Thursday 18 December 2014

Stem Cells Faulty in Duchenne Muscular Dystrophy

Stem Cells Faulty in Duchenne Muscular Dystrophy
Thursday, 18 December 2014

Like human patients, mice with a form of Duchenne muscular dystrophy undergo progressive muscle degeneration and accumulate connective tissue as they age. Now, researchers at the Stanford University School of Medicine have found that the fault may lie at least partly in the stem cells that surround the muscle fibres.

They've found that during the course of the disease, the stem cells become less able to make new muscle and instead begin to express genes involved in the formation of connective tissue. Excess connective tissue – a condition called fibrosis – can accumulate in many organs, including the lungs, liver and heart, in many different disorders. In the skeletal muscles of people with muscular dystrophy, the fibrotic tissue impairs the function of the muscle fibres and leads to increasing weakness and stiffness, which are hallmarks of the disease.

The researchers discovered that this abnormal change in stem cells could be inhibited in laboratory mice by giving the animals a drug that is already approved for use in humans. The drug works by blocking a signalling pathway involved in the development of fibrosis. Although much more research is needed, the scientists are hopeful that a similar approach may one day work in children with muscular dystrophy.

"These cells are losing their ability to produce muscle, and are beginning to look more like fibroblasts, which secrete connective tissue," said Thomas Rando, MD, PhD, professor of neurology and neurological sciences.

"It's possible that if we could prevent this transition in the muscle stem cells, we could slow or ameliorate the fibrosis seen in muscular dystrophy in humans."

A paper describing the researchers' findings will be published Dec. 17 in Science Translational Medicine. Rando, the paper's senior author, is director of the Glenn Laboratories for the Biology of Aging and founding director of the Muscular Dystrophy Association Clinic at Stanford. Former postdoctoral scholar Stefano Biressi, PhD, is the lead author. Biressi is now at the Centre for Integrative Biology at the University of Trento in Italy.

A devastating disease
Duchenne muscular dystrophy is a devastating disease that affects about 1 in every 3,600 boys born in the United States. Patients usually experience severe, progressive muscle weakness that confines them to a wheelchair in early adolescence and eventually leads to paralysis. It's caused by mutations in the dystrophin gene, which encodes the dystrophin protein. The dystrophin protein serves to connect muscle fibres to the surrounding external matrix. This connection stabilizes the fibres, enhancing their strength and preventing injury. Sufferers are nearly always boys because the dystrophin gene is located on the X chromosome. (Girls would need to inherit two faulty copies, which is unlikely because male carriers often die in early adulthood.)

Under normal conditions, muscle stem cells respond to muscle damage by dividing into cells, one of which becomes new muscle, while the other remains a stem cell. However, in the mice missing the dystrophin gene, the muscle stem cells slowly assume a different fate. They begin to resemble fibroblasts instead of muscle-making machines.

To conduct the research, Biressi and Rando used a strain of laboratory mice in which the muscle stem cells were engineered to glow with a fluorescent light when treated with a drug called tamoxifen. They then bred the mice with another strain in which the dystrophin gene is mutated, and followed the fate of the cells over time.

The researchers found that the expression of myogenic genes, which are associated with the regeneration of muscle in response to injury, was nearly completely lacking in many of the muscle stem cells in the mice after just 11 months, while the expression of fibrotic genes had increased compared with that of control animals. The cells from the dystrophic animals were also oddly located: Rather than being nestled next to the muscle fibres, they had begun to move away into the spaces between tissues.

The role of a signalling pathway
Rando and Biressi knew that a similar, but much less pronounced, accumulation of connective tissue in muscle fibres occurs during normal aging. That process is governed by signalling proteins, which include the Wnt and TGF-beta protein families. Wnt plays a critical role in embryonic development and cancer; TGF-beta controls cell division and specialization. They wondered whether blocking the Wnt/TGF-beta pathway in the dystrophic mice would inhibit fibrosis in the animals' muscles.

The researchers turned to a drug called losartan, which is used to treat high blood pressure. Losartan inhibits the expression of the genes for TGF-beta types 1 and 2. The researchers thought it would probably interrupt the signalling pathway that leads the muscle stem cells astray.

Treating the mice with losartan, they found, did in fact prevent the muscle stem cells from expressing fibrosis-associated genes and partially maintained their ability to form new muscle.

"This scar tissue, or fibrosis, leaves the muscle less elastic and impairs muscle function," Rando said.

"So we'd like to understand why it happens, and how to prevent it. It's also important to limit fibrosis to increase the likelihood of success with other possible therapies, such as cell therapy or gene therapy."

Next steps
Because TGF-beta type 1 plays many roles throughout the body, the researchers are now working to find ways to specifically inhibit TGF-beta type 2, which is involved in the transition of the muscle stem cells. They're also interested in learning how to translate the research to other diseases.

"Fibrosis seems to occur in a vicious cycle," Rando said.

"As the muscle stem cells become less able to regenerate new muscle, the tissue is less able to repair itself after damage. This leads to fibrosis, which then further impairs muscle formation. Understanding the biological basis of fibrosis could have a profound effect on many other diseases."

Contact: Krista Conger
.........


For more on stem cells and cloning, go to CellNEWS at

Tuesday 16 December 2014

New Technology Directly Reprograms Skin Fibroblasts for a New Role

Penn study has implications for new skin disease treatments
Tuesday, 16 December 2014

Dermal fibroblasts are directly reprogrammed to
pigmented melanocytes by three transcription
factors (SOX10, MITF and PAX3).
Credit: Ruifeng Yang, Perelman School of
Medicine, University of Pennsylvania. 
As the main component of connective tissue in the body, fibroblasts are the most common type of cell. Taking advantage of that ready availability, scientists from the Perelman School of Medicine at the University of Pennsylvania, the Wistar Institute, Boston University School of Medicine, and New Jersey Institute of Technology have discovered a way to repurpose fibroblasts into functional melanocytes, the body's pigment-producing cells. The technique has immediate and important implications for developing new cell-based treatments for skin diseases such as vitiligo, as well as new screening strategies for melanoma. The work was published this week in Nature Communications.

The new technique cuts out a cellular middleman. Study senior author Xiaowei "George" Xu, MD, PhD, an associate professor of Pathology and Laboratory Medicine, explains.

"Through direct reprogramming, we do not have to go through the pluripotent stem cell stage, but directly convert fibroblasts to melanocytes. So these cells do not have tumourigenicity."

Changing a cell from one type to another is hardly unusual. Nature does it all the time, most notably as cells divide and differentiate themselves into various types as an organism grows from an embryo into a fully-functional being. With stem cell therapies, medicine is learning how to tap into such cell specialization for new clinical treatments. But controlling and directing the process is challenging. It is difficult to identify the specific transcription factors needed to create a desired cell type. Also, the necessary process of first changing a cell into an induced pluripotent stem cell (iPSC) capable of differentiation, and then into the desired type, can inadvertently create tumours.

Xu and his colleagues began by conducting an extensive literature search to identify 10 specific cell transcription factors important for melanocyte development. They then performed a transcription factor screening assay and found three transcription factors out of those 10 that are required for melanocytes: SOX10, MITF, and PAX3, a combination dubbed SMP3.

"We did a huge amount of work," says Xu.

"We eliminated all the combinations of the other transcription factors and found that these three are essential."

The researchers first tested the SMP3 combination in mouse embryonic fibroblasts, which then quickly displayed melanocytic markers. Their next step used a human-derived SMP3 combination in human foetal dermal cells, and again melanocytes (human-induced melanocytes, or hiMels) rapidly appeared. Further testing confirmed that these hiMels indeed functioned as normal melanocytes, not only in cell culture but also in whole animals, using a hair-patch assay, in which the hiMels generated melanin pigment. The hiMels proved to be functionally identical in every respect to normal melanocytes.

Xu and his colleagues anticipate using their new technique in the treatment of a wide variety of skin diseases, particularly those such as vitiligo for which cell-based therapies are the best and most efficient approach.

The method could also provide a new way to study melanoma.

By generating melanocytes from the fibroblasts of melanoma patients, Xu explains: "we can screen not only to find why these patients easily develop melanoma, but possibly use their cells to screen for small compounds that can prevent melanoma from happening."

Perhaps most significantly, say the researchers, is the far greater number of fibroblasts available in the body for reprogramming compared to tissue-specific adult stem cells, which makes this new technique well-suited for other cell-based treatments.

Contact: Karen Kreeger
.........


For more on stem cells and cloning, go to CellNEWS at

Monday 15 December 2014

Cell Biologists Discover On-off Switch for Key Stem Cell Gene

Discovery may propel advances in regenerative medicine
Sunday, 14 December 2014

Consider the relationship between an air traffic controller and a pilot. The pilot gets the passengers to their destination, but the air traffic controller decides when the plane can take off and when it must wait. The same relationship plays out at the cellular level in animals, including humans. A region of an animal's genome - the controller - directs when a particular gene - the pilot - can perform its prescribed function.

These are images of mouse embryonic stem cells
which grow in a round colony of cells (A) and
express Sox2 (B), shown in red. Sox2 control
region-deleted cells have lost the typical
appearance of embryonic stem cells (C) and do
not express Sox2 (D). The DNA is shown in blue
in B and D. Credit: Jennifer Mitchell/University
of Toronto. 
A new study by cell and systems biologists at the University of Toronto (U of T) investigating stem cells in mice shows, for the first time, an instance of such a relationship between the Sox2 gene which is critical for early development, and a region elsewhere on the genome that effectively regulates its activity. The discovery could mean a significant advance in the emerging field of human regenerative medicine, as the Sox2 gene is essential for maintaining embryonic stem cells that can develop into any cell type of a mature animal.

"We studied how the Sox2 gene is turned on in mice, and found the region of the genome that is needed to turn the gene on in embryonic stem cells," said Professor Jennifer Mitchell of U of T's Department of Cell and Systems Biology, lead investigator of a study published in the December 15 issue of Genes & Development.

"Like the gene itself, this region of the genome enables these stem cells to maintain their ability to become any type of cell, a property known as pluripotency. We named the region of the genome that we discovered the Sox2 control region, or SCR," said Mitchell.

Since the sequencing of the human genome was completed in 2003, researchers have been trying to figure out which parts of the genome made some people more likely to develop certain diseases. They have found that the answers are more often in the regions of the human genome that turn genes on and off.

"If we want to understand how genes are turned on and off, we need to know where the sequences that perform this function are located in the genome," said Mitchell.

"The parts of the human genome linked to complex diseases such as heart disease, cancer and neurological disorders can often be far away from the genes they regulate, so it can be difficult to figure out which gene is being affected and ultimately causing the disease."

It was previously thought that regions much closer to the Sox2 gene were the ones that turned it on in embryonic stem cells. Mitchell and her colleagues eliminated this possibility when they deleted these nearby regions in the genome of mice and found there was no impact on the gene's ability to be turned on in embryonic stem cells.

"We then focused on the region we've since named the SCR as my work had shown that it can contact the Sox2 gene from its location 100,000 base pairs away," said study lead author Harry Zhou, a former graduate student in Mitchell's lab, now a student at U of T's Faculty of Medicine.

"To contact the gene, the DNA makes a loop that brings the SCR close to the gene itself only in embryonic stem cells. Once we had a good idea that this region could be acting on the Sox2 gene, we removed the region from the genome and monitored the effect on Sox2."

The researchers discovered that this region is required to both turn Sox2 on, and for the embryonic stem cells to maintain their characteristic appearance and ability to differentiate into all the cell types of the adult organism.

"Just as deletion of the Sox2 gene causes the very early embryo to die, it is likely that an abnormality in the regulatory region would also cause early embryonic death before any of the organs have even formed," said Mitchell.

"It is possible that the formation of the loop needed to make contact with the Sox2 gene is an important final step in the process by which researchers practicing regenerative medicine can generate pluripotent cells from adult cells."

"Though the degree to which human embryonic stem cells possess this feature is not entirely clear, by understanding how another complex organism's genome works we ultimately learn more about how our own genome works," said Zhou.

Contact: Sean Bettam
.........


For more on stem cells and cloning, go to CellNEWS at

Wednesday 3 December 2014

Not All Induced Pluripotent Stem Cells are Made Equal

Not All Induced Pluripotent Stem Cells are Made Equal
Wednesday, 03 December 2014

Mick Bhatia, Scientific Director, McMaster Stem
Cell and Cancer Research Institute, Canada
Research Chair in Human Stem Biology,
Professor, Department of Biochemistry and 
Biomedical Science, McMaster University.

Scientists at McMaster University have discovered that human stem cells made from adult donor cells "remember" where they came from and that's what they prefer to become again.

This means the type of cell obtained from an individual patient to make pluripotent stem cells, determines what can be best done with them. For example, to repair the lung of a patient with lung disease, it is best to start off with a lung cell to make the therapeutic stem cells to treat the disease, or a breast cell for the regeneration of tissue for breast cancer patients.

Pluripotency is the ability stem cells have to turn into any one of the 226 cell types that make up the human body. The work challenges the previously accepted thought that any pluripotent human stem cell could be used to similarly generate the same amount of mature tissue cells.

This finding, published today in the prestigious science journal Nature Communications, will be used to further drug development at McMaster, and potentially improve transplants using human stem cell sources.

Human blood cells in a dish. Cells were
reprogrammed to stem cells from blood and were
10x more effective than using skin as a starting 
tissue. 

The study was led by Mick Bhatia, director of the McMaster Stem Cell and Cancer Research Institute. He holds the Canada Research Chair in Human Stem Cell Biology and he is a professor in the Department of Biochemistry and Biomedical Sciences of the Michael G. DeGroote School of Medicine.

"It's like the stem cell we make wants to become a doctor like its grandpa or an artist like its great-grandma," said Bhatia.

"We've shown that human induced pluripotent stem cells, called iPSCs, have a memory that is engraved at the molecular/genetic level of the cell type used to make them, which increases their ability to differentiate to the parent tissue type after being put in various stem cell states.”

"So, not all human iPSCs are made equal," Bhatia added.

"Moving forward, this means that iPSC generation from a specific tissue requiring regeneration is a better approach for future cellular therapies. Besides being faster and more cost-efficient in the development of stem cell therapy treatments, this provides a new opportunity for use of iPSCs in disease modelling and personalized drug discovery that was not appreciated before."

Neural cells generated from patient-specific stem 
cells. 
Small quantities of tissue would need to be harvested from the patient; for example, a few millilitres of blood or a small skin punch biopsy. From there, the harvested cells can be put into various stem cell states where they make copies of themselves indefinitely. The trickier part has been to get them out of copy function model and into differentiation mode to produce the cells needed for researchers to test drug therapies with no harm to patients, said Bhatia.

The McMaster Stem Cell and Cancer Research Institute is the only Canadian university team continuing to work exclusively with the more fragile human stem cells rather than mouse stem cells for its research, and that has furthered the work to future clinical impact. By contrast, the iPSCs of mice, which are widely used in stem cell research, have no memory, the authors note.

"So, if you only studied the mouse alone, you'd never uncover this opportunity," said Bhatia.

In a previous discovery, Bhatia and his team discovered how to make human blood from adult human skin. This meant that patients needing blood would be able to have blood created from a patch of their own skin to provide transfusions.

With these new findings, "our starting block has changed," said Bhatia, adding that now researchers "can make better tailored human stem cells for therapies because we've got a more efficient way of making higher quality and quantity of cells. For example, our team has shown that the stem cells that come from blood in the first place make blood 10 times better."

Contact: Veronica McGuire
.........


For more on stem cells and cloning, go to CellNEWS at