Cartilage Cells from Cow Knee Joints Grow New Cartilage Tissue in Laboratory


A research team from Umeå University in Sweden has used cartilage cells isolated from the knee joints of cows engineer joint-specific cartilage. Such a technique might lead to a novel stem cell-based tissue engineering treatment for osteoarthritis.

Hyaline cartilage is a specific type of cartilage found at joints where bones come together. Hyaline cartilage is a tough, pliable shock absorber, but because it is poorly supplied by blood vessels its capacity to regenerate is also poor. Knee injuries and the everyday wear-and-tear wear down cartilage tissue and might lead to a condition called osteoarthritis. In Sweden along, 26.6 percent of all people age 45 years or older were diagnosed with osteoarthritis. According to the Centers for Disease Control, in the United States, osteoarthritis affects 13.9% of adults aged 25 years and older and 33.6% (12.4 million) of those older than 65 in 2005; an estimated 26.9 million US adults in 2005 up from 21 million in 1990 (believed to be conservative estimate). Serious osteoarthritis cases can involve the loss of practically the entire cartilage tissue in the joint. Osteoarthritis causes pain and immobility in patients, but it also burdens society with accumulated medical costs.

“There is currently no good cure for osteoarthritis,” says Janne Ylärinne, doctoral student at the Department of Integrative Medical Biology. “Surgical treatments may help when the damage to the cartilage is relatively minor, whereas joint replacement surgery is the only available solution for people with larger cartilage damage. However, artificial joints only last for a couple of decades, making the surgery unsuitable for young persons. So we need a more permanent solution.”

Fortunately, tissue engineering might provide way to successfully treat osteoarthritis. Ylärinne and his colleagues developed new methods to produce cartilage-like “neotissues” in the laboratory.

Normally, tissue engineering methods that grow cartilage use cartilage-making cells, signaling molecules such a growth factors, and some sort of three-dimensional scaffold that acts as an artificial support system that makes the culture system more realistic for the cells. Unfortunately, such protocols are difficult, inexact, and generate respectable variation in what they produce. Consequently, it is also unclear whether stem cells or primary cells are best suited for cartilage tissue engineering experiments.

In these experiments, Ylärinne and others used primary cow chondrocytes (cartilage-making cells from cows) to which they successfully devised improved methods for growing cartilage tissue in a laboratory environment. The cartilage made by Ylärinne and others is similar to that normally present in the human joints.

Bovine cartilage made in laboratory

In the future, protocols like this one might help the development of neocartilage production for actual cartilage repair. If this protocol or others like it can be adapted to stem cells rather than primary cartilage cells, then perhaps these cells can be grown to provide unlimited amount of material for tissue engineering. However, despite the hopefulness of this research, more research is needed to improve the tissue quality and make it more structurally similar to the hyaline cartilage found at human joints.

VM202 is a Safe, Beneficial Treatment for Limb Ischemia


The Korean biotechnology company ViroMed Co., Ltd. has announced the publication of a Phase 2 study that evaluated their VM202 product in patients with critical limb ischemia. This study involved 52 patients in the United States and showed that VM202 is not only safe, but also produced significant clinical benefits.

VM202 is a plasmid (small circle of DNA) that encodes the human hepatic growth factor (HGF) gene. When injected into muscles, VM202 is readily taken up by nearby cells that then quickly synthesize the two isoforms of HGF. Heightened HGF concentrations can treat ischemic cardiovascular diseases by inducing the formation of new blood vessels (angiogenesis). These new collateral vessels increase blood flow and tissue perfusion in the sick tissue, which effectively treats any tissue ischemia.

VM202

Severe obstruction of the arteries that feed the extremities (hands, feet and legs) is the cause of critical limb ischemia (CLI). The term “ischemia” refers to the starvation of a tissue for oxygen. The lack of sufficient blood flow to an organ or tissue can cause severe pain and even skin ulcers, sores, or gangrene. CLI-induced pain can awaken the patient during the night, and, therefore, is called “rest pain.” Rest pains often occur in the leg and is usually temporarily relieved by dangling the leg over the bed or getting up and walking.

CLI does not improve on its own. It is a severe condition that requires immediate by a vascular surgeon or vascular specialist.

Look at the right side of these angiograms and you will see that a vessel is obstructed and blood is not flowing through it. This is an example of Critical Limb Ischemia.

Look at the right side of these angiograms and you will see that a vessel is obstructed and blood is not flowing through it. This is an example of Critical Limb Ischemia.

In this Phase 2 study, patients were divided into three groups, one of which received a placebo treatment, the second of which received a low-dose treatment VM202, and a third group that received a high-dose of VM202.

Both patient groups that received VM202 showed improvement compared to the placebo group, but patients in the higher-dose group showed significantly better ulcer healing and higher tissue oxygen levels than the placebo group. For example, 62 percent of the ulcers healed in patients treated with high-dose VM202 compared to only 11 percent of ulcers in patients who were treated with the placebo. Also, 71 percent of patients who received the high-dose VM202 showed improved oxygen concentrations in their tissues, compared to only 33 percent of patients who were treated with the placebo.

Emerson C. Perin, Director of the Stem Cell Center at the Texas Heart Institute and the principal investigator of this Phase 2 study, said: “These positive results are exciting, and VM202 shows great promise for treating patients with this debilitating disease who often have limited therapeutic options. We are looking forward to conducting a phase III trial to better understand the potential of this novel approach, especially in treating non-healing ulcers, which is a serious symptom that often leads to amputation because of the lack of medical therapies available.”

ViroMed has already been granted an IND or Investigational New Drug approval by the USFDA to initiate a Phase 3 study in diabetic patients who suffer from non-chronic ischemic foot ulcers. This study will enroll 300 subjects who will be divided into a VM202 group and a placebo group. The treatment regiment will mimic that of this smaller Phase 2 study and will only follow patients for seven months. This time, ViroMed is interested in determining if VM202 helps wound closure, which will constitute the primary efficacy endpoint on this new study.

Godspeed ViroMed!!

Killing off Senescent Cells Extends the Life and Improves the Health of Laboratory Mice


Our bodies contain a mixture of cells that grow and others whose growing days are long since over. Such cells are called “senescent cells” and there is some indication that accumulations of worn out cells that have given up the ghost can contribute to the onset of age-related diseases.

A new study from the Mayo Clinic in Rochester, Minnesota has shown that eliminating senescent cells can extend the healthy lives of lab mice. These results constitute one of the first direct demonstrations that treatments that specifically target these deadbeat cells, either by killing them off of blocking their deleterious effects, might provide a new strategy to combat age-related diseases in human patients.

Aging is a fact of life for animals. Aging bodies contain cells that have lost the ability to divide. These senescent cells build up throughout our bodies and release molecules that can potentially harm nearby tissues. Diseases of advanced age, like type 2 diabetes, kidney failure, and heart disease, have been linked to the presence of large numbers of senescent cells.

Two Mayo Clinic molecular biologists, Darren Baker and Jan van Deursen, devised an ingenious way to test the relationship of accumulations of quiescent cells with age-related diseases. They engineered laboratory mice with a construct they called “ATTAC.” This construct expresses the FK506-binding-protein–caspase 8 (FKBP–Casp8) fusion protein and green fluorescent protein (GFP) under the control of a promoter that is only active when cells are senescent. The ATTAC construct is not harmful to cells, but if ATTAC-containing cells that have become senescent are hit with a drug called AP20187, they die. Baker and van Deursen injected one-year-old mice with AP20187 twice a week starting at one year of age. They injected a control group of mice with buffer only.

The AP20187 treatments extended the median lifespan in both male and female mice. To make sure that nothing strange was going on with the genetic backgrounds of these mice, they conducted these experiments in mice strains with two distinct genetic backgrounds, but the results were the same. The engineered mice that were injected with AP20187 showed consistent clearance of quiescent cells and extended, healthier life spans.

The AP20187-injected mice showed clearance of fat cells, their kidneys functioned at a higher level, and their hearts were more resilient to stress. These same mice also showed more energetic behaviors, since they explores their cages more and they developed cancers at later ages. There was also evidence of less inflammation in the AP20187-injected mice.  These mice had their lifespans extended by 20–30%. These results were published in the journal Nature on February 3rd of 2016.

Despite the rather fancy genetics involved with this experiment, the design is somewhat easy to follow and the results have a ring of credibility to them. “We think these [quiescent] cells are bad when they accumulate. We remove them and see the consequences,” says Baker. “That’s how I try to explain it to my kids.”

In 2011, Baker and van Deusen and others investigated mice that harbor a mutation that greatly accelerates aging. This mutation mimics the human genetic disease “progeria,” which is a rare, fatal genetic condition characterized by accelerated aging in children. The name of the condition, progeria, comes from Greek and means “prematurely old.” Classic progeria is also called “Hutchinson-Gilford Progeria Syndrome,” which was named after Dr. Jonathan Hutchinson and Dr. Hastings Gilford, who first described it.

In their 2011 Nature paper, Baker and others showed that removing senescent cells in mice with an engineered type of progeria benefited those mice. Therefore, this 2016 paper is a follow-up to that 2011 study.

On the coattails of these experiments, Baker, van Deusen and others in their laboratories are beating the bushes for drugs that can directly eliminate senescent cells, or, at least, stop them from secreting the damaging factors that do so much damage to nearby tissues. In fact, van Deursen has co-founded a company that has licensed patents to develop such drugs.

According to Dominic Withers, a clinician-scientist who studies ageing at Imperial College London, the experiments and Baker, van Deusen and their colleagues, “gives you confidence that senescent cells are an important target.” Withers also said: “I think that there is every chance this will be a viable therapeutic option.”

G-CSF Fails to Improve Long-Term Clinical Outcomes in REVIVAL-2 Trial


Granulocyte-Colony Stimulating Factor (G-CSF) is a glycoprotein (protein with sugars attached to it) that signals to the bone marrow to produce granulated white blood cells (specifically neutrophils), and to release stem cells and progenitor cells into the peripheral circulation.

This function of G-CSF makes it a candidate treatment for patients who have recently experienced a heart attack, since the release of stem cells from the bone marrow could, in theory, bring more stem cells to the damaged heart to heal it. Additionally, G-CSF is known to induce the proliferation and enhance the survival of heart muscle cells.

In several experiments with laboratory animals showed that G-CSF treatments after a heart attack significantly reduced mortality (Moazzami K, Roohi A, and Moazzimi B. Cochrane Database Systematic Reviews 2013; 5: CD008844. However, in a clinical trial known as the REVIVAL-2 trial, a double-blind, placebo-controlled study, G-CSG treatment failed to influence the performance of the heart six months after administration.

Now Birgit Steppich and others have published a seven-year follow-up of the subjects in the original REVIVAL-2 study to determine if G-CSF had long-term benefits that were not revealed in the short-term study. These results were published in the journal Thrombosis and Haemostasis (115.4/2016).

Of the initially enrolled 114 patients, 106 patients completed the seven-year follow-up. The results of this trial showed that G-CSF treatment for five days in successfully revascularized heart attack patients did not alter the incidence of death, recurrent heart attacks, stroke, or secondary adverse heart events during the seven-year follow-up.

These results are similar to those of the STEMMI trial, which treated patients with G-CSF for six days 10-65 hours after the reperfusion. In a five-year follow-up of 74 patients, there were no differences in the occurrence of major cardiovascular events between the G-CSF-treated group and the placebo group (Achili F, et al., Heart 2014; 100: 574-581).

Therefore, it appears that even though G-CSF worked in laboratory rodents that had suffered heart attacks, this treatment does not consistently benefit human heart attack patients. Although why it does not work will almost certainly require more insights than we presently possess.

Using CXCR4 to Make Stem Cells Stay Put: Regenerating Intervertebral Discs


The migration of several different types of stem cells is regulated by a receptor called “CXCR4” and the molecule that binds to this receptor, SDF-1. SDF-1 is a powerful summoner of white blood cells. During early development, SDF-1 mediates the migration of hematopoietic cells from fetal liver to bone marrow and plays a role in the formation of large blood vessels. During adult hood, SDF-1 plays an important role in making new blood vessels by recruiting endothelial progenitor cells (EPCs) from the bone marrow. Consequently, SDF-1 has a role in tumor metastasis where cancer cells that express the receptor CXCR4 are attracted to metastasis target tissues that release SDF-1. SDF-1 also attracts mesenchymal stem cells and helps them suppress the breakdown of bone.

Hopefully, I have convinced you that SDF-1 and its receptor CXC4 are important molecules. Can overexpression the CXCR4 receptor improve the retention of stem cells within an injured tissue?

Xiao-Tao Wu and Feng Wang from Zhongda Hospital in Nanjing, China and their colleagues have used this CXCR4 receptor/SDF-1 system to test this question in the damaged spinal cord.  This work was published in the journal DNA and Cell Biology (doi:10.1089/dna.2015.3118).

Isolated MSCs were treated with genetically engineered viruses to so that would overexpress the CXCR4 receptor. In order to track these cells under medical imaging scans, the MSCs were also labeled with superparamagnetic iron oxide (SPIO). Next, rabbits that had suffered injuries to their intervertebral discs that lie between the vertebrae were given infusions of these labeled, genetically engineered MSCs. Images of the spine were taken at 0, 8, and 16 weeks after the surgery. The degeneration of the damaged intervertebral discs were also evaluated by disc height (damaged, degenerating intervertebral discs tend to shrink and lose height).

The SPIO-labeled CXCR4-MSC could be detected within the intervertebral discs by MRI 16 weeks post-transplantation. The MSCs that had been engineered to overexpress CXCR4 showed better retention within the discs, relative to implanted MSCs that had not been engineered to overexpress CXCR4.

Did the implanted MSCs affect the integrity of the intervertebral discs? Indeed they did. Compared to the control group, loss of disc height was slowed in the animals that received the CXCR4-overexpressing MSCs. Also, the genetically engineered MSCs seemed to make more cartilage-specific materials, like the giant molecule aggrecan and type II collagen. There is a caveat here, since there is no indication that measured protein directly; only mRNAs. Until the quantities of these molecules can be directly shown to increase in the disc, the increases in these cartilage-building molecules can be said to be presumptive, but not proven.

From these experiments, it seems reasonable to conclude that CXCR4 overexpression promoted MSC retention within the damaged intervertebral discs and the increased stem cell retention enhanced stem cell-based disc regeneration. Therefore this SDF-1/CXCR4 signaling pathway might be a way to drive stem cell migration and infiltration within degenerated intervertebral discs.

Noninvasive, Targeted, and Non-Viral Ultrasound-Mediated GDNF-Plasmid Delivery for Treatment of Parkinson’s Disease


A growth factor called Glial cell line-derived neurotrophic factor (GDNF) has the remarkable ability to supports the growth and survival of dopamine-using neurons. Dopamine-using neurons are the cells that die off in Parkinson’s disease (PD). Providing GDNF to dopamine-using neurons can help them survive , but getting GDNF genes into the central nervous system relies on invasive intracerebral injections in order to pass through the blood-brain barrier.

Typically, genes are placed into the central nervous system by means of genetically engineered viruses. Viruses, however, are often recognized by the immune system and are destroyed before they can deliver their genetic payload. Therefore, non-viral gene delivery that can pass through the blood-brain barrier is an attractive alternative, since it is non-invasive. Unfortunately, such a high-yield technique is not yet available.

A new study by workers in the laboratories of Hao-Li Liu from Chang Gung University and Chih-Kuang Yeh from National Tsing Hua University, Taiwan has utilized a novel, non-viral gene delivery system to deliver genes into the central nervous system.

In this study, Lui and Yeh and their research teams used tiny bubbles made from positively-charged molecules to carry genes across the blood brain barrier. These bubbles formed stable complexes with GDNF genes, and when the skulls of laboratory animals were exposed to focused ultrasound, the bubble-gene complexes permeated the blood brain barrier and induced local GDNF expression.

(A) Schematic of GDNFp-cMBs and mechanism for controlled gene transfection of GDNFp-cMBs into brain triggered by FUS. (B) Left: Microscope bright-field images; middle: TEM images; right: PI staining image of cMBs and GDNFp-cMBs. (C) Size distributions of cMBs, GDNFp-cMBs and nMBs. (D) Zeta potential of nMB and cMB before and after adding GDNFp. (E) DNA loading efficiency of GDNFp onto nMB and cMB. The left axis was the amount of GDNFp bound onto MBs (solid line). The right axis was the GDNFp loaded efficiency onto MBs (dotted line). Single asterisk, p < 0.05, versus nMBs. Data were analyzes by Student’s paired t-test presented as mean ± SEM (n = 6 per group).

(A) Schematic of GDNFp-cMBs and mechanism for controlled gene transfection of GDNFp-cMBs into brain triggered by FUS. (B) Left: Microscope bright-field images; middle: TEM images; right: PI staining image of cMBs and GDNFp-cMBs. (C) Size distributions of cMBs, GDNFp-cMBs and nMBs. (D) Zeta potential of nMB and cMB before and after adding GDNFp. (E) DNA loading efficiency of GDNFp onto nMB and cMB. The left axis was the amount of GDNFp bound onto MBs (solid line). The right axis was the GDNFp loaded efficiency onto MBs (dotted line). Single asterisk, p < 0.05, versus nMBs. Data were analyzes by Student’s paired t-test presented as mean ± SEM (n = 6 per group).

In fact, this technique outperformed intracerebral injection in terms of targeted GDNF delivery. The amount of GDNF expressed in these laboratory animals that received the GDNF gene/microbubbles + ultrasound protocol was significantly higher than those animals that had genes directly injected into their brains. Furthermore, these higher levels of GDNF genes increased the levels of neuroprotection from PD. Animals that had a form of PD and had received nonviral GDNF gene therapy showed reduced disease progression and restored behavioral function.

This interesting study explores the potential of using ultrasound-induced passage through the blood brain barrier to bring genes into the central nervous system. This noninvasive technique successfully delivered genes into the brain to delay the effects of, and possibly treat, a neurodegenerative disease.

This study was published in Scientific Reports 6, Article number: 19579 (2016), doi:10.1038/srep19579.

 

Stem Cell-Derived Retinal Grafts Integrate into Damaged Monkey Retinas


Retinal degenerations are the leading cause of blindness and fixing a defective retina is not an easy task.

Fortunately, a model system in nonhuman primates that has been used to test retinal replacement with stem cell-derived retinal cells has seen some success. In several experiment in small animals, retinal transplantations helped blind animals regain their sight. However, small laboratory rodents are not terribly good model systems for human eye problems.

To address the clinical relevancy of this transplantation system, Shirai and colleagues confirmed in rats and in macaques that transplantion of human embryonic stem cell (hESC)–derived retinas integrate into the already-existing retina and develop as fully mature retinal grafts.

In this paper, Shirai and others established the developmental stage at which embryonic stem cell-derived retinal cells could integrate into the retina and replace damaged cells. By transplanting cells into nude rats that do not have the ability to reject transplanted tissue, they refined their cell-based technique to heal damaged retinas. Then they took their refined technique into macques to treat two newly established monkey models of retinal degeneration.

In the first model system, Shirai et al. exposed one group monkeys to retina-damaging chemicals, and the other group had their retinas damaged by lasers. In both cases, the result was photoreceptor degeneration. Anywhere from 46 to 109 days after injury, the human embryonic stem cell-derived retinal sheets were implanted into the damaged retinas.

The retinal grafts integrated into the primate eyes and continued to differentiate into cone and rod cells, which are the two types of photoreceptor cells in the retina. Functional studies are still being conducted, but if vision can be improved, but these new macaque models confirm the clinical potential of stem cell–derived grafts for retinal blindness that results from photoreceptor degeneration.

See H. Shirai et al., Transplantation of human embryonic stem cell-derived retinal tissue in two primate models of retinal degeneration. Proc. Natl. Acad. Sci. U.S.A. 113, E81–E90 (2015).