New Neuron Formation Required for Maintenance of Olfactory Nerves in Mice

For many years, scientists and neurologists were convinced that neurons in the brain only formed during early development, and after that it was simply impossible for new neurons to be formed.  More recent work, however, has shown this to be largely untrue, since several regions of the brain possess resident stem cell populations that can divide to replenished damaged neurons and even augment learning and memory.  The capacity of neural stem cell populations to regenerate the central nervous system is a continuing field of intense research, and scientists at the National Institutes of Health (NIH) have reported one region of the central nervous system that can form new brain cells; the mouse olfactory system, which processes smells.  This work appeared in the October 8 issue of the Journal of Neuroscience.

“This is a surprising new role for brain stem cells and changes the way we view them,” said Leonardo Belluscio, Ph.D., a scientist at NIH’s National Institute of Neurological Disorders and Stroke (NINDS) and lead author of the study.

The olfactory bulb is at the front of the brain (shown as “A” in he picture below), and is rather small in humans, but somewhat larger in other animals.  This structure receives information directly from the nose about volatile odors.  Neurons in the olfactory bulb sort through this smelly information and relay neural signals to the rest of the brain.  This is the point at which we become aware of the smells in our surroundings.  The loss of the sense of smell is sometimes an early symptom in a variety of neurological disorders, including Alzheimer’s and Parkinson’s diseases.

 

Neurogenesis is the process by which neuroprogenitor cells are produced in the subventricular zone deep in the brain.  After birth, these cells migrate to the olfactory bulb, which becomes the final location of these cells.  Once they arrive at the olfactory bulb. the neuroprogenitor cells divide, differentiate, and form connections with existing cells to become integrated into the neural circuitry in the olfactory bulb and elsewhere.

Dr. Belluscio studies the olfactory system, and for this study, he collaborated with Heather Cameron, Ph.D., a neurogenesis researcher at the NIH’s National Institute of Mental Health.  The goal of this study was to better understand how the continuous addition of new neurons affects the neural organization of the olfactory bulb.  They used two different types of genetically engineered laboratory mice that had specifically genes knocked out.  Consequently, these mice lacked the specific stem cell populations that generate the new neurons during adulthood, without affecting the other olfactory bulb cells.  Previously, this remarkable level of specificity had not been achieved.

Belluscio and his coworkers had previously shown that plugging the nostrils of the animals so that they are not subject to olfactory stimulation causes the axonal extensions of the olfactory neurons to dramatically spread out and lose the precise network of connections with other cells that are normally observed under normal conditions.  They also showed that this widespread disrupted circuitry could re-organize itself and restore its original precision once the sensory deprivation was reversed.  Therefore, Belluscio and his team temporarily plugged a nostril in their lab animals to block olfactory sensory information from entering the brain.  However, if laboratory animals that do not produce new neuroprogenitors are subjected to this type of manipulation, once the nose is unblocked, new neurons are prevented from forming and entering the olfactory bulb, and, therefore, the neural circuits remain in disarray. “We found that without the introduction of the new neurons, the system could not recover from its disrupted state,” said Dr. Belluscio.

Further examination showed that elimination of the formation of adult-born neurons in mice that did not experience sensory deprivation also caused the organization of the olfactory bulb organization began to degenerate, eventually resembling the pattern observed in animals prevented from receiving sensory information from the nose.  Belluscio and his team also noticed that the extent of stem cell loss was directly proportional to the degree of disorganization in the olfactory bulb.

According to Belluscio, circuits of the adult brain are thought to be rather stable and that introducing new neurons alters the existing circuitry, causing it to re-organize. “However, in this case, the circuitry appears to be inherently unstable requiring a constant supply of new neurons not only to recover its organization following disruption but also to maintain or stabilize its mature structure. It’s actually quite amazing that despite the continuous replacement of cells within this olfactory bulb circuit, under normal circumstances its organization does not change,” he said.

Dr. Belluscio and his colleagues think that these new neurons in the olfactory bulb are important for the maintenance of activity-dependent changes in the brain, which help animals adapt to a constantly varying environment.

“It’s very exciting to find that new neurons affect the precise connections between neurons in the olfactory bulb. Because new neurons throughout the brain share many features, it seems likely that neurogenesis in other regions, such as the hippocampus, which is involved in memory, also produce similar changes in connectivity,” said Dr. Cameron.

The underlying basis of the connection between neurological disease and changes in the olfactory system is also unknown but may come from a better understanding of how the sense of smell works. “This is an exciting area of science,” said Dr. Belluscio, “I believe the olfactory system is very sensitive to changes in neural activity and given its connection to other brain regions, it could lend insight into the relationship between olfactory loss and many brain disorders.”

New Standard of Care for Umbilical Cord Blood Transplants

New research led by John Wagner, Jr., M.D., director of the Pediatric Blood and Marrow Transplantation program at the University of Minnesota and a researcher in the Masonic Cancer Center, University of Minnesota, has established a new standard of care for children who suffer from acute myeloid leukemia (AML).

In a recent paper in the New England Journal of Medicine, Wagner and his coworkers compared clinical outcomes in children who suffered from acute leukemia and myelodysplastic syndrome who received transplants of either one unit or two units of partially matched cord blood. This large study was conducted at multiple sites across the United States, between December 2006 and February 2012. Coordinating the study was the Blood and Marrow Transplant Clinical Trials Network (BMT CTN) in collaboration with the Pediatric Blood and Marrow Transplant Consortium and the Children’s Oncology Group.

Umbilical cord blood provides a wonderfully rich source of blood-forming stem cells, and has been demonstrated to benefit many diseases of the blood or bone marrow, as in the case of leukemia and myelodysplasia, or bone marrow failure syndromes, hemoglobinopathies, inherited immune deficiencies and certain metabolic diseases. For leukemia patients cord blood offers several advantages since there is no need for strict tissue-type matching (human leukocyte antigen or HLA matching) or for a prolonged search for a suitable donor.

Wagner and others discovered that the survival rates of children who received either one or two units of umbilical cord blood were about the same, but, overall, their recorded survival rates were better than those reported in prior published reports. These higher survival rates, therefore, represent a new standard of care for pediatric patients, for whom there is often an adequate single cord blood unit, and for adults for usually require double units, since a single unit with an adequate number of blood-forming stem cells simply may not exist at time.

“Based on promising early studies using two cord blood units in adults for whom one unit is often not sufficient, we designed this study in order to determine if the higher number of blood forming stem cells in two cord blood units might improve survival,” explained Wagner. “What we found, however, was that both treatment arms performed very well with similar rates of white blood cell recovery and survival.”

Children with blood cancers who receive transfusions of umbilical cord blood show quantifiable clinical benefits even though the blood may not match their own tissue types. The reason stems from the immaturity of cord blood stem cells and their ability to suppress rejection from the immune system. This is an important aspect of umbilical cord blood transplants, since patients who cannot find a matched unrelated donor also benefit from cord blood transplants. However, cord collection from the placenta after birth often results in a limited number of blood-forming stem cells, which decreases the potential benefits of cord blood. The “double UCB approach” was pioneered at the University of Minnesota as a strategy to overcome this inherent limitation in the use of umbilical cord blood.

Despite the similarities in survival rates between children who received one unit or two units of cord blood, some differences were noted. Children transplanted with a single cord unit had faster recovery rates for platelets and lower risks of Graft Versus Host Disease; a condition in which the transplanted donor blood immune cells attack the patient’s body, which causes several complications.

“This is helpful news for physicians considering the best treatment options for their patients,” said Joanne Kurtzberg, M.D., chief scientific officer of the Robertson Clinical and Translational Cell Therapy Program, director of the Pediatric Blood and Marrow Transplant Program, co-director of the Stem Cell Laboratory and director of the Carolinas Cord Blood Bank at Duke University Medical Center. “We found children who have a cord blood unit with an adequate number of cells do not benefit from receiving two units. This reduces the cost of a cord blood transplant for the majority of pediatric patients needing the procedure. However, for larger children without an adequately dosed single cord blood unit, using two units will provide access to a potentially life-saving transplant.”

“The involvement of multiple research partners was instrumental to the success of the study completion,” added Dennis Confer, M.D., chief medical officer for the National Marrow Donor Program® (NMDP)/Be The Match® and associate scientific director for CIBMTR. “This trial is a testament to the importance of the BMT CTN and the collaboration of partners like the Children’s Oncology Group.”

Mary Horowitz, M.D., M.S., chief scientific director of CIBMTR and professor of medicine at the Medical College of Wisconsin, concurred. “Because of this tremendous collaboration, we were able to expand the scale of this research to multiple transplant centers across the United States and Canada. And the results will undoubtedly improve clinical practice, and most importantly, patient outcomes.”

Interestingly, in this study, patients who received cord blood with significant HLA mismatches showed no detrimental effects on their outcomes. Future studies will examine a closer look at how the HLA match within the cord blood unit impacts outcomes for patients, particularly those within minority populations.

Small Human Stomach Organoids Made From Induced Pluripotent Stem Cells

A new study published in the international journal Nature describes, for the first time, the use of human pluripotent stem cells to create a three-dimensional stomach-like mini-organ. This is the beginning of what might become an unprecedented tool for examining the genesis of diseases such as stomach cancer to diabetes.

Jim Wells and his colleagues at Cincinnati Children’s Hospital Medical Center used human pluripotent stem cells, which are made from mature human cells through a combination of genetic engineering and cell culture techniques, to grow a their miniature stomachs. Wells’ group then used their mini-stomachs also known as gastric organoids, in collaboration with scientists from University of Cincinnati College of Medicine, to study the infection of stomach tissue by the bacterium Helicobacter pylori, which causes peptic ulcer disease and stomach cancer.

According to Wells, a scientist in the divisions of Developmental Biology and Endocrinology at Cincinnati Children’s, this is the first time anyone has succeeded in making three-dimensional human gastric organoids (hGOs). This achievement may present new opportunities for drug discovery, modeling early stages of stomach cancer and studying some of the factors that give rise to obesity related diabetes. This work also represents the first time researchers have produced three-dimensional human embryonic foregut, which is a good starting point for generating other foregut organ tissues such as the lungs and pancreas. “Until this study, no one had generated gastric cells from human pluripotent stem cells (hPSCs),” Wells said. “In addition, we discovered how to promote formation of three-dimensional gastric tissue with complex architecture and cellular composition.”

a, Schematic representation of a typical antral gland showing normal cell types and associated molecular markers. b–g, Immunofluorescent staining demonstrated that day-34 hGOs consisted of normal cell types found in the antrum, but not the fundus. The hGO epithelium contained surface mucous cells that express MUC5AC (b, left), similar to the P12 mouse antrum (b, right), but not ATP4B-expressing parietal cells (c, left) that characterize the fundus (c, right). SOX9+ cells were found at the base of the hGO epithelium (d, left), similar to the progenitor cells found in the P12 antrum (d, right). Furthermore, hGOs contained MUC6+ antral gland cells (e) and LGR5-expressing cells (yellow arrow) (f). Boxed regions in b–f are shown as high magnification images below (b, c, d) or to the right (e, f) of the original. g, Day-34 hGOs also contained endocrine cells (SYP) that expressed the gastric hormones GAST, SST, GHRL and serotonin (5-HT). Scale bars, 100 μm (original images in b–f) and 20 μm (magnified images in b–f and g). Marker expression data are representative from a minimum of 10 independent experiments, except LGR5-eGFP data, which is a representative example from two separate experiments. DAPI, 4′,6-diamidine-2-phenylindole.

Wells’ gastric organoids are a significant advance in gastroenterological research because distinct differences in development and architecture of the adult stomach limit the reliability of mouse models for studying human stomach development and disease.

As a research tool, human gastric organoids may help clarify other unknown features of the stomach, such as identifying those biochemical processes in the gut that allow gastric-bypass patients to become diabetes-free soon after surgery before losing significant weight. Medical conditions such as obesity-fueled diabetes and the metabolic syndrome are of great interest to public health workers, given the explosion of global cases in the last few decades. A major challenge to addressing these and other medical conditions that involve the stomach has been a relative lack of reliable laboratory model systems to accurately recapitulate human biology.

The key to growing human gastric organoids was to identify the developmental steps involved in normal stomach formation. Manipulation of these processes in a cell culture system drove human pluripotent stem cells to form immature stomach tissue. In culture and over the course of a month, these steps resulted in the formation of 3D human gastric organoids that were around 3mm (1/10th of an inch) in diameter. Wells and his colleagues also used this approach to identify steps that go awry when the stomach does not form correctly.

In collaboration with his colleagues, Kyle McCracken, an MD/PhD graduate student in Wells’ laboratory, and Yana Zavros, PhD, a researcher at UC’s Department of Molecular and Cellular Physiology, Wells showed that his gastric organoids were rapidly infected by H. pylori bacteria. Within 24 hours of inoculation, the bacteria had triggered significant biochemical changes to the organ, and the human gastric organoids faithfully mimicked the early stages of H. pylori-induced gastric disease. McCracken also noticed activation of a cancer gene called c-Met, which is one of the first stages in the induction of stomach cancer, an important long-term sequel to peptic ulcer disease. McCracken was also surprised by the rapid spread of infection in the tissues of his human gastric organoids.

a, Day-34 hGOs contained a zone of MKI67+ proliferative cells similar to the embryonic (E18.5) and postnatal (P12) mouse antrum. b, Using hGOs to model human-specific disease processes of H. pylori infection. Pathogenic (G27) and attenuated (ΔCagA) bacteria were microinjected into the lumen of hGOs and after 24 h, bacteria (both G27 and ΔCagA strains) were tightly associated with the apical surface of the hGO epithelium. c, Immunoprecipitation (IP) for the oncogene c-Met demonstrates that H. pylori induced a robust activation (tyrosine phosphorylation (pTyr)) of c-Met, and this is a CagA-dependent process. Furthermore, CagA immunoprecipitated with c-Met, suggesting that these proteins interact in hGO epithelial cells. Phosphorylated c-Met (phos. c-MET) and CagA control lysates were not immunoprecipitated but used to confirm molecular masses. The molecular mass markers are indicated (130 and 170 kilodaltons (kDa)) and shown in Extended Data Fig. 9c. IB, immunoblotting. d, Within 24 h, H. pylori infection caused a CagA-dependent twofold increase in the number of proliferating cells in the hGO epithelium, measured by 5-ethynyl-2′-deoxyuridine (EdU) incorporation. *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per condition, data representative of 4 independent experiments. Scale bars, 100 μm (a) and 20 μm (b). Error bars represent s.e.m.

There is a relative dearth of literature on how the human stomach developments, which was a significant impediment to Wells’ research. Wells and his coworkers had to use a combination of published works and studies from his own lab, to answer a number of basic developmental questions about how the stomach forms. Over the course of two years, by experimenting with different factors to drive the formation of the stomach, Wells and his colleagues came upon a protocol that resulted in the formation of 3D human gastric tissues in culture.

a, Schematic representation of the in vitro culture system used to direct the differentiation of pluripotent stem cells into three-dimensional gastric organoids. b, Defining molecular domains of the posterior foregut in E10.5 mouse embryos with Sox2, Pdx1 and Cdx2; Sox2/Pdx1, antrum (a); Sox2, fundus (f); Pdx1, dorsal and ventral pancreas (dp and vp); Pdx1/Cdx2, duodenum (d). c, Posterior foregut spheroids exposed for three days to retinoic acid (2 μM) exhibited >100-fold induction of PDX1 compared to control spheroids, measured by qPCR at day 9. *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per condition, data representative of 4 independent experiments. d, Time course qPCR analysis of antral differentiation (according to protocol detailed in Fig. 2a) demonstrated sequential activation of SOX2 at day 6 (posterior foregut (FG) endoderm), followed by induction of PDX1 at day 9 (presumptive antrum). Day-9 antral spheroids had a 500-fold increase in SOX2 and a 10,000-fold increase in PDX1 relative to day-3 definitive endoderm (DE). *P < 0.05; two-tailed Student’s t-test; n = 3 biological replicates per time point, data representative of 2 independent experiments. The pancreatic marker PTF1A was not significantly increased. e, Stereomicrographs showing morphological changes during growth of gastric organoids. By 4 weeks, the epithelium of hGOs exhibited a complex folded and glandular architecture (arrows). D, day. f, Comparison of mouse stomach at E18.5 and day-34 hGOs. Pdx1 was highly expressed in the mouse antrum but excluded from the fundus. Human gastric organoids expressed PDX1 throughout the epithelium and exhibited morphology similar to the late gestational mouse antrum (arrows). Scale bars, 100 μm (b and f) and 250 µm (e). Error bars represent s.d.

Wells emphasized importance of basic research for the eventual success of this project, adding, “This milestone would not have been possible if it hadn’t been for previous studies from many other basic researchers on understanding embryonic organ development.”

While this does represent a terrific stride toward better model systems for gastric research and pathology, these gastric organoids are very immature and lack several of the cell types found in mature stomach tissue. For example, these organoids lack chief cells, which secrete the stomach enzyme pepsin (in an inactive form called pepsinogen), and parietal cells, which produce stomach acid. This is significant because chronic inflammation of the stomach can cause loss of parietal cells, which decreases chief cell differentiation and induce chief cells to transdifferentiate back into neck cells. This leads to overproduction of mucus cells. This mucus cell metaplasia is known as spasmolytic polypeptide expressing metaplasia (SPEM) that seems to be a precancerous condition for the stomach. Also if parietal cells are lost, mature chief cells do not form. This seems to imply that parietal cells secrete factors that lead to differentiation of chief cells, so if lost. These gastric organoids also do not make ECL cells or enterochromaffin-like cells, which secrete histamine, one of the most important regulators of stomach acid production. A prolonged stimulation of these ECL cells causes increased numbers of them. This is especially important in gastrinomas, which are tumors in which there is an excessive secretion of the stomach hormone gastrin, one of the key factors contributing to Zollinger-Ellison syndrome.  The hallmark of this disease is ulceration of the stomach and upper small intestine (duodenum) as a result of excessive and unregulated secretion of gastric acid.  Most commonly, hypergastrinemia is the result of these gastrin-secreting tumors or gastrinomas that develop in the pancreas or duodenum.  Thus, in only this short discussion, we have noted several diseases of the stomach that cannot be modeled with this particular system because these stomach-specific cells are not present.

Therefore, while this is a fantastic model system for stomach development and H. pylori infection, more work remains in order to make a stomach model that more accurately models the adult stomach.

“In Body” Muscle Regeneration

Researchers at Wake Forest Baptist Medical Center’s Institute for Regenerative Medicine have hit upon a new strategy for tissue healing: mobilizing the body’s stem cells to the site of injury. Thus harnessing the body’s natural healing powers might make “in body” regeneration of muscle tissue is a possibility.

Sang Jin Lee, assistant professor of Medicine at Wake Forest, and his colleagues implanted small bits of biomaterial scaffolds into the legs of rats and mice. When they embedded these scaffolds with proteins that mobilize muscle stem cells (like insulin-like growth factor-1 or IGF-1), the stem cells migrated from the muscles to the bioscaffolds and formed muscle tissue.

“Working to leverage the body’s own regenerative properties, we designed a muscle-specific scaffolding system that can actively participate in functional tissue regeneration,” said Lee. “This is a proof-of-concept study that we hope can one day be applied to human patients.”

If patients have large sections of muscle removed because of infections, tumors or accidents, muscle grafts from other parts of the body are typically used to restore at least some of the missing muscle. Several laboratories are trying the grow muscle in the laboratory from muscle biopsies that can be then transplanted back into the patient. Growing muscle on scaffolds fashioned from biomaterials have also proven successful.

Lee’s technique overcomes some of the short-comings of these aforementioned procedures. As Lee put it, “Our aim was to bypass the challenges of both of these techniques and to demonstrate the mobilization of muscle cells to a target-specific site for muscle regeneration.”

Most tissues in our bodies contain a resident stem cell population that serves to regenerate the tissue as needed. Lee and his colleagues wanted to determine if these resident stem cells could be coaxed to move from the tissue or origin, muscle in this case, and embeds themselves in an implanted scaffold.

In their first experiments, Lee and his team implanted scaffolds into the leg muscles of rats. After retrieving them several weeks later, it was clear that the muscle stem cell population (muscle satellite cells) not only migrated into the scaffold, but other stem cell populations had also taken up residence in the scaffolds. These scaffolds were also contained an interspersed network of blood vessels only 4 weeks aster transplantation.

In their next experiments, Lee and others laced the scaffolds with different cocktails of proteins to boost the stem cell recruitment properties of the implanted scaffolds. The protein that showed the most robust stem cell recruitment ability was IGF-1. In fact, IGF-1-laced scaffolds had four times the number of cells as plain scaffolds and increased formation of muscle fibers.

“The protein [IGF-1] effectively promoted cell recruitment and accelerated muscle regeneration,” said Lee.

For their next project, Lee would like to test the ability of his scaffolds to promote muscle regeneration in larger laboratory animals.

Pretreatment of Mesenchymal Stem Cells with Melatonin Improves Their Healing Properties in Animals with Strokes

The transplantation of mesenchymal stem cells or MSCs as they as affectionately known, does indeed benefit patients who have had a stroke. Unfortunately, the benefits of MSC transplantation if is limited by inability of these cells to survive after they are implanted into a low-oxygen environment. When a person suffers from a stroke, a blood vessel that feeds the brain has been blocked, and this blockage results in the death of particular cells in the brain. The affected areas of the brain, however, have been deprived of oxygen, and the transplantation of new cells into these areas can result in the prompt death of the implanted cells.

Fortunately, previous studies have revealed that pretreatment of the implanted cells with the hormone melatonin can increase the survival of MSCs that were implanted into kidneys that suffered oxygen deprivation. Therefore, could melatonin pretreatment also improve MSC survival in the case of strokes?

A new study by Guo-Yuan Yang and his colleagues at the Med-X Research Institute in Shanghai, China has examined the effects of melatonin pretreatment on the survival of MSCs that were implanted into the brains of laboratory animals that suffered a stroke.

In a nutshell, Yang and his colleagues showed that melatonin pretreatment greatly increased survival of cultured MSCs when these cells were subjected to low-oxygen conditions. Then when they went whole hog and transplanted their melatonin-pretreated MSCs into the brains of animals that had suffered a stroke, they once again observed that these cells survived at a substantially higher rate than their untreated counterparts. Melatonin-pretreated MSCs also further reduced bleeds into the brain (infarction) and improved the behavioral outcomes of the laboratory animals.

When Yang’s group examined the molecules secreted by the melatonin-treated MSCs, they discovered that the melatonin-pretreated MSCs made a lot more blood-vessel-promoting proteins (such as vascular endothelial growth factor or VEGF), and nerve cell-promoting molecules. Not surprisingly, the rats implanted with melatonin-pretreated MSCs shows significantly more new blood vessels formed, new neurons formed, and better looking brains in general.

Melatonin treatment increased the levels of two signaling molecules, p-ERK1/2, in MSCs. These particular signaling molecules are linked to higher survival rates. When Yang and his crew blocked melatonin signaling by treating cells with as drug called luzindole, these positive effects were reversed and when another drug called U0126, which prevents ERK from becoming phosphorylated was also applied to the cells, it completely reversed the protective effects of melatonin.

These results show that melatonin improves MSC survival and function. Furthermore, melatonin does this by activating the ERK1/2 signaling pathway. Therefore, mesenchymal cells pretreated by melatonin may represent a viable approach to enhance the beneficial effects of stem cell therapy for strokes, and maybe other conditions too? Well shall see. Stay tuned…..

Toxic stem cells to fight tumors

A Harvard team has developed special stem cells that secrete toxins that kill cancer cells, and cause no harm to healthy ones.

“Now, we have toxin-resistant stem cells that can make and release cancer-killing drugs,” Khalid Shah, a co-author of the study and the director of the Molecular Neurotherapy and Imaging Lab at Massachusetts General Hospital and Harvard Medical School, said in an official statement.

According to Shah, experiments in mice have proven very successful.

During the tests, the main part of the brain tumor was surgically removed, followed by the application of stem cells that were placed at the site of the tumor embedded in a biodegradable gel to kill the remaining cancerous cells.

Once within the cancer cell, the toxin disrupts its ability to synthesize proteins, killing it in a matter of days.

“After doing all of the molecular analysis and imaging to track the inhibition of protein synthesis within brain tumors, we do see the toxins kill the cancer cells,” he declared.

Shah said that the toxins that kill cancer have been used to treat a few types of blood cancers. However, these toxins were not effective dealing with solid tumors because these cancers are not as accessible and the toxins in the stem cells don’t have enough time to kill the cancer, since they only have a short half-life.

However, the new modified stem cells developed by Shah’s team change this limitation. “Now, we have toxin-resistant stem cells that can make and release cancer-killing drugs,” he said.

The study, published in the journal Stem Cells, possibly represents a breakthrough in cancer research, since it kills cancer cells while keeping remaining, healthy cells intact.

Scientists have applied for approval from the FDA to start the clinical trials of the method.

Experts praised the study as “the future” of cancer research.

“This is a clever study, which signals the beginning of the next wave of therapies. It shows you can attack solid tumors by putting minipharmacies inside the patient which deliver the toxic payload direct to the tumor,” Chris Mason, a professor of regenerative medicine at University College London, who was not participating in the study, told the BBC.

Human articular cartilage defects can be treated with nasal septum cells

A report from collaborating research teams from the University and the University Hospital of Basel specifies that cells isolated from the nasal septum cartilage can adapt to the environment the knee and repair articular cartilage defects. The ability of nasal cartilage cells to self-renew and adapt to the joint environment is associated with the expression of genes know as HOX genes. This research was published in the journal Science Translational Medicine in combination with reports of the first patients treated with their own nasal cartilage.

Lesions in articular or joint-specific cartilage is a degenerative that tends to occur in older people or younger athletes who engage in impact-heavy sports. Sometimes people who have experienced accidents can also suffer from cartilage lesions. Cartilage lesions present several challenges for orthopedic surgeons to repair. These surgeries are often complicated, and the recovery times are also long. However, Prof. Ivan Martin, professor of tissue engineering, and Prof. Marcel Jakob, Head of Traumatology, from the Department of Biomedicine at the University and the University Hospital of Basel have presented a new treatment option for cartilage lesions that includes the use of nasal cartilage cells to replace cartilage cells in joints.

When grown in cell culture, cartilage cells extracted from the nasal septum (also known as nasal chondrocytes) have a remarkable ability to generate new cartilage tissue after their growth in culture. In an ongoing clinical study, the Basal research group have taken small biopsies (6 millimeters in diameter) from the nasal septa of seven of 25 patients below the age of 55 years. After isolating the cartilage cells from these cartilage samples, they cultured these cells and expanded them and applied them to a three-dimensional scaffold in order to engineer a cartilage graft with a specific size (30 x 40 millimeters).

Martin and his colleagues used these very cartilage grafts to treat the cartilage lesions in human patients. After removing the damaged cartilage tissue from the knee of several patients, their knees were treated with the engineered, tailored tissue from their noses.

Two previous experiments demonstrated the potential efficacy of this procedure. First, a previous clinical study conducted in cooperation with plastic surgeons and the Basel group used the same method to successfully reconstruct nasal wings affected by tumors.

Secondly, a preclinical study with goats whose knees were implanted with nasal cartilage cells showed that these cells were not only compatible with the knee-joint, but also successfully reconstituted the joint cartilage. Lead author of this study, Karoliina Pelttari, and her colleagues were quite surprised that the implanted nasal cartilage cells, which originate from a completely different set of embryonic cell types than the knee-joint were compatible. Nasal septum cells develop from neuroectodermal cells, which also form the nervous system and their self-renewal capacity is attributed to their lack of expression of some homeobox (HOX) genes. However, these same HOX genes are expressed in articular cartilage cells that are formed by mesodermal cells in the embryo.

“The findings from the basic research and the preclinical studies on the properties of nasal cartilage cells and the resulting engineered transplants have opened up the possibility to investigate an innovative clinical treatment of cartilage damage,” says Prof. Ivan Martin about the results. Several studies have confirmed that human nasal cells maintain their capacity to grow and form new cartilage despite the age of the patient. This means that older people could also benefit from this new method, as could patients with large articular cartilage defects.

The primary target of the ongoing clinical study at the University Hospital of Basel is to confirm the safety, efficacy and feasibility of nasal cartilage grafts transplanted into joints, the clinical effectiveness of this procedure, from the data presently in hand, is highly promising.

Patient’s Own Stem Cells Treat Rare Neurological Disorder

Stiff-Person syndrome is a rare neurological disease that, for all intents and purposes, looks like an autoimmune disease. It is characterized by muscular rigidity that tends to come and go. This rigidity occurs in the muscles of the trunks and limbs. Patients with Stiff-Person syndrome also have an enhanced sensitivity to stimuli such as noise, touch, and emotional distress, and various stimuli may cause the patient to experience painful muscle spasms that cause abnormal postures and stiffening. Stiff-Person syndrome or SPS is more common in women than in men and SPS patients often suffer from other autoimmune conditions in addition to SPS (for example, pernicious anemia, diabetes, vitiligo, and thyroiditis). Unfortunately, the precise cause of SPS is not known, but again, it looks like an autoimmune condition.

A research team at Ottawa Hospital Research Institute has made a breakthrough in the successful treatment of SPS using bone marrow stem cell transplants. The medical director at the Ottawa Hospital Research Institute, Dr. Harold L. Atkins, who is also a physician in the Blood and Bone Marrow Transplant Program at The Ottawa Hospital and an associate professor at the University of Ottawa has used bone marrow transplants to two female SPS patients into remission.

SPS can leave patients bedridden and in severe pain, but thanks to Atkins and his team, the progression of the disease in these women has ceased, allowing both women to regain their previous function and leaving them well enough to return to work and normal everyday activities.

Adkins and his group published this case study in JAMA Neurology, which is produced by the Journal of the American Medical Association. This is the first documented report that taking stem cells from a person’s own body can produce long-lasting remission of stiff person syndrome.

“We approach these cases very carefully and are always aware that there have just been a few patients treated and followed for a short time,” says Dr. Atkins. Atkins and his extracted bone marrow stem cells from each woman, and then used chemotherapy to eliminate their immune systems. Once their immune system were reliably eliminated, both women had their own stem cells returned to their bodies in order to reconstitute their immune systems. This procedure essentially gives the immune system a “do-over.”.

“By changing the immune system, one hopes to put the stiff person syndrome into remission,” adds Dr. Atkins. “Seeing these two patients return to their normal lives is really every physicians dream.”

This very procedure, which is known as an “autologous stem cell transfer” or ASCT has been used to successfully treat people who suffer from autoimmune diseases such as multiple sclerosis, scleroderma, and systemic lupus erythematosis. Atkins and his team used high-doses of chemotherapy and antibodies that specifically bind lymphocytes to rid the women’s bodies of their rogue immune cells before their immune systems were regenerated using their own stem cells. Adkins an his colleagues viewed this as a viable treatment option based strategies that had been used to treat other autoimmune diseases.

Patient 1 was diagnosed with stiff person syndrome in 2005 at age 48 after experiencing leg stiffness and several falls. After her treatment, her symptoms disappeared and she was fully mobile again six months after receiving the stem cell transplant procedure in 2009.

Patient 2 was diagnosed with stiff person syndrome in 2008 at age 30. She had stopped working and driving, and had moved back in with her parents before her stem cell transplant in 2011. Also, she has been able to return to her work and previous activities, and has not had any stiff person syndrome symptoms in more than a year.

“The results achieved by Dr. Atkins and his team through this innovative treatment show how research at The Ottawa Hospital can lead to life-changing and, even life-saving care,” says Dr. Duncan Stewart, Chief Executive Officer and Scientific Director of the Ottawa Hospital Research Institute. “Translating research into better care for patients is what we’re all about at the research institute.”

A More Efficient Way to Make Induced Pluripotent Stam cells

Mark Stadtfeld and his colleagues at the NYU Longone Medical Center has devised a new method for making induced pluripotent stem cells that greatly increases efficiency at which these cells are made.

Induced pluripotent stem cells or iPSCs are made from mature, adult cells by mean of a combination of genetic engineering and cell culture techniques. In short, the expression of four genes is forced in adult cells; Oct4, Sox2, Klf4, and c-Myc or OSKM. The proteins encoded by these four genes cooperatively work to drive a fraction of the cells into an immature state that resembles that of embryonic stem cells. These cells are them grown in cell culture systems that select for those cells that can grow continuously and form colonies of cells derived from progenitor cells. These cell colonies are them repeated isolated a re-cultured until an iPSC line has been established.

Unfortunately, this process is rather inefficient and tedious, since less than one percent or so of the reprogrammed cells actually undergo successful reprogramming. Additionally, it can take several weeks to properly establish an iPSC line. Thus, stem cell scientists have been looking at several different ways to boost the efficiency of this process.

Stadtfeld and his coworkers tried to add compounds to the cultured cells to determine if the culture conditions could actually augment the efficiency of the reprogramming process. “We especially wanted to know if these compounds could be combined to obtain stem cells at high-efficiency,” said Stadtfeld.

The compounds to which Stadtfeld was referring were two cell signaling proteins called Wnt and TFG-beta. Both of these compounds regulate a host of cell growth processes. Stadtfeld wanted to try regulating both of these pathways at the same time, in addition to providing cells with ascorbic acid, which is also known as vitamin C. Even vitamin C is more popularly known as an antioxidant, vitamin C also can remodel chromatin (that tight structure into which cells package their DNA).

When mouse skin fibroblasts were treated with OSKM and a compound that activates Wnt signaling, the efficiency of iPSC derivation increased slightly. The same thing was observed if fibroblasts were treated with OSKM and a compound that inhibits TGF-beta signaling or vitamin C. However, when all three of these compounds were combined, OSKM-engineered fibroblasts were reprogrammed at an efficiency of close to 80 percent. When different cell types were used as the starting cell, such as blood progenitor cells, the efficiency jumped to close to 100 percent; a result that was also observed if liver progenitor cells were used as the starting cell.

Stadtfeld is confident that these dramatic increases in iPSC derivation should improve future studies with iPSCs, since his protocol should make iPSC derivation more predictable. “It’s just a lot easier this way to study the mechanisms that govern reprogramming, as well as detect any undesired features that might develop in iPSCs,” he said.

Vitamin C and the two compounds used to manipulate the Wnt and TGF-β pathways have been widely used in research and have few unknown or hazardous effects. However, OKSM has in some cases caused undesired features in iPSCs, such as increased mutation rates. Stadtfeld believes that by making iPSC induction more rapid and efficient, his new technique might also make the resulting stem cells safer. “Conceivably it reduces the risk of abnormalities by smoothening out the reprogramming process,” Dr. Stadtfeld says. “That’s one of the issues we’re following up.”

Embryonic Stem Cell-Derived Retinal Cells Treat Blindness in Eye Patients

Embryonic stem cells are derived from human embryos, can only grow in culture indefinitely, and have the ability to potentially differentiate into any adult cell type in the human body.  Because cell and tissues made from embryonic stem cells bear the same tissue types as the embryos from which they were derived, they will be rejected by the immune system patient.  However, there are sites in our bodies were the immune system does not go, and that includes the central nervous system and the eyes.  This is the reason why clinical trials with embryonic stem cell-derived cells have focused, to date, on spinal cord injuries and eye diseases.

Several clinical trials have examined the ability of retinal pigmented epithelial (RPE) cells made from embryonic stem cells to treat patients with dry macular degeneration or an inherited eye disease called Stargardt’s disease.  Data from these trials has been reported in an article in the medical journal The Lancet, and accordingly, none of the treated patients showed tumor formation or immunological rejection of the implants and, most impressively perhaps, partial blindness was reversed in about half of the eyes that received transplants.

The results might re-energize the quest to harness embryonic stem cells for human medicine.  Dr. Anthony Atala of the Wake Forest Institute for Regenerative Medicine called the work “a major accomplishment” in an accompanying commentary on the article.

RPE cells lie just behind the photoreceptor cells in the retina of our eyes.  Photoreceptors have their ends hurried in the RPE layer.  This arrangement exists for a very good reason; the photoreceptors are exposed to high intensities of light and they suffer respectable amounts of oxidative damage.  The components of the photoreceptors cells are made in the very lowest parts of the RPEs and then are eventually pushed to the ends of the cells.  At the end of the photoreceptor cells, the RPEs relieve the photoreceptors of their photodamaged parts and gobble them down, and recycle the cellular components.  Thus, RPE cells serve a photoreceptor cell repair and service cells.  If the RPE cells begin to die, the photoreceptors are not long the this work either.

In the case of dry macular degeneration, which accounts for 90 percent of diagnosed cases of macular degeneration, the light-sensitive photoreceptor cells of the macula (the portion of the retina were the day vision is the sharpest) slowly break down. Damage to the macula causes blurring or spotty loss of central vision and yellowish cellular deposits called drusen (extracellular waste products from metabolism) form under the retina between the retinal pigmented epithelium (RPE) layer and a basement membrane called Bruch’s membrane, that supports the retina. An increase in the size and number of drusen is associated with the death of RPE and, consequently, photoreceptor cells, and is sometimes the first sign of dry macular degeneration.

Mutations in several genes have been identified in families with dry macular degeneration that increase the risk for dry macular degeneration.  These include the SERPING1 gene, those genes that encode the complement system proteins  factor H (CFH), factor B (CFB) and factor 3, and fibulin-5.  Additionally, some environmental and behavioral factors also influence the risk a person will develop macular degeneration.  These include smoking, exposure to blue light, ingestion of a high-fat diet, elevated blood pressure and serum cholesterol levels, and low vitamin D levels.

Stargardt’s disease is an inherited, juvenile form of macular degeneration that is caused by mutations in the ABCR gene.  The protein encoded by this gene is a waste metabolite transporter, and defects in this protein cause the build up of a toxic metabolite called lipofuscin in the RPE cells, which leads to their demise and the death of the photoreceptors.

In this study, the main goal was to assess the safety of the transplanted cells. The study “provides the first evidence, in humans with any disease, of the long-term safety and possible biologic activity” of cells derived from embryos, said co-author Dr. Robert Lanza, chief scientific officer of Advanced Cell Technology, which produced the cells and funded the study.

Nine patients with Stargardt’s disease and nine with dry age-related macular degeneration received implants of the retinal cells in one eye. The other eye served as a control.  Four eyes developed cataracts and two became inflamed, probably due to the patients’ age (median: 77) or the use of immune-supressing transplant drugs.

The implanted RPE cells survived in all 18 patients, most of whose vision improved.  In those with macular degeneration, treated eyes saw a median of 14 additional letters on a standard eye chart a year after receiving the cells, with one patient gaining 19 letters. The untreated eyes got worse, overall. The Stargardt’s patients had similar results.

In real-life terms, patients who couldn’t see objects under 12 feet (4 meters) tall can now see normal-size adults.

The vision of one 75-year old rancher who was blind in the treated eye (20/400) improved to 20/40, enough to ride horses again, Lanza said.  Others became able to use computers, read watches, go to the mall or travel to the airport alone for the first time in years.

While calling the results “encouraging,” stem cell expert Dusko Ilic of Kings College London, who was not involved in the work, warned that even if the larger clinical trial planned for later this year is also successful, “it will take years before the treatment becomes available.”

Other cell types can also form RPE cells and these include induced pluripotent stem cells, mesenchymal stem cells from fat (Ophthalmic Res. 2012;48 Suppl 1:1-5), adult retinal stem cells (Pigment Cell Melanoma Res. 2011 Feb;24(1):233-40), and iris pigmented epithelial cells (Prog Retin Eye Res. 2007 May;26(3):302-21).  We do not need to destroy embryos to treat eye diseases with stem cells.

Nose Stem Cells Help Bulgarian Man Walk With Braces

Darek Fidyka, a 38-year-old Bulgarian man, was severely injured by a stab wound in 2010 and consequently lost the ability to walk.

Now, a new procedure using stem cells from his nose has given him the ability to walk with the help of braces.

Olfactory ensheathing cells or OECs (also known as olfactory ensheathing glial or OEGs) are found in the olfactory system, inside the skull and in the covering of cells that lines the roof of the nose. OECs share similarities to other glial cells like Schwann cells, astrocytes, and oligodendrocytes. OECs can aid the extension of neural projections known as axons from the nasal tissue to the olfactory glomeruli. OECs can do this because they secrete several interesting neurotrophic factors and cell adhesion molecules and migrate along with the regenerating axons. Because of these properties, OECs can escort axonal extension through glial scars that are made in a spinal cord after a spinal cord injury. These scars inhibit the outgrowth of new axons but OECs can allow regenerating axons to bridge these glial scars.

An advantage of OECs is that they can coexist with astrocytes, the cells that contribute to the formation of the glial scar, and even seem to prevent the out-of-hand response astrocytes have in response to injury in which they synthesize a host of molecules that inhibit axon regeneration called “inhibitory proteoglycans.”

The pioneering technique used in this procedure, according to Geoffrey Raisman, a professor at University College London’s (UCL) institute of neurology, used OECs to construct a kind of bridge between two stumps of the damaged spinal column.

“We believe… this procedure is the breakthrough which, as it is further developed, will result in a historic change in the currently hopeless outlook for people disabled by spinal cord injury,” said Riesman, who led this research project.

Raisman, who is a spinal injury specialist at UCL, collaborated with neurosurgeons at Wroclaw University Hospital in Poland to remove one of Fidyka’s olfactory bulbs, which give people their sense of smell, and transplant his olfactory ensheathing cells (OECs) in combination with. olfactory nerve fibroblasts (ONFs) into the damaged spinal cord areas. Following 19 months of treatment, Fidyka recovered some voluntary movement and some sensation in his legs.

The Nicholls Spinal Injury Foundation, a British-based charity which part-funded the research, said in statement that Fidyka was continuing to improve more than predicted, and was now able to drive and live more independently.

OECs have been used before to treat spinal cord injury patients. I refer you to chapter 27 in my book, The Stem Cell Epistles, to learn more about these. The novel technique in this paper is the additional use of nasal fibroblasts and the construction of a bridge between the two damaged remnants of the spinal cord.

The reason OECs were recruited to treat spinal cord injuries is that when axons that carry information about smells are damaged, the neuron simply regenerates its atonal extension, which grows into the olfactory bulbs. OECs facilitate this process by re-opening the surface of the olfactory bulbs in order for the new axons to enter them. Thus Raisman and others have the notion that transplanted OECs in the damaged spinal cord could equally facilitate the regeneration of severed nerve fibers.

Raisman also added that the technique used in this case, that is bridging the spinal cord with nerve grafts from the patient, had been used in animal studies for years, but was never used in a human patient in combination with OECs.

“The OECs and the ONFs appeared to work together, but the mechanism between their interaction is still unclear,” he said in a statement about the work.

Several spinal cord injury experts who were not directly involved in this work said its results offered some new hope. However, they were also quick to add that more work needed to be done to precisely determine what had led to this success. More patients must be successfully treated with this procedure before its potential can be properly assessed.

“While this study is only in one patient, it provides hope of a possible treatment for restoration of some function in individuals with complete spinal cord injury,” said John Sladek, a professor of neurology and pediatrics at the University of Colorado School of Medicine in the United States.

Raisman and his team now plan to repeat the treatment technique in between three and five spinal cord injury patients over the next three to five years. “This Nose will enable a gradual optimization of the procedures,” he told Reuters.

Stem Cell Transplant from Gut Repairs Damaged Gut in Mice with Inflammatory Bowel Disease

Even though a stem population has been identified and studied in the gastrointestinal tract, Wellcome Trust Researchers have identified a new source of GI-based stem cells that have the ability to repair damage from inflammatory bowel disease when transplanted into mice.  This work comes to us from the Wellcome Trust-Medical Research Council Cambridge Stem Cell Institute at the University of Cambridge and BRIC at the University of Copenhagen, Denmark.  This work could translate into patient-specific regenerative therapies for inflammatory bowel diseases such as ulcerative colitis.

Adult tissues contain specialized stem cells populations that maintain individual tissues and organs.  Adult stem cells tend to be restricted to their tissue of origin and also tend to have the ability to differentiate into a limited subset of adult cell types.  Stem cells found in the gut, for example, typically can typically contribute to the replenishment of the gut whereas stem cells in the skin will only contribute to maintenance of the skin.

When examining the developing intestinal tissue in a mouse embryos, Kim Jensen and her team discovered stem cell population hat were quite different from those adult stem cells that have been described in the gut.  These cells actively divided and also could be grown in the laboratory over long periods of time without undergoing differentiation into mature cells.  Under specific culture conditions, however, these cells could be induced to differentiate into mature intestinal tissue.

When these cells were transplanted into mice that suffered from an inflammatory bowel disease, The implanted stem cell attached to the damaged areas within the intestine, and began to integrate into the existing tissue, within three hours of implantation.

The lead researcher in this study, Dr. Kim Jensen, a Wellcome Trust researcher and Lundbeck foundation fellow, said: “We found that the cells formed a living plaster over the damaged gut. They seemed to respond to the environment they had been placed in and matured accordingly to repair the damage.

“One of the risks of stem cell transplants like this is that the cells will continue to expand and form a tumour, but we didn’t see any evidence of that with this immature stem cell population from the gut.”

Cells with similar characteristics were isolated from both mice and humans.  Jensen’s team also generated similar cells by reprogramming adult human cells to make induced Pluripotent Stem Cells (iPSCs) that were also grown under the appropriate culture conditions.

“We’ve identified a source of gut stem cells that can be easily expanded in the laboratory, which could have huge implications for treating human inflammatory bowel diseases. The next step will be to see whether the human cells behave in the same way in the mouse transplant system and then we can consider investigating their use in patients,” added Dr Jensen.

Human Umbilical Mesenchymal Stem Cells Decreases Dextran Sulfate Sodium-Induced Colitis in Mice

Ulcerative colitis is one of the Inflammatory Bowel Diseases (IBDs) that features chronic inflammation of the large intestine. This is an autoimmune disease that features constant attacks by the immune system on the intestinal mucosae, and the inner layer of the large intestine undergoes constant damage and healing, which increases the risk of the patient to developing colorectal carcinoma.

Mesenchymal stem cells have the capability to suppress inflammation, which makes them promising tools for treating diseases like ulcerative colitis. Unfortunately, the lack of reproducible techniques for harvesting and expanding MSCs has prevented bone marrow- and umbilical cord blood-derived MSCs from being routinely used in clinical situations.

However, a study that was published in the journal Clinical and Experimental Pharmacology and Physiology has used Wharton’s jelly derived umbilical MSCs (UMSCs) to treat mice in which an experimental form of ulcerative colitis was induced. Dextran sulfate sodium (DSS) induced colitis in mice has many of the pathological features of ulcerative colitis in humans.

When mice treated with DSS were also given Wharton’s jelly derived UMSCs showed significant diminution of the severity of colitis. The structure of the tissue in the colon looked far more normal and the types of molecules produced by inflammation were significantly reduced. In addition, transplantation of UMSCs reduced the permeability of the intestine and also increased the expression of tight junction proteins, which help knit the colonic cells together and maintain the structural integrity of the colon. These results show that the anti-inflammatory properties of UMSCs and their capacity to regulate tight junction proteins ameliorates ulcerative colitis.

U of Pitt Team Discovers Stem Cells in the Esophagus

Even though several studies have been unsuccessful at identifying a stem population in the esophagus, a study from the University of Pittsburgh has discovered a stem cell pool that services the esophagus. Researchers from the University of Pittsburgh School of Medicine have published an animal report in the journal Cell Reports that might lead to new insights into the development and treatment of esophageal cancer and a precancerous condition known as Barrett’s esophagus.

In the US, more than 18,000 people will be diagnosed with esophageal cancer in 2014 and almost 15,500 people will die from it, according to numbers generated by the American Cancer Society. The precancerous condition known as Barrett’s esophagus is characterized by tissue changes in the lining of the esophagus in which the esophageal lining begins to resemble the tissue architecture of the intestine. Barrett’s esophagus is usually a long-term consequence of gastro-esophageal reflux disease or GERD.

“The esophageal lining must renew regularly as cells slough off into the gastrointestinal tract,” said senior investigator Eric Lagasse, Pharm.D., Ph.D., associate professor of pathology, Pitt School of Medicine, and director of the Cancer Stem Cell Center at the McGowan Institute for Regenerative Medicine. “To do that, cells in the deeper layers of the esophagus divide about twice a week to produce daughter cells that become the specialized cells of the lining. Until now, we haven’t been able to determine whether all the cells in the deeper layers are the same or if there is a subpopulation of stem cells there.”

Lagasse and his team grew small explants of esophageal tissue in culture. These esophageal “organoids” from mice were then used to conduct experiments that were used to identify and track the different cells in the basal layer of the tissue. In these organoids, Lagasse and others found a small population of cells that divide more slowly, are less mature, can differentiate into several different types of esophageal-specific cell types, and have the ability to self-renew. The ability to self-renew is a defining feature of stem cells.

“It was thought that there were no stem cells in the esophagus because all the cells were dividing rather than resting or quiescent, which is more typical of stem cells,” Dr. Lagasse noted. “Our findings reveal that there indeed are esophageal stem cells, and rather than being quiescent, they divide slowly compared to the rest of the deeper layer cells.”

Lagasse and his team would now like to examine human esophageal tissues from patients with Barrett’s esophagus in order to determine if such patients show evidence of esophageal stem cell dysfunction.

“Some scientists have speculated that abnormalities of esophageal stem cells could be the origin of the tissue changes that occur in Barrett’s disease,” Dr. Lagasse said. “Our current and future studies could make it possible to test this long-standing hypothesis.”

New Stem Cell Technology to Form Blood Vessels and Treat Peripheral Artery Disease

How to make new blood vessels for patients who need them? Researchers at the University of Indiana University School of Medicine have developed a new therapy for illnesses such as peripheral artery disease. Diseases such a peripheral artery disease can lead to skin problems, gangrene and sometimes amputation.

Our bodies have the ability to repair blood vessels and creating new ones, because of a cell type called “endothelial colony-forming cells.” Unfortunately, these cells tend to lose their ability to proliferate and form new blood vessels as patients age or develop diseases like peripheral arterial disease, according to Mervin C. Yoder Jr., M.D., who is the Richard and Pauline Klingler Professor of Pediatrics at IU and leader of the research team.

Physicians can prescribe drugs that improve blood flow to patients with peripheral artery disease, but if the blood vessels are reduced in number or function, the benefits from such drugs are minimal. A better treatment might be to introduce “younger,” more effective endothelial colony forming into the affected tissues. In this case, such a treatment would jump-start the creation of new blood vessels. Gathering such cells, however is rather difficult, since endothelial colony-forming cells are somewhat difficult to find in adults, especially in those with peripheral arterial disease. Fortunately, endothelial colony-forming cells are rather numerous in umbilical cord blood.

Yoder and his colleagues published their work in the journal Nature Biotechnology, and they have reported that they have developed a potential therapy by using patient-specific induced pluripotent stem cells (iPSCs). Induced pluripotent stem cells are pluripotent stem cells that are derived from normal adult cells by means of genetic engineering and cell culture techniques. Once an iPSC line has been derived from a patient, they can potentially be differentiated into any adult cells type, including endothelial colony-forming cells.

In this paper, Yoder and his research team developed a novel methodology to differentiate iPSCs into cells with the characteristics of the endothelial colony-forming cells that are found in umbilical cord blood. These laboratory-generated endothelial colony-forming cells were injected into mice, and they proliferated and generated human blood vessels that nicely restored blood flow to damaged tissues in mouse retinas and limbs

Another problem addressed in this paper was growing endothelial colony-forming cells from umbilical cord in culture so that they can achieve sufficient numbers for therapies. In this paper, Yoder and his team designed a cell culture system that was able to dramatically expand these iPSC-derived endothelial colony-forming cells in culture from one founding cell to 100 million new cells in a little less than three months.

“This is one of the first studies using induced pluripotent stem cells that has [sic] been able to produce new cells in clinically relevant numbers — enough to enable a clinical trial,” Dr. Yoder said. According to Yoder, the next steps will be to reach solidify an agreement with a facility approved to produce cells for use in human testing. Additionally, Yoder would like to treat more than just peripheral artery disease, since he and his colleagues are evaluating the potential uses of these cells to treat diseases of the eye and lungs that involve blood flow problems.

Conditioning Stem Cells to Survive in the Heart

After a heart attack, the heart is a very inhospitable place for implanted stem cells. These cells have to deal with low oxygen levels, marauding white blood cells, toxins released from dead or nearly-dead cells, and other nasty things.

Getting cells to survive in this place is essential if the cells are going to provide any healing to he heart. Fortunately, a Chinese group has discovered that growing cells in inhospitable conditions before implantation greatly improves their survival. Now, this same group from Emory University School of Medicine in Atlanta, Georgia has shown that a small molecule can do the same thing.

This work, published in Current Stem Cell Research and Therapy, centers upon a pathway in cells controlled by a protein called the hypoxia-inducible factor or HIF. This protein regulates those genes that allow cells to withstand low-oxygen and other stressful conditions. HIF is composed of two parts: an oxygen-sensitive inducible HIF-1α subunit and a constitutive HIF-1β subunit. During nonstressful conditions, the alpha subunit is constantly being degraded after it is made because it is modified by a enzymes called prolyl hydroxylase (PHD) enzymes. In the presence of low oxygen conditions, PHD enzymes are inhibited and HIF-1α increases in concentration. The HIFα/β heterodimer forms and is stabilized, and translocates to the nucleus where it activates target genes.

It turns out that small molecules can inhibit PHD enzymes and induce the low-oxygen status in cells without subjecting them to rigorous culture conditions. For example, dimethyloxalylglycine (DMOG) can inhibit PHD enzymes and produce in cells the types of responses normally observed under low-oxygen conditions.

In this paper, Ling Wei and colleagues cultured mesenchymal stem cells from bone marrow with or without 1 mM DMOG for 24 hours in complete culture medium before transplantation. These cells were then transplanted into the hearts of rats 30 minutes after those rats had suffered an experimentally-induced heart attack. They then measured the rates of cell death 24 hours after engraftment, and heart function, new blood vessel formation and infarct size 4 weeks later.

In DMOG-preconditioned bone marrow MSCs (DMOG-BMSCs), the expression of survival and blood-vessel-making factors were significantly increased. In comparison with control cells.  DMOG-BMSCs also survived better and enhanced the formation of new blood vessels in culture and when implanted into the heart of a living animal.

C to H, Angiogenesis was inspected using vWF staining (red) in heart sections from MI, C-BMSC
and DMOG-BMSC groups 4 weeks after MI. Hoechst staining (blue) shows the total cells. I. Summary of total tube length measured in experiments A and B. The total tube length in C-BMSC group was arbitrarily presented as 1. N = 3 independent measurements. J, Summary of total vessel density in different groups of in vivo experiments. N = 8 animals in each group.

Transplantation of DMOG-BMSCs also reduced heart infarct size and promoted functional benefits of the cell therapy.

Effect of BMSCs transplantation on ischemia-induced infarct formation. Heart infarct area and scar formation were determined using Masson’s
Trichrome staining 4 weeks after MI. A to C. Images of representative infarcted hearts from a MI control
rat, a MI rat received C-BMSCs, and a MI rat received DMOG-BMSCs. D. Transplantation of BMSCs
reduced heart infarction formation, the protective effects were significantly greater with transplantation of DMOG-BMSCs. N = 5 rats in each group.

Thus, this paper shows that targeting an oxygen sensing system in stem cells such as PHD enzymes (prolyl hydroxylase) provides a new promising pharmacological approach for enhanced survival of BMSCs.  This procedure also increases paracrine signaling, augments the regenerative activities of these cells, and, ultimately, and improves functional recovery of the heart as a result of cell transplantation therapy for the heart after a heart attack.  This is only a preclinical study, but the data is strong, and hopefully new clinical trials will bear this out.

CAR Immune Cells to Treat Childhood Cancers

In clinical trials, cancer treatments that use genetically modified versions of a patient’s own cells to specifically target the disease have remarkable results. The next step for these companies that spent enormous amounts of time, capital, and intellectual energy inventing and designing these treatments is to get them into hospitals despite their enormous price tags.

In two separate clinical trials, one sponsored by the Swiss company Novalis AG and another by the Seattle-based biotech company Juno Therapeutics Inc., close to 90% of all patients saw their leukemia completely disappear after being given experimental “CAR” or “chimeric antigen receptor” T-cell therapies.

Both trials examined small numbers of patients (22 children in the Novartis trial and 16 adults in the Juno trial). These patients had acute lymphoblastic leukemia, which is the most common childhood cancer. All of them had also not responded to the available standard treatments. Consequently, both companies are now conducting larger trials.

“CAR T cells are probably one of the most exciting concepts and fields to come out in cancer in a very, very long time,” says Dr. Daniel DeAngelo, a Boston-based hematologist and associate professor of medicine at Harvard Medical School, who wasn’t involved in either study.

Usman Azam, head of cell and gene therapies at Novartis, calls the therapies “critically important” for Novartis. “I think that a cure for cancers such as leukemia and lymphoma through a CAR technology is plausible,” said Dr. Azam in an interview with The Wall Street Journal. “Our job is to get this into patients as soon as we feasibly can.”

Novatis created a new research unit headed by Dr. Azam. Novartis’ rationale is to accelerate the advent of CAR T-Cell Therapy to medical markets. The U.S. Food and Drug Administration (US FDA) granted Novartis’ leading CAR therapy “breakthrough” designation in July of 2014. Presently Novartis wants to file it with regulators in 2016.

CAR therapies use the patient’s own immune system to fight the cancer, but with a genetic-engineering twist. “Immunotherapies,” culture immune cells from the patient and manipulate them in culture to sensitize them to the cancer. CAR therapies extract T-cells, which are disease-fighting white blood cells, from a patient’s blood. These T-cells are then genetically engineered and grown in a laboratory for around 10 days and reintroduced into the patient.

The T-cells are usually infected with a hamstrung virus that can introduce genes into cells but cannot productively infect them. These recombinant viruses endows the T-cells with genes that encode chimeric antigen receptors, or CARs. CARS bind specifically to proteins on the surface of malignant cancer cells. Once attached to the cancer cells, the T-cells can kill them very effectively.

Both Novartis and Juno are tapping academic scientists to develop their treatments. For example, Novartis has teamed with the University of Pennsylvania and Juno has formed a formal relationships with scientists at Memorial Sloan-Kettering Cancer Center in New York, Seattle Children’s Hospital and the Fred Hutchinson Cancer Research Center, which is also in Seattle.

Even though Novartis and Juno will probably be the first to bring their immunotherapies to the market, other companies are also in the hunt to bring similar therapies to medical markets. Pfizer Inc., Kite Pharma Inc., and Celgene Corp., which is working in collaboration with Bluebird Bio Inc. all are developing competing strategies.

“Competition will keep all of the companies involved on their toes,” said Hans Bishop, Juno’s chief executive.

Unfortunately, CAR therapies still have a few unanswered questions surrounding them. For example: “How long do they last?” Given the small numbers of patients who have been treated with these treatments to date, it is very hard to tell with the available data. Another confounding factor is that those patients in the previous clinical trials whose cancer went into remission after the CAR therapies then became eligible for stem-cell transplants, which can also prolong survival.

Secondly, a potentially dangerous side effect called “cytokine-release syndrome,” shows the therapy is working, but can cause a sharp drop in blood pressure and a surge in the heart rate. The deaths of two patients in a Juno-backed Sloan-Kettering trial in March caused a temporary halt in the study because of worries over these particular adverse reactions.  “Patients need to be healthy enough to combat that side effect,” says Mr. Bishop, who thinks it is now manageable. Patients are once again being recruited for this trial, and patients with a risk of heart failure are excluded, and the modified cell dose for patients with very advanced leukemia also has been lowered.

But largest hurdle of all will probably be the cost of these therapies. Since they are a genetically engineered product, CAR T-cells are very complex to manufacture; each batch is composed of unique, personalized T-cells that were made from a patient’s own blood cells. The inability to mass-produce CAR T-cells will definitely increase the price companies charge for them.

“What we’re talking about here is a single, very expensive therapy that’s used once for a specific patient and is not generalizable,” says Dr. Malcolm Brenner, director of the Center for Cell and Gene Therapy at the Texas Children’s Hospital in Houston, who, in MArch, signed an agreement to commercialize his own CAR research with Celgene.

Novartis and Juno both insist that it is too early to speculate on the price of the treatment, but Dr. Usman agrees the challenge is getting the manufacturing process to “a viable level where it’s both affordable and attractive.”

Citigroup believes CAR therapies could cost in excess of $500,000 per patient, which it notes is roughly in line with the cost of a stem cell transplant, even though most analysts think it is too early to estimate potential revenue or price.

“This technology needs to be widely developed and accessible to patients,” says Dr. DeAngelo. “If the cost is going to be a hindrance, it’s going to be a really sad day.”

Scalability and cost are one reason Pfizer is taking a different approach to this field. “We would like to take it to the next level, where CAR therapies become a more standardized, highly controlled treatment,” said Mikael Dolsten, Pfizer’s head of global research and development.

Working with French biotech Cellectis SA, Pfizer wants to develop a generic CAR therapy for use in any patient. While this will certainly lower the cost of the treatment, since it is the result of a mass-produced, off-the-shelf-product, this work is still at the preclinical stages and may not work in humans.

Global head of health-care research at Société Générale, Stephen McGarry, thinks that the revolutionary treatments being developed by Novartis and Juno could justify “astronomical” prices, he believes health-care payers and patients will probably protest such high prices. “When you look at the initial data with the Novartis therapy, you’re getting cures in some kids—what do you charge for that?” he asks.

Cells from placentas safe for patients with multiple sclerosis

A new Phase I clinical trial has demonstrated that Multiple Sclerosis (MS) patients were able to safely tolerate treatment with cells cultured from human placental tissue.  The results of this study were recently published in the journal Multiple Sclerosis and Related Disorders.  This pioneering study was conducted by researchers at Mount Sinai, Celgene Cellular Therapeutics, which is a subsidiary of Celgene Corporation, and collaborators at several other institutions, including the Swedish Neuroscience Institute in Seattle, WA, MultiCare Health System-Neuroscience Center of Washington, London Health Sciences Centre at University Hospital in London, the Clinical Neuroscience Research Unit at the University of Minnesota, the University of Colorado Denver, The Ottawa Hospital Multiple Sclerosis Clinic, and the MS Comprehensive Care Center at SUNY.

Even though this clinical trial was designed solely to determine the safety of this treatment, the data collected from the participating patients suggested that a preparation of cultured cells called PDA-001 may repair damaged nerve tissues in patients with MS.  PDA-001 cells resemble “mesenchymal,” stromal stem cells, which are found in many tissues of the body.  However, in this study, the cells were grown in cell culture systems, which means that one donor was able to supply enough cells for several patients.

“This is the first time placenta-derived cells have been tested as a possible therapy for multiple sclerosis,” said Fred Lublin, MD, Director of the Corinne Goldsmith Dickinson Center for Multiple Sclerosis, Professor of Neurology at Icahn School of Medicine at Mount Sinai and the lead investigator of the study. “The next step will be to study larger numbers of MS patients to assess efficacy of the cells, but we could be looking at a new frontier in treatment for the disease.”

MS is a chronic autoimmune disease.  The body’s immune system attacks the insulating myelin sheath that surrounds and protectively coats the nerve fibers in the central nervous system.  The myelin sheath greatly improves the speed at which nerve impulses pass through these nerves and without the myelin sheath, nerve impulse conduction becomes sluggish, and the nerves also eventually die off.  Long-term, MS causes extensive nerve malfunction and can lead to paralysis and blindness.  MS usually begins as an episodic condition called “relapsing-remitting MS” or RRMS.  Patients will have occasional outbreaks of nerve malfunction, pain, or numbness.  However, many MS patients will see their condition evolves into a chronic condition with worsening disability called “secondary progressive MS” or SPMS.

This Phase I trial examined 16 MS patients, 10 of whom had  RRMS and six of whom were diagnosed with SPMS and were between the ages of 18 and 65.  Six patients were given a high dose of the placental-based cell line PDA-001, and another six were given a lower dose.  The remaining four patients were given placebos.  Dr. Lubin noted that alteration of the immune system by any means can cause MS to worsen in some patients.  Therefore, all participating subjects were given monthly brain scans over a six-month period to ensure they did not acquire any new or enlarging brain lesions, which are indicative of worsening MS activity.  However, none of the subjects in this study showed any paradoxical worsening on MRI and after one year.  The majority had stable or improved levels of disability.

“We’re hoping to learn more about how placental stromal cells contribute to myelin repair,” said Dr. Lublin. “We suspect they either convert to a myelin making cell, or they enhance the environment of the area where the damage is to allow for natural repair. Our long-term goal is to develop strategies to facilitate repair of the damaged nervous system.”

Skin Cells Can Be Engineered into Pulmonary Valves for Pediatric Patients

Stem cells researchers from the University of Maryland School of Medicine in Baltimore have designed a treatment that takes a child’s skin cells, reprograms them to function as heart valvular cells and then utilizes these cells to engineer a pulmonary valve that can grow with the patient.

“Current valve replacements cannot grow with patients as they age, but the use of a patient-specific pulmonary valve would introduce a ‘living’ valvular construct that should grow with the patient. Our study is particularly important for pediatric patients who often require repeated operations for pulmonary valve replacements,” said lead author David L. Simpson, Ph.D., The study is published in the current issue of Annals of Thoracic Surgery.

In the heart, the “pulmonary valve” is located between the right ventricle and the pulmonary artery, which takes blood from the right side of the heart to the lungs. This crescent-shaped valve aids in moving blood from the heart into the lungs.

According to data from the Society of Thoracic Surgeons (STS) Congenital Heart Surgery Database, close to 800 patients experience pulmonary valve failure and could benefit from bioengineered patient-specific pulmonary valves. The STS Database collects information from more than 95% of hospitals in the United States and Canada that perform pediatric and congenital heart surgery. Numbers compiled from these hospitals show that approximately 3,200 patients underwent pulmonary valve replacement during a four-year period from January 2010 to December 2013.

Although the study was conducted outside the body, the next step will be implanting the new valves into patients to test their durability and longevity.

“We created a pulmonary valve that is unique to the individual patient and contains living cells from that patient. That valve is less likely to be destroyed by the patient’s immune system, thus improving the outcome and hopefully increasing the quality of life for our patient,” said senior co-author Sunjay Kaushal, M.D., Ph.D.. “In the future, it may be possible to generate this pulmonary valve by using a blood sample instead of a skin biopsy.”

David Simpson added that he hopes the study will encourage additional research in tissue engineering and entice more people to enter the field, “Hopefully, growing interest and research in this field will translate more quickly into clinical application.”

Tonsil-Based Stem Cells To Repair the Liver

Byeongmoon Jeong and colleagues report in the journal ACS Applied Materials & Interfaces that injections of stem cells from tonsils, a body part we don’t need, can repair damaged livers without the need for surgery. The liver rids the body of toxins, makes blood proteins, and metabolizes a goodly number of molecules from our food. Liver failure is a deadly condition and a liver transplant is often the only option to restore the patient to health. Unfortunately there is a need for available organs for transplantation, Also, liver transplantation presents certain risks and also is extremely expensive.

A promising alternative to liver transplantation is the implantation of liver cells. Adult stem cells can be used to make new liver cells, and bone marrow-based stem cells have been used, but they these cells have inherent limitations. Recently, scientists have identified another stem cell source that can be used for this purpose from tonsils. Every year, thousands of tonsillectomies are performed to remove tonsils, and the extirpated tonsils are discarded. Now, however, these throw-away tissues could have a new purpose. Scientists have devised ways to grow tonsil-based stem cells on a three-dimensional scaffold that simulates living liver tissue.

Jeong’s team encapsulated tonsil-derived stem cells in a heat-sensitive liquid that solidifies into a gel at body temperature. To these cells ensconced in this gel, they added protein growth factors to stimulate the stem cells to differentiate into liver cells. The stem cells differentiated into liver cells, degraded the scaffold, and formed functioning liver cells. Jeong and others think that with a little tweaking, this procedure could potentially provide an injectable tissue engineering technique to treat liver disease without surgery.

See Seung-Jin Kim, Min Hee Park, Hyo Jung Moon, Jin Hye Park, Du Young Ko, Byeongmoon Jeong. Polypeptide Thermogels As a 3D Culture Scaffold for Hepatogenic Differentiation of Human Tonsil-derived Mesenchymal Stem Cells. ACS Applied Materials & Interfaces, 2014; 140905122318006 DOI:10.1021/am504652y.