Neural Stem Cells Regenerate Axons in Severe Spinal Cord Injury

New relay circuits, formed across sites of complete spinal transaction, result in functional recovery in rats

Friday, 14 September 2012

In a study at the University of California, San Diego and VA San Diego Healthcare, researchers were able to regenerate "an astonishing degree" of axonal growth at the site of severe spinal cord injury in rats. Their research revealed that early stage neurons have the ability to survive and extend axons to form new, functional neuronal relays across an injury site in the adult central nervous system (CNS).

The study also proved that at least some types of adult CNS axons can overcome a normally inhibitory growth environment to grow over long distances. Importantly, stem cells across species exhibit these properties. The work will be published in the journal Cell on September 14.

The scientists embedded neural stem cells in a matrix of fibrin (a protein key to blood clotting that is already used in human neuron procedures), mixed with growth factors to form a gel. The gel was then applied to the injury site in rats with completely severed spinal cords.

"Using this method, after six weeks, the number of axons emerging from the injury site exceeded by 200-fold what had ever been seen before," said Mark Tuszynski, MD, PhD, professor in the UC San Diego Department of Neurosciences and director of the UCSD Center for Neural Repair, who headed the study.

"The axons also grew 10 times the length of axons in any previous study and, importantly, the regeneration of these axons resulted in significant functional improvement."

In addition, adult cells above the injury site regenerated into the neural stem cells, establishing a new relay circuit that could be measured electrically.

"By stimulating the spinal cord four segments above the injury and recording these electrical stimulation three segments below, we detected new relays across the transaction site," said Tuszynski.

To confirm that the mechanism underlying recovery was due to formation of new relays, when rats recovered, their spinal cords were re-transected above the implant. The rats lost motor function – confirming formation of new relays across the injury.

The grafting procedure resulted in significant functional improvement: On a 21-point walking scale, without treatment, the rats score was only 1.5; following the stem cell therapy, it rose to 7 – a score reflecting the animals' ability to move all joints of affected legs.

Results were then replicated using two human stem cell lines, one already in human trials for ALS.

"We obtained the exact results using human cells as we had in the rat cells," said Tuszynski.

The study made use of green fluorescent proteins (GFP), a technique that had never before been used to track neural stem cell growth.

"By tagging the cells with GFP, we were able to observe the stem cells grow, become neurons and grow axons, showing us the full ability of these cells to grow and make connections with the host neurons," said first author Paul Lu, PhD, assistant research scientist at UCSD's Center for Neural Repair.

"This is very exciting, because the technology didn't exist before."

According to the researchers, the study makes clear that early-stage neurons can overcome inhibitors present in the adult nervous system that normally work to maintain the elaborate central nervous system and to keep cells in the adult CNS from growing aberrantly.

Source: University of California - San Diego 

Contact: Debra Kain

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ZenMaster

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

http://cellnews-blog.blogspot.com/

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Molecular Beacons Light Up Stem Cell Transformation

Molecular Beacons Light Up Stem Cell Transformation

Wednesday, 12 September 2012

A novel set of custom-designed "molecular beacons" allows scientists to monitor gene expression in living populations of stem cells as they turn into a specific tissue in real-time. The technology, which Brown University researchers describe in a new study, provides tissue engineers with a potentially powerful tool to discover what it may take to make stem cells transform into desired tissue cells more often and more quickly. That's a key goal in improving regenerative medicine treatments.

"We're not the inventors of molecular beacons but we have used it in a way that hasn't been done before, which is to do this in long-term culture and watch the same population change in a reliable and harmless way," said graduate student Hetal Desai, lead author of the paper published online Sept. 5, 2012, in the journal Tissue Engineering Part A.

A population of fat-derived stem cells

expresses the bone-specific COL1A1 gene.

Green fluorescence from a “molecular

beacon” shows increasing expression from

day nine, left, through day 11 and day 14.

By day 16, right, expression begins to wane.

Credit: Darling Lab/Brown University.

In their research, Desai and corresponding author Eric Darling, assistant professor of biology in the Department of Molecular Pharmacology, Physiology, and Biotechnology, designed their beacons to fluoresce when they bind to mRNA from three specific genes in fat-derived stem cells that are expressed only when the stem cells are transforming into bone cells.

Throughout 21 days of their development, the cells in the experiments remained alive and unfettered, Desai said, except that some populations received a chemical inducement toward becoming bone and others did not. Over those three weeks, the team watched the populations for the fluorescence of the beacons to see how many stem cells within each population were becoming bone and the timing of each gene expression milestone.

The beacons' fluorescence made it easy to see a distinct pattern in that timing. Expression of the gene ALPL peaked first in more than 90 percent of induced stem cells on day four, followed by about 85 percent expressing the gene COL1A1 on day 14. The last few days of the experiments saw an unmistakably sharp rise in expression of the gene BGLAP in more than 80 percent of the induced stem cells.

Each successive episode of gene expression ramped up from zero to the peak more quickly, the researchers noted, leading to a new hypothesis that the pace of the stem cell transformation, or "differentiation" in stem cell parlance, may become more synchronized in a population over time.

"If you could find a way to get them on this track earlier, you could get the differentiation faster," Darling said.

Meanwhile the stem cell populations that were not induced with bone-promoting chemicals, showed virtually no beacon fluorescence or expression of the genes, indicating that the beacons were truly indicators of steps along the transformation from stem cell to bone.

Beacons don't affect cells

Desai said the team took extra care to design beacons that would not alter the cells' development or functioning in any way. While the beacons do bind to messenger RNA produced in gene expression, for example, they do not require adding any genes to the stem cells' DNA, or expressing any special proteins, as many other fluorescence techniques do.

The team performed several experiments using the beacons in conventionally developing bone cells to make sure that they developed normally even as the beacons were in operation. While some scientists design RNA-based probes to purposely interfere with gene expression, this team had the opposite intent.

"You know that song 'Hold on Loosely but Don't Let Go?'" Desai said.

"That's sort of the theme song for this. There's a set of rules for interference RNA, and we essentially did the opposite of what those rules said you should do."

Toward quicker healing

Now that the beacons' performance in indicating milestones of stem cell differentiation has been demonstrated, Darling said, the technology can be applied to studying the process in a wide variety of cells and under a variety of other experimental conditions.

In the case of tissue engineering, he said, the beacons can aid experiments seeking to determine what conditions (inducing chemicals or otherwise) are most effective in converting the most stem cells to desired tissues most quickly. They could help tissue engineers learn the best timing for adding an inducing chemical. They might also provide a way for tissue engineers to identify and harvest only those cells that are converting to the desired tissue.

"They are becoming bone cells and if you enrich for them and you get rid of all the ones that aren't becoming bone cells, it stands to reason that you will have a better product at the end," Darling said.

More broadly, Darling added, molecular beacons are proving useful in a wide variety of gene expression studies.

"The reason we are looking at this technique is that we wanted something we could use on any cell, look at any gene and not affect that cell while we are looking at it," Darling said.

"If this is acting as we believe it is, we can really look at any gene that we want. It seems like a very versatile tool."

Source: Brown University 

Contact: David Orenstein

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ZenMaster

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

http://cellnews-blog.blogspot.com/

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Neonatal Heart Stem Cells May Help Mend Kids' Broken Hearts

Cardiac stem cells from newborns show stronger regenerative ability than adult stem cells

Tuesday, 11 September 2012

Researchers at the University of Maryland School of Medicine, who are exploring novel ways to treat serious heart problems in children, have conducted the first direct comparison of the regenerative abilities of neonatal and adult-derived human cardiac stem cells. Among their findings: cardiac stem cells (CSCs) from newborns have a three-fold ability to restore heart function to nearly normal levels compared with adult CSCs. Further, in animal models of heart attack, hearts treated with neonatal stem cells pumped stronger than those given adult cells. The study is published in the September 11, 2012, issue of Circulation.

"The surprising finding is that the cells from neonates are extremely regenerative and perform better than adult stem cells," says the study's senor author, Sunjay Kaushal, M.D., Ph.D., associate professor of surgery at the University of Maryland School of Medicine and director, paediatric cardiac surgery at the University of Maryland Medical Center.

"We are extremely excited and hopeful that this new cell-based therapy can play an important role in the treatment of children with congenital heart disease, many of whom don't have other options."

Dr. Kaushal envisions cellular therapy as either a stand-alone therapy for children with heart failure or an adjunct to medical and surgical treatments. While surgery can provide structural relief for some patients with congenital heart disease and medicine can boost heart function up to two percent, he says cellular therapy may improve heart function even more dramatically.

"We're looking at this type of therapy to improve heart function in children by 10, 12, or 15 percent. This will be a quantum leap in heart function improvement."

Heart failure in children, as in adults, has been on the rise in the past decade and the prognosis for patients hospitalized with heart failure remains poor. In contrast to adults, Dr. Kaushal says heart failure in children is typically the result of a constellation of problems: reduced cardiac blood flow; weakening and enlargement of the heart; and various congenital malformations. Recent research has shown that several types of cardiac stem cells can help the heart repair itself, essentially reversing the theory that a broken heart cannot be mended.

Stem cells are unspecialized cells that can become tissue- or organ-specific cells with a particular function. In a process called differentiation, cardiac stem cells may develop into rhythmically contracting muscle cells, smooth muscle cells or endothelial cells. Stem cells in the heart may also secrete growth factors conducive to forming heart muscle and keeping the muscle from dying.

To conduct the study, researchers obtained a small amount of heart tissue during normal cardiac surgery from 43 neonates and 13 adults. The cells were expanded in a growth medium yielding millions of cells. The researchers developed a consistent way to isolate and grow neonatal stem cells from as little as 20 milligrams of heart tissue. Adult and neonate stem cell activity was observed both in the laboratory and in animal models. In addition, the animal models were compared to controls that were not given the stem cells.

Dr. Kaushal says it is not clear why the neonatal stem cells performed so well. One explanation hinges on sheer numbers: there are many more stem cells in a baby's heart than in the adult heart. Another explanation: neonate-derived cells release more growth factors that trigger blood vessel development and/or preservation than adult cells.

"This research provides an important link in our quest to understand how stem cells function and how they can best be applied to cure disease and correct medical deficiencies," says E. Albert Reece, M.D., Ph.D., M.B.A., vice president for medical affairs, University of Maryland; the John Z. and Akiko K. Bowers Distinguished Professor; and dean, University of Maryland School of Medicine.

"Sometimes simple science is the best science. In this case, a basic, comparative study has revealed in stark terms the powerful regenerative qualities of neonatal cardiac stem cells, heretofore unknown."

Insights gained through this research may provide new treatment options for a life-threatening congenital heart syndrome called hypoplastic left heart syndrome (HLHS). Dr. Kaushal and his team will soon begin the first clinical trial in the United States to determine whether the damage to hearts of babies with HLHS can be reversed with stem cell therapy. HLHS limits the heart's ability to pump blood from the left side of the heart to the body. Current treatment options include either a heart transplant or a series of reconstructive surgical procedures. Nevertheless, only 50-60 percent of children who have had those procedures survive to age five.

According to the American Heart Association, congenital heart disease may affect approximately one in 100 children. In the United States, more than 1 million adults are living with congenital heart defects.

Source: University of Maryland Medical Center 

Contact: Bill Seiler

Reference:

A strong regenerative ability of cardiac stem cells derived from neonatal hearts

Simpson DL, Mishra R, Sharma S, Goh SK, Deshmukh S, Kaushal S.

Circulation. September 11, 2012

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ZenMaster

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

http://cellnews-blog.blogspot.com/

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