Category Archives: Massachusetts Stem Cells

Doubts have surfaced about a landmark paper claiming that human embryos were cleared of a deadly mutation using genome editing. In an article1 posted to the bioRxiv preprint server on 28 August, a team of prominent stem-cell scientists and geneticists question whether the mutation was actually fixed.

The 2 August Nature paper2, led by reproductive biologist Shoukhrat Mitalipov at the Oregon Health and Science University in Portland, described experiments in dozens of embryos to correct a mutation that causes a heart condition called hypertrophic cardiomyopathy.

In contrast to previous human-embryo editing studies, Mitalipovs team reported a high success rate at correcting a disease-causing mutation in a gene. The team claimed that the CRISPRCas9 genome editing tool was able to replace a mutant version of the MYBPC3 gene carried by sperm with a normal copy from the egg cell, yielding an embryo with two normal copies. Mitalipovs team also introduced a healthy version of the gene along with the CRISPR machinery, but they found that the corrected embryos had shunned it for the maternal version.

But there is reason to doubt whether this really occurred, reports a team led by Dieter Egli, a stem-cell scientist at Columbia University in New York City, and Maria Jasin, a developmental biologist at Memorial Sloan Kettering Cancer Center in New York City. George Church, a geneticist at Harvard Medical School in Boston, Massachusetts, is another co-author.

In their bioRxiv paper, Egli and Jasin and their co-authors say that there is no plausible biological mechanism to explain how a genetic mutation in sperm could be corrected based on the eggs version of the gene. More likely, they say, Mitalipovs team failed to actually fix the mutation and were misled into thinking they had by using an inadequate genetics assay. Egli and Jasin declined to comment because they say they have submitted their article to Nature.

The critique levelled by Egli et al. offers no new results but instead relies on alternative explanations of our results based on pure speculation, Mitalipov said in a statement.

But other scientists contacted by Nature's news team shared the Egli team's concerns. (Natures news team is editorially independent of its journal team.) Reproductive biologist Anthony Perry at the University of Bath, UK, says that after fertilization, the genomes of the egg and sperm reside at opposite ends of the egg cell, and each is enshrouded in a membrane for several hours. This fact, Perry says, would make it difficult for CRISPR-Cas9 to fix the sperms mutation based on the eggs version of the gene, using a process called homologous recombination. Its very difficult to conceive how recombination can occur between parental genomes across these huge cellular distances, he says.

Egli and Jasin raise that issue in their paper. They suggest that Mitalipovs team was misled into believing that they had corrected the mutation by relying on a genetic assay that was unable to detect a far likelier outcome of the genome-editing experiment: that CRISPR had instead introduced a large deletion in the paternal gene that was not picked up by their genetic assay. The Cas9 enzyme breaks DNA strands, and cells can attempt to repair the damage by haphazardly stitching the genome together, often resulting in missing or extra DNA letters.

That explanation makes sense, says Gatan Burgio, a geneticist at the Australian National University in Canberra. In my view Egli et al. convincingly provided a series of compelling arguments explaining that the correction of the deleterious mutation by self repair is unlikely to have occurred.

Another possibility Eglis team raise is that the embryos were produced without a genetic contribution from sperm, a process known as parthenogenesis. Mitalipovs team showed that the paternal genome was present in only 2 out of the 6 embryonic stem cell lines they made from gene-edited embryos.

Robin Lovell-Badge, a developmental biologist at the Francis Crick Institute in London, says that it is possible that there is a novel or unsuspected biological mechanism at work in the very early human embryo that could explain how Mitalipovs team corrected the embryos genomes in the manner claimed. He would first like to hear from Mitalipov before passing judgement. It simply says that we need to know more, not that the work is unimportant, Lovell-Badge says of Egli and Jasins paper.

In the statement, Mitalipovs said his team stands by their results. We will respond to their critiques point by point in the form of a formal peer-reviewed response in a matter of weeks.

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Doubts raised about CRISPR gene-editing study in human embryos - Nature.com

From left to right: Richard Stone, a doctor at Dana-Farber Cancer Institute in Boston, poses with Peter Karalekas (center), 76, and Matthew Churitch, 22. Churitch donated stem cells to Karalekas two years ago, and he visited Dana-Farber with Karalekas earlier this summer. (Courtesy photo)

BOSTON -- After winding his way through Massachusetts, Connecticut, New Hampshire and Maine for 76 years, Peter Karalekas has a proclamation: He's a Southerner now.

He still lives in Kittery, Maine, just about an hour from the Lowell middle school where he taught for 21 years.

He has no plans to move.

Rather, Karalekas considers himself a Southerner because of his stem cells.

He never exactly felt all that sick.

Karalekas worked tirelessly for decades, first as a teacher and coach at the James S. Daley Middle School in Lowell and then as the owner of a half-dozen T-Bones restaurants across New Hampshire.

Even despite the 12-hour days, seven days a week, in the grind of the restaurant industry, Karalekas felt healthy and rarely fell ill.

Peter Karalekas, left, a 76-year-old former Lowellian, smiles during his first meeting with Matthew Churitch, 22, of Nashville, Tennessee, who helped save Karalekas life by donating stem cells. (Courtesy photo)

The two, who do not have children, moved to Kittery 17 years ago.

Everything started to change in 2014.

Karalekas recalls being "short-winded," but he had very few other symptoms when he was diagnosed with myelodysplastic syndrome, a rare type of cancer in which the bone marrow is damaged and cannot produce enough blood cells.

The prognosis was not good.

"They said the only thing that would save me was a stem cell transplant," Karalekas said. "Otherwise, I had a couple of months to live, because my cells were all dropping drastically.

He went onto a registry, hoping for a donor to pop up, but doctors told him it could take from six months to two years to find the right match. Even with a transplant, Karalekas said, his chances of success were "30 to 40 percent."

The call came four weeks later.

Matthew Churitch got his call quickly, too.

He joined the National Marrow Donor Program's Be the Match Registry in 2014, the summer between his freshman and sophomore years at Clemson University. His mother had been on the registry to donate for years. Churitch's decision was simple: When a friend was diagnosed with leukemia, he knew he should sign up, too.

He did the requisite cheek swab, unsure if he would ever even be contacted to donate. By the time he had finished the following semester, he got the call.

A match was found.

Churitch went through several more levels of testing and preparation to donate stem cells to a stranger. He went to Clemson's student health center to have blood drawn.

He returned to his native Nashville, Tennessee, going to a medical center 10 days in a row to receive shots in his stomach that would stimulate his bone marrow and prepare his cells for transplant.

He sat for eight hours, a needle in each arm as his stem cells were filtered out so they could be transferred to Boston.

"Getting the shots isn't fun," he said. "You're pretty sore afterward for a few weeks. But knowing that the person on the other end is in hundreds and hundreds times more pain than any donor would ever go through -- that kind of pushed me through."

Karalekas and Churitch first connected via an anonymous letter, per the transplant registry's rules, updating Churitch on Karalekas's lengthy, isolated recovery. They were able to speak directly after a year.

Churitch dialed Karalekas' number on a lengthy walk to class, took a deep breath and hit the call button. Moments later, both men were crying and laughing.

"That was really awesome, just being able to hear his voice and recognize that there's somebody else on the other end of this," Churitch said. "A lot of people don't get the chance to connect with their recipients or their donors."

Karalekas wanted more. He told his wife early on that he wanted to meet his "angel from heaven," so when Churitch graduated Clemson earlier this year, Karalekas paid to bring the 22-year-old and his mother to New England.

In late June, Karalekas and his wife pulled into a pickup lane at Logan International Airport in Boston.

"I got out of the car, I charged over, and I gave them both a huge hug," Karalekas said.

Karalekas showed Churitch and his mother around for five days.They went on a private tour of Fenway Park; they wandered the historic streets of Portsmouth, New Hampshire; they visited Dana-Farber together to meet the team that treated Karalekas.

Both families quickly bonded. Karalekas recalls his brother George asking Churitch about his portable phone charger, expressing curiosity about how convenient it was. A few weeks later, a brand-new portable charger arrived at George's door, a gift from Churitch.

In January, Karalekas and his wife will vacation in Arizona and will cheer on Churitch's mother -- without Churitch even present -- in the Phoenix Marathon.

Donor and recipient talk every week.

"It's like we're a very, very close-knit family now," Karalekas said. "He's the son we never had."

Churitch is now in his first year at the University of South Carolina School of Medicine Greenville with hopes of becoming a physician. He hopes to use Karalekas's experience as inspiration for any patients facing future hardship, and he hopes that others, especially young people, will see their success and join the registry.

"You never know where that will take you," he said. "You can gain a friend for life, impact somebody and their family in need."

Karalekas said he feels he has a new life: His chances of beating the disease are now 97 percent, he says, up from the 30 percent or 40 percent when he started treatment. Thanks to the transplant from a handsome, athletic college student in Tennessee.

"I said, 'I'm a Southerner now,'" Karalekas said. "My stem cells are 99 percent this gentleman. I'm 99 percent him."

Follow Chris on Twitter @ChrisLisinski.

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For Lowell native, stem cell match becomes a match as friends - Lowell Sun

Juno Therapeutics immunotherapy JCAR017 eliminated a brain tumor in a patient whose lymphoma had spread to her brain, researchers at Massachusetts General Hospital Cancer Centerreported.

The team wrote about the case, which involved a 68-year-old woman withprimary refractory diffuse large B-cell lymphoma (DLBCL), in theNew England Journal of Medicine.The article was titled Anti-CD19 CAR T Cells in CNS Diffuse Large-B-Cell Lymphoma.

Researchers said the patient had failed to respond to chemotherapy with etoposide, doxorubicin, cyclophosphamide with vincristine, and prednisone with Rituxan (rituximab). Doctors then tried four additional therapies, including a stem cell transplant but all failed to stop the cancer.

The women then enrolled in a clinical trial that was testing JCAR017 (NCT02631044) against cancer. The treatment involves using immune-system T-cells to target cancer originating in another component of the immune system, B-cells.

The immunotherapys chimeric antigen receptor (CAR) T-cells go after the protein CD19, which appears on the surface of almost all cancers originating in B-cells. The treatment allows the T-cells to recognize and destroy the cancer cells.

At the time of the clinical trial, the patient was not on an immunosuppressive therapy.

She also did not have graft-versus-host disease (GVHD), indicating that her body was not rejecting the stem cell transplant. Just before she started the trial, a scan showed that she had a new brain lesion, suggesting that the stem cell transplant was not working. A different kind of scan confirmed the lesion.

The patient started the trial with a high dose of chemotherapy that eliminated parts of her immune system and primed her body to receive CAR T-cells. Then she received JCAR017. She experienced no adverse events from the procedure, including no release of inflammatory cytokines, no neurotoxic effects, and no GVHD.

Amazingly, a month later, two kinds of brain scans showed complete remission of her cancer.

A blip occurred two months later, when scans showed her cancer had returned. But after an incisional biopsy of the tumor, which isa very minor procedure, the tumor started receding again without needing further therapy. Researchers said the shrinking correlated with an expansion in the number of CAR T-cells in her system.

A month later, another scan showed complete remission again. This time the response was durable: The patient continues to be in remission at 12 months.

Many cancer therapies are unable topass through the blood-brain barrier to treat brain cancers. But scientists have found CAR T-cells in cerebrospinal fluid, indicating they can pass through the barrier, which the body uses to prevent harmful invaders from getting to the brain.

Brain involvement in DLBCL carries a grave prognosis, and the ability to induce a complete and durable response with conventional therapies is rare, Dr. Jeremy Abramson, of the Massachusetts General Hospital Cancer Center, said in a press release.

In addition, all available CAR T-cell trials have excluded patients with central nervous system involvement, said Abramson, who wrote the New England Journal of Medicine article. This result has implications not only for secondary DLBCL like this case, but also for primary central nervous system lymphoma, for which treatment options are similarly limited after relapse and few patents are cured.

In an intriguing finding, the CAR T-cells expanded in the patient again months after the initial administration of JCAR017. This implied that something about the biopsy procedure had triggered an expansion of the CAR T-cell population that led to the regression of the tumor.

Typically the drugs we use to fight cancer and other diseases wear off over time, Abramson said. This spontaneous re-expansion after biopsy highlights this therapy as something entirely different, a living drug that can re-expand and proliferate in response to biologic stimuli.

The team said that studying ways of reactivating CAR T-cells could make them even more effective.

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Lymphoma Patient's Brain Tumor Disappeared After She Received JCAR017, Study Reports - Lymphoma News Today

Two years in the making, Asymmetrex's stem cell counting technology now improves manufacturing of therapeutic stem cells.

Boston, MA (PRWEB) August 29, 2017

Today, Asymmetrex, the Massachusetts based stem cell biotechnology innovator, begins offering a new contract service to support the cell therapy industrys production of therapeutic adult tissue stem cells. Stem cells are a small sub-population of specialized cells that function to renew and repair the organs and tissues of children and adults. Stem cells from a healthy donor can be transplanted to restore or repair diseased or damaged organs and tissues in patients who are sick or injured.

Stem cell transplant treatments do not require expansion of donor stem cells when a donor with a compatible immune system is available for a patient. However, in general, there are many more patients in need of a transplant than there are immune compatible donors. So, the future ability of stem cell medicine to address the full degree of medical need depends on developing the ability to expand initial donor stem cell populations into larger numbers without loss of their curative properties. Expanded numbers of quality adult tissue stem cells are also needed to support statistically sound clinical trials for continued development of stem cell transplant therapies.

Asymmetrexs new advance for cell therapy suppliers is the result of the companys unique proprietary expertise in adult tissue stem cell culture. Its recently patented AlphaSTEM Test technology, which is a means to count adult tissues stem cells specifically for the first time, is based on the earlier discovery that during expansion in culture, ironically, adult tissue stem cells are lost as a result of their intrinsic tissue renewal property. Under typical culture conditions, stem cells continuously produce non-stem cells that subsequently undergo significant, though limited, multiplication. However, as in the body, stem cells do this while not multiplying their own number. The result of these continued natural processes in culture is progressive dilution and loss of the stem cells with each subsequent expansion culture.

Recently, Asymmetrex demonstrated that not only can its AlphaSTEM Test technology be used to count and certify the quality of adult tissue stem cells, but it can also be used to establish unique culture conditions projected to reduce stem cell dilution significantly during expansion. Asymmetrexs Director, James L. Sherley, M.D., Ph.D., shares, Two years ago when we achieved specific counting, I expected that we would be able to find conditions for increasing stem cell number, too; but even I didnt anticipate the projected huge savings in production costs! The companys projections indicate that it can achieve production of twice as many stem cells as several companies have reported, but require only 5% or less of their production cost. The required culture time is also reduced significantly.

The new service has been developed first for human mesenchymal stem cells, which are found in a variety of tissues including bone marrow, adipose (fat), umbilical cord, and amniotic fluid. These cells are an ideal first focus, as there are many companies worldwide working on their production to supply hundreds of stem cell transplantation clinical trials each year. Asymmetrex plans to also investigate application of the method to other types of adult tissue stem cells, which have been largely dismissed for expansion because their donor populations are not as proliferative as mesenchymal stem cells. Sherley says that blood stem cells are a very important member of this latter group on the Asymmetrex horizon.

About Asymmetrex

Asymmetrex, LLC is a Massachusetts life sciences company with a focus on developing technologies to advance stem cell medicine. Asymmetrexs founder and director, James L. Sherley, M.D., Ph.D., is an internationally recognized expert on the unique properties of adult tissue stem cells. The companys patent portfolio contains biotechnologies that solve the two main technical problems quantification and production that have stood in the way of successful commercialization of human adult tissue stem cells for regenerative medicine and drug development. In addition, the portfolio includes novel technologies for isolating cancer stem cells and producing induced pluripotent stem cells for disease research purposes. Asymmetrex markets the first technology for determination of the dose and quality of tissue stem cell preparations (the AlphaSTEM Test) for use in stem cell transplantation therapies and in pre-clinical assays for drug safety. The same technology underpins Asymmetrex's contract service for optimizing manufacturing processes for therapeutic adult tissue stem cells.

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Asymmetrex Introduces New Contract Service For Producing ... - PR Web (press release)

By Gunjan SinhaAug. 23, 2017 , 9:00 AM

UTRECHT, THE NETHERLANDSBy her 50th birthday, Els van der Heijden felt sicker than ever. Born with the hereditary disorder cystic fibrosis (CF), she had managed to work around her illness, finishing college and landing a challenging job in consulting. But Van der Heijden, who lives in a small Dutch town, says she always felt "a dark cloud hanging over my head." When she began feeling exhausted and easily out of breath in 2015, she thought it was the beginning of the end.

Then she read a newspaper article about a child with CF named Fabian whose life had been saved after scientists grew a "mini-organ" from a tissue sample snipped from his colon, one organ that CF affects. Doctors had used the mini-organ to test ivacaftor (Kalydeco), a drug so expensive that Dutch insurers refuse to cover it without evidence that it will help an individual CF patient. No such data existed for Fabian, whose CF was caused by an extremely rare mutation. But his minigut responded to ivacaftor, and he improved within hours of taking it. His insurance eventually agreed to pay for the drug.

Van der Heijden's doctor arranged to have a minigut made for her as well; it responded to a drug marketed as Orkambi that combines ivacaftor and another compound, lumacaftor. Within weeks after she began taking that combination, "I had an enormous amount of energy," she says. "For the first time ever, I felt like my body was functioning like it should."

The life-altering test was developed in the lab of Hans Clevers, director of the Hubrecht Institute here. More than a decade ago, Clevers identified a type of mother cell in the gut that can give birth to all other intestinal cells. With the right nutrition, his team coaxed such stem cells to grow into a 3D, pencil tip-sized version of the gut from which it came. The minigut was functionally similar to the intestine and replete with all its major cell typesan organoid.

That was the start of a revolution. Clevers and others have since grown organoids from many other organs, including the stomach, pancreas, brain, and liver. Easy to manipulate, organoids are clarifying how tissues develop and repair injury. But perhaps most exciting, many researchers say, is their ability to model diseases in new ways. Researchers are creating organoids from tumor cells to mimic cancers and introducing specific mutations into organoids made from healthy tissue to study how cancer arises. And as Clevers's lab has shown, organoids can help predict how an individual will respond to a drugmaking personalized medicine a reality. "It is highly likely that organoids will revolutionize therapy of many severe diseases," says Rudolf Jaenisch, a stem cell scientist at the Massachusetts Institute of Technology in Cambridge.

For Clevers, the bonanza has come as a surprise. A basic biologist at heart, he says he never had real-world applications in mind. "I was always driven by curiosity," he says. "For 25 years we published papers with no practical relevance for anyone on this planet."

Organoids can be used to study how pathogens interact with human tissues. In this lung organoid grown in Hans Clevers's lab, cells colored green are infected with respiratory syncytial virus.

NORMAN SACHS

On a bright July morning at the Hubrecht Institute, Clevers listens patiently to presentations during a weekly lab meeting. One postdoc presents data on her efforts to develop an organoid model for small-cell lung cancer; another reports progress on culturing hormone-secreting organoids from human gut tissue. Whenever their research questions strike him as uninspired, Clevers urges them to be more ambitious: "Why don't you pursue something you don't know?" he asks.

"Hans is capable of raising questions that are not contaminated by the anticipated answer," says Edward Nieuwenhuis, chairman of pediatrics at University Medical Center Utrecht (UMCU) and a good friend. "He has a better nose than most for sniffing around and finding interesting stuff," says Ronald Plasterk, who co-directed the Hubrecht lab with Clevers from 2002 to 2007 and is now the Dutch Minister of the Interior and Kingdom Relations. That approach has earned Clevers many awards. In June, for example, he was inducted into the Orden Pour le Mrite, an elite German order with just 80 members worldwide.

Clevers began his career studying immune cells as a postdoc at the Dana-Farber Cancer Institute in Boston. He landed his first job at UMCU's clinical immunology department in 1989, where he quickly became department head. Most of the work was clinical, such as leukemia diagnostics and blood work for transplants. "But my research interests were always much more basic than the environment that I was in," he says.

In early work, he identified a key molecule, T cell-specific transcription factor 1 (TCF-1), that signals the immune cells known as T lymphocytes to proliferate. Later he found that TCF-1 is part of the larger Wnt family of signaling molecules that's important not only for immune responses, but also for embryonic development and tissue repair. In 1997, his lab team discovered that mice lacking the gene for one of those signals, TCF-4, failed to develop pockets in their intestinal lining called crypts. Soon after, a study with Bert Vogelstein at Johns Hopkins University in Baltimore, Maryland, showed that TCF-4 also helps initiate human colon cancer. Fascinated, Clevers switched his focus from the immune system to the gut.

Inspired by a flurry of research on stem cells at the time, Clevers began hunting for intestinal stem cells. More than 50 years ago, researchers deduced that rodent crypts produce many cells that survive only a few days, suggesting some unidentified, longer-lived source for the cells.

After almost a decade of tedious experiments, Clevers's postdoc Nick Barker struck gold in 2007: He discovered that cells carrying a receptor named LGR5 give rise to all cells in mouse intestines and that molecules in the Wnt pathway signal those cells to divide. Barker later found LGR5-positive cells in other organs as well. In some, the cells were always active; in others, such as the liver, they multiplied only when tissues sensed injury.

At the time, culturing stem cells was notoriously hard, but after combing through previous lab experiments, another postdoc in Clevers's lab, Toshiro Sato, concocted a mix of growth factors that coaxed the gut stem cells to replicate in a dish. He hoped to see a flat layer of cells. But what emerged in 2009 from a single LGR5-positive cell was "a beautiful structure that surprised and intrigued me," says Sato, now at Keio University in Tokyo: a 3D replica of a gut epithelium. The structure self-organized into crypts and finger-shaped protrusions called villi, and it began making its own biochemicals. A paper about the feat was rejected several times before being published. Clevers recalls: "No one wanted to believe it."

Soon, the lab began culturing LGR5-positive cells and growing organoids from the stomach, liver, and other organs. "It was an exciting time, and I really felt like we were on the frontiers of discovery," says another postdoc at the time, Meritxell Huch, now at the Gurdon Institute in Cambridge, U.K. "But we certainly didn't think we were opening a new field."

Organoids, lab-grown miniature versions of organs, are transforming science and medicine. Researchers have grown them from many different organs; they have also created organoids from tumor cells to mimic cancers.

V. ALTOUNIAN/SCIENCE

Captivated by stem cells and their potential to regenerate tissues, other labs were starting to make organoids. A few months before Sato's 2009 paper, Akifumi Ootani, a postdoc in Calvin Kuo's group at Stanford University in Palo Alto, California, reported using a different strategy to grow gut organoids. Kuo's method starts with tissue fragments rather than individual stem cells and grows them in a gel partly exposed to air instead of submerged in nutrient medium. Around the same time, Yoshiki Sasai of the RIKEN Center for Developmental Biology in Kobe, Japan, cultured the first brain organoids, starting not with adult stem cells but with embryonic stem cells. Other researchers grew organoids from induced pluripotent stem cells, which resemble embryonic stem cells but are grown from adult cells.

The various methods create different kinds of organoids, each with advantages and drawbacks. Kuo's organoids contain a mix of cell types, which enables "observation of higher-order behaviors such as muscle contraction," he says. Because those organoids include stroma, a scaffold of connective tissue essential for tumor growth, they may prove better for studying therapies that target the stroma, such as cancer immunotherapy. Clevers's mix of growth factors grows organoids consisting primarily of epithelial cells, so his technique doesn't work for the brain and other organs with few or no epithelial cells. Nor can his organoids be used to test drugs targeting blood vessels or immune cells because organoids have neither.

Both methods can generate organoids from individual patients, producing a personalized minigut in just 1 to 3 weeks. (Although Clevers's organoids originate from adult stem cells, isolating those cells isn't necessary; culturing a tissue fragment with the right nutrients is enough.) The methods are reproducible, and the organoids remain genetically stable in culture; they can also be stored in freezers for years.

In 2013, Clevers and others founded a nonprofit, Hubrecht Organoid Technology (HUB), to market applications. Clevers first proposed using organoids for tissue transplants, says HUB Managing Director Rob Vries. Studies showed that healthy organoids implanted in mice with diseased colons could repair injury. "But we bagged the idea because there were too many regulatory hurdles and the chance of success was low," Vries says.

The idea of enlisting organoids to treat CF came from Jeffrey Beekman, a researcher at UMCU who studies that disease. All Dutch newborns are screened for CF, and colon biopsy samples are taken from babies who test positive. The tissue is tested to gauge how dysfunctional the defective gene is and then stored. Growing organoids from those samples would be relatively simple, argued Beekman, who has since spearheaded the project.

CF can arise from more than 2000 mutations in one gene, which cripple the ion channels that move salt and water through cell membranes. The disease affects all tissues, but the primary symptom is excess mucus in the lungs and gut, causing chest infections, coughing, difficulty breathing, and digestive problems.

Ivacaftor and the combination drug lumacaftor and ivacaftor, both marketed by Vertex Pharmaceuticals in Boston, restore the ion channels' function. But the drugs don't work equally well for everyone, and they have been tested and approved only for people with the most common mutations, together accounting for roughly half of all CF patients. Vertex, which declined to answer questions for this story, has been reluctant to spend millions on trials in patients with rare mutations because the potential payoff is small. And with the price tagboth drugs cost between 100,000 and 200,000 per year in Europehealth services and insurance companies have been unwilling to pay for the medicines for people with those untested mutations.

Van der Heijden falls into that category because only two other people in the Netherlands share her mutation. But when organoids grown from her gut were exposed to lumacaftor and ivacaftor, the organoids swelled like normal gut tissue, a sign that the defective protein was working and that salt and water were flowing through. The result helped persuade Vertex to give her the drug through a compassionate-use program, without payment. (Regulatory agencies require her to be monitored in a clinical trial.) Her side effects included fatigue, nausea, and diarrhea, but after a few months, "it was as if someone opened the curtain and said, Look, the sun is there, come out and play," she says. "And I did."

Cystic fibrosis patient Els van der Heijden received a new drug combination based on organoid tests. Within weeks, "I had an enormous amount of energy," she says.

TESSA NEDEREND

In collaboration with Vertex, HUB has tested ivacaftor on organoids grown from CF patients who had taken part in a clinical trial of that drug. The study confirmed that organoids can predict who will respond to the drug.

HUB has also tested ivacaftor on organoids from 50 patients with nine rare mutations. On the basis of the results, insurers agreed to pay for the drug in six more Dutch patients, and Vertex is following up with the first clinical trial of ivacaftor in CF patients with rare mutations. Meanwhile, HUB is building a biobank, financed by Dutch health insurers, containing organoids from all 1500 Dutch CF patients for testing both existing drugs and new candidates.

"This is the next big thing in CF research," says Eitan Kerem, head of pediatrics at Hadassah Medical Center in Jerusalem, who is building a similar biobank and has launched a trial in patients with rare mutations. Organoids are especially useful because no great animal models for CF exist, Kerem says; ferrets and pigs are sometimes used, but "they are expensive and not available to most researchers."

Drug and biotech companies are now striking deals with HUB to explore organoids in other diseases. The success with CF suggests that they can model other single-gene disorders, such as -1 antitrypsin deficiency, which causes symptoms primarily in the lungs and liver. Some companies are also testing failed drugs on organoids and comparing the results with animal and clinical data, hoping to find ways to predict and avoid such failures.

Cancer is also a major target. By growing organoids from tumor samples, researchers can create minitumors and use them to study how cancer develops or to test drugs. Soon after the minigut paper came out in 2009, David Tuveson, who heads the cancer center at Cold Spring Harbor Laboratory in New York, began prodding Clevers to develop organoids for pancreatic cancer, which is notoriously hard to treat. Existing cell culture models were not very realistic, Tuveson says, and creating genetically engineered mice took up to a year, compared with up to 3 weeks for pancreatic cancer organoids.

The organoids have already helped clarify new pathways that lead to pancreatic cancer, Tuveson says, and unpublished data suggest that they will help researchers predict which treatments will be most effective. He and Clevers are trying to make the organoids resemble real cancer more closely by adding stroma and immune cells. The Hubrecht lab is also involved in two trials to assess whether colon cancer organoids grown from individual patients can predict drug response.

Charles Sawyers of Memorial Sloan Kettering Cancer Center in New York City is trying to make prostate cancer organoids, but he says they are finicky. Organoids from primary tumors generally don't grow; those from metastatic tissue sometimes do, but normal cells often outgrow cancer cells. "They seem to need a lot of tender love and care, and there is no method to the madness," says Sawyers, who has succeeded with only 20 patients so far.

But Sawyers discovered that he could easily grow organoids from normal prostate tissue"it just works beautifully," he saysand then use gene-editing techniques such as CRISPR to study any cancer mutation he wants. "Is this a tumor suppressor gene? Is this an oncogene? Does it collaborate with geneXY? You can play the kind of games on the scale that you always wanted to," he says. As Kuo puts it, "We can build cancer from the ground up."

Other cancer researchers want in, too. Tuveson received so many requests for organoid training that he began hosting regular workshops at his laboratory. In 2016, the U.S. National Cancer Institute launched a scheme to develop more than 1000 cell culture models, including organoids, for researchers around the world to use, together with Cancer Research UK in London, the Wellcome Trust Sanger Institute in Hinxton, U.K., and HUB.

Using personalized organoids to treat cancer still faces hurdles. Organoid culture time, which varies by cancer, must be shortened, and the cost, a few thousand dollars per patient, needs to come down. Also, cancers accumulate genetic mutations as they progress, which could mean that an organoid grown from a patient's cancer early on might not reflect its later state. Nevertheless, "from my perspective it's the most transformative advance in cancer research that I know of," Tuveson says.

If all of that excites Clevers, he rarely shows it. He avoids emotional language while discussing his research, preferring instead to describe and explain. Even close friends sometimes find his pragmatism puzzling. "He talks about his research like someone talking about screwing in a screw," Nieuwenhuis says.

Clevers says he gets his high from "the satisfaction of finding something novel," regardless of practical applications. Recent experiments, for instance, suggest that when an organ lacks LGR-5-positive cells, differentiated cells may be able to "dedifferentiate" and repair tissuesa radical change from the one-way street toward specific identities that stem cells were thought to travel. "Some organs may not have a professional stem cell at all," Clevers says, with a hint of wonder. But when asked how he felt when he saw his findings have profound benefits for patients such as Fabian and Els van der Heijden, he simply says, "I did not expect that."

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ORGANOID - Science Magazine

Laboratory mice have made valuable contributions to biological research for centuries, at first as a model to study broad questions such as blood circulation and respiration, and then, increasingly to home in on more-specific questions in health and medicine. In particular, researchers hit on the idea in the 1980s to replace a mouses immune system with that of a humans.

These humanized mice have been useful models in some situations, such as the study of the human immune response to HIV infection or the transplantation of certain types of tissue. But recent research bycardiologist and stem cell expertJoseph Wu, MD, PhD, together with former postdoctoral scholars Nigel Kooreman, MD, and Patricia de Almeida, PhD, and graduate student Jonathan Stack, DVM, suggest that these mice dont adequately mimic the human immune response to stem cell transplantation.

They described their research today in Cell Reports.According to our release:

the Stanford researchers found that, unlike what would occur in a human patient, the humanized mice are unable to robustly reject the transplantation of genetically mismatched human stem cells. As a result, they cant be used to study the immunosuppressive drugs that patients will likely require after transplant. The researchers conclude that the humanized mouse model is not suitable for studying the human immune response to transplanted stem cells or cells derived from them.

The researchers also collaborated with Dale Greiner, PhD, from the University of Massachusetts Medical School, and Leonard Shultz, PhD, from the Jackson Laboratory.Greiner and Shultz helped to pioneer the use of humanized mice in the 1990s to model human diseases and they provided the mice used in the study.

Wu and his colleagues caution against using the current humanized mice as models for human stem cell transplantation, and urge the development of optimized models for use in clinical decision-making.

Many in the fields of pluripotent stem cell research and regenerative medicine are pushing the use of the humanized mice to study the human immune response. But if we start to make claims using this model, assuming that these cells wont be rejected by patients, it could be worrisome, Kooreman said. Our work clearly shows that, although there is some human immune cell activity, these animals dont fully reconstitute the human immune system.

Previously: When mice mislead, medical research lands in the trap, Fortune teller: Mice with humanized livers predict HCV drug candidates behavior in humansand Stroke of luck: Stem cell transplants show strong signs of efficacy in clinical safety trial for strokePhoto by Jakub Solovsky

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Current humanized mice not good models for studying stem cell transplants, say researchers - Scope (blog)

A type of mouse widely used to assess how the human immune system responds to transplanted stem cells does not reflect what is likely to occur in patients, according to a study by researchers at the Stanford University School of Medicine. The researchers urge further optimization of this animal model before making decisions about whether and when to begin wide-scale stem cell transplants in humans.

Known as humanized mice, the animals have been engineered to have a human, rather than a murine, immune system. Researchers have relied upon the animals for decades to study, among other things, the immune response to the transplantation of pancreatic islet cells for diabetes and skin grafts for burn victims.

However, the Stanford researchers found that, unlike what would occur in a human patient, the humanized mice are unable to robustly reject the transplantation of genetically mismatched human stem cells. As a result, they cant be used to study the immunosuppressive drugs that patients will likely require after transplant. The researchers conclude that the humanized mouse model is not suitable for studying the human immune response to transplanted stem cells or cells derived from them.

In an ideal situation, these humanized mice would reject foreign stem cells just as a human patient would, said Joseph Wu, MD, PhD, director of Stanfords Cardiovascular Institute and professor of cardiovascular medicine and of radiology. We could then test a variety of immunosuppressive drugs to learn which might work best in patients, or to screen for new drugs that could inhibit this rejection. We cant do that with these animals.

Wu shares senior authorship of the research, which was published Aug. 22 in Cell Reports, with Dale Greiner, PhD, professor in the Program in Molecular Medicine at the University of Massachusetts Medical School, and Leonard Shultz, PhD, professor at the Jackson Laboratory. Former postdoctoral scholars Nigel Kooreman, MD, and Patricia de Almeida, PhD, and graduate student Jonathan Stack, DVM, share lead authorship of the study.

Although these mice are fully functional in their immune response to HIV infection or after transplantation of other tissues, they are unable to completely reject the stem cells, said Kooreman. Understanding why this is, and whether we can overcome this deficiency, is a critical step in advancing stem cell therapies in humans.

Humanized mice are critical preclinical models in many biomedical fields helping to bring basic science into the clinic, but as this work shows, it is critical to frame the question properly, said Greiner. Multiple laboratories remain committed to advancing our understanding and enhancing the function of engrafted human immune systems.

Greiner and Shultz helped to pioneer the use of humanized mice in the 1990s to model human diseases and they provided the mice used in the study.Understanding stem cell transplants

The researchers were studying pluripotent stem cells, which can become any tissue in the body. They tested the animals immune response to human embryonic stem cells, which are naturally pluripotent, and to induced pluripotent stem cells. Although iPS cells can be made from a patients own tissues, future clinical applications will likely rely on pre-screened, FDA-approved banks of stem cell-derived products developed for specific clinical situations, such as heart muscle cells to repair tissue damaged by a heart attack, or endothelial cells to stimulate new blood vessel growth. Unlike patient-specific iPS cells, these cells would be reliable and immediately available for clinical use. But because they wont genetically match each patient, its likely that they would be rejected without giving the recipients immunosuppressive drugs.

Humanized mice were first developed in the 1980s. Researchers genetically engineered the mice to be unable to develop their own immune system. They then used human immune and bone marrow precursor cells to reconstitute the animals immune system. Over the years subsequent studies have shown that the human immune cells survive better when fragments of the human thymus and liver are also implanted into the animals.

Kooreman and his colleagues found that two varieties of humanized mice were unable to completely reject unrelated human embryonic stem cells or iPS cells, despite the fact that some human immune cells homed to and were active in the transplanted stem cell grafts. In some cases, the cells not only thrived, but grew rapidly to form cancers called teratomas. In contrast, mice with unaltered immune systems quickly dispatched both forms of human pluripotent stem cells.

The researchers obtained similar results when they transplanted endothelial cells derived from the pluripotent stem cells.

A new mouse model

To understand more about what was happening, Kooreman and his colleagues created a new mouse model similar to the humanized mice. Instead of reconstituting the animals nonexistent immune systems with human cells, however, they used immune and bone marrow cells from a different strain of mice. They then performed the same set of experiments again.

Unlike the humanized mice, these new mice robustly rejected human pluripotent stem cells as well as mouse stem cells from a genetically mismatched strain of mice. In other words, their newly acquired immune systems appeared to be in much better working order.

Although more research needs to be done to identify the cause of the discrepancy between the two types of animals, the researchers speculate it may have something to do with the complexity of the immune system and the need to further optimize the humanized mouse model to perhaps include other types of cells or signaling molecules. In the meantime, they are warning other researchers of potential pitfalls in using this model to screen for immunosuppressive drugs that could be effective after human stem cell transplants.

Many in the fields of pluripotent stem cell research and regenerative medicine are pushing the use of the humanized mice to study the human immune response, said Kooreman. But if we start to make claims using this model, assuming that these cells wont be rejected by patients, it could be worrisome. Our work clearly shows that, although there is some human immune cell activity, these animals dont fully reconstitute the human immune system.

The researchers are hopeful that recent advances may overcome some of the current models limitations.

The immune system is highly complex and there still remains much we need to learn, said Shultz. Each roadblock we identify will only serve as a landmark as we navigate the future. Already, weve seen recent improvements in humanized mouse models that foster enhancement of human immune function.

This article has been republished frommaterialsprovided byStanford Medicine. Note: material may have been edited for length and content. For further information, please contact the cited source.

Reference:

Kooreman, N. G., Almeida, P. E., Stack, J. P., Nelakanti, R. V., Diecke, S., Shao, N., . . . Wu, J. C. (2017). Alloimmune Responses of Humanized Mice to Human Pluripotent Stem Cell Therapeutics. Cell Reports, 20(8), 1978-1990. doi:10.1016/j.celrep.2017.08.003

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Mouse Model of Human Immune System Inadequate for Stem Cell ... - Technology Networks

Leah Nash/NYT/Redux/eyevine

Reproductive biologist Shoukhrat Mitalipov and his team used genome editing to correct a gene that causes a potentially fatal heart condition in humans.

An international team of researchers has used CRISPRCas9 gene editing a technique that allows scientists to make precise changes to genomes with relative ease to correct a disease-causing mutation in dozens of viable human embryos. The study represents a significant improvement in efficiency and accuracy over previous efforts.

The researchers targeted a mutation in a gene called MYBPC3. Such mutations cause the heart muscle to thicken a condition known as hypertrophic cardiomyopathy that is the leading cause of sudden death in young athletes. The mutation is dominant, meaning that a child need inherit only one copy of the mutated gene to experience its effects.

In the gene-editing experiment, published online today in Nature1, the embryos were not destined for implantation.

The team also tackled two safety hurdles that had clouded discussions about applying CRISPRCas9 to gene therapy in humans: the risk of making additional, unwanted genetic changes (called off-target mutations) and the risk of generating mosaics in which different cells in the embryo contain different genetic sequences. The researchers say that they have found no evidence of off-target genetic changes, and generated only a single mosaic in an experiment involving 58 embryos.

Several teams in China have already reported using CRISPRCas9 to alter disease-related genes in human embryos. Work is also under way in Sweden and the United Kingdom to use the technique to study the early stages of human embryo development. That research is aimed at understanding basic reproductive and developmental biology, as well as unpicking some of the causes of early miscarriages.

For the latest Nature paper, embryo experiments were conducted in the United States and led by Shoukhrat Mitalipov, a reproductive-biology specialist at the Oregon Health and Science University in Portland. The United States does not allow federal money to be used for research involving human embryos, but the work is not illegal if it is funded by private donors.

In February, an influential report by the US National Academics of Science, Engineering, and Medicine concluded that scientists should be allowed to use gene editing in human embryos for research. The report also said that, ultimately, it may be acceptable to use the technique to alter embryos destined for implantation, if the goal was to treat a devastating disease and if there were no other reasonable alternatives.

Mitalipovs team took several steps to improve the safety of the technique. The CRISPR system requires an enzyme called Cas9, which cuts the genome at a site targeted by an RNA guide molecule. Typically, researchers wishing to edit a genome will insert DNA encoding CRISPR components into cells, and then rely on the cells' machinery to generate the necessary proteins and RNA. But Mitalipovs team instead injected the Cas9 protein itself, bound to its guide RNA, directly into the cells.

Reporter Shamini Bundell investigates a new development in the gene editing of human embryos.

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Because the Cas9 protein degrades faster than the DNA that encodes it, the enzyme is left with less time to cut DNA, says genome engineer Jin-Soo Kim of the Institute for Basic Science in Daejeon, South Korea, and a co-author on the study. Cas9 is rapidly degraded, he says. There is little time for off-target mutations to accumulate.

Even so, Kim notes that the CRISPRCas9 error rate can vary depending on which DNA sequence is being targeted. The MYBPC3 mutation, in particular, was predicted to produce relatively few opportunities for off-target cutting.

Just because the team did not find off-target changes does not mean that the changes aren't there, cautions Keith Joung, who studies gene editing at the Massachusetts General Hospital in Boston. "Although this is likely the widest examination of off-target effects in genome-edited human embryos performed to date," he says, "these investigators would need to do much more work if they wanted to define with certainty whether off-target effects do or do not occur in this context."

The researchers also attempted to reduce the risk of mosaics by injecting the CRISPRCas9 components into the egg at the same time as they injected the sperm to fertilize it. This is earlier in development than previous human embryo editing experiments had tried2, and studies in mouse embryos have shown that the technique can eliminate mosaics when the fathers genome is targeted3.

In an experiment Mitalipov's group performed in 58 human embryos fertilized with sperm carrying the MYBPC3 mutation, 42 were successfully edited to contain two normal copies of the MYBPC3 gene. Only one was a mosaic. By comparison, the team found that 13 of 54 treated embryos were mosaics when the CRISPRCas9 machinery was injected 18 hours after fertilization.

The low rate of mosaics and the unusually high efficiency of gene editing make the study stand out, says stem-cell biologist Fredrik Lanner of the Karolinska Institute in Stockholm, who co-authored a commentary accompanying the article. Additional testing is needed to show that the low rate of mosaics holds true for other gene-editing targets, but for now, he says, "it's a huge step in that direction".

Lanner is also editing genes in human embryos, as a way of learning more about developmental biology. But he notes that in Sweden, it would be illegal for him to create embryos solely for the sake of research. Instead, he must use surplus embryos from fertility clinics (created using eggs that have already been fertilized), putting the kind of study that Mitalipov's team did in which CRISPRCas9 machinery is introduced at the same time as sperm out of reach.

The efficiency of gene editing in the Nature paper is exciting, says stem-cell biologist George Daley of Boston Childrens Hospital in Massachusetts. It puts a stake in the ground that this technology is likely to be operative, he says. But its still very premature.

Daley worries that the success reported in the paper could motivate a clinician to try the technique before it has been fully tested. He points to an experimental technique called mitochondrial replacement therapy, which aims to treat embryos for a disorder that disables energy-generating cell structures called mitochondria. Last September, news broke that a doctor had performed the technique in a fertility clinic in Mexico, even though many experts believed it was not yet ready for clinical practice. Since then, reports have rolled in of other clinicians performing the technique.

Developmental biologist Robin Lovell-Badge of the Francis Crick Institute in London shares those concerns. But he notes that worries about designer babies children who have been genetically enhanced, rather than merely correcting disease-causing mutations may be eased somewhat by the new paper. In their experiments, Mitalipovs team provided a strand of DNA to serve as a template for rewriting the disease-causing mutation. But, surprisingly, the embryos did not use the template the researchers provided. Instead, the embryos used the mothers DNA as a guide to repair the MYBPC3 mutation carried by the fathers sperm.

This isnt a clear step towards a designer baby, says Lovell-Badge. This suggests that you couldnt add anything that wasnt already there.

See the article here:
CRISPR fixes disease gene in viable human embryos - Nature.com

Yale University has teamed up with cancer biotech X4 Pharmaceuticals to work on its therapy for WHIM syndrome, a rare genetic disorder that plays havoc with the immune system but has no approved treatment.

The Cambridge, Massachusetts, biotechwhich specializes in drugs targeting the CXCR4 receptorsays it will help Yale investigate the molecular mechanisms behind WHIM syndrome, specifically the role that CXCR4 mutations may play in the disease. CXCR4 is the receptor for the chemokine CXCL12, and research has suggested that the interplay of the receptor and its ligand can downplay immune responses.

In patients with WHIM syndrome, reduced immunity makes them vulnerable to a range of symptoms, notablyas the acronym indicateswarts, hypogammaglobulinemia (low antibody levels), infections, and myelokathexis (a severe deficiency in white blood cells).

Some patients are treated off-label with GCSF drugs such as filgrastim to improve white blood cell counts, while antibiotics and infusions of immune globulin are used to try to reduce infections. Still, there are no drugs available to treat the underlying disease mechanism.

X4 was launched with financial backing from Genzyme founder Henri Termeer before his death earlier this year, and came out of stealth mode in 2015 around lead drug X4P-001, a CXCR4 inhibitor that is being tested in phase 1/2 trials in clear cell renal cell carcinoma (ccRCC), melanomaand other solid tumors.

The biotech has always had ambitions outside of cancer, however, and started a phase 2/3 trial of a low-dose formulation of X4P-001 in WHIM syndrome January, with the aim of enrolling 33 patients ages 13 or older with the disorder. It is hoping to complete the trial in August 2019.

Linking with Yale gives X4 the opportunity to collaborate with Joo Pedro Pereira, Ph.D., an associate professor of immunobiology who is involved with Yale's stem cell and cancer centers. His research focuses on the process that generates many different cell types including all immune cells, and its role in conferring immunity.

"The incorrect positioning of immune cells in primary and secondary immune organs due to CXCR4 mutations has been well documented," said Pereira.

"This research will elucidate the fundamental mechanisms that lead to chronic impairment of the immune system, particularly of long-term immunity, as a result of aberrant immune cell positioning and trafficking."

The number of patients with WHIM is hard to estimate, but X4 reckons that there could be several thousand people worldwide affected with the disorder.

Another group with WHIM syndrome in its sights is the National Institute of Allergy and Infectious Diseases (NIAID), which is running a study comparing Sanofi Genzyme's Mozobil (plerixafor) with filgrastim that is due to generate results in 2021.

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X4 joins hands with Yale on rare disease program - FierceBiotech

Inflammation, in moderation? Increased levels of TGF-1, secreted by astrocytes, may fuel the progression of ALS by blocking the ability of key immune cells to help heal injured motor neurons and promote their survival (see April 2015, May 2015 news). [Courtesy of Endo et al., 2015. Cell Reports.]

Now, a research team led by Harvard Universitys Qiao Zhou in Massachusetts report that reactive astrocytes may promote the destruction of motor neurons in co-culture by secreting TGF-1 (Tripathi et al., 2017). The study found that increased levels of TGF-1, produced by wild-type or SOD1 G93A reactive astrocytes isolated from mice, induced the cytoplasmic aggregation of key proteins, impaired autophagy and reduced the survival of human embryonic stem cell-derived motor neurons.

The study is published on June 29 in Stem Cell Reports.

The findings build on previous work led by Nagoya Universitys Koji Yamanaka in Japan, which found that the progression of ALS may be mediated by a TGF-1-mediated mechanism (see April 2015 news; Endo et al., 2015).

Together, the results suggest that reducing excess levels of TGF-1 may be a potential approach to slow progression of the disease.

References

Tripathi P, Rodriguez-Muela N, Klim JR, de Boer AS, Agrawal S, Sandoe J, Lopes CS, Ogliari KS, Williams LA, Shear M, Rubin LL, Eggan K, Zhou Q. Reactive Astrocytes Promote ALS-like Degeneration and Intracellular Protein Aggregation in Human Motor Neurons by Disrupting Autophagy through TGF-1. Stem Cell Reports. 2017 Jun 29. [PubMed].

Endo F, Komine O, Fujimori-Tonou N, Katsuno M, Jin S, Watanabe S, Sobue G, Dezawa M, Wyss-Coray T, Yamanaka K. Astrocyte-Derived TGF-1 Accelerates Disease Progression in ALS Mice by Interfering with the Neuroprotective Functions of Microglia and T Cells. Cell Rep. 2015 Apr 15 [PubMed].

Further Reading

Kunis G, Baruch K, Rosenzweig N, Kertser A, Miller O, Berkutzki T, Schwartz M.IFN--dependent activation of the brains choroid plexus for CNS immune surveillance and repair. Brain. 2013 Nov;136(Pt 11):3427-40. [PubMed].

Kunis G, Baruch K, Miller O, Schwartz M. Immunization with a Myelin-Derived Antigen Activates the Brains Choroid Plexus for Recruitment of Immunoregulatory Cells to the CNS and Attenuates Disease Progression in a Mouse Model of ALS. J Neurosci. 2015 Apr 22;35(16):6381-93. [PubMed].

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TGF-1: ALS Astrocytes' Secret Sauce? - ALS Research Forum