Bone marrow stem cells used to repair damaged retina

University of Florida (UF) researchers have programmed bone marrow stem cells to repair damaged retinas in mice, suggesting a potential treatment for one of the most common causes of vision loss in older people.

The success in repairing a damaged layer of retinal cells in mice implies that blood stem cells taken from bone marrow can be programmed to restore a variety of cells and tissues, including ones involved in cardiovascular disorders such as atherosclerosis and coronary artery disease.

“To our knowledge, this is the first report using targeted gene manipulation to specifically programme an adult stem cell to become a new cell type,” said Dr Maria B. Grant, a professor of pharmacology and therapeutics at UF’s College of Medicine. “Although we used genes, we also suggest you can do the same thing with drugs – but ultimately you would not give the drugs to the patient, you would give the drugs to their cells. Take the cells out, activate certain chemical pathways, and put the cells back into the patient.”

In a paper which was due to appear in the September 2009 issue of the journal Molecular Therapy, scientists describe how they used a virus carrying a gene that gently changed cultured adult stem cells from mice into retinal cells. Only after the stem cells were reintroduced into the mice did they completely transform into the desired type of vision cells, apparently taking environmental cues from the damaged retinas.

After studying the celltransformation process, scientists were able to bypass the gene manipulation step entirely and instead use chemical compounds that mirrored environmental conditions in the body, thus pointing the stem cells toward their ultimate identities as vision cells.

“First we were able to show you can overexpress a protein unique to a retinal cell type and trick the stem cell into thinking it is that kind of cell,” said Grant, who collaborated with Edward Scott, the director of the Program in Stem Cell Biology and Regenerative Medicine at UF’s McKnight Brain Institute.

“As we proceeded, we found we could activate the stem cells by mimicking the body’s natural signalling channels with chemicals. This implies a whole new field of stem cell research that uses drug manipulation rather than genetic manipulation to send these immature cells along new pathways,” Grant said.

Scientists chose to build retinal pigment epithelial cells, which form the outer barrier of the retina. In addition to being very specialised to identify, RPE cells are faulty in many retinal diseases, including agerelated macular degeneration and some forms of blindness related to diabetes.

“This work applies to 85% of patients who have age-related macular degeneration,” Grant said. “There are no therapies for this devastating disease.”

The researchers removed blood stem cells from the bone marrow of mice, modified the cells in cultures, and injected them back into the animals’ circulatory systems. From there, the stem cells were able to home in on the eye injury and become retinal cells. At 28 days after receiving the modified stem cells, mice that had previously demonstrated no retinal function were no different than normal mice in electrical measures of their response to light.



Gene therapy for blindness remains positive one year on

Three young adults who received gene therapy for a blinding eye condition remained healthy and maintained previous visual gains one year later, according to an August online report in Human Gene Therapy. One patient also noticed a visual improvement that helped her perform daily tasks, which scientists describe in an 13 August letter to the editor in the New England Journal of Medicine.

These findings have emerged from a phase I clinical trial supported by the National Eye Institute (NEI) at the US National Institutes of Health, and conducted by researchers at the University of Pennsylvania, Philadelphia, and the University of Florida, Gainesville. This is the first study that reports the one-year safety and effectiveness of successful gene therapy for a form of Leber congenital amaurosis (LCA), a currently untreatable hereditary condition that causes severe vision loss and blindness in infants and children.

“These results are very significant because they represent one of the first steps toward the clinical use of gene therapy for an inherited form of blindness,” said NEI director Paul A. Sieving, MD, PhD.

The three patients in the study-aged 22, 24 and 25-have been legally blind since birth due to a specific form of LCA caused by mutations in the RPE65 gene. The protein made by this gene is a crucial component of the visual cycle. The RPE65 protein is necessary for the production of a retina-specific form of vitamin A that is required for the lightsensitive photoreceptor cells to function. Mutations in the RPE65 gene prevent this production, which halts the visual cycle and blocks vision.

The RPE65 disease offers an opportunity for treatment in that it leaves some photoreceptors intact. In this study, researchers pinpointed an area of intact photoreceptors in the retina of each patient. They injected healthy copies of the RPE65 gene under the retina in this area in an attempt to repair the visual cycle.

One year after the procedure, the therapy had not provoked an immune response in the eye or in the body. Though the patients' visual acuity, or ability to read letters on an eye chart, remained unchanged, all three patients could detect very dim lights that they were unable to see prior to treatment. This visual benefit provides evidence that the newly introduced RPE65 gene is functional and is increasing the light sensitivity of the retina.

● References 1. Cideciyan AV, et al. (2009) Human RPE65 Gene Therapy for Leber Congenital Amaurosis: Persistence of Early Visual Improvements and Safety at 1 Year. Human Gene Therapy, vol. 20, no. 9; published online August 2009, ahead of print (doi: 10.1089/ hum.2009.086). 2. Cideciyan AV, et al. Vision 1 Year after Gene Therapy for Leber's Congenital Amaurosis. N Engl J Med 2009; 361:725- 727.



Researchers open new way to reprogramme cells

A research team comprised of faculty at Massachusetts, USbased Worcester Polytechnic Institute’s (WPI) Life Sciences and Bioengineering Center (LSBC) and investigators at CellThera, a private company also located at the LSBC, has discovered a novel way to turn on stem cell genes in human fibroblasts (skin cells) without the risks associated with inserting extra genes or using viruses.

This discovery opens a new avenue for reprogramming cells that could eventually lead to treatments for a range of human diseases and traumatic injuries by coaxing a patient’s own cells to repair and regenerate the damaged tissues.

The research team reported its findings in the paper “Induction of Stem Cell Gene Expression in Adult Human Fibroblasts without Transgenes,” published online 21 July 2009 as a “fast track” paper from the journal Cloning and Stem Cells.

“We show that by manipulating culture conditions alone, we can achieve changes in fibroblasts that would be beneficial in development of patient-specific cell therapy approaches,” the authors wrote in the paper.

Early on, the emerging field of regenerative medicine focused on pluripotent embryonic stem cells. In the pluripotent state, several genes are known to be active, helping to control the stem cells. These genes, including OCT4, SOX2 and NANOG, are accepted as markers of pluripotency because they are active in stem cells, but become dormant once the stem cells begin to differentiate and head down the path to developing into a specific kind of cell type and tissue.

While the study of embryonic stem cells continues to yield important knowledge, research teams around the world are also working to change, or reprogramme, fully-differentiated cells like skin cells, back to a more pluripotent state. Called induced pluripotent stem cells (iPS), these reprogrammed cells could be used to regenerate tissue without some of the problems associated with embryonic stem cells, including ethical questions and the potential for embryonic stem cells to be rejected by a patient's immune system or to grow out of control and cause tumours.

The first induced pluripotent stem cells were created in 2007 by Shinya Yamanaka's team at Kyoto University in Japan, which inserted extra copies of four known stem cell genes, including OCT4 and SOX2, into human skin cells. Those genes began expressing proteins that changed the skin cells back to a more pluripotent state.

This technique, which has since been repeated by other labs and refined to the point were fewer additional genes are needed to achieve reprogramming, was a major scientific breakthrough. Its potential for use in human therapies is limited, however, because inserting new genes into adult cells, either directly or by using viruses to carry the genetic payload, can cause a host of problems.

In the current study, the team at WPI and CellThera turned on the existing, yet dormant, stem cell genes OCT4, SOX2 and NANOG already in the skin cells by lowering the amount of atmospheric oxygen the cells were exposed to, and by adding a protein called fibroblast growth factor 2 (FGF2) to the culture medium.

Furthermore, once the stem cell genes were activated and began expressing proteins, the team found those proteins migrated back into the nucleus of the skin cells, precisely as would occur in induced pluripotent stem cells. “This was an exciting observation,” said Raymond Page, PhD, research assistant professor of biology and biotechnology at WPI and lead author on the paper. “Having these proteins localise to the nucleus is the first step of reprogramming these cells.”

Even more surprising, the team found that the stem cell genes OCT4, SOX2 and NANOG were not completely dormant in untreated skins cells, as was presumed. Those genes were, in fact, sending out messages, but those messages were not being translated into the proteins that do the work of making cells pluripotent.

“This was quite unexpected,” said Tanja Dominko, DVM, PhD, associate professor of biology and biotechnology at WPI and president of CellThera. “Not only does this data force us to rethink what the true markers of pluripotency may be, it suggests there is a natural mechanism at work in these cells regulating the stem cell gene expression. That opens a whole new line of inquiry.”



DNA mutations linked to diabetes

Genes that regulate the energy consumption of cells have a different structure and expression in type II diabetics than they do in healthy people, according to a new study from the Swedish medical university Karolinska Institutet published in Cell Metabolism. The researchers believe that these ‘epigenetic mutations’ might have a key part to play in the development of the disease.

Type II diabetes is characterised by a lower sensitivity to insulin in muscles and organs, and a reduced ability to consume energy in the form of glucose. Heredity and environmental factors (e.g. exercise) are both involved in the disease pathogenesis, but scientists are still unclear as to the mechanisms behind it.

A research group at Karolinska Institutet has now shown that genes in the muscle cells of diabetics are chemically modified through what is known as DNA methylation. They found that in muscles cells taken from patients with earlyonset diabetes, a gene designated as PGC-1 was modified and had reduced expression. PGC-1 controls other genes that regulate the metabolism of glucose by the cell.

The team has also demonstrated that DNA methylation occurs rapidly, when cells from healthy people are exposed to certain factors associated with diabetes, such as raised levels of free fatty acids and cytokines. DNA methylation is a form of epigenetic regulation, a process involving chemical modifications that are imposed externally on genes and that alter their activity without any change to the underlying DNA sequence.

“This type of epigenetic modification might be the link that explains how environmental factors have a longterm influence on the development of type II diabetes,” says Juleen Zierath, who led the study. “It remains to be seen whether the DNA methylation of this gene can be affected by, say, dietary factors.”

● Reference: “Non-CpG Methylation of the PGC-1 Promoter through DNMT3B Controls Mitochondrial Density”, Romain Barrès, Megan E. Osler, Jie Yan, Anna Rune, Tomas Fritz, Kenneth Caidahl, Anna Krook and Juleen R. Zierath, Cell Metabolism, 2 September 2009.



Medsol partners with KFSH for regional genetic testing

Medsol laboratories, a part of Gulf Healthcare International, is offering a new line of genetic tests in the MENA via a partnership with Saudi Arabiabased King Faisal Specialist Hospital and Research Centre (KFSH&RC). The tests will be available in association with the CAP accredited Saudi Diagnostics Limited (SDL), owned by KFSH&RC. Medsol provides pathology testing services via a network of branded laboratories, including independent and hospital laboratories across MENA and will offer the genetic tests throughout its 14 laboratories across the region.

The availability of molecular genetics testing is particularly important in the Gulf region with the widespread prevalence of hereditary disease and common practice of consanguinity.

Genetic tests being offered at Medsol laboratories include:

● Cystic Fibrosis Single Mutation
● Factor V Leiden
● Familial Mediterranean Fever
● Fragile X Syndrome (PCR & Southern Blot Analysis)
● Hereditary Hemochromatosis
● Huntington Disease
● 5,10- Methylenetetrahydrofolate Reductase (MTHFR) Deficiency
● Mitochondrial Myopathy Encephalopathy Lactic Acidosis/Stroke-like Episodes (MELAS)
● Multiple endocrine Neoplasa (MEN 2)
● Myotonic Dystrophy
● Prothrombin 20210 G – A Mutation
● Sickle Celll Anemia (HbS, HbC)

Samples will be collected by Medsol and sent to SDL for detailed analysis. The turnaround time for tests in the specialist area of genetics ranges from several weeks to a few months.

                                   
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