June 1, 2010 (Vol. 30, No. 11)

Cutting-Edge Research Seeks to Expand Range of Applications for Reparative Technology

The regenerative medicine field is a hotbed of innovative research. The recent “Repairing the Body” conference, sponsored by Cranfield Health, showcased some of the cutting-edge work being done within industry and academia. The topics discussed ranged from immunological intervention to stem cell and other reparative therapies.

High-content screening is capable of embracing the biological complexities inherent in stem cell applications, according to Edward Ainscow, Ph.D., associate director of AstraZeneca’s advanced science and technology laboratory (ASTL).

Regenerative medicine is a focus area for the company’s new opportunities group, and ASTL’s high-content screening infrastructure is helping advance developments. AstraZeneca’s molecule libraries are now being screened for potential regenerative medicine applications in diabetic retinopathy, myocardial infarction, and osteoporosis using mesenchymal stem cells from adipose tissue, which can give rise to multiple cell types.

“We find these cells are a good workhorse and model tool for looking at how we can modify the differentiation pathways of stem cells,” said Dr. Ainscow. The researchers are using the HD IncuCyte™—a microscope in an incubator—to monitor differentiation in different media through studying changes in cellular morphology. This setup shows an increase in striated morphology as the stem cells differentiate into cardiomyocytes.

High-content screening also allows monitoring of the expression of differentiation markers. “High-content screening is well suited to regenerative medicine because it allows profiling of multiple events at the cellular and subcellular level,” added Dr. Ainscow. “Emerging developments in in vitro cellular systems will increase the opportunities in this area.” 

University of Manchester researchers are studying tissue regeneration in Xenopus tropicalis tadpoles.

The demand for total joint replacement from people under 65 suffering from osteoarthritis is likely to increase dramatically by 2030, explained Alan Getgood, M.D., clinical research associate at the University of Cambridge. Some of these “young people with old knees” develop traumatic osteoarthritis as a result of sport and recreational injuries to the cruciate anterior ligament and meniscal cartilages.

Dr. Getgood believes that these injuries could be addressed by using biological treatment at an early stage. Accordingly, he is working with the U.K. Technology Strategy Board and Orthomimetics (now part of the Belgian regenerative medicine company TiGenix) on Chondromimetic, a collagen scaffold that can be used to “plug” articular cartilage lesions, thereby helping regenerate articular cartilage by providing a structure for cells to attach and produce new tissue.

“This is potentially a fantastic biological carrier,” said Dr. Getgood. Current approaches to repair these injuries such as surgical microfracture and cellular therapy tend to produce a fibrous tissue that has a shorter life than natural hyaline cartilage, which has a more complex structure.

Dr. Getgood has been looking at how the addition of various biological factors such as mesenchymal stem cells from bone marrow, platelet-rich plasma, and recombinant growth factors to Chondromimetic might improve the regeneration of the articular tissue. Recombinant human fibroblast growth factor 18 (rhFGF18) (which Merck Serono licensed from Zymogenetics) looks particularly promising as it produces repair tissue that is more like hyaline cartilage.

An arthroscopic view (keyhole surgery) inside the knee: The outer smooth surface is articular cartilage of the knee femoral condyle. The inner white area is the Chondromimetic plug inserted into a cartilage defect. The grey rod is a probe used to examine the surface of the knee. [University of Cambridge]

Wound healing is an important application area in regenerative medicine. “We would like to find novel ways of healing disfiguring wounds arising from congenital conditions, accidents, and disease,” noted Enrique Amaya, Ph.D., The Healing Foundation professor of tissue regeneration at the University of Manchester.

“All organisms have quite remarkable wound-healing capacities that are present in the embryo but lost during adulthood.”  To study how this capacity might be maintained during adulthood, Dr. Amaya is applying his long-time interest in using the embryo of  the West African frog Xenopus tropicalis as a model organism.

Frog embryos have many advantages—all the organs can be visualized, allowing many events to be monitored; the embryos can be produced in large numbers; and they are accessible at all stages of their development. There are also many genetic similarities between frogs and humans—one of the main conclusions that was made following the recent sequencing of the X. tropicalis genome.

Embryos heal quickly and completely, with no scarring, while adult healing is slow, incomplete, and produces a scar. Using frog embryos, Dr. Amaya set out to discover if the difference arises because embryos do not mount an inflammatory response to injury, while adults do. He learned that embryos do, in fact, produce inflammatory cells on injury. “But we think these cells differ from those produced by adults.” As a result, they have been studying the marker proteins expressed by these cells.

In other research, Dr. Amaya’s team is looking at tissue regeneration in the tadpole where an injured tail will regenerate in a functional (if not perfect) fashion. Microarray studies show which genes are induced on injury and during the regeneration.

University of Manchester researchers are studying tissue regeneration in Xenopus tropicalis tadpoles.

Spinal Cord Repair

Another area generating a lot of attention is the application of regenerative medicine to spinal cord repair. James Fawcett, M.D., chairman of the Cambridge Centre for Brain Repair, pointed out that spontaneous recovery occurs after injury but is limited, and repair capacity declines with age. He has used an animal model to study some of the molecules involved in plasticity of spinal cord neurons. Potential treatments are now being developed with Acorda Therapeutics.

Neurodegenerative Disease

Patients suffering from neurodegenerative disease may find hope in regenerative medicine. Huntington disease (HD) is a rare disease involving the massive loss of striatial neurons. It is a candidate for stem cell therapy and also an interesting model. There has been background work on cell transplantation in HD and it is known, from animal studies, that grafted cells can alleviate functional deficits.

The developmental and speciation stage of the donor tissue is crucial, according to Nick Allen, Ph.D., reader in genetics at the University of Wales, Cardiff. The differentiation of human embryonic stem cells into neural cells is well established, and Dr. Allen’s group has developed a chemically defined medium that decreases the number of contaminant cells.

“We now have good protocols with defined media and drugs to control the signalling pathways.” The team is currently looking at scale-up and reproducibility, using small bioreactors, with collaborators in Paris. “These human embryonic stem cells differentiate very nicely and give functional neurons.”  They are trying to manipulate the signaling pathways to give the specific cells that would be of use in HD, and assessment of grafts of such neurons has begun.

Transgenic animal models of HD are a cumbersome platform for drug discovery. There is a clear need for in vitro models. “We aim to generate disease-relevant cells for screening,” said Dr. Allen, describing work his team has done on cells derived from embryos arising from pre-implantation diagnosis IVF studies for HD.

In an EU program, the Cardiff team is looking at cells from embryos that have tested positive for HD. Currently, the transcriptome of these cells is being studied and a number of genes are being validated.

However, work with IVF-derived HD embryos is limited by availability of the cells, so Dr. Allen’s group is also looking at induced pluripotent cells derived from fibroblasts of patients with HD. The advantage of this approach is that there is a clinical history attached to the patient, which is not so in the pre-implantation diagnosis IVF cells. There are also genome-wide association studies under way in HD populations. “There are very exciting times ahead as we develop sophisticated cell models together with clinical information.”

Stem Cell Therapy

Meanwhile, Myrtle Gordon, Ph.D., emeritus professor in experimental hematology, division of investigative science at Imperial College London, studies a population of stem cells called OmniCytes for treating liver damage. OmniCytes express CD34 and account for around 1% of CD34+ cells in bone marrow.

OmniCytes can become albumin-producing liver cells and have been transplanted into liver-damaged mice. “We now realize that mechanisms other than repopulation give functional improvement—there are secreted factors,” Dr. Gordon explained. OmniCytes inhibit cell death in rat liver cells, he added, as shown by biochemical and histological studies.

A large number of OmniCytes can be collected from leukapheresis, which is easier than bone marrow collection. There is no need for prolonged culture, and the required number of cells for transplant are available in less than three hours. “We are fairly encouraged by OmniCytes as a potential source for stem cell therapy. They can be useful in limiting damage and provide a bridge to liver transplantation.” 

A Phase I trial administering OmniCytes into the portal vein or hepatic artery has been carried out as an autologous therapy with the aid of Hammersmith Hospital in  London. A Phase II trial is under way with patients with alcoholic liver cirrhosis.

The next stage is to develop an off-the-shelf therapy and, for this, the researchers have optimized the expansion process. This will be applied in patients with liver failure. OmniCytes also differentiate into other cells; potential applications include diabetes, cardiovascular indications, and bone repair.

Finally, Clare Blackburn, Ph.D., head of thymus development and regeneration group at the MRC Centre for Regenerative Medicine at the University of Edinburgh, spoke about an approach to repair the immune system.

The thymus is one of the first organs to degenerate, with volume being lost early in life, which is reflected in a decrease of T-cell output. This increases susceptibility to infection and autoimmune disease and decreases response to vaccines. However, the thymus can regenerate, although attempts to do so have been fraught with drawbacks.

Dr. Blackburn’s group has identified the epithelial progenitor cells of the thymus  which, when transferred to athymic mice, will make a functional thymus. “This raises the question of whether in vitro stem cells can break the bottleneck of the supply of tissue in thymus transplant.” For this, one would need to grow thymic cells in vitro or generate them from ES cells.

Dr. Blackburn is currently focused on discovering the cellular hierarchies of the cells that generate and maintain the fetal and mature thymus.  

A number of researchers believe that stem cell therapy offers hope for people suffering from neurodegenerative disorders such as Huntington disease. [iStockphoto]

Susan Aldridge, Ph.D. ([email protected]), is a freelance science and medical writer specializing in biotechnology, pharmaceuticals, chemistry, medicine, and health.

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