Scientists Successfully Expand Bone Marrow-Derived Stem Cells in Culture

Fully functional hematopoietic stem cells (or blood-forming) are successfully proliferating amongst other bone marrow-derived cells in a culture dish. 

ScienceDaily (Sep. 12, 2011) — All stem cells -- regardless of their source -- share the remarkable capability to replenish themselves by undergoing self-renewal. Yet, so far, efforts to grow and expand scarce hematopoietic (or blood-forming) stem cells in culture for therapeutic applications have been met with limited success.

Now, researchers at the Stowers Institute for Medical Research teased apart the molecular mechanisms enabling stem cell renewal in hematopoietic stem cells isolated from mice and successfully applied their insight to expand cultured hematopoietic stem cells a hundredfold.

Their findings, which will be published in the Sept. 15, 2011, edition of Genes & Development, demonstrate that self-renewal requires three complementary events: proliferation, active suppression of differentiation and programmed cell death during proliferation.

"The previous efforts so far to grow and expand scarce hematopoietic stem cells in culture for therapeutic applications have been met with limited success," says Stowers investigator Linheng Li, Ph.D., who led the study. "Being able to tap into stem cell's inherent potential for self-renewal could turn limited sources of hematopoietic stem cells such as umbilical cord blood into more widely available resources for hematopoietic stem cells," he adds while cautioning that their findings have yet to be replicated in human cells.

The transplantation of human hematopoietic stem cells isolated from bone marrow is used in the treatment of anemia, immune deficiencies and other diseases, including cancer. However, since bone marrow transplants require a suitable donor-recipient tissue match, the number of potential donors is limited.

Hematopoietic stem cells isolated from umbilical cord blood could be a good alternative source: Readily available and immunologically immature, they allow the donor-recipient match to be less than perfect without the risk of immune rejection of the transplant. Unfortunately, their therapeutic use is limited since umbilical cord blood contains only a small number of stem cells.

Although self-renewal is typically considered a single trait of stem cells, Li and his team wondered whether it could be pulled apart into three distinct requirements: proliferation, maintenance of the undifferentiated state, and the suppression of programmed cell death or apoptosis. "The default state of stem cells is to differentiate into a specialized cell types," explains postdoctoral researcher and first author John Perry, Ph.D. "Differentiation must be blocked in order for stem cells to undergo self-renewal."

Proliferation of stem cells in an undifferentiated state, however, calls tumor suppressor genes into action. These genes help prevent cancer by inducing a process of cell death known as apoptosis. "Consequently, self-renewal of adult stem cells must also include a third event, the active suppression of apoptosis," says Perry.

To test their hypothesis, Perry and his colleagues isolated hematopoietic stem cells from mice and analyzed two key genetic pathways -- the Wnt/β-catenin and PI3K/Akt pathways. Wnt proteins had been identified as "self-renewal factors," while PI3K/Akt activation had been shown to induce proliferation and promote survival by inhibiting apoptosis.

Surprisingly, activation of the Wnt/β-catenin pathway alone blocked differentiation but eventually resulted in cell death, while activation of the PI3K/Akt pathway alone increased differentiation but facilitated cell survival. Only when both pathways were activated, did the pool of hematopoietic stem cells start expanding. "This demonstrated both pathways had to cooperate to promote self-renewal," says Perry.

Although altering both pathways drives self-renewal of hematopoietic stem cells, it also permanently blocks their ability to mature into fully functional blood cells. To sidestep the differentiation block and generate normal, functioning hematopoietic stem cells usable for therapy, the Stowers scientists used small molecules to reversibly activate both the Wnt/β-catenin and PI3K/Akt pathways in culture.

"We were able to expand the most primitive hematopoietic stem cells, which, when transplanted back into mice gave rise to all blood cell types throughout three, sequential transplantation experiments," says Li. "If similar results can be achieved using human hematopoietic stem cells from sources such as umbilical cord blood, this work is expected to have substantial clinical impact."

Researchers who also contributed to the work include Xi C. He, Ryohichi Sugimura, Justin C. Grindley and Jeffrey S. Haug at the Stowers Institute for Medical Research and Sheng Ding in the Gladstone Institute of Cardiovascular Disease at the University of California, San Francisco.

The work was funded in part by the Stowers Institute for Medical Research and the Leukemia & Lymphoma Society.

New Method Could Help Prevent Osteoarthritis

ScienceDaily (Sep. 12, 2011) — A new method is set to help doctors diagnose osteoarthritis at such an early stage that it will be possible to delay the progression of the disease by many years, or maybe even stop it entirely.

The improved method takes three-dimensional images of the joint, in which disease-related cartilage changes are easily seen as different colours.

The joint disease osteoarthritis is one of our most common chronic diseases and one of the primary causes of disability for people around the world.

"Osteoarthritis often attacks the knee and hip joints and breaks down the impact absorbing cartilage found there. For those affected, the progression of the disease usually takes many years, with gradually increasing pain which often leads to disability," says Carl Siversson, who has just defended his thesis in Medical Radiation Physics at Lund University in Sweden.

One of the problems with osteoarthritis has been diagnosing and monitoring the disease before symptoms become evident. It has therefore been difficult to change or delay the course of the disease. A few years ago, researchers from Lund University and Harvard Medical School developed a method to measure the degree of osteoarthritis using an MRI scanner, even at a very early stage. The method is called dGEMRIC (delayed gadolinium-enhanced MRI of cartilage).

"This was major progress, but one problem was that the measurements could only be performed in a limited part of the cartilage. We have now improved the method so that we can study all the cartilage in the joint at once. We have achieved this by solving the problem of how to correct all the irregularities in the MRI images," says Carl Siversson.

The improved method has now been tested both on healthy individuals and on individuals with osteoarthritis, and the results show that the disease can now be monitored in ways that were not previously possible, according to Carl Siversson.

"Now we are continuing our work to make the method easy for doctors to use in their practice. Our hope is that the method will also be significant for future drug development," says Carl Siversson, who after completing his PhD will continue his research at Harvard Medical School in Boston, USA.

The new method is described in Carl Siversson's thesis Three-dimensional T1 quantification techniques for assessment of cartilage quality using dGEMRIC:http://www.lunduniversity.lu.se/o.o.i.s?id=24732&postid=2064122

'Trojan Horse' Particle Sneaks Chemotherapy in to Kill Ovarian Cancer Cells

Cancer cell (red and blue nucleus) that ingested two large microparticles (green). 

ScienceDaily (Sep. 12, 2011) — A common chemotherapy drug has been successfully delivered to cancer cells inside tiny microparticles using a method inspired by our knowledge of how the human immune system works. The drug, delivered in this way, reduced ovarian cancer tumours in an animal model by 65 times more than using the standard method. This approach is now being developed for clinical use.

The research was funded by, among others, the Biotechnology and Biological Sciences Research Council (BBSRC) Follow-on Fund -- funding for 'proof of concept' at the very early stage of turning research outputs into a commercial proposition -- and carried out at Queen Mary, University of London. The study will be published next week in the journal, Biomaterials.

Dr Davidson Ateh, who worked on the research at Queen Mary, University of London and set up the start-up company, BioMoti, that will develop the technology for clinical use said "It's like we've made a re-enactment of the battle of Troy but on the tiniest scale. In Troy, the Greeks fooled the Trojans into accepting a hollow horse full of soldiers -- we've managed to trick cancer cells into accepting drug-filled microparticles."

Dr Ateh and colleagues found that by coating tiny microparticles of around a hundredth the diameter of a human hair with a special protein called CD95, they could in fact trigger cancer cells into ingesting these particles. Not only that, but the particles could deliver a dose of a common chemotherapy drug called paclitaxel.

The key to their success is that CD95 attaches to another protein called CD95L, which is found much more commonly on the surface of cancer cells than it is on normal healthy cells.

Once attached, the cancer cells ingest CD95 and the microparticle with it.

Inside the cell, the microparticle can unload its chemotherapy cargo, which kills the cell to reduce the size of the tumour.

Dr Ateh added "Other researchers had already noticed that cancer cells may use this CD95-CD95L arrangement to avoid being destroyed by the immune system, which is why they display more CD95L than normal cells. We've managed to turn this to our advantage and hijack the cancer cell's own trick-of-the-trade to get our 'Trojan Horse' through the gate."

Co-author and Professor of Gynecological Oncology at Queen Mary, University of London, Iain McNeish commented "Chemotherapy is still the main way that we treat ovarian cancer, which can be particularly aggressive and difficult to treat. Anything we can do to concentrate the treatment in tumour cells and at the same time protect healthy cells is a good thing. This is an elegant method and if it works in a clinical setting as well as we hope it will patients could experience a better treatment with fewer side effects."

Principal investigator and Professor of Pathology at Queen Mary, University of London, Joanne Martin added "There are lots of drugs that we would like to deliver into cells -- not just chemotherapies -- and this could work for those too. For example, there are new classes of drugs to treat tumours, such as biological therapies. If we could load biological therapies into the same CD95-coated particles, then there's no reason why they couldn't also be delivered in this way."

The researchers are now advancing these studies and BioMoti is seeking to attract larger and established pharmaceutical companies to partner the technology, dubbed OncoJanTM in reference to the earlier Trojan horse, for the clinical development of new treatments in specific types of cancer.

Professor Douglas Kell, Chief Executive, BBSRC said "BBSRC funds a great deal of work that is aimed at improving our fundamental understanding of biological processes. When there are opportunities to use this understanding to the benefit of people, it is vitally important to seize and make the most of them. This is a good example where research into the human immune system has led to a new technology that has the potential to underpin health and wellbeing into later life."

Link Between High Cholesterol and Alzheimer's Disease Revealed in New Study

ScienceDaily (Sep. 12, 2011) — People with high cholesterol may have a higher risk of developing Alzheimer's disease, according to a study published in the September 13, 2011, issue of Neurology®, the medical journal of the American Academy of Neurology.

"We found that high cholesterol levels were significantly related to brain plaques associated with Alzheimer's disease," said study author Kensuke Sasaki, MD, PhD, of Kyushu University in Fukuoka, Japan.

For the study, the cholesterol levels were tested for 2,587 people age 40 to 79 who had no signs of Alzheimer's disease. Then they examined 147 autopsied people who died after a long observation period (10 to 15 years). Of those, 50 people, or 34 percent, had been diagnosed with dementia before death.

The autopsies looked for plaques and tangles in the brain, both known to be trademark signs of Alzheimer's disease. Plaques are an accumulation of a form of the protein amyloid, which occurs between nerve cells. Tangles are an accumulation of a different protein, called tau, which occurs inside nerve cells.

People with high cholesterol levels, defined by a reading of more than 5.8 mmol/L, had significantly more brain plaques when compared to those with normal or lower cholesterol levels. A total of 86 percent of people with high cholesterol had brain plaques, compared with only 62 percent of people with low cholesterol levels.

The study found no link between high cholesterol and the tangles that develop in the brain with Alzheimer's disease.

In addition to high cholesterol increasing the risk of Alzheimer's disease, Sasaki previously found that insulin resistance, a sign of diabetes, may be another risk factor for brain plaques associated with Alzheimer's disease.

"Our study clearly makes the point that high cholesterol may contribute directly or indirectly to plaques in the brain," Sasaki said, "but failed treatment trials of cholesterol-lowering drugs in Alzheimer's disease means there is no simple link between lowering cholesterol and preventing Alzheimer's."

This study was supported by the Japan Society for the Promotion of Science and the Japanese Ministry of Health, Labor and Welfare.

X-Ray Protein Probe Leads to Potential Anticancer Tactic

ScienceDaily (Sep. 12, 2011) — Researchers at Emory University School of Medicine have identified a new type of potential anticancer drug. The compound, named FOBISIN, targets 14-3-3 proteins, important for the runaway growth of cancer cells.

X-rays induce FOBISIN to become bonded to the 14-3-3 protein, suggesting a anticancer therapy tactic.

The researchers were using X-rays to see how FOBISIN fits into the clamp-shaped 14-3-3 protein structure. Unexpectedly, the X-rays induced the compound to be permanently bonded to the protein. The finding suggests that compounds like FOBISIN can be used in combination with radiation to trigger potent anticancer activity.

The results were published online Sept. 9 in Proceedings of the National Academy of Sciences Early Edition.

Senior author Haian Fu, PhD, has been studying 14-3-3 proteins for two decades. He is professor of pharmacology and of hematology and oncology at Emory University School of Medicine, and the director of the Emory Chemical Biology Discovery Center.

"Targeting 14-3-3 proteins could be especially valuable because they can impact multiple pathways critical for cancer cell growth," he says. "14-3-3 proteins have been shown to be dysregulated in a number of cancer types, including lung cancer and breast cancer."

14-3-3 proteins act as adaptors that clamp onto other proteins. Fu and co-workers Jing Zhao, postdoctoral fellow, and Yuhong Du, assistant professor and associate director of the Discovery Center, sorted through thousands of chemicals to find one (FOBISIN: Fourteen-three-three Binding Small moleculeInhibitor) that prevents 14-3-3 from interacting with its partners. 14-3-3 proteins are found in mammals, plants and fungi. In humans, they come in seven varieties, and FOBISIN appears to inhibit interactions by all seven.

A 14-3-3 proteins' ability to clamp depends on whether the target protein is phosphorylated, a chemical modification that regulates protein function. FOBISIN's inhibitory power also requires the presence of phosphorylation in the molecule.

Fu's group teamed up with the laboratory of Xiaodong Cheng, PhD, co-senior author, professor of biochemistry and a Georgia Research Alliance Eminent Scholar, to examine how FOBISIN fits into its targets.

Scientists use X-rays as a tool to probe protein structure. If a protein and a drug that targets it can be crystallized together, the X-ray diffraction pattern from the crystals reveals the 3D arrangement of the atoms and how the drug interacts with the protein. Research assistant professor John Horton, PhD, and research associate Anup Upadhyay, PhD, in the Cheng laboratory used synchrotron X-ray radiation from the Advanced Photon Source at Argonne National Laboratory for this purpose.

"In this case, the X-rays had an unexpected effect: they caused FOBISIN to become covalently attached to the 14-3-3 protein," Cheng says.

The finding suggests that compounds like FOBISIN could be developed as "pro-drugs" that upon exposure to radiation, permanently stick to and inhibit their targets. A common strategy in fighting cancer is to combine drugs and radiation so that the drugs increase cells' sensitivity to radiation. Here, the radiation would activate the drug.

"These compounds could be used in combination with other strategies to enhance the tumor selectivity of the treatment," Fu says.

The research was funded by the U.S. National Institutes of Health, the Georgia Cancer Coalition, and the Georgia Research Alliance.