory machinery inside embryonic stem cells,” adds Martin Pera, Ph.D., director of the Center for Stem Cell and Regenerative Medicine at the Keck School of Medicine. “His team has identified a chemical that controls a critical switch that enables stem cells to multiply indefinitely in the laboratory. These findings will help lead to the development of new techniques to propagate pure populations of embryonic stem cells on a large scale, an essential prerequisite to the successful development of stem cell based therapies.”

Tomoyuki Miyabayashi, Jia-Ling Teo, Masashi Yamamoto, Michael McMillan, Cu Nguyen, Michael Kahn, “Wnt/ß-catenin/CBP signaling maintains long-term murine embryonic stem cell pluripotency.” PNAS Early Edition, Mar. 19, 2007, http://www.pnas.org/.
 

BUFFALO, N.Y. — The problem of efficiently delivering drugs, especially those that are hydrophobic or water-repellant, to tumors or other disease sites has long challenged scientists to develop innovative delivery systems that keep these drugs intact until reaching their targets.

Now scientists in the University at Buffalo’s Institute for Lasers, Photonics and Biophotonics and Roswell Park Cancer Institute have developed an innovative solution in which the delivery system is the drug itself.

They describe for the first time, in Molecular Pharmaceutics, a drug delivery system that consists of nanocrystals of a hydrophobic drug.

The system involves the use of nanocrystals measuring about 100 nanometers of pure HPPH, (2-devinyl-2-(1′-hexyloxyethyl) pyropheophorbide), a photosensitizer currently in Phase I/II human clinical trials at RPCI for treating various types of cancer.

The UB researchers found that the nanocrystals of HPPH were taken up by tumors in vivo, with efficacy comparable to conventional, surfactant-based delivery systems.

A patent has been filed on this work.

“In this case, the drug itself acts as its own carrier,” said Haridas Pudavar, Ph.D., UB research assistant professor of chemistry and a co-author.

The nanocrystals present a major advantage over methods of delivery involving other carriers, according to Paras Prasad, Ph.D., SUNY Distinguished Professor in the Department of Chemistry in UB’s College of Arts and Sciences, executive director of the institute and a co-author.

Because other delivery systems, especially those containing surfactants, commonly used with HPPH and many other drugs, may add to the toxicity in the body, they have been considered imperfect solutions.

“Unlike formulations that require separate delivery systems, once this drug is approved, no additional approvals will be needed,” said Prasad.

“Our published data in animal models demonstrate no difference in drug activity with the nanocrystal formulation,” said Ravindra Pandey, Ph.D., Distinguished Professor of Biophysical Sciences at RPCI and a co-author on the paper.

“This is a case where the easiest formulation works the best,” added Indrajit Roy, Ph.D., UB research assistant professor of chemistry and another co-author.

The researchers found that because HPPH is amphiphillic, i.e., partially soluble in water and oil, nanocrystals of it will self-assemble, that is, in solution the molecules aggregate, but not into such big clusters that they settle to the bottom.

“It’s a controlled formation of a colloidally stable suspension of nanosized crystals,” explained Tymish Ohulchanskyy, Ph.D., UB senior research scientist and a co-author.

The researchers originally were investigating nanocrystals as a delivery method for hydrophobic dyes in bioimaging applications, another promising use for nanocrystals that they continue to pursue.

Further in vivo studies with HPPH nanocrystals are being conducted by scientists at UB and RPCI, including Pandey and Allan R. Oseroff, M.D., Ph.D., chair of the department of dermatology at RPCI and in UB’s School of Medicine and Biomedical Sciences.

The UB/RPCI team is exploring the use of the same technique for delivering other hydrophobic drugs, including those used in chemotherapy.

Additional co-authors on the paper are Koichi Baba, Ph.D., former postdoctoral research associate in the UB Department of Chemistry, and Yihui Chen, Ph.D., postdoctoral research associate at RPCI.

The nanocrystal research was supported by the National Institutes of Health, the John R. Oishei Foundation and UB’s New York State Center of Excellence in Bioinformatics and Life Sciences with additional support from RPCI.

In related work, the UB researchers have achieved improved depth penetration of HPPH using two-photon photodynamic therapy, research that recently was published in the Journal of the American Chemical Society.

The University at Buffalo is a premier research-intensive public university, the largest and most comprehensive campus in the State University of New York.

A scientist from San Diego has published some intriguing research suggesting that stem cells taken from hair follicles may someday be used to restore nerve damage.

Dr. Robert M. Hoffman of the San Diego-based AntiCancer, Inc discovered that a certain protein marker called Nestim, which is present in neural stem cells, is also present in hair follicle stem cells.   This suggests that stem cells from hair follicles have the same properties as stem cells from nerve cells.

Stem cells are a type of undifferentiated (generic) cell that can be turned into a number of specialized cells like muscle cells, neurons etc.   Stem cells can be used to replace damaged specialized cells that cannot be regenerated.   For example, stem cells can turn themselves into nerve cells and can be used to restore a damaged spinal cord and allow a paralyzed person to restore some or all of their previous function.

In the resulting studies done on mice, it was demonstrated that hair follicle stem cells can differentiate into blood vessels and neural tissue after being transplanted to a layer of skin in the mice.   Researchers also found that the hair follicle stem cells, when implanted into a region of a severed sciatic or tibial nerve in the mice’s leg, greatly enhanced the rate of nerve regeneration and the restoration of nerve function.  The improvement was so great that the mice even regained the ability to walk normally after treatment with the hair follicle stem cells.

Based on his research, Dr Hoffman concluded that hair follicle stem cells provide an effective and accessible source of stem cells for the treatment of peripheral nerve injury.

Reference: “Expert Opinion on Biological Therapy Journal” - March 2007, Vol. 7, No. 3, Pages 289-291

Newswise — For cells that hold so much promise, stem cells’ potential has so far gone largely untapped. But new research from Rockefeller University and Howard Hughes Medical Institute scientists now shows that adult stem cells taken from skin can be used to clone mice using a procedure called nuclear transfer. The findings are reported in the Feb. 12 online edition of the Proceedings of the National Academy of Sciences.

Embryonic stem cells have received the most press for their potential to generate healthy cells and tissues that could replace damaged or diseased organs.

“Scientists are well-aware that tissue derived from someone else’s embryonic stem cells would be recognized as foreign and rejected by the patient,” says senior co-author Elaine Fuchs, the Rebecca Lancefield Professor at Rockefeller and a Howard Hughes Medical Institute investigator. “This is one of the reasons why scientists have focused so much attention toward using nuclear transfer, which would allow us to use adult stem cells from the same patient rather than those harvested from an unrelated embryo.”

Fuchs and her colleagues tested the method in adult stem cells taken from the skin of mice.

Using purification methods developed in Fuchs’ Laboratory of Mammalian Cell Biology and Development, postdocs Valentina Greco and Géraldine Guasch isolated stem cells from the mice’s hair follicles. They gave these stem cells to Jinsong Li, a postdoc in Rockefeller’s Laboratory of Developmental Biology and Neurogenetics, headed by senior co-author Peter Mombaerts. To execute the nuclear transfer procedure, Li took unfertilized mouse oocytes and replaced the nucleus of each oocyte with a nucleus from these adult skin stem cells.

A main hurdle in nuclear transfer with adult cells has been its efficiency — out of a hundred attempts, only a handful may succeed — with reported success rates never reaching into double digits. “The efficiency of nuclear transfer is very low,” says Li. “Using purified adult skin stem cells as our source of nuclei, we have found that higher nuclear transfer efficiencies can be achieved.”

Greco, Guasch and Li compared the cloning efficiency of adult skin stem cells with that of more differentiated skin cells and also with cumulus cells — the cells that surround a developing oocyte and have traditionally been the preferred cell type for nuclear transfer. The stem cells gave the best efficiency, yielding 19 pups, nine of which grew up into normal, healthy, breeding adult mice.

This is not the first time scientists have tried to use adult stem cells to clone mice. Experiments using adult hematopoetic stem cells — the cells in the bone marrow that all blood cells are derived from — were reported last year. But their conclusions were confusing, says Mombaerts, and there are no reports on using adult stem cells for reproducible cloning of mice that survive until adulthood. By using cells from the same mouse and performing the experiments on the two successive days, the Rockefeller scientists could directly compare adult stem cells with other cell types.

Nuclear transfer can also be used to make embryonic stem cell lines, a process which can be done in a tissue culture dish and which is simpler and more efficient than generating a cloned mouse. Although this procedure has not yet successfully generated human embryonic stem cell lines, once technological hurdles are overcome, it may be possible in the future to use a patient’s skin stem cells to tailor make embryonic stem cell lines, circumventing the problem of immune rejection.

Such stem cells might also be used to study a variety of different diseases, for which patient tissue is often hard to come by.

“There are many diseases, such as liver, pancreatic and neurodegenerative disorders where researchers are only able to obtain affected tissue from autopsies,” says Fuchs.

If on the other hand, scientists are able to generate embryonic stem cells from the skin of a patient, for example an Alzheimer’s patient, these embryonic stem cells might be used in the laboratory to enable scientists to generate neurons and study the neurodegenerative process.

Newswise — Scientists know that a better understanding of how proteins bond could lead to more effective treatments for genetic disorders and other life-threatening conditions.

Now, a pair of Florida State University researchers’ new theory has been proven to accurately predict the association rate for proteins. Their theory is outlined in the February issue of the scientific journal Structure.

“A protein can have multiple targets or can be targeted by multiple molecules,” said Professor Huan-Xiang Zhou, who serves on the faculty of FSU’s School of Computational Science and department of physics. “Rapid association between proteins is crucial in a wide array of biological processes, such as the utilization of and defense against toxins; the activation of receptor proteins on cell membranes by growth hormones; and the regulation of actin polymerization, which influences the physical structure of living cells. The association rate thus plays a critical role in the overall health of the organism.”

Mutations are one factor that can disrupt quick association between proteins and lead to disease, he said.

“For example, Wiskott-Aldrich syndrome, a pediatric genetic disorder characterized by eczema, immune deficiencies and low blood-platelet counts, can be traced to mutations on the Wiskott-Aldrich syndrome protein,” Zhou said. “Normally, fast association of the protein with other biomolecules is critical for the creation of proper cell structures. The failure of the protein to associate quickly, then, is the root cause of the condition.”

In their Structure paper, Zhou and graduate student Razmi Alsallaq put forth a new theory that has been proven to accurately predict the association rate for proteins by developing a theoretical model for the association process. A central component of the model is the transition state, a phase that two associating proteins go through before finally becoming a specific complex. The rate prediction is broken into two parts: how much the rate would be if the proteins find each other purely through random motion, and how much electrical attraction increases the rate.

“This theory opens numerous opportunities for further study,” Zhou said.

“For example, we now can begin to uncover the molecular bases of large variations in association rate among proteins. It also might be possible to design proteins with the desired association rate.”

Attila Szabo, chief of the Theoretical Biophysical Chemistry section of the National Institutes of Health, described the Structure paper as “the most comprehensive investigation yet conducted of protein-protein association rate. It provides convincing evidence that the remarkable simplification of the calculation of association rates between proteins, proposed by Zhou and coworkers, really works.”

The Structure paper can be viewed online at http://www.structure.org/.

Tissue Engineered Products Could be on the Market in as Little as Five Years
 
SAN DIEGO — Gene therapy and tissue engineering conjure up thoughts of futuristic science fiction. But this biotechnology is developing rapidly and could be at your nearby orthopaedic surgeon’s surgical suite before you know it, according to Regis O’Keefe, MD, Ph.D., Professor of Orthopaedics with the University of Rochester Medical Center in New York State and spokesperson for the American Academy of Orthopaedic Surgeons. “Gene therapy using stem cells is a lot closer to clinical use in orthopaedics than most people think,” said Dr. O’Keefe. “These tissue engineered products could be on the market in five to ten years.”

The goal of tissue engineering is to create living tissue to replace or repair diseased tissue. Tissue engineered products for orthopaedics, may facilitate repair or serve as a “functional replacement.” There are countless applications in orthopaedics — replacement for bone, cartilage, muscle and ligament loss and to increase or promote bone formation in spinal fusions and with some fractures. Biological approaches are being used to improve muscle healing for sports injuries including menisci and ligament injuries.

Researchers have found that muscle stem cells are more plentiful than bone marrow stem cells, for example. “Muscle cells have emerged as promising vehicles for gene therapy and tissue engineering in the musculoskeletal system,” said Johnny Huard, Ph.D., Associate Professor in the Department of Orthopaedic Surgery, Molecular Genetics and Biochemistry, Bioengineering, and Pathology at the University of Pittsburgh School of Medicine, and is also the Director of the Stem Cell Research Center at Children’s Hospital of Pittsburgh. One study showed that 95% of muscle stem cells were incorporated into the bone.

O’Keefe points out that the #1 reason that people go to the doctor is for issues with the musculoskeletal system. In a society where many “baby boomers” expect to stay active longer with a high quality of life, physical fitness has become a priority. “A person cannot be fit from a cardiovascular and pulmonary standpoint without having a musculoskeletal system that is healthy and can support increased activity,” added Dr. O’Keefe. “There is a tremendous opportunity to use tissue engineering for osteoporosis, osteoarthritis and bone repair to help keep that musculoskeletal system in good condition.”

Philadelphia – In recent years, scientists have discovered that a family of enzymes called sirtuins can dramatically extend life in organisms as diverse as yeast, worms, and flies. They may also be able to control age-associated metabolic disorders, including obesity and type II diabetes.

Naturally occurring substances have been shown to activate sirtuins, including a constituent of red wine called resveratrol – although an individual would need to drink about two cases of wine a day to derive a clinically effective dose of resveratrol. Still, the findings have energized a number of scientific groups and biotechnology companies, all of which are now eagerly searching for drug candidates able to boost sirtuin activity. The public-health benefits of such an “anti-aging” drug would be substantial – as would the economic returns.

Now, a new study from scientists at The Wistar Institute points to another strategy for activating sirtuins to unleash their anti-aging powers. A report on the research appears in the February 9 edition of Molecular Cell, and a podcast interview with the study’s senior author, Ronen Marmorstein, Ph.D., a professor in the Gene Expression and Regulation Program at Wistar, is available on the Institute’s web site (http://www.wistar.org/podcast/pr/DrRonenMarmorstein/CreatingAntiAgingDrugs.mp4).

Using the techniques of structural biology, the Wistar team demonstrated that a component of the common vitamin B3, also known as niacin, binds to a specific site on the sirtuin molecule to inhibit its activity. This observation suggests that drugs designed to prevent the vitamin B3 component, nicotinamide, from binding at this site could have the effect of activating sirtuins. Any such drug would, in essence, inhibit the inhibitory effect of nicotinamide. As in mathematics, the two negatives would create a positive result – activation of sirtuins. “Our findings suggest a new avenue for designing sirtuin-activating drugs,” says Marmorstein. “The jury is still out as to whether a drug of this kind might result in longer life in humans, but I’m equally excited by the possibility that such interventions might help counteract age-related health problems like obesity and type II diabetes.”

The nicotinamide binding site may be a particularly attractive drug target for other reasons too, according to Marmorstein. His group now hopes to use rational drug design techniques to create such a drug.

“Many drugs have unwanted side effects because in addition to the intended target, the drugs also hit other biologically active molecules that you don’t want to affect,” he says. “This nicotinamide-binding site we’ve identified appears to be unique to the sirtuins, so that if we’re able to design a molecule to target it, it should be very specific for these sirtuin molecules.”

Marmorstein’s research on sirtuins also links to a long line of observations concerning calorie-restricted diets and longevity.

“People have known for some time that low-calorie diets result in life extension in many organisms, but they didn’t know why,” he says. “Recent research has shown that the connection works at least in part through these sirtuin molecules.”

The lead author on the Molecular Cell study is Brandi D. Sanders at The Wistar Institute. Kehao Zhao, Ph.D., formerly at Wistar and now at the Novartis Institutes for Biomedical Research Inc. in Cambridge, Massachusetts, is also a coauthor, as is James T. Slama, Ph.D., with the College of Pharmacy at the University of Toledo, Ohio. Funding to support the research was provided by the National Institutes of Health and the Commonwealth Universal Research Enhancement Program of the Pennsylvania Department of Health.

The Wistar Institute is an international leader in biomedical research, with special expertise in cancer research and vaccine development. Founded in 1892 as the first independent nonprofit biomedical research institute in the country, Wistar has long held the prestigious Cancer Center designation from the National Cancer Institute. Discoveries at Wistar have led to the creation of the rubella vaccine that eradicated the disease in the U.S., rabies vaccines used worldwide, and a new rotavirus vaccine approved in 2006. Wistar scientists have also identified many cancer genes and developed monoclonal antibodies and other important research tools. Today, Wistar is home to eminent melanoma researchers and pioneering scientists working on experimental vaccines against flu, HIV, and other diseases. The institute works actively to transfer its inventions to the commercial sector to ensure that research advances move from the laboratory to the clinic as quickly as possible. The Wistar Institute: Today’s Discoveries – Tomorrow’s Cures. On the web at www.wistar.org.

Newswise — The layers of mucus that protect sensitive tissue throughout the body have an undesirable side effect: they can also keep helpful medications away. To overcome this hurdle, Johns Hopkins researchers have found a way to coat nanoparticles with a chemical that helps them slip through this sticky barrier.

During experiments with these coated particles, the researchers also discovered that mucus layers have much larger pores than previously thought, providing a doorway that should allow larger and longer-acting doses of medicine to reach the protected tissue.

The team’s findings were reported this week in the Early Online Edition of Proceedings of the National Academy of Sciences.

The discoveries are important because mucus layers, which trap and help remove pathogens and other foreign materials, can block the localized delivery of drugs to many parts of the body, including the lungs, eyes, digestive tract and female reproductive system. Because of these barriers, doctors often must prescribe pills or injections that send drugs through the entire body, an approach that can lead to unwanted side effects or doses that are too weak to provide effective treatment.

“Mucus barriers evolved to serve a helpful purpose: to keep things out,” said Justin Hanes, an associate professor of chemical and biomolecular engineering who supervised the research. “But if you want to deliver medicine in a microscopic particle, they can also keep the drugs from getting through. We’ve found a way to keep helpful nanoparticles from sticking to mucus, and we learned that the openings in the mucus ‘mesh’ are much larger than most people expected. These findings set the stage for a new generation of nanomedicines that can be delivered directly to the affected areas.”

To get its particles past the mucus, Hanes’ team studied an unlikely model: viruses. Earlier research led by Richard Cone, a professor in the Department of Biophysics at Johns Hopkins, had established that some viruses are able to make their way through the human mucus barrier. Hanes and his colleagues decided to look for a chemical coating that might mimic the characteristics of a virus.

“We found that the viruses that got through had surfaces that were attracted to water, and they had a net neutral electrical charge,” said Samuel K. Lai, a Johns Hopkins chemical and biomolecular engineering doctoral student from Canada and Hong Kong who was lead author of the journal article. “We thought that if we could coat a drug-delivery nanoparticle with a chemical that had these characteristics, it might not get stuck in the mucus barrier.”

To make their nanoparticles behave like viruses, the researchers coated them with polyethylene glycol, PEG, a non-toxic material commonly used in pharmaceuticals. PEG dissolves in water and is excreted harmlessly by the kidneys.

The researchers also considered the size of their nanoparticles. Previous studies indicated that even if nanoparticles did not stick to the mucus, they might have to be smaller than 55 nanometers wide to pass through the tiny openings in the human mucus mesh. (A human hair is roughly 80,000 nanometers wide.) Using high-resolution video microscopy and computer software, the researchers discovered that their PEG-coated 200-nanometer particles could slip through a barrier of human mucus.

They then conducted further tests to see how large their microscopic drug carriers could be before they got trapped in the mesh. Larger nanoparticles are more desirable because they can release greater amounts of medicine over a longer period of time. “We wanted to make the particles as large as possible,” said Hanes, who also serves as director of therapeutics for the Institute for NanoBioTechnology at Johns Hopkins. “The shocking thing was how fast the particles that were 500 nanometers wide moved through the mucus mesh. The work suggests that the openings in the mucus barrier are much larger than originally expected by most. And we were also surprised to find that the larger nanoparticles (200 and 500 nanometers wide) actually moved through the mucus layer more quickly than the smaller ones (100 nanometers wide).”

This has important implications, Hanes said, because a 500-nanometer particle can be used to deliver medicine to a targeted area, released over periods of days to weeks. Larger particles also allow a wider array of drug molecules to be efficiently encapsulated. He and his colleagues believe this system has great potential in the delivery of chemotherapy, antibiotics, nucleic acids and other treatment directly to the lungs, gastrointestinal tract and cervicovaginal tract.

Through Johns Hopkins Technology Transfer, the team has applied for patents covering this process.