High-Quality Cartilage Production from Pluripotent Stem Cells


High-quality cartilage has been produced from pluripotent stem cells by workers in the laboratory of Sue Kimber and her team in the Faculty of Life Sciences at The University of Manchester. Such success might be used in the future to treat the painful joint condition osteoarthritis.

Kimber and her colleagues used strict laboratory conditions to grow and transform embryonic stem cells into cartilage cells known as chondrocytes.

Professor Kimber said: “This work represents an important step forward in treating cartilage damage by using embryonic stem cells to form new tissue, although it’s still in its early experimental stages.” Kimber’s research was published in Stem Cells Translational Medicine.

During the study, the team analyzed the ability of embryonic stems cells to become cartilage precursor cells. Kimber and her colleagues then implanted these pre-chrondrocytes into cartilage defects in the knee joints of rats. After four weeks, the damaged cartilage was partially repaired and following 12 weeks a smooth surface, which looked very similar to normal cartilage, was observed. More detailed studied of this newly regenerated cartilage demonstrated that cartilage cells from embryonic stem cells were still present and active within the tissue.

Developing and testing this protocol in rats is the first step in generating the information required to run such a study in people with arthritis. Before such a clinical trial can be run, more data will need to be collected in order to check that this protocol is effective and that there are no toxic side-effects.

However, Kimber and her coworkers say that this study is very promising as not only did this protocol generate new, healthy-looking cartilage but also importantly there were no signs of any side-effects such as growing abnormal or disorganized, joint tissue or tumors. Further work will build on this finding and demonstrate that this could be a safe and effective treatment for people with joint damage.

Chondrocytes created from adult stem cells are being used on an experimental basis, but, to date, they cannot be produced in large amounts, and the procedure is expensive.

With their huge capacity to proliferate, pluripotent stem cells such as embryonic stem cells and induced pluripotent stem cells can be manipulated to form almost any type of mature cell. Such cells offer the possibility of high-volume production of cartilage cells, and their use would also be cheaper and applicable to a greater number of arthritis patients, the researchers claim.

“We’ve shown that the protocol we’ve developed has strong potential for developing large numbers of chondrogenic cells appropriate for clinical use,” added Prof Kimber. “These results thus mark an important step forward in supporting further development toward clinical translation.”

Osteoarthritis affects more than eight million people in the UK alone, and is a major cause of disability. It and occurs when cartilage at the ends of bones wears away causing joint pain and stiffness.

Director of research at Arthritis Research UK Dr Stephen Simpson added: “Current treatments of osteoarthritis are restricted to relieving painful symptoms, with no effective therapies to delay or reverse cartilage degeneration. Joint replacements are successful in older patients but not young people, or athletes who’ve suffered a sports injury.

“Embryonic stem cells offer an alternative source of cartilage cells to adult stem cells, and we’re excited about the immense potential of Professor Kimber’s work and the impact it could have for people with osteoarthritis.”

New Gene Therapy for Hemophilia


According to a multi-year, ongoing study, a new kind of gene therapy for hemophilia B could be safe and effective for human patients.

“The result was stunning,” said Timothy Nichols, MD, director of the Francis Owen Blood Research Laboratory at the University of North Carolina School of Medicine and co-senior author of the paper. “Just a small amount of new factor IX necessary for proper clotting produced a major reduction in bleeding events. It was extraordinarily powerful.”

Nichols published his work in the journal Science Translational Medicine, in which he showed that a genetically engineered retrovirus could successfully transfer new factor IX (clotting) genes into animals with hemophilia B to dramatically decrease spontaneous bleeding. To date, the new therapy has proven safe.

A new FDA-approved hemophilia treatment lasts longer than a few days but patients still require injections indefinitely at least once or twice a month. This new gene therapy only requires hemophilia patients to receive a one-time dose of new clotting genes instead of a lifetime of multiple injections of recombinant factor IX. This new gene therapy approach would involve a single injection and could potentially save money and provide a long-term solution to a life-long condition. A major potential advantage of this new gene therapy approach is that it uses lentiviral vectors, to which most people do not have antibodies that would reject the vectors and make the therapy less effective.

In human clinical studies, approximately 40 percent of the potential participants with hemophilia have antibodies in their blood against adeno-associated virus (AAV), which precludes them from entering AAV trials for hemophilia gene therapy treatment. Thus more people could potentially benefit from the lentivirus gene therapy approach.

Hemophilia is a bleeding disorder in which people lack a clotting factor. Therefore they bleed much more easily than people without the disease. People with hemophilia often bleed spontaneously into joints, which can be extremely painful and crippling. Spontaneous bleeds into soft tissues are also common and can be fatal if not treated promptly. Hemophilia A affects about one in 5,000 male births. These patients do not produce enough factor VIII in the liver. This leads to an inability to clot. Hemophilia B affects about one in 35,000 births; these patients lack factor IX.

The new method detailed in the Science Translational Medicine paper was spearheaded by Luigi Naldini, PhD, director of the San Raffaele Telethon Institute for Gene Therapy. Naldini and Nichols developed a way to use a lentivirus, a large retrovirus, to deliver factor IX genes to the livers of three dogs that have a naturally occurring form of hemophilia. They removed the genes involved in viral replication. “Essentially, this molecular engineering rendered the virus inert,” Nichols said. “It had the ability to get into the body but not cause disease.” This process turned the virus into a vector – simply a vehicle to carry genetic cargo.

Unlike some other viral vectors that have been used for gene therapy experiments, the lentiviral vector is so large that it can carry a large payload – namely, the clotting factor IX genes that people with hemophilia B lack. (This approach could also be used for hemophilia A where the FVIII gene is considerably larger.)

These viral vectors were then injected directly into the liver or intravenously. After more than three years, the three dogs in the study experienced zero or one serious bleeding event each year. Before the therapy, the dogs experienced an average of five spontaneous bleeding events that required clinical treatment. Importantly, the researchers detected no harmful effects.

“This safety feature is of paramount importance,” Nichols said. “Prior work elsewhere during the early 2000s used retroviruses for gene therapy to treat people with Severe Combined Immunodeficiency, but some patients in clinical trials developed leukemia.” Newer retroviral vectors, though, have so far proved safe for SCID patients.

To further demonstrate the safety of this new hemophilia treatment, Nichols and Naldini used three different strains of mice that were highly susceptible to developing complications, such as malignancies, when injected with lentiviruses. Fortunately, Nichols, Naldini and their coworkers found no harmful effects in these mice. Thus manipulating lentiviruses and converting them into lentiviral vectors made them safe for gene therapy.

“Considering the mouse model data and the absence of detectible genotoxicity during long-term expression in the hemophilia B dogs, the lentiviral vectors have a very encouraging safety profile in this case,” Nichols said.

This gene therapy approach requires more work before it can be used in human trials. For instance, researchers hope to increase the potency of the therapy to decrease spontaneous bleeding even more while also keeping the therapy safe.

Before the treatment, the hemophilia dogs had no sign of factor IX production. After the treatment, they exhibited between 1 and 3 percent of the production found in normal dogs. This slight increase was enough to substantially decrease bleeding events.

Nichols wants to try to boost factor IX production to between 5 and 10 percent of normal while still remaining safe. This amount of factor IX expression could potentially eliminate spontaneous bleeding events for people with hemophilia B.

Umbilical Cord Blood Mesenchymal Stem Cells Relieve the Symptoms of Interstitial Cystitis by Activating the Wnt Pathway and EGF Receptor


Interstitial tissue refers to the tissue that lies between major structures in an organ. For example, the tissue between muscles is an example of interstitial tissue.

Interstitial cystitis, otherwise known as painful bladder syndrome is a chronic condition that causes bladder pressure, bladder pain and sometimes pelvic pain, ranging from mild discomfort to severe pain.

The bladder is a hollow, muscular organ that stores urine and expands until it is full, at which time it signals the brain that it is time to urinate, communicating through the pelvic nerves. This creates the urge to urinate for most people. In the case of interstitial cystitis, these signals get mixed up and you feel the need to urinate more often and with smaller volumes of urine than most people. Interstitial cystitis most often affects women and can have a long-lasting impact on quality of life. Unfortunately no treatment reliably eliminates interstitial cystitis, but medications and other therapies may offer relief. There is no sign of bacterial infection in the case of bacterial cystitis.

A new study evaluated the potential of umbilical cord blood-derived mesenchymal stem cells or (UCB-MSCs) to treat interstitial cystitis (IC). In this study, Dr. Miho Song and colleagues from the Asan Medical Center, Seoul, South Korea, established a rat model of IC in 10-weeks-old female Sprague-Dawley rats by instilling 0.1M HCl or PBS (sham). After 1-week, human UCB-MSCs (IC+MSCs) or PBS (IC) were directly injected into the submucosal layer of the bladder.

To clarify this part of the experiment, the urinary bladder is made of several distinct tissue layers: a) The innermost layer of the bladder is the mucosa layer that lines the hollow lumen. Unlike the mucosa of other hollow organs, the urinary bladder is lined with transitional epithelial tissue that is able to stretch significantly to accommodate large volumes of urine. The transitional epithelium also provides protection to the underlying tissues from acidic or alkaline urine; b) Surrounding the mucosal layer is the submucosa, a layer of connective tissue with blood vessels and nervous tissue that supports and controls the surrounding tissue layers; c) The visceral muscles of the muscularis layer surround the submucosa and provide the urinary bladder with its ability to expand and contract. The muscularis is commonly referred to as the detrusor muscle and contracts during urination to expel urine from the body. The muscularis also forms the internal urethral sphincter, a ring of muscle that surrounds the urethral opening and holds urine in the urinary bladder. During urination, the sphincter relaxes to allow urine to flow into the urethra.

Bladder histology

Now a single subcutaneous injection of human UCB-MSCs significantly attenuated the irregular and decreased voiding interval in the IC group. In addition, the denudation of the epithelium that is characteristic of IC and increased inflammatory responses, mast cell infiltration, neurofilament production, and angiogenesis observed in the IC bladders were prevented in the IC+MSC group. Therefore, the injected UBC-MSCs prevented the structural changes in the bladder associated with the pathology of IC.

How did these cells do this? Further examination showed that the injected UCB-MSCs successfully engrafted to the stromal and epithelial tissues of the bladder and activated the Wnt signaling cascade. In fact, if the Wnt activity of these infused cells was blocked, the positive effects of the UCB-MSCs were also partially blocked. Additionally, activation of the epidermal growth factor receptor (EGFR) also helped UCB-MSCs heal the bladder. If the activity of the EGF receptor was inhibited by small molecules, then the benefits of MSC therapy were also abrogated. Also if both the Wnt pathway and EGFR were inhibited, the therapeutic capacities of UCB-MSCs were completely wiped out.

These data show the therapeutic effects of MSC therapy against IC in an orthodox rat animal model. However, this study also elucidates the molecular mechanisms responsible for these therapeutic effects. Our findings not only provide the basis for clinical trials of MSC therapy to IC, but also advance our understanding of IC pathophysiology.

Stem Cells Lurk in Tumors and Can Resist Treatment


Regenerative medicine seeks to train stem cells to transform into nearly any kind of cell type. Unfortunately, this ability that makes stem cells so useful also is cause for concern in cancer treatments. Malignant tumors contain resident stem cells, which prompts worries among cancer experts that the cells’ transformative powers help cancers escape treatment.

Data from new research shows that the threat posed by cancer stem cells is more prevalent than previously thought. Until now, stem cells had been identified only in aggressive, fast-growing tumors. However, a mouse study at Washington University School of Medicine in St. Louis has revealed that slow-growing tumors also have treatment-resistant stem cells.

Brain tumor stem cells (orange) in mice express a stem cell marker (green). Researchers at Washington University School of Medicine in St. Louis are studying how cancer stem cells make tumors harder to kill and are looking for ways to eradicate these treatment-resistant cells. Credit: Yi-Hsien Chen
Brain tumor stem cells (orange) in mice express a stem cell marker (green). Researchers at Washington University School of Medicine in St. Louis are studying how cancer stem cells make tumors harder to kill and are looking for ways to eradicate these treatment-resistant cells. Credit: Yi-Hsien Chen

In mice, low-grade brain cancer stem cells were less sensitive to anticancer drugs. When compared to healthy stem cells, tumor-based stem cells from brain tumors, revealed the reasons behind their resistance to treatments, which points to new therapeutic strategies.

“At the very least, we’re going to have to use different drugs and different, likely higher dosages to make sure we kill these tumor stem cells,” said senior author David H. Gutmann, MD, PhD, the Donald O. Schnuck Family Professor of Neurology.  Their data were published in the March 12 edition of Cell Reports.

First author Yi-Hsien Chen, who is a senior postdoctoral research associate in Gutmann’s laboratory, used a mouse model of neurofibromatosis type 1 (NF1), which forms low-grade brain tumors, to identify cancer stem cells and demonstrate that they could form tumors when transplanted into normal, cancer-free mice.

Neurofibromatosis type I is caused by mutations in the NF1 genes, and such mutations affect about 1 in every 2,500 babies. Neurofibromatosis type I can cause an array of physical problems, including brain tumors, impaired vision, learning disabilities, behavioral problems, heart defects and bone deformities.

In children with NF1 mutations, the most common brain tumor is optic gliomas. Treatment for NF1-related optic gliomas usually includes drugs that inhibit a cell growth pathway originally identified by Gutmann. In laboratory tests conducted as part of the new research, it took 10 times the dosage of these drugs to kill the low-grade cancer stem cells.

Compared with healthy stem cells from the brain, cancer stem cells made multiple copies of a protein called Abcg1 that helps those cells survive stress.

“This protein blocks a signal from inside the cells that should make them more vulnerable to treatment,” Gutmann explained. “If we can identify a drug that disables this protein, it would make some cancer stem cells easier to kill.”

Even though these laboratory mice were bred to model NF1 optic gliomas, Gutmann and others said that their findings could be applied more broadly to other brain tumors.

“Because stem cells haven’t differentiated into specialized cells, they can easily activate genes to turn on new developmental programs that allow the cells to survive cancer treatments,” said Gutmann, who directs the Washington University Neurofibromatosis Center. “Based on these new findings, we will have to develop additional strategies to keep these tumors from evading our best treatments.”

Bone Marrow Stem Cell Treatment Plus Immunosuppression are Superior to Immunosuppression Alone in Multiple Sclerosis Patients


Multiple Sclerosis (MS) is a debilitating autoimmune disease in which the immune system attacks elements of the central nervous system. There are different types of MS, but more progressive cases can leave patients unable to walk and may require rather extreme immunosuppressive treatments that can predispose a patient to illness and cancer.

However, a new study that was published in the journal Neurology has shown that stem cell transplantation could be a more effective therapy in severe cases of multiple sclerosis (MS) than the drug mitoxantrone.

Mitoxanthone is a “type II topoisomerase inhibitor” that disrupts DNA synthesis and DNA repair by inserting between the bases in DNA. Mitoxanthone can cause nausea, vomiting, hair loss, heart damage, and suppression of the immune system. Some side effects may have delayed onset. Heart damage (cardiomyopathy) is a particularly concerning effect with this drug, since it is irreversible. Therefore, because of the risk of cardiomyopathy, mitoxantrone carries a limit on the cumulative lifetime dose, which is based on the body surface area of patients.

Mitoxantrone
Mitoxantrone

Because MS is an immune-mediated disorder, and because immune cells are made by stem cells in the bone marrow, bone marrow transplants (hematopoietic stem cell transplantation), which are routinely used in the treatment of leukemia and lymphoma, are being considered as a treatment for MS.

A clinical trial conducted by Giovanni Mancardi from the University of Genova, Italy designed a randomized phase II clinical trial study that included 21 MS patients, whose average age was 36 and whose disability due to the disease had worsened in the previous year despite the fact that the patients were under conventional medication treatment. The average disability level of the participants was represented by the need of a crutch or cane to walk. The goal of the study was to determine the efficacy of intense immunosuppression followed by either a bone marrow transplant with the patient’s own bone marrow, or mitoxantrone (MTX) in MS disease activity.

Giovanni Mancardi
Giovanni Mancardi

All participants in this clinical trial received immune-suppressive medication. MTX was given to 12 of the patients while the remaining 9 received hematopoietic stem cells harvested from their own bone marrow. After treatment with MTX, the stem cells were intravenously reintroduced into their donors and the stem cells migrated back to the bone marrow where they generated new immune cells. All participants were followed-up for a period of up to four years after their treatment.

“This process appears to reset the immune system,” said the lead study author Dr. Giovanni Mancardi. “With these results, we can speculate that stem cell treatment may profoundly affect the course of the disease.”

Mancardi and his team found that treatment of MS patients with robust immunosuppression followed by stem cell treatment resulted in a significantly higher decrease in disease progression in comparison with MTX treatment alone. MS patients under stem cell treatment reduced the number of new areas of brain damage (T2 lesions) by 79% compared to patients under MTX treatment. Another type of lesion seen in MS patients – gadolinium-enhancing lesions – were not detected in patients under stem cell treatment during the study, whereas 56% of patients receiving MTX exhibited at least one new gadolinium-enhancing lesion.

Mancardi and his team concluded that an intense immunosuppression followed by autologous hematopoietic stem cell transplantation is more efficient than MTX to reduce MS activity in severe cases.

“More research is needed with larger numbers of patients who are randomized to receive either the stem cell transplant or an approved therapy, but it’s very exciting to see that this treatment may be so superior to a current treatment for people with severe MS that is not responding well to standard treatments,” concluded study author Dr. Mancardi.

Gene Discoveered That Drives Fertility in Male Mice


Workers in the laboratory of Richard Freiman, associate professor of medical science at Brown University have discovered a specific gene in human males that seems to be essential to sperm production later in life.

A paper published in the journal Stem Cells details how the loss of a protein called TAF4b in male mice causes premature infertility. According to Freiman, mutations that prevent the continuous production of TAF4b leave mice incapable of sustaining spermatogenesis after only a few months of sexual maturity.

This study began when Freiman’s team discovered that TAF4b was expressed at high levels in the ovaries and the testes. Later, Freiman and his colleagues used homologous recombination to specifically modify the TAF4b gene. Freiman explained that homologous recombination can “knock specific genes out of the mouse genome,” after which you can “examine the mice that are born and see what function the gene serves in normal development.” Such experiments showed that male mice whose TAF4b gene was synthetically modified so that it expressed TAF4b initially, but did not sustain its expression were only fertile for a month or two, whereas mice with intact TAF4b remained fertile for several years.

“Cells that are involved in initial fertility are different than cells involved in subsequent rounds of sperm production. The first set undergoes meiosis and become sperm by a direct route, but the other set develops into precursor cells that become stem cells,” Freiman said. “What we hypothesized about our mice is that they’re able to go through this initial round of spermatogenesis, but they can’t make the stem cell population, so they can’t set themselves up for long-term fertility,” he added.

Since humans have a TAF4b gene that is very similar to the mouse gene, the results of Freiman’s laboratory might be applicable to human fertility, said Eric Gustafson, a postdoctoral research fellow in Freiman’s laboratory and first author of the paper.

These results interact with another study that was published last year that examined a population of four infertile brothers in eastern Turkey; each of whom each had a homozygous mutation in their TAF4b gene similar to the one created in the mice. According to Gustafson, these men had very low or no sperm counts. “So we think these genes have many similar, if not identical functions in humans. What we learn about in the mouse gene may be used to address or diagnose reproductive defects in humans as well,” he added.

With couples having children later and later in life, this study has important implications for family planning. “If we could learn how this process is regulated normally, clinicians might be able to devise better strategies to either monitor or even intervene with cases of infertility,” said Freiman. To give an example, Freiman suggested that if scientists could detect the mutation in teenage boys early on, then doctors could freeze their patients’ sperm for later in life.

The study also has important outcomes in terms of stem cell research, said Professor of Biology Gary Wessel, who was not involved in the study. “This research shows us that this particular transcription factor, TAF4b, is involved in the transcription process involved in maintaining the stem cell itself,” Wessel said. “As a consequence, it now gives the investigators a more careful view of what stem cell decisions are like,” he added.

The study’s results are relevant to all stem cell research, Wessel said. “Everything in biology is connected. If you make any kind of breakthrough, it’s going to have ripple effects throughout the entire discipline.”

How does TAF4b affect fertility? That’s the next goal of Freiman’s research. Freiman said. “We now know that TAF4b performs this function, but we don’t know how it does it,” he added. “Once we figure that out, it might reveal new areas of intervention for fertility preservation.”

NG2-Expressing Neural Lineage Cells Derived from Embryonic Stem Cells Penetrate Glial Scar and Promote Axonal Outgrowth After Spinal Cord Injury


After a spinal cord injury, resident stem cells in the spinal cord contribute to the production of a glial scar that is rich in chondroitin sulfate proteoglycan (CSPG). The glial scar is a formidable barrier to axonal regeneration in the injured spinal cord, since CSPG actively repels growing axonal growth cones. Even though the glial scar seals off the spinal cord from further damage from inflammation, the long-term effects of the glial scar are to prevent regeneration of spinal nerves, which have the ability to regenerate in culture.

The major components of the site of injury include myelin debris, the scar-forming astrocytes, activated resident microglia and infiltrating blood-borne immune cells, chondroitin sulfate proteoglycans (CSPGs) and other growth-inhibitory matrix components. All of them are potential targets for therapeutic intervention. Many of the interventions can be optimized by considering the beneficial aspects of the scar tissue and fine-tuning the optimal time window for their application. Each target and the strategies directed at its modulation are shown.
The major components of the site of injury include myelin debris, the scar-forming astrocytes, activated resident microglia and infiltrating blood-borne immune cells, chondroitin sulfate proteoglycans (CSPGs) and other growth-inhibitory matrix components. All of them are potential targets for therapeutic intervention. Many of the interventions can be optimized by considering the beneficial aspects of the scar tissue and fine-tuning the optimal time window for their application. Each target and the strategies directed at its modulation are shown.

New work by Sudhakar Vadivelu, in the laboratory of John McDonald at the International Center for Spinal Cord Injury, Hugo W. Moser Research Institute at the Kennedy Krieger Institute, Baltimore, Maryland has discovered new ways to breach the glial scar. Vadivelu and colleagues used a cell culture system that tested the ability of particular cells to help growing axonal growth cones penetrate glial scar material. This culture system showed that embryonic stem cell-derived neural lineage cells (ESNLCs) with prominent expression of nerve glial antigen 2 (NG2) survived, and passed through an increasingly inhibitory gradient of CSPG. These cells also expressed matrix metalloproteinase 9 (MMP-9) at the appropriate stage of their development, which helped poke holes in the CSPG. The outgrowth of axons from ESNLCs followed the NG2-expressing cells because the migrating cells chiseled pathways through the CSPG for the outgrowth of new axons.

To confirm these results in a living animal, Vadivelu and others transplanted embryonic stem cell-derived ESNLCs directly into the cavities of a contused spinal cord of laboratory animals 9 days after injury. One week later, implanted ESNLCs survived and expressed NG2 and MMP-9. The axons of these neurons had grown through long distances (>10 mm), although they preferred to grow across white rather than gray matter.

These data are consistent with CSPG within the injury scar acting as an important impediment to neuronal regeneration, but that NG2+ progenitors derived from ESNLCs can alter the microenvironment within the injured spinal cord to allow axons to grow through such a barrier. This beneficial action seems to be due, in part, at least, to the developmentally-regulated expression of MMP-9. Vadivelu and others conclude from these data that it might be possible to induce axonal regeneration in the human spinal cord by transplanting ESNLCs or other cells that express NG2.