Artifical Blood Vessels Made From Thermoplastic Polyurethane Polymers


Wherever we find some of the worse medical events – heart attacks, strokes, pulmonary embolisms, we find blocked blood vessels. Obstructed blood vessels are a lurking time bomb in our bodies and they usually have to be replaced. Blood vessel replacement requires cutting another blood vessel from another part of the body or the implantation of artificial vascular prostheses.

A new option might emerge in the future, however. Vienna University of Technology, in collaboration with the Vienna Medical University developed artificial blood vessels that were fabricated from specialized elastomer material that have excellent mechanical properties. After implantation, these artificial blood vessels are dissolved and replaced by the body’s own blood vessels. At the end of the healing process, natural, fully functional blood vessels are once again in place. The technique works quite well in tissue cultures systems, but now it has been shown to successfully regenerate blood vessels in laboratory animals, specifically rats.

Atherosclerotic vascular disorders, in which blood vessels are obstructed by cholesterol-filled plaques, are one of the most common causes of death in industrialized countries. Typically, patients are treated with a bypass operation, and for such procedures, blood vessels are extirpated from another part of the patient’s body and used to replace the damaged vessel. This creates a new wound and a new area of the body with less than optimal blood supply that must heal. Also, the transplanted vessel rarely has the properties necessary to thrive in its new location.

This new strategy to replace diseased blood vessels is the result of a fruitful collaboration between Vienna University of Technology (or TU Wien, which is short for Technische Universität Wien) and the Medical University of Vienna. Hopefully the success of this research will cause artificially manufactured vessels to be used more frequently in future.

To make an artificial blood vessel, the most important thing is to start with the right material. The material must be compatible with body tissue, and pliable enough to be formed into a small diameter tube that is not easily blocked by blood clots.

Extensive work at TU Wien has resulted in the development of new polymers. “These are so-called thermoplastic polyurethanes,” explains Robert Liska from the Institute of Applied Synthetic Chemistry of TU Wien.  “By selecting very specific molecular building blocks we have succeeded in synthesizing a polymer with the desired properties.”

In order to generate artificial blood vessels from their thermoplastic polyurethanes, TU Wien materials scientists spun polymer solutions in an electrical field. This allowed them to form very fine threads and that could be wound into a spool. “The wall of these artificial blood vessels is very similar to that of natural ones,” says Heinz Schima of the Medical University of Vienna. The thermoplastic polyurethanes form a polymer fabric that is slightly porous and allows a small amount of blood to leak through it. This also enriches the blood vessel wall with growth factors, which encourages the migration of endothelial progenitor cells. Martina Marchetti-Deschmann at TU Wien studied the interaction between the thermoplastic polyurethane material and blood by using spatially resolved mass spectrometry.

This new technology has already proven to successfully form functional blood vessels in rats. “The rats’ blood vessels were examined six months after insertion of the vascular prostheses,” says Helga Bergmeister of MedUni Vienna. “We did not find any aneurysms, thromboses or inflammation. Endogenous cells had colonized the vascular prostheses and turned the artificial constructs into natural body tissue.” In fact, the body’s own blood vessel-forming tissues re-grew significantly faster than expected, which shortened the degradation period of the plastic tubes and their replacement with the body’s own endothelial cells. TU Wein and the Medical University of Vienna are making further adaptations to the material.

A few more preclinical trials are necessary before the artificial blood vessels can be used in human clinical trials. However, based on the results so far, the research team is very confident that the new method will prove itself for use in humans in a few years’ time.

This project was recently awarded PRIZE prototype funding from Austria Wirtschaftsservice (AWS).

Stem Cell Treatments for Stroke: A Tale of Two Trials


Two different clinical trials that examined the efficacy of stem cell treatments after a stroke have yielded very different results.

“Stroke” refers to a serious medical condition that occurs when the blood supply to the brain is disrupted by blockage of or injury to blood vessels that supply the brain with blood. Strokes cause a loss of, or reduction in, brain function. There are two main types of strokes. Ischemic stroke accounts for about 80% of strokes. Ischemic strokes result from the cessation of blood flow to an area of the brain because of a blood clot. Hemorrhagic stroke occurs if there is a leakage of blood into the brain because a blood vessel has burst. The bleed into the brain increases the pressure on the brain and leads to brain damage.

In the two clinical trials discussed in this post, both treatments were designed to address ischemic strokes, which disrupt the blood supply to the brain and starve brain cells of oxygen, causing them to die. Brain scans of patients who have suffered from an ischemic stroke may reveal large areas of damaged brain tissue. People who have had a stroke may experience difficulties with speech and language, orientation and movement, or memory. Such problems can be permanent or temporary.

Any advances in the treatment of stroke are particularly Currently, the only available treatment is to administer anti-clotting agents to dissolve the clot that has blocked the blood flow to the brain. Unfortunately, this treatment must be provided early and only a small proportion of patients get to hospital in time to be treated in this way.

There are no existing treatments for an ischemic stroke beyond the initial acute phase. However, rehabilitation can alleviate the disabilities caused by a stroke.

The European Stroke Organization released the results of large clinical trials on the treatment of strokes with CTX0E03 human neural stem cells. The PISCES trial, as it is known, is a phase I trial, and such trials usually involve giving a small number of people a new treatment to see if it is safe. Phase I trials are not designed to test if the treatment is effective, so any positive results from a Phase I trial should be treated with some caution.

This study examined the safety and tolerability of a stem cell therapy called ReN001 in the treatment of ischemic stroke.

11 males with long-term disability between 6 and 60 months after a stroke. None of the patients showed any cell-related or immunological adverse events. Patients did show sustained reductions in neurological impairment and spasticity compared to their stable pre-treatment baseline performance.

This clinical trial is a win for ReNeuron, the company that developed, makes and markets, ReN001. A Phase II is being planned.

A second clinical trial examined the efficacy of Athersys, Inc. MultiStem treatment for ischemic stroke. This phase II trial was designed to evaluate the efficacy of the product in stroke patients. 65 patients were treated with MultiStem and 61 were given the placebo. Unfortunately, even though the MultiStem treatment was safe as well tolerated, the cell therapy did not produce any statistically significant differences at 90 days in patients compared with a placebo.

Even though the data was disappointing, a second look at the data showed something interesting. When the 27 stroke patients who had received the MultiStem treatment 24-26 hours after the stroke were compared with all the other patients, it became clear that these patients did the best. Therefore, this trial seems to indicate that the window of treatment for MultiStem after a stroke is 24-36 hours and after 36 hours it works no better than placebo.

“Unfortunately, we just didn’t have the window right for this study,” Athersys Chief Operating Officer William Lehmann Reuters News Service. “We believe investors should see this as a sign that MultiStem works.”

The MultiStem treatment was also associated with lower rates of mortality and life threatening adverse effects, infections and lung-related events. Nine patients who had received the placebo died during the 90 day period (14.8%) while only four who received the MultiStem treatment died (6.2%).

The CEO of Athersys, Gil Van Bokkelen, said: “While the trial did not achieve the primary or component secondary endpoints, we believe the evidence indicating that patients who received MultiStem treatment early appeared to exhibit meaningfully better recovery is important and promising.”

This randomized, double-blind, placebo-controlled Phase 2 study is being conducted at sites in the United States and the United Kingdom.

Fat-Based Stem Cells Speed the Healing of Bed Sores in Animals


Pressure ulcers, which are also knows as bedsores (or decubitus ulcers) are localized injuries to the skin that can also include the underlying tissue that usually occur as a result of pressure, or pressure in combination with rubbing or friction. They tend to occur some sort of bony prominence such as elbows, hips, shoulders, ankles, back of the head, and other such places. More than 2.5 million patients each year in the U.S. require treatment for pressure ulcers, and the elderly are at particularly high risk for these lesions. Currently, therapies for pressure ulcers consist of conservative medical management for shallow lesions and aggressive debridement and surgery for deeper lesions.

Jeffery Gimble and his colleagues from the Tulane University School of Medicine in New Orleans, Louisiana, used a mouse model for pressure ulcers to test the ability of fat-derived stromal/stem cell treatment to accelerate and enhance the healing of pressure ulcers.  The dorsal skin of both young (2 months old) and old (20 months old) C57BL/6J female mice was pinched between external magnets for 12 hours over 2 consecutive days. This treatment initiated a pressure ulcer, and one day after induction of the pressure ulcers, some of these mice were injected with fat-derived stromal stem cells that had been isolated from healthy mice that were of the same genetic lineage as the injured mice. However, the donor mice were genetically engineered to express a green fluorescent protein in all their tissues. Other mice were treated with injections of saline-treated controls.

The mice that were injected with fat-derived stromal/stem cells displayed a cell-concentration-dependent acceleration of wound closure. The cell-injected mice also showed improved epidermal/dermal architecture, increased fat deposition, and reduced inflammation at the sites of injury. Interestingly, these fat-derived stem cell-induced improvements occurred in both young and elderly mice. However, the gene expression profile of genes involved in the making of blood vessels, regulating the immune system, and tissue repair differed according to the age of the mice, with younger mice making more of these genes that their older counterparts. These results are consistent with clinical reports of the improved skin architecture after fat grafting in patients with thermal injuries.

This current proof-of-principle study sets the stage for clinical translation of the transplantation of fat-based stem cells as a treatment of pressure ulcers.

MS Patients in Phase 1 Stem Cell Trial Show Improvement


Phase 1 clinical studies are designed to determine the proper dosage of an agent and to assess the safety of a drug. Phase 1 studies are not designed to determine if the patients who take the drug or agent benefit from it. Therefore, it is highly gratifying to see a medical agent produce distinct improvements in a phase 1 study.

The Tisch MS Research Center of New York (Tisch MSRCNY) has announced in an April 23rd press release that patients enrolled in their FDA-approved Phase I trial using autologous neural stem cells in the treatment of multiple sclerosis (MS) show significant improvements. These results were presented during the Multiple Sclerosis Highlights in the Field session at the 67th American Academy of Neurology (AAN) Annual Meeting in Washington, D.C.

MS is a chronic autoimmune disease of the central nervous system caused by attacks against the myelin sheath by the patient’s own immune system. The destruction of the myelin sheath causes systemic neurodegeneration. MS affects more than 2.3 million people worldwide.

In its interim analysis of their data, Tisch MSRCNY researchers reported that six of the nine patients treated with stem cells show increased motor strength, improved bladder function and an enhanced quality of life. Significantly, these treatments are well tolerated and, to date, no serious adverse events were reported.

“This preliminary data is encouraging because in addition to helping establish safety and tolerability, the trial is yielding some positive therapeutic results even at this early stage,” said Dr. Saud A. Sadiq, Chief Research Scientist at Tisch MSRCNY and the study’s principal investigator. Sadiq cautioned that these results result from an interim analysis and definitive conclusions will only be made upon completion of the trial.

The Tisch MSRCNY study investigates a pioneering regenerative strategy that utilizes stem cells harvested from the patient’s own bone marrow.  Specifically, a special stem cell population called “MSC-NPs” or mesenchymal stem cell-derived neural progenitors are isolated from bone marrow and used in this clinical trial.  MSC-NPs represent a neural subpopulation of bone marrow-derived MSCs with reduced mesodermal pluripotency and minimized risk of ectopic differentiation.  In preclinical studies in laboratory mice afflicted with “experimental autoimmune encephalomyelitis” (an excellent model system for MS), Tisch MSRCNY scientists established that three doses of MSC-NPs delivered intrathecally (IT) resulted in improved neurological function associated with suppression of local inflammatory response and trophic support for damaged cells at lesion sites.

Once the MSC-NPs were isolated from the patient’s bone marrow stem cell, they were injected intrathecally, that is, into the cerebrospinal fluid surrounding the spinal cord, in 20 participants who meet the inclusion criteria for the trial. This is an open label safety and tolerability study, which means that both the physicians and patients know what treatments that are giving and receiving in contrast to blinded studies. All clinical activities in this study will be are conducted at Tisch MS Research Center of New York or at affiliated International Multiple Sclerosis Management Practiced. The interim analysis reports on the first nine patients who received at least one treatment of stem cells.

Study patient Vicky Gill, a married mother of two whose husband, Michael, also has MS, has already experienced the positive benefits of the therapy. “For the past six years, I would fall frequently, had very limited movement in my legs and always walked with my cane. After just two stem cell treatments, I am now gaining sensation and function I thought was totally lost, have not had any recent falls and at times don’t need a cane at all.”

The patients in this trial will receive three rounds of injections at three-month intervals. Safety and efficacy parameters will be evaluated in all patients through regular follow-up visits. Dr. Sadiq plans to continue and complete the Phase I study and if these positive trends continue, move on to a multi-center Phase II efficacy trial.

Cardio3 BioSciences Announces First Patient Enrollment in New CART Therapy Trial


The European cell therapy company Cardio3 BioSciences (C3BS) announced the enrollment of the first patient in its Phase I clinical trial to evaluate the Company’s lead CAR T-Cell therapy. This CART cell therapy is called “NKG2D CAR T-Cell” and will be tested in blood cancer patients with acute myeloid leukemia (AML) or multiple myeloma (MM). In the coming days T lymphocytes will be isolated from patients’ peripheral blood, cultured and genetically engineered to express the chimeric antigen receptor. Then these NKG2D CAR T-Cells will be infused into the patients.

NKG2D CAR T-Cells express a chimeric antigen receptor that was constructed by using the native sequence of non-engineered natural killer cell (NK cell) receptors. This receptor has the ability to target a broad range of solid tumors and blood cancers by targeting specific molecules present on cell surfaces of numerous types cancers. NKG2D CAR T-Cell is a potential new treatment option for patients with solid tumors such as breast, colorectal, lung, liver, ovarian and bladder cancer, in addition to the blood cancers targeted in this trial. The concepts that undergird this clinical trial were discovered at Dartmouth College by Professor Charles Sentman, and has been published in numerous peer-reviewed publications such as Journal of Immunology, Cancer Research and Blood.

NKG2D CAR T-Cell received an Investigational New Drug (IND) clearance, under the name CM-CS1, from the U.S. Food and Drug Administration (FDA) in July 2014 for the Phase I clinical trial in blood-borne cancers.

Dr. Christian Homsy, CEO of Cardio3 BioSciences, said: “We are extremely pleased to initiate enrollment of the first Phase I trial of our CAR T-Cell therapy program with lead product candidate NKG2D CAR T-Cell, in-line with our previously disclosed clinical development plan. As AML and MM are two underserved blood cancer subtypes, there is a clear need for new, viable treatment options. To date, NKG2D CAR T-Cell therapies have demonstrated the prevention of tumor development and increased survival in preclinical animal models, suggesting that NKG2D CAR T-Cell has the potential to be one such therapy.”

Cardio3 BioSciences expects to complete the study in mid-2016 and will provide updates as the trial advances. Because it is a Phase I trial, it will assess the safety and feasibility of NKG2D CAR T-Cell as primary endpoints, with secondary endpoints including clinical effectiveness. If the trial is successful, however, it might provide alternative therapies for patients with a variety of cancers.

Making Purkinje Cells in a Culture Dish


The beating heart is functionally divided into two levels; an upper set of chamber and a lower set of chambers.  The heart beat originates in upper chambers, but that signal to beat must be relayed to the lower chambers of the heart.  However does this signal get to the bottom of the heart?  The answer is that there is an extension cord that relays the signal to beat from the top of the heart to the lower chambers of the heart.  This extension cord comes in the form of a conduction system that consists of modified cells that do not contract, but conduct electrochemical signals from the upper chambers of the heart to the lower chambers.

conduction_system_of_the_heart1322840773280

The beat originates in the upper part of the left atrium (upper chamber) in the so-called pacemaker or sinoatrial node.  The signal to beat spreads rapidly across the atrial tissue and the a transmission node called the atrioventricular node at the bottom of the heart.  Once the signal to beat gets to the atrioventricular node, that signal goes to the conductive tissues in the septum of the heart called the “bundle of His” or atrioventricular bundle.  From there, the signal splits into the left and right bundle branches that swing around bottom of the heart into the ventricle walls.  Tiny extensions of the conduction system called “purkinje fibers” move into the walls of the ventricles.  These purkinje fibers function to control cardiac action potentials essential for a consistent heartbeat.

Several studies suggest that dysfunctional purkinje fibers are a potential source of arrhythmias in several heart syndromes.  However, how purkinje fiber dysfunction is responsible for causing arrhythmias has not been fully studied.  In order to begin this studying the role of purkinje fibers in arrhythmias, the laboratory of Glenn I. Fishman (New York University School of Medicine, USA) has generated an engineered mouse embryonic stem cell (ESC) line which can generate huge numbers of purkinje fiber cells.  Normally, when embryonic stem cells are differentiated into heart muscle cells (cardiomyocytes), purkinje cells constitute a very minor, even rare cell sub‐population (see Maass K, Shekhar A, Lu J, et al. Isolation and characterization of embryonic stem cell-derived cardiac purkinje cells. Stem Cells 2015;33:1102-1112).

According to previous expression studies, Fishman and others utilized ESCs that expressed Green Fluorescent Protein (GFP) under the control of the Cntn2 promoter.  The Cntn2 gene encodes the Contacting-2 protein, which marks those cells that will differentiate into Purkinje fiber conduction network cells (Pallante BA, et al. Circ Arrhythm Electrophysiol 2010;3:186-194;Kim EE, et al. The Journal of clinical investigation 2014;124:5027-5036).  Cntn2, however is not a perfect marker because it is also expressed in certain neuronal cells.  Therefore an additional marker was used; “MHCα‐mCherry.”  MHCα‐mCherry expressed a very brightly colored protein under the control of the myosin heavy chain gene promoter.  Because the alpha-myosin heavy chain is a heart muscle-specific protein, this brightly-colored protein is only expressed in heart-specific cells. Any cells that express both the Cntn2-GFP and the MHCα‐mCherry are almost certainly purkinje fiber cells.

Fishman and his team differentiated mouse ESCs into purkinje fiber cells and characterized the parallel activation of αMHC and Cntn2 in the developing murine heart.  ESC-derived purkinje fibers made up around 2% of the cell population at 4 weeks, and appeared long and pointy.  They also expressed a range of proteins similar to that of endogenous purkinje fiber cells, such as Cntn2, Troponin T (in a sarcomeric pattern, no less), and the conduction‐system specific connexin40 gap junctional protein.  Further analysis demonstrated the heightened expression of many genes associated with the cardiac conduction system, such as Nkx2‐5, Connexin40, HCN4, CACNA1G, Scn5a, and SCN10A.  The use of patch-clamping showed that these cultured cells  had similar electrophysiological properties to that of endogenous PCs; a highly important characteristic.

In combination with ESC-derived sinoatrial cells (see Scavone A, et al. Circulation research 2013;113:389-398), pacemaker cells (see Morikawa K, et al. Pacing Clin Electrophysiol 2010;33:290-303), and atrial‐like cardiomyocytes (Josowitz R, et al. PLoS One 2014;9:e101316), the creation of PC cells in this study may represent an extremely exciting step towards cell therapy for the failing heart. These data represent a useful strategy for the production of a large amount of a useful cell type from a heterogeneous cardiac cell population, which may be used to inform on diverse study areas including developmental biology, disease pathogenesis and anti‐arrhythmic drug screening. The authors themselves hope that using patient-specific fibroblasts and a direct reprogramming process, PCs may be used to treat heritable, acquired and post‐surgical damage to the heart’s conduction system in a patient-tailored manner.

Using Cord Blood Stem Cells to “Re-educate” White Blood Cells and Treat Hair Loss


Alopecia areata (AA) is an autoimmune disease that targets the hair follicles. It affects the quality of life and self-esteem of patients because they lose their hair. Is there a way to treat this disease without suppressing the immune system?

Yong Zhao and from Tianhe Stem Cell Biotechnologies in Shandong, China and his collaborators used a so-called “Stem Cell Educator therapy” in which they took the patient’s blood and circulated it through a closed-loop system that separated mononuclear cells from the whole blood, and then allowed those cells to briefly interact with adherent human cord blood-derived multipotent stem cells (CB-SC). After this interaction, the mononuclear cells were returned to the patient’s circulation. This procedure uses the cord blood cells to “educate” the white blood cells of the patient to not attack the patient’s hair follicles.

In an open-label, phase 1/phase 2 study, nine patients with severe AA received one treatment with the Stem Cell Educator therapy. These patients were about 20 years old and had lost their hair, on the average, about 5 years ago.

All these patients experienced improved hair regrowth and quality of life after receiving Stem Cell Educator therapy.  Furthermore, analyses of immune cells from the blood of treated patients showed that the types of immune cells that attack tissues decreased and the number of cells that regulate the immune response increased. Also, investigations of hair follicles in the treated patients revealed that the restored hair follicles expressed a ring of transforming growth factor beta 1 (TGF-β1) around the hair follicles. TGF-β1 is a secreted molecule that down-regulates the immune response and prevents immune cells from attacking your own tissue. The fact that the hair follicles secreted all this TGF-β1 shows that the restored hair follicles had steeled them against the immune system.

How did the cord blood cells do this? By culturing white blood cells with cord blood cells in cell culture, Zhao and others showed that the human cord blood-derived multipotent stem cells induced white blood cells to increase their expression of molecules that are known to tame self-destructive white blood cells. Thus the cord blood stem cells secrete regulatory molecules that change the character of the immune cells so that they no longer attack the hair follicles.

These clinical data demonstrate the safety and efficacy of the Stem Cell Educator therapy for the treatment of AA. This is a very innovative approach that can produce lasting improvement in hair regrowth in subjects with moderate or severe AA.

UTMB Galveston Physicians Build Lungs in Laboratory


As a bioreactor bubbles and whirls a pair of living lungs slowly takes shape. Is this a scene from Shelly’s Frankenstein? No. Instead it is an everyday occurrence in a laboratory at the University of Texas Medical Branch (UTMB) Galveston National Laboratory. The star of this “lung show” is a little pig named “Harry.” Little Harry has the distinction of being the first patient to be surgically implanted with a laboratory-built lung, and both the doctor and patient are doing just fine.

Dr. Joan Nichols of UTMB’s Galveston National Laboratory put it this way: “We build lungs here.” Nichols continued: “That’s pretty much what it’s become in the last six months or so, is a little factory to build lungs.”

The lung is a uniquely sculpted organ. Therefore, UTMB medical researchers required a pattern or scaffold upon which they could build lungs. They began with lungs acquired from dead animals and humans and spent more than a year perfecting protocols to isolate the lungs without damaging them and then remove all the cells. After the decellularization process, Nichols and her group were left with nothing but the elastic protein structure that serves as the skeleton of the lung. These lung skeletons were then incubated in a bioreactor that constant bathes the lung tissue in fresh culture medium and oxygen with lung cells extracted from living creatures. The lungs cells adhered to the lung skeleton and grew until they thoroughly covered it to create a new pair of lungs.

Bioengineered lungs
Bioengineered Lungs in a Bioreactor

 

Dr. Joaquin Cortiella, the director of UTMB’s Laboratory of Regenerative and Nano Medicine, likened this entire process to engineering a building: “You basically have a scaffold and then you build on top of that to create the building.”

Nichols and her colleagues have used this procedure to create both animal and human lungs. A freshly minted set of pig lungs were transplanted into Harry the pig. Harry’s healthy recovery from lung transplantation surgery indicates that this procedure experiment and potentially opens up the prospect of implanting new laboratory-built lungs into people.

“It’s the first time it’s ever been done, where we’ve taken a lung and it’s inside of the lung cavity of this pig,” Cortiella said.

Bioengineered lungs could vastly expand the number of organs available for those patients who require transplants, and this is especially the case in children, according to Cortiella. Cortiella’s experience in pediatric medicine is his main motivation for taking on this research project; he sometimes had to watch babies die from lung ailments.

“I also have lung disease,” Cortiella said. “I have pulmonary fibrosis. Breathing is a difficult thing for me at times. And so, for me, I appreciate the fact that there are not enough lungs out there to give to everybody who needs them.

“And so, if we develop something that can actually be tailor-made for somebody – or at least, have something available that we can transplant into people that are on the waiting list – the less people will die waiting for them,” he said.

Harry the pig is doing well, but in order to get a fuller picture of how well the bioengineered lung interacted with the rest of the Harry’s body, he will have to be euthanized for more detailed tissue examinations.

Previously, research groups have attempted to use synthetic materials instead of lung skeletons derived from living lungs. Unfortunately, none of the synthetic materials that were tried provided adequate structural support to make a living lung. Nichols thinks that doctors may eventually be able to make replacement organs with 3-D printers.

“Someday?” she said. “Someday, we are going to use these techniques to bio-engineer organs for people that need them.”

Investigational “CART” Cells, A Personalized Cellular Cancer Therapy is Well Tolerated By Patients


Chimeric Antigen Receptor T cells or CART cells are genetically modified versions of a patients’ own immune cells that expressed molecules that specifically bind tumor cells and mark them for destruction.  A host of animal experiments have demonstrated the safety and effectiveness of CART cells for treating tumors, but getting a therapy to work in animals is different than getting it to work in human patients.

CAR-Engineered_T-Cell_Adoptive_Transfer

Thus, the recent news that patients treated with CART cells made from their own T cells are tolerating them well is very welcome news.  Equally welcome is the news that the infused CART cells successfully traveled to those tumors they were designed to attack in an early-stage trial for mesothelioma and pancreatic and ovarian cancers at the Perelman School of Medicine at the University of Pennsylvania. Data from these trials adds to an already growing body of research that shows that CAR T cell technology shows remarkable promise for fighting tumors.  These interim results will be presented at the American Association for Cancer Research (AACR) Annual Meeting 2015, April 18-22.

“The goal of this phase I trial was to study the safety and feasibility of CART-meso cells in patients with mesothelin-expressing tumors,” says Janos L. Tanyi, MD, PhD, an assistant professor of Gynecologic Oncology. “We found no major adverse events associated with the treatment, which suggests that the patients tolerated it very well. But importantly, the T cells successfully targeted the patients’ tumor sites and survived in the blood stream for up to 28 days.”

The data that Tanyi will present at this conference will consist of scans and measurements acquired from five different patients; two of whom are suffering from ovarian cancer, two who have epithelial mesothelioma, and one with pancreatic cancer.  All five patients agreed to received the new investigational CART cell therapy.  Significantly, all the patients who received this therapy had cancers that stopped responding to conventional treatments.

CAR T cells are made from each patient’s T lymphocytes that are extracted from blood by a process known as “apheresis.”  T lymphocytes are isolated from the blood cells by cell sorting and then genetically modified to secrete a special protein that identifies and attacks tumor cells.  In this case, the cells were genetically engineered to target those cancer cells that express a protein called Mesothelin on their surfaces.  The engineered protein secreted by the engineered T cells could identify and kill them the tumor cells.  Even though Mesothelin is also found on the surfaces of the pleura (membranes that surround the lungs), the peritoneum (the lining that surrounds the abdominal cavity), and the pericardium (the scar that surrounds the heart),a variety of tumors express Mesothelin at such high levels that they are much more likely to be attacked by the CAR T cells that the normal tissues.

The preliminary results suggest the T cells did not attack normal tissues, but these patients must be followed up annually for 15 years in order to more closely observe the persistence of the CART-meso cells, their potential antitumor activity, and to better characterize their safety profiles.  Because the CAR T cells to not last indefinitely in the bloodstream, their ability to attack normal tissue should, theoretically at least, be minimal.

3D Printer Makes the Tiniest Human Organs Ever Made


Through the use of 3-D printers, mini human organs can be made in all kinds of shapes and sizes. A new experiment by tissue engineers from Wake Forest University has made tiny beating hearts that beat in sync, and another pulsing heart that fused with a spherical, liver.

These printed, mini-organs were by Anthony Atala and his team at the Wake Forest Institute for Regenerative Medicine in Winston-Salem, North Carolina. They represent the first step in developing an entire human body on a chip. The mini-hearts were made by reprogramming human skin cells into heart cells, which were then clumped together in a cell culture. A 3-D printer was then used to give them the desired shape and size, which in this case was a sphere of tissue with a diameter of 0.25 millimeters.

The development of these miniature organs was motivated by a desire to make model systems that mimic the function of life-size organs. Eventually, such a system could create mini-organs that could be linked up to form an entire organ system that could be used to test new treatments or probe the effects of chemicals and viruses.

The production of these mini-organs could potentially serve an alternative to animal testing, which is usually rather costly and doesn’t always produce results that are applicable to humans.

Further work on these mini-organs could also discover ways to expand these organs and make them life-size that they can be used for organ transplants.

3-D Printing to Make Replacement Body Parts


Advances in three-dimensional (3D) printing have produced a swell of interest in artificial organs that are designed to replace, or even enhance, human tissues.

3-D printed organs

At the Inside 3D Printing conference in New York on April 15–17, 2015, researchers from academia and industry are gathering to discuss the growing interest in using three-dimensional (3D) printing to make replacement body parts. Although surgeons are already using 3D-printed metal and plastic implants to replace bones, researchers are looking ahead to printing organs using cells as “ink.”  All the structures shown here were all 3D printed at Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, and include a rudimentary proto-kidney (top left), complete with living cells.

Printed organs, such as a prototype outer ear that was developed by researchers at Princeton University in New Jersey and Johns Hopkins University in Baltimore, Maryland, will be featured at the conference.  This ear is printed from a range of materials: a hydrogel to form an ear-shaped scaffold, cells that will grow to form cartilage, and silver nanoparticles to form an antenna (see M.S. Mannoor et al. Nano Lett. 13, 2634−2639; 2013. This is just one example of the increasing versatility of 3D printing.

This New York meeting, which is being advertised as the largest event in the industry, will provide exposure for a whole world of devices and novelties. But it will also feature serious discussions on the emerging market for printed body parts.

The dream of bioprinting is to print organs that can be used for transplant. For example, at the Wake Forest Baptist Medical Center in Winston-Salem, North Carolina, researchers are developing a 3D-printed kidney. The project is in its early stages and the kidney is far from functional and some doubt that researchers will ever be able to print such a complex organ. Perhaps a more achievable near-term goal might be to print sheets of kidney tissue that could be grafted onto existing kidneys.

Printed replacement for skull

Printed structures made of hard metal or polymers are already on the market for people in need of an artificial hip, finger bone or facial reconstruction. This skull implant (grey) was made by Oxford Performance Materials of South Windsor, Connecticut, and was approved by US regulators in 2013. It is made of a polymer meant to encourage bone growth, to aid integration of the implant into the surrounding skeleton. The company also sells implants for facial reconstruction and for replacing small bones in the feet and hands.

3-D printed lung tree

One of the key advantages of using 3D printing for surgical implants is the opportunity to model the implant to fit the patient. This airway splint (shown on the right branch of the model trachea) was designed by researchers at the University of Michigan in Ann Arbor to fit an infant with a damaged airway. The splint was made out of a material that is gradually absorbed by the body as the airway heals. The research team benefited from the concentration of 3D-printing expertise that has built up in Michigan because of the US automobile industry, which uses the technology for printing prototypes and design samples.

The business of 3-D printing also includes titanium replacement hip joints, which can be tailored to fit individual people, and made-to-order polymer bones to reconstruct damaged skulls and fingers. Printed body parts brought in US $537 million last year, up about 30% on the previous year, says Terry Wohlers, president of Wohlers Associates, a business consultancy firm in Fort Collins, Colorado, that specializes in 3D printing.

3-D printed prosthetics

3D printing can also be used to generate cheap — and creative — prostheses.  A prosthetic hand can cost thousands of dollars, which is a burdensome expense when fitting it to a growing child.  Jon Schull founded a company called e-NABLE that provides free printed prosthetics to those in need, harnessing the efforts of hundreds of volunteers who own consumer-grade 3D printers. “When people get tired of printing Star Wars figurines, they give us a call,” he says.  The cost of materials for a printed prosthesis is about US $35.

3-D animal prosthesis

Also, 3-D printed prostheses are not just for humans.  For example, a duck named Buttercup was born with its left foot turned backwards.  The Feathered Angels Waterfowl Sanctuary in Arlington, Tennessee, arranged for the fowl to receive a new foot, complete with a bendable ankle.  Also in the an eagle, a box turtle and a handful of dogs also have been fitted with 3-D printed prostheses.

Scientists are looking ahead to radical emerging technologies that use live cells as ‘ink’, assembling them layer-by-layer into rudimentary tissues, says Jennifer Lewis, a bioengineer at Harvard University in Cambridge, Massachusetts. Bioprinting firm Organovo of San Diego, California, already sells such tissues to researchers aiming to test experimental drugs for toxicity to liver cells. The company’s next step will be to provide printed tissue patches to repair damaged livers in humans, says Organovo’s chief executive, Keith Murphy.

Lewis hesitates to say that 3D printing will ever yield whole organs to relieve the shortage of kidneys and livers available for transplant. “I would love for that to be true,” she says. “But these are highly complicated architectures.”

Induced Pluripotent Stem Cells from Bone Cancer Patients Provide Crucial Insights into the Genesis of Bone Cancer


A team of Mount Sinai researchers have utilized induced pluripotent stem cells (iPSCs) to elucidate the genetic changes that seem to convert a well-known anti-cancer signaling gene into a driver bone cancers. When it comes to bone cancers, the survival rate has not improved in 40 years despite advances in treatment. Since this study might provide new targets and suggest new strategies for attacking such cancers. it represents a welcome addition to the cancer literature.

This study, which was published in the journal Cell, revolves around iPSCs, which were discovered in 2006 by Nobel laureate Shinya Yamanaka. iPSCs use genetic engineering and cell culture techniques to reprogram mature, adult cells to become like embryonic stem cells. These iPSCs are “pluripotent,” which means that they are able to differentiate into any adult cell type and can also divide in culture indefinitely.

For therapeutic purposes, iPSCs can be derived from a patient’s own cells, differentiated into the cells the patient needs to be replaced, and then implanted into the patient’s body to augment tissue healing or even organ reconstruction. Since iPSCs can be successfully differentiated into heart muscle, nerve cells, bone, and other cell types, they have the potential advance the field of regenerative medicine by leaps and bounds.

iPSCs have already made their presence known in the clinic by serving as model systems for research and diagnosis. The new Mount Sinai study used iPSCs to construct an accurate model of a genetic disease “in a dish.” The culture dishes contain self-renewing patient-specific iPSCs or a specific cell line that enable in-depth study diseases that are driven by each person’s genetic differences. When matched with patient records, iPSCs and iPSC-derived target cells have the ability to help physicians predict a patient’s prognosis and whether or not a given drug will be effective for him or her.

In this study, skin cells from healthy patients and patients with a genetic disease called Li-Fraumeni syndrome were isolated and reprogrammed into patient-specific iPSC lines. These iPSCs were then differentiated into bone-making cells (osteoblasts), which are the cells where particular rare and common bone cancers start. Li-Fraumeni syndrome greatly predisposes patients to a variety of cancers in several different types of tissues.

The patient-derived osteoblasts were then tested for their tendencies to become tumor cells and to make bone. This particular bone cancer model did a better job of recapitulating the characteristics of bone cancer than previously used mouse or cellular models.

LFS iPSCs for stem cell production

“Our study is among the first to use induced pluripotent stem cells as the foundation of a model for cancer,” said lead author Dung-Fang Lee, PhD, a postdoctoral fellow in the Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai. “This model, when combined with a rare genetic disease, revealed for the first time how a protein known to prevent tumor growth in most cases, p53, may instead drive bone cancer when genetic changes cause too much of it to be made in the wrong place.”

The Mount Sinai disease model research uses a simple fact of human life as its basis: human genes undergo mutations at a certain rate that tends to increase as we age, and the formation of new mutations in relentless and constant. Some mutations make no difference, a few some confer advantages, and others cause disease. Beyond inherited mutations that contribute to cancer risk, the combination of random, accumulated DNA changes in our cells as we age also increase our cancer risk.

The current study focused on those genetic pathways involved in Li-Fraumeni Syndrome or LFS, a rare genetic disease that causes high risk for many cancers in affected families. Osteosarcoma (bone cancer) is a common cancer observed in LFS patients and many of them are diagnosed before the age of 30. Additionally, osteosarcoma is the most common type of bone cancer in all children, and after leukemia, the second leading cause of cancer death for them.

 

Importantly, about 70 percent of LFS families have a mutation in their copy of a genes called TP53, which encodes the p53 protein. P53 is a “the tumor suppressor,” which means that it functions to preserve the integrity of the genome and keep the rate of cell division in check. Common forms of osteosarcoma, which are driven by somatic or inherited mutations, have also been closely linked by past studies to defects in p53 when mutations interfere with the ability of the protein to function properly.

p53
Crystal Structure of p53 protein bound to DNA

 

Rare genetic diseases like LFS provide excellent model systems because they tend to result from a change in a single gene, instead of the diverse and overlapping mutations observed in common diseases, and, in this case, more common, non-inherited bone cancers. The LFS-iPSC based modeling highlights the contribution of p53 alone to osteosarcoma.

By analyzing iPSC lines, and bone cancer driven by p53 mutations in LFS patients, the Munt Sinai research team showed, for the first time, that the LFS bone cancer results from an overactive p53 gene. Too much p53 in osteoblasts dampens the function of a gene, H19, and a related protein, decorin, that would otherwise help stem cells differentiate into normal osteoblasts.

The inability of cells to differentiate makes them vulnerable to genetic mistakes that drive cancer, since more “stemness” means a tendency toward rapid, abnormal growth, like that observed in tumors. One tragic feature of osteosarcoma is the rapid, error-prone production of weaker bone by cancerous bone-making cells, where a young person surprisingly breaks a bone to reveal undiagnosed, advanced cancer.

Dung-Fang Lee and his colleagues discovered that the H19 gene seems to control a network of interconnected genes that fine-tune the balance between cell growth and resistance to growth. Decorin is a protein that is part of connective tissue like bone, but that also plays a signaling role, interacting with growth factors to slow the rate that cells divide and multiply, unless turned off by too much p53.

“Our experiments showed that restoring H19 expression hindered by too much p53 restored “protective differentiation” of osteoblasts to counter events of tumor growth early on in bone cancer,” said co-author, Ihor Lemischka, PhD, Director of The Black Family Stem Cell Institute within the Icahn School of Medicine. “The work has implications for the future treatment or prevention of LFS-associated osteosarcoma, and possibly for all forms of bone cancer driven by p53 mutations, with H19 and p53 established now as potential targets for future drugs.”

A New Way to Prepare Fat-Based Stem Cells to Treat Wounds


An Italian laboratory headed by Dr. Raposio at the University of Parma has designed a simple and fast technique for preparing fat-based stem cells for use in the clinic.

Fat contains an alternative source of mesenchymal stem cells with characteristics similar to those found in bone marrow, but the fat-based stem cells are easier to isolate and have been shown to be effective enhancers of wound healing.

Raposio and his colleagues used fat contributed by liposuction patients. Each patient provided about 80 cubic centimeters of fat in liposuction procedures that were collected under anesthesia. Once the cells from this fat were isolated, they were mixed with platelet-rich plasma (PRP) that had been previously collected. Mixing PRP with stem cells enhances the capabilities of the fat-based stem cells and generates a concoction called “e-PRP.”  This simple procedure that consisted of fat collection, stem cell collection and mixing the cells with PRP to make e-PRP quickly made a produce that was ready for grafting onto wounds on the skin.

Detailed analyses of the cells isolated from the fat showed that they consisted of about 50,000 fat-based mesenchymal stem cells or ASCs. They represented about 5% of all cells in the sample. The remaining cells were blood-derived cell and blood vessel-making endothelial cells.

The significance of this procedure lies in the fact that most of the protocols used to isolate stem cells from fat take about two hours and require animal-derived reagents. However, the number of ASCs isolated with this new procedure is sufficient for application to wounds without the need of expanding the cells in culture. Also, this new procedure does not require serum or animal-derived reagents, and it takes only 15 minutes.

Thus this method of ASC isolation is innovative, feasible, and represents an advance in the stem cell-based treatment of chronic wounds.

Amniotic Fluid Stem Cells Make Robust Blood Vessel Networks


The growth of new blood vessels in culture received in new boost from researchers at Rice University and Texas Children’s Hospital who used stem cells from amniotic fluid to promote the growth of robust, functional blood vessels in healing hydrogels.

These results were published in the Journal of Biomedical Materials Research Part A.

Engineer Jeffrey Jacot thinks that amniotic fluid stem cells are valuable for regenerative medicine because of their ability to differentiate into many other types of cells, including endothelial cells that form blood vessels. Amniotic fluid stem cells are taken from the discarded membranes in which babies are encased in before birth. Jacot and others combined these cells with an injectable hydrogel that acted as a scaffold.

In previous experiments, Jacot and his colleagues used amniotic fluid cells from pregnant women to help heal infants born with congenital heart defects. Amniotic fluids, drawn during standard tests, are generally discarded but show promise for implants made from a baby’s own genetically matched material.

“The main thing we’ve figured out is how to get a vascularized device: laboratory-grown tissue that is made entirely from amniotic fluid cells,” Jacot said. “We showed it’s possible to use only cells derived from amniotic fluid.”

Researchers from Rice, Texas Children’s Hospital and Baylor College of Medicine combined amniotic fluid stem cells with a hydrogel made from polyethylene glycol and fibrin. Fibrin is the proteins formed during blood clots, but it is also used for cellular-matrix interactions, wound healing and angiogenesis (the process by which new vessels are made). Fibrin is widely used as a bioscaffold but it suffers from low mechanical stiffness and is degraded rapidly in the body. When fibrin was combined with polyethylene glycol, the hydrogel became much more robust, according to Jacot.

Additionally, these groups used a growth factor called vascular endothelial growth factor to induce the stem cells to differentiate into endothelial cells. Furthermore, when induced in the presence of fibrin, these cells infiltrated the native vasculature from neighboring tissue to make additional blood vessels.

When mice were injected with fibrin-only hydrogels, thin fibril structures formed. However if those same hydrogels were infused with amniotic fluid stem cells that had been induced with vascular endothelial growth factor, the cell/fibrin hydrogel concoctions showed far more robust vasculature.

In similar experiments with hydrogels seeded with bone marrow-derived mesenchymal cells, once again, vascular growth was observed, but these vessels did not have the guarantee of a tissue match. Interestingly, seeding with endothelial cells didn’t work as well as the researchers expected, he said.

Jacot and others will continue to study the use of amniotic stem cells to build biocompatible patches for the hearts of infants born with birth defects and for other procedures.

Scientists Use Stem Cells to Grow Three-Dimensional Mini Lungs.


In research done in several laboratories, lung tissue was derived from flat cell culture systems or by growing cells on scaffolds made from donated organs.

Now in a new study published in the online journal eLife, a multi-institution team has defined a culture system for generating self-organizing human lung organoids, which are three-dimensional structures that mimic the structure and complexity of human lungs.

“These mini lungs can mimic the responses of real tissues and will be a good model to study how organs form, change with disease, and how they might respond to new drugs,” said study senior author Jason R. Spence, Ph.D., an assistant professor of internal medicine and cell and developmental biology at the University of Michigan Medical School.

Spence and his colleagues successfully grew structures that resembled both the large airways or bronchi and small lung sacs, known as alveoli.

These mini lung structures were developed in a cell culture system. Therefore, they lack several components of the human lung, including blood vessels, which are a critical component of gas exchange during breathing.

Despite that, these cultured organoids can serve as a unique research model system for researchers as they grind out basic science ideas that are turned into clinical innovations. These three-dimensional mini-lungs should be an excellent complement to research in liver laboratory animals.

Traditionally, the behavior of cells has been investigated in the laboratory in two-dimensional culture systems where cells are grown in thin layers on cell-culture dishes. Most cells in the body, however, exist in a three-dimensional environment as part of complex tissues and organs. Tissue engineered have been trying to re-create these environments in the laboratory by successfully generating small version of particular organs known as organoids, which serve as models of the stomach, brain, liver and human intestine. The advantage of growing three-dimensional structures of lung tissue, according to Dr. Spence, is that the organization of organoids bears greater similarity to the human lung.

To make these lung organoids, researchers at the U-M’s Spence Lab and colleagues from the University of California, San Francisco; Cincinnati Children’s Hospital Medical Center; Seattle Children’s Hospital and University of Washington, Seattle manipulated several of the cell signaling pathways that control the formation of organs.

First, stem cells were induced to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and several other internal organs. Second, the group activated two important development pathways (FGF and WNT signaling ) that are stimulate endoderm to form three-dimensional tissue. By inhibiting two other key development pathways at the same time (BMP and TGFβ signaling), the endoderm became tissue that resembles the early lung found in embryos.

In the laboratory, this early culture-derived lung-like tissue spontaneously formed three-dimensional spherical structures as it developed. Afterwards, they had to expand these structures and develop them into lung tissue. In order to do this, Spence and his colleagues and collaborators exposed the cells to additional proteins involved in lung development (FGF and Hedgehog).

After all this manipulation, the resulting lung organoids survived in the laboratory for over 100 days.

“We expected different cells types to form, but their organization into structures resembling human airways was a very exciting result,” said author Briana Dye, a graduate student in the U-M Department of Cell and Developmental Biology.

While this type of experiment is remarkable, this is only the beginning of lung tissue engineering.  These mini-lungs  will hopefully serve and new model systems for drug testing and researching genetic diseases that affect the lungs, such as cystic fibrosis, sarcoidosis, or inherited forms of emphysema.  It will be a while before scientists can make replacement lungs for human patients, but these experiments by Spence and others are a remarkable start.

A Patient’s Own Stem Cells Treats Their Crohn’s Disease


Stem cells isolated from the fat of patients with Crohn’s disease, an inflammatory disease of the bowel, relieved them from fistulas, which are a common, and potentially dangerous side effect of the disease. This is according to the results of a phase 2 clinical trial published in the latest issue of STEM CELLS Translational Medicine (SCTM).

Patients with Crohn’s disease suffer from a painful, chronic disease in which the body’s immune system attacks its own gastrointestinal tract. In Crohn’s patients, inflammation within the bowel can sometimes extend completely through the intestinal wall and create a what is known as a “fistula.”. Fistulas are abnormal connections between the intestine and another organ or even the skin. If left untreated, a fistula can become infected and form an abscess that can be life threatening.

Chang Sik Yu, M.D., Ph.D., of the Asan Medical Center in Seoul, Korea, who is a senior author of the SCTM paper, describes the results of a clinical trial that was conducted in collaboration with four other hospitals in South Korea. According to Dr. Yu: “Crohn’s fistula is one of the most distressing diseases as it decreases patient’s quality of life and frequently recurs. It has been reported to occur in up to 38 percent of Crohn’s patients and over the course of the disease, 10 to 18 percent of them must undergo a proctectomy, which is a surgical procedure to remove the rectum.”

Overall, the treatments currently available for Crohn’s fistula remain unsatisfactory because they fail to achieve complete closure, lower recurrence of the fistulas and do not limit adverse effects, Dr. Yu said. Given the challenges and unmet medical needs in Crohn’s fistula, attention has turned to stem cell therapy as a possible treatment.

Several studies, including those undertaken by Dr. Yu’s team, have shown that mesenchymal stem cells (MSCs) do indeed improve Crohn’s disease and Crohn’s fistula. Their phase II trial enrolled 43 patients for a term of one year, over the period from January 2010 to August 2012. These patients received injections of their own fat-based MSCs, and 82 percent of them experienced complete closure of fistula eight weeks after the final ASC injection. 75 percent of the trial participants remained fistula-free two years later.

“It strongly demonstrated MSCs derived from ASCs are a safe and useful therapeutic tool for the treatment of Crohn’s fistula,” Dr. Yu said.

The latest study was intended to evaluate the long-term outcome by following 41 of the original 43 patients for yet another year. Dr. Yu reported, “Our long-term follow-up found that one or two doses of autologous ASC therapy achieved complete closure of the fistulas in 75 percent of the patients at 24 months, and sustainable safety and efficacy of initial response in 83 percent. No adverse events related to ASC administration were observed. Furthermore, complete closure after initial treatment was well sustained.”

“These results strongly suggest that autologous ASCs may be a novel treatment option for Crohn’s fistulae,” he said.

“Stem cells derived from fat tissue are known to regulate the immune response, which may explain these successful long-term results treating Crohn’s fistulae with a high risk of recurrence,” said Anthony Atala, M.D., Editor-in-Chief of STEM CELLS Translational Medicine and director of the Wake Forest Institute for Regenerative Medicine.

Dead Heart Muscle Regrown in Rodents


If you cut a piece of tissue from the heart of a salamander or zebrafish, they wild simply grow new heart tissue. Unfortunately, humans are unable to easily regenerate heart cells, and this males it difficult to recover from the permanent damage caused by heart attacks.

Fortunately, life scientists from the Weizmann Institute of Science in Israel and the Victor Chang Institute in Sydney have discovered a way to stimulate heart muscle cells in mammals to grow. This finding could have major implications for future heart attack sufferers.

Even though human blood, hair and skin cells renew themselves throughout life, cell division in the heart comes to a virtual standstill shortly after birth, according to Prof. Richard Harvey, from the Victor Chang cardiac research institute, and one of the authors of this research. Harvey said, “So there’s always been an intense interest in the mechanism salamanders and fish use which makes them capable of heart regeneration, and one thing they do is send their cardiomyocytes, or muscle cells, into a dormant state, which they then come out of to go into a proliferative state, which means they start dividing rapidly and replacing lost cardiomyocytes.”

Harvey continued: “There are various theories why the human heart can not do that, one being that our more sophisticated immune system has come at a cost, and because human cardiomyocytes are in a deeper state of quiescence, that has made it very difficult to stimulate them to divide.”

Today, for the first time in history, more people in developing countries die from strokes and heart attacks than infectious diseases. Fortunately there are cost-effective ways to save lives

By studying mice, Harvey and his colleagues found a way to overcome that regenerative barrier – at least in the rodents.

Harvey and others found that by stimulating a cell signaling pathway in the heart that is driven by a hormone called neuregulin, heart muscle cells divided in a spectacular way in both adolescent and adult mice. In humans, neuregulin expression is usually muted about one week after birth, and by about 20 weeks after birth in mice.

By triggering of the neuregulin pathway following a heart attack in mice, Harvey and others induced the replacement of lost muscle, which repaired the heart to a level close to that prior to the heart attack. Harvey said that he and other scientists should be able to determine with in the next five years if it is possible to replicate these results in humans.

“This is such a significant finding that it will harness research activities in many labs around the world, and there will be much more attention now on how this neuregulin-response could be maximised,” Harvey said.

“We will now examine what else we can use, other than genes, to activate that pathway, and it could be that there are already drugs out there, used for other conditions and regarded as safe, that can trigger this response in humans.”

When one of the blood vessels that provide blood to the heart muscle becomes blocked, the patient suffers a heart attack. Heart attacks or “myocardial infractions” cause billions of cardiomyocytes to die. Even if you survive a heart attack, you usually experience diminished quality of life because of it.

“The dream is that one day we will be able to regenerate damaged heart tissue, much like a salamander can regrow a new limb if it is bitten off by a predator,” Harvey said.

Molecular biologist Gabriele D’Uva lead this research, which was published in the scientific journal Nature Cell Biology.

Mesenchymal Stem Cells from Neonatal Thymus Helps Make New Blood Vessels


The thymus is an organ that sits over the top of the heart and it plays a pivotal role in the development of T-lymphocytes. The thymus is a very durable organ that can readily regenerate if it is injured. This regenerative ability is largely due to it high level of vascularization (lots of blood vessels). This vascularization is due to a robust population of resident mesenchymal stem cells that supports blood vessel formation in the damaged thymus. The process of blood vessel formation is called “angiogenesis.” The angiogenic potential of these thymus-based mesenchymal stem cells might hold excellent potential for regenerative therapies.

Thymus_lg

As it turns out, neonatal surgeries tend to generate thymus tissue that is usually thrown out as medical waste. Ming-Sing Si from Mott’s Children Hospital in Ann Arbor, Michigan and colleagues isolated mesenchymal stem cells from these surgically-derived neonatal thymuses and tested their ability to stimulate blood vessels in an experimental setting.

Discarded thymus tissue was obtained from the University of Michigan, and this tissue was minced, degraded with enzymes, and cultured. The mesenchymal stem cells (MSCs) moved from the thymus tissue onto the culture dishes. These thymus-based MSCs grew like gangbusters in culture and could be passaged over 30 times.

Discarded human neonatal thymus tissue is a source of mesenchymal stromal cells (MSCs). (A): Discarded human neonatal thymus tissue during pediatric cardiac surgery. (B): Minced thymus tissue prior to plating. (C): Cells migrating from thymus tissue fragments during explant culture at 10 days. (D): Clonogenicity of thymus MSCs at 2 weeks (representative of 7 donors). (E): Colony-forming efficiency of thymus MSCs. (F): Averaged cumulative population doubling of thymus MSCs (n = 4) over 9 weeks of culture. Abbreviation: CFU-F, fibroblastic colony-forming unit.
Discarded human neonatal thymus tissue is a source of mesenchymal stromal cells (MSCs). (A): Discarded human neonatal thymus tissue during pediatric cardiac surgery. (B): Minced thymus tissue prior to plating. (C): Cells migrating from thymus tissue fragments during explant culture at 10 days. (D): Clonogenicity of thymus MSCs at 2 weeks (representative of 7 donors). (E): Colony-forming efficiency of thymus MSCs. (F): Averaged cumulative population doubling of thymus MSCs (n = 4) over 9 weeks of culture. Abbreviation: CFU-F, fibroblastic colony-forming unit.

When these thymus-based MSCs were combined with human umbilical vein endothelial cells, within one day, the cells formed an extensive network of blood vessels.

Thymus mesenchymal stromal cells (MSCs) cooperate with human umbilical vein endothelial cells (HUVECs) to form a network in a two-dimensional angiogenesis assay. (A): Monolayer appearance of HUVECs after 48 hours of culture on fibrin hydrogel. (B): Thymus MSCs clustered together after 24 hours of culture on fibrin hydrogel. (C): Combining HUVECs with thymus MSCs (2:1) resulted in the appearance of interconnected tubules at 24 hours. Scale bars = 100 μm. Results are representative of two independent experiments.
Thymus mesenchymal stromal cells (MSCs) cooperate with human umbilical vein endothelial cells (HUVECs) to form a network in a two-dimensional angiogenesis assay. (A): Monolayer appearance of HUVECs after 48 hours of culture on fibrin hydrogel. (B): Thymus MSCs clustered together after 24 hours of culture on fibrin hydrogel. (C): Combining HUVECs with thymus MSCs (2:1) resulted in the appearance of interconnected tubules at 24 hours. Scale bars = 100 μm. Results are representative of two independent experiments.

Gene expression studies showed that culturing thymus MSCs with human umbilical vein endothelial cells (HUVECs) caused the HUVECs to express a variety of blood vessel-specific genes.  These thymus-based MSCs were also able to induce blood vessels if the cells were wadded up into a ball (spheroids).

To top it all off, Si and others implanted thymus-based MSCs underneath the skin of nude mice.  They used hydrogels with no cells, hydrogels plus HUVECs, hydrogels plus thymus-based MSCs, and hydrogels with thymus-based MSCs plus HUVECs.  The control implants and the HUVEC implants showed no blood vessels.  HUVECs make very good blood vessels, but they have to be directed to do so.  Both the thymus-based MSCs and the MSCs plus HUVECs showed extensive integration into the host tissue with lots of blood vessels.

Thymus mesenchymal stromal cells (MSCs) incite angiogenesis in vivo. Fibrin constructs without spheroids (control) or with 500 spheroids with 600 human umbilical vein endothelial cells (HUVECs) per spheroid, 200 thymus MSCs per spheroid, or 600 HUVECs plus 200 thymus MSCs per spheroid were generated (n = 3 per group) and were implanted subcutaneously for 14 days in NOD-SCID mice. Explanted constructs were photographed (edges traced in A–D) and processed for histology. (A): Controls did not manifest local reaction. (B): HUVEC constructs appeared avascular. (C): Thymus MSC constructs were integrated and caused increased adjacent vascularization. (D): HUVEC plus thymus MSC constructs were integrated and surrounded by a host vascular response and appeared to have vessels within. (E–H): Construct hematoxylin and eosin staining. Scale bars = 50 μm. (E): Avascular tissue invasion of control construct. Scale bar = 100 μm. (F): HUVEC construct with adjacent cellularity and vascularity between panniculus carnosus muscle layer (∗) and construct. “Ghost” (†) of the prior locations of spheroid and necrotic spheroid (‡) were present in internal regions of all constructs with spheroids. (G): Thymus MSC construct with increased adjacent cellularity and vascularity. (H): HUVEC plus thymus MSC construct with increased vascularization within the construct. (I): Manual measurement of vessel density demonstrates significant differences by two-way analysis of variance. Control and HUVEC constructs had minimal adjacent vascularization. Thymus MSC constructs promoted the greatest adjacent response, whereas HUVEC plus thymus MSC constructs contained the greatest vessel density within the construct. (J, K): Immunohistochemical staining with human-specific CD31 monoclonal antibody revealed that only constructs with HUVEC plus thymus MSCs contained CD31-positive luminal structures with blood cells. Scale bar = 20 μm. Abbreviations: C, controls; H, human umbilical vein endothelial cell constructs; T, thymus mesenchymal stromal cell construct.
Thymus mesenchymal stromal cells (MSCs) incite angiogenesis in vivo. Fibrin constructs without spheroids (control) or with 500 spheroids with 600 human umbilical vein endothelial cells (HUVECs) per spheroid, 200 thymus MSCs per spheroid, or 600 HUVECs plus 200 thymus MSCs per spheroid were generated (n = 3 per group) and were implanted subcutaneously for 14 days in NOD-SCID mice. Explanted constructs were photographed (edges traced in A–D) and processed for histology. (A): Controls did not manifest local reaction. (B): HUVEC constructs appeared avascular. (C): Thymus MSC constructs were integrated and caused increased adjacent vascularization. (D): HUVEC plus thymus MSC constructs were integrated and surrounded by a host vascular response and appeared to have vessels within. (E–H): Construct hematoxylin and eosin staining. Scale bars = 50 μm. (E): Avascular tissue invasion of control construct. Scale bar = 100 μm. (F): HUVEC construct with adjacent cellularity and vascularity between panniculus carnosus muscle layer (∗) and construct. “Ghost” (†) of the prior locations of spheroid and necrotic spheroid (‡) were present in internal regions of all constructs with spheroids. (G): Thymus MSC construct with increased adjacent cellularity and vascularity. (H): HUVEC plus thymus MSC construct with increased vascularization within the construct. (I): Manual measurement of vessel density demonstrates significant differences by two-way analysis of variance. Control and HUVEC constructs had minimal adjacent vascularization. Thymus MSC constructs promoted the greatest adjacent response, whereas HUVEC plus thymus MSC constructs contained the greatest vessel density within the construct. (J, K): Immunohistochemical staining with human-specific CD31 monoclonal antibody revealed that only constructs with HUVEC plus thymus MSCs contained CD31-positive luminal structures with blood cells. Scale bar = 20 μm. Abbreviations: C, controls; H, human umbilical vein endothelial cell constructs; T, thymus mesenchymal stromal cell construct.

These MSCs show low expression of human leukocyte antigen class I, which, translated, means that these cells are unlikely to be recognized by the patient’s immune system.  Therefore, these cells could be donated to patients whose resident MSCs are of poor quality or do not have enough of their own MSCs for therapeutic processes.

This paper shows that discarded neonatal thymus contains large numbers of resident MSCs that can be isolated and cultured by a standard explant method.  These MSCs have all the characteristics of traditional MSCs, but have more robust growth characteristics in culture.  These thymus MSCs also possess outstanding proangiogenesis qualities that should be further tested and considered as promoters of tissue and organ regeneration in tissue engineering strategies.

Stem Cells from Fat Improve Blood Vessel Responses after Injury


When tissues are injured, the blood vessels that feed them are often shocked and damaged as well. “Vasoactivity” refers the ability of blood vessels to dilate or constrict. When tissues are harmed, blood vessels tend to shrink in order to squelch blood loss at the site of damage. This same response, however, and deprive the damaged tissues of much-needed oxygen and lead to “ischemia,” which is the insufficient supply of blood and oxygen to an organ.

James B. Hoying and his colleagues at the University of Louisville in Kentucky used the “stromal vascular fraction” or SVF from fat in order to treat damaged blood vessels to determine if they could mitigate the decrease in vasoactivity as a result of injury.

The SVF refers to the stem fraction from fat after the fat has been minced, digested with enzymes, and centrifuged (it’s more complicated than that, but this is a short summary). The cells that remain include mesenchymal stromal cells, growth factors, immune cells, pre-fat cells and fat cells, blood-cell-making stem cells, and blood vessel-making cells (endothelial cells). The SVF, therefore, contains a cocktail of cell types and growth factors that are available for regenerative medicine.

Hoying and his team discovered that when fluorescent SVF cells were injected into a laboratory mouse, they cells distributed to a variety of tissues. Further and more detailed examinations showed that these cells were finding their ways into organs and tissues because they traveled through the circulatory system and could be found in the walls of blood vessels.

Next, the composition of the SVF was examined. About 25% of the cells in the SVF were endothelial cells, 22% were various types of blood cells, 20% were “CD11b” cells, which means that these cells had a protein called CD11b on their cell surfaces. That protein was formerly canned “Mac-1” and is was normally found on the surfaces of phagocytic cells called macrophages. Therefore, this CD11b faction could very well be macrophages, but other cell types have this protein on their surfaces as well.

Macrophages

Next, Hoying and others injected these SVF-derived cells into the large leg vein (saphenous) of the leg. Such injections consistently caused these vessels to relax and dilate. Secondly, the SVF-derived cells caused the vessels to relax in a CD11b-dependent manner. In other words, the more CD11b cells there were in the SVF preparation, the greater the amount of vasoactivity they induced. If fractions were depleted of their CD11b, they could not induce vasoactivity.

When Hoying and others examined the SVF-treated vessels, they saw CD11b+ cells lining the inner layer of the vessels. Thus these cells were getting right up against the inside of the vessel and signaling to the underlying smooth muscle to relax.

Finally, Hoying and others clamped the saphenous veins of laboratory mice. Such clamping will induce tissue ischemia and inflammation in the vessels. Can SVF cells calm the inflammation and make the vessels more vasoactive? The answer is an unqualified yes.  See below.  The veins from SVF-treated animals show signficantly greater dilation than those from untreated or CD11b-depleted SVF-treated animals.

SVF cells relax vasomotor tone in inflamed saphenous arteries. (A): Schematic of the experimental plan involving the cell treatment of locally inflamed (cuffed) saphenous arteries of mice injected with syngeneic adipose SVF cells constitutively expressing luciferase and GFP reporter transgenes or SVF cells depleted of CD11b+ cells. Also shown is a gross view and a histological cross-section of a cuffed saphenous artery. (B): Hematoxylin and eosin-stained histological cross-sections of normal (noncuffed) and cuffed mouse saphenous arteries untreated or injected with SVF cells or SVF-11bΔ cells. Rightmost panels: Higher magnification images of the adjacent images. Scale bars = 25 μm in the left and right columns and 100 μm in the middle column. (C): Lumen diameters of untreated (n = 9) and cell-injected cuffed saphenous arteries measured from histological sections. Cell treatments included complete SVF cell isolates (C + SVF, n = 7) or SVF isolates depleted of CD11b+ cells (C + SVF-11bΔ, n = 7). Data are shown as the mean ± SEM; ∗, p < .05, determined by one-way analysis of variance. (D): Visualization of luciferase-positive SVF cells within histological paraffin sections of cuffed saphenous arteries from untreated, SVF-injected, and SVF-11bΔ-injected mice via immunostaining for luciferase. Brown stain indicates positive luciferase immune-staining and the presence of SVF cells. Tissues were harvested 1 week after cell delivery. Scale bars = 100 μm. Abbreviations: C, cuff; GFP, green fluorescent protein; PE, polyethylene; SVF, stromal vascular fraction; SVF-11bΔ, CD11b+ cell-depleted adipose SVF cells.
SVF cells relax vasomotor tone in inflamed saphenous arteries. (A): Schematic of the experimental plan involving the cell treatment of locally inflamed (cuffed) saphenous arteries of mice injected with syngeneic adipose SVF cells constitutively expressing luciferase and GFP reporter transgenes or SVF cells depleted of CD11b+ cells. Also shown is a gross view and a histological cross-section of a cuffed saphenous artery. (B): Hematoxylin and eosin-stained histological cross-sections of normal (noncuffed) and cuffed mouse saphenous arteries untreated or injected with SVF cells or SVF-11bΔ cells. Rightmost panels: Higher magnification images of the adjacent images. Scale bars = 25 μm in the left and right columns and 100 μm in the middle column. (C): Lumen diameters of untreated (n = 9) and cell-injected cuffed saphenous arteries measured from histological sections. Cell treatments included complete SVF cell isolates (C + SVF, n = 7) or SVF isolates depleted of CD11b+ cells (C + SVF-11bΔ, n = 7). Data are shown as the mean ± SEM; ∗, p < .05, determined by one-way analysis of variance. (D): Visualization of luciferase-positive SVF cells within histological paraffin sections of cuffed saphenous arteries from untreated, SVF-injected, and SVF-11bΔ-injected mice via immunostaining for luciferase. Brown stain indicates positive luciferase immune-staining and the presence of SVF cells. Tissues were harvested 1 week after cell delivery. Scale bars = 100 μm. Abbreviations: C, cuff; GFP, green fluorescent protein; PE, polyethylene; SVF, stromal vascular fraction; SVF-11bΔ, CD11b+ cell-depleted adipose SVF cells.

This an interesting and exciting finding not only because of the ability of these fat-based cells to maintain vasoactivity even under pro-inflammatory conditions, but because it is the macrophage cell population that is doing the work.  In most stem preparations, macrophages are excluded.  This paper shows that macrophages have greater therapeutic capabilities than previously thought, and should also be tested for sanative properties.

Using Polio Virus to Kill Deadly Brain Tumors


If you spend any time with people in retirement homes, they will sometimes tell you stories about their childhood and the dreaded “summer plague” known as polio. During the summer, children would go to ponds and lakes to swim in order to cool off from the summer heat. In those bodies of water, polio viruses would lurk, waiting to infect their new host. In most cases, infected people would experience a very flu-like disease that never went any further. In other cases, the flu-like disease might be more severe. Even in these people, the virus would be shed from the body by the digestive system and contaminate sewage water.

In rare cases, the central nervous system would be affected, but the aftermath of the disease would vary substantially.  Some  might some numbness for a little while. In a fraction of cases, they might actually experience paralysis that did not go away. The extent of this paralysis could vary tremendously. In some cases, people might retain the ability to walk, but with a limp. In other cases, they might not be able to walk at all. And in more severe cases, they might lose the ability to breathe on their own and require an iron lung to breathe for them. Polio struck young and old, male and female, rich and poor alike and was no respecter of persons.

polio_life_cycle

The polio vaccines made by Jonas Salk (a formalin-killed vaccine) and the live vaccine made by Albert Sabin essentially eradicated polio in many countries and saved untold of millions of lives from suffering. In fact, Albert Sabin gave away the rights to his vaccine even though those rights could have made him a millionaire. Because his live vaccine could be given in a sugar cube, it was extremely easy and inexpensive to administer to large populations.

Albert Sabin
Albert Sabin

Given this history, why would clinicians reinstate a deadly virus to fight cancer? The answer is that polio viruses is a highly lytic virus, but it can be genetically manipulated to specifically attack cancer cells.

First a bit about the molecular biology of polio virus.  Polio virus is a member of a group of RNA viruses called the “picornaviruses.”  The name of this group comes from “pico” meaning small, “RNA” to refer to the type of nucleic acid found in the virus, and the virus to indicate the type of infectious agent that it happens to be.  Viruses are nucleic acid molecules encases in a protein capsid.  When ingested from contaminated water by drinking or simply putting your hands in your mouth, the polio virus binds to a cell surface protein called CD155 found in the intestinal walls.  It enters these cells and uncoats.  The RNA genome of the polio virus is then translated by ribosomes in the cell into a large protein.  This is a key feature of picornaviruses; their viral RNA genomes can serve as messenger RNAs that are directly translated into protein right after emerging from their capsids.

polio molecular biology

This large protein has the ability to process itself.  That processing comes in the form of clipping pieces of the protein into small pieces.  These smaller pieces have specific functions.  The first pieces are creatively called P1, P2 and P3 (you have to love those biochemists and their ability to dream up creative names – yes that was a joke).  Eventually, the viral proteinases (enzymes that clip proteins) 2A and 3C further process these three precursor proteins to form the viral capsid proteins VP1-4 (formed from P1) and the viral replication proteins 2A, 2B, 2C (formed from P2), 3A, 3B, 3C, and 3D (formed from P3). As mentioned before, 2A and 3C are proteinases, 3B is a protein called VPg, and 3D is RNA-dependent RNA polymerase that replicates the viral RNA into copies that are packaged into viral capsids.

The whole infection process is insidiously effective because there is a piece of RNA at the very front of the poliovirus genome called the IRES, which stands for the internal ribosome entry site.  This sequence of 400 to 500 bases directs the viral translation initiation step in a manner independent of whether or not there is a special cap-structure on the front end of the RNA.  It allows poliovirus RNAs to be effectively recognized by the host cell and the cell’s own mRNAs to not be well-recognized because the polio 2A protease degrades elements of the cell’s own translation machinery, which prevents the cell from recognizing its own mRNAs.

As it turns out, if you swap this IRES with IRESs from other types of viruses, you can change the types of cells that polio virus will infect.  In 2000, Eckard Wimmer and his group from the State University of New York at Stony Brook showed that substituting the polio virus IRES for that of the IRES from the common cold virus (Rhinovirus) allowed poliovirus to grow in cultured brain tumor cells (see Gromeier M, et al., Proc Natl Acad Sci U S A. 2000, 97(12): 6803–6808).  Since these tumors expressed CD155, the receptor for poliovirus, they could be infected with it.  Wimmer and his team made attenuated strains of these viruses and used them in non-human primates that had brain tumors.  When injected directly into the tumor, the viruses infected only the tumor cells, and grew poorly, but the immune response against the virus and the infected cells caused the tumors to aggressively shrink.

So Gromeier and his team collaborated with neuro-oncologists to use their engineered polio viruses to treat human patients with glioblastomas.  These are very aggressive cancers that usually end up killing the patient.  in a clinical trial, of 22 people enrolled in the trial, half are doing well, and several are considered to be in remission, which is pretty much unheard of for glioblastomas.

The news show 60 Minutes even did a piece on this treatment and interviewed two patients with aggressive glioblastomas were treated by this polio virus.  Their tumors have essentially disappeared.  In fact, the first person who was ever treated with this treatment is now cancer free.

While this is a small study, it was supposed to be a Phase I study that only determined safe dosages and safety parameters.  you do not expect patients to improve much in Phase I studies because you are still tweaking the treatment.  These results are astonishing.  Also, because it uses the patient’s own immune response against the infected cells it does not depend on massive alterations of the patient’s physiology.

This is a remarkable finding.  I hope it can be developed into something mainstream that turns out to be safe and effective.