In order to use human pluripotent stem cells, they must match the tissue types of the patient. Otherwise the patient’s immune system will reject any implanted cells, unless they are administered into areas of the body outside the long reach of the immune system like the eye or the brain.
However, induced pluripotent stem cells (iPSCs) are made from the patient’s own cells, by using a combination of genetic engineering and cell culture techniques and any cells that differentiated from an established iPSCs line will match the tissue types of the patient and not be rejected. Unfortunately, iPSCs take a while to make and the quality of the cell line varies from person to person. Therefore, using these cells in a clinical setting represents some issues.
A new technique that was developed at the EURAC Center for Biomedicine simplifies the method for iPSC derivation from blood cells. Instead of having to use specific reagents with isolated white blood cells, which contain a nucleus and DNA, as opposed to red blood cells, which have no nucleus, the EURAC Center for Biomedicine method does not require such reagents. This reduces cost, the time required to derive iPSCs, and the complexity of the procedure. The new EURAC Center for Biomedicine protocol will also work well on blood samples that were previously collected and preserved by freezing in a blood bank. Because it is highly convenient to store samples in this fashion, this technique is highly feasible.
This technique uses no viruses, and can readily make iPSCs to study illnesses in the laboratory. Alessandra Rossini and her colleagues succeeded in making iPSC lines from several different patients with a variety of maladies. This technique might revolutionize iPSC-making protocols.
Even though it is now possible to fabricate organs and tissues in the laboratory, the vast majority of these structures can be made in isolation without compromising their functionality. Brain cells are quite different, since they required connections known as synapses with other brain cells. Synapses are also responsible for the functional interactions between different regions of the brain. Even though nerve cells (neurons) can be made in the laboratory, engineering connections between neurons is not trivial. Furthermore, some laboratories have used pluripotent stem cells to make portions of the brain in the laboratory, but getting those portions to properly connect with other regions of the brain has proven stultifying.
A new study by William Freed at the National Institutes of Health and his colleagues has designed a way to successfully grow multiple brain structures in the laboratory that form proper connections with each other in culture. This report is the first of its kind.
In particular, Freed and his co-workers defined a culture system for human pluripotent stem cells that produced connected human midbrain and neocortex.
The midbrain houses dopaminergic neurons (mDAs). These neurons use the neurotransmitter dopamine to signal to other neurons that reside elsewhere in the brain. Abnormalities of mDAs or connections between mDAs and other neurons are thought to play intimate roles in disorders like schizophrenia, Parkinson disease, attention-deficit disorder, Roulette’s syndrome, Lesch-Nyhan syndrome, and maybe even eating disorders.
Unfortunately, studying neocortical neurons and mDAs in isolation reveal little about the connections between them or their interactions. However, this new data from Freed that shows that it is possible to grow and interconnect these two types of neurons in culture provides neuroscientists with a powerful model system for examining this system and the abnormalities that afflict it.
The encourage connections between the two neuronal populations, mDAs and neocortical neurons were grown in special containers called “ibidi wound healing” dishes. Ibidi wound healing dishes contain two chambers separated by a removable barrier. Neocortical neurons were grown on one side and mDAs were grown on the other side. Both neuron populations were derived from human pluripotent stem cells. Once the cell cultures had properly formed, the barrier was removed and the two cell populations formed synapses across the barrier.
Freed is eager to examine human pluripotent stem cells derived from patients with neurological disorders that have been traced to abnormalities with connections between mDAs and other neuronal populations to study if neurons made from these patient’s cells properly synapse.
Clearly, this model system has great potential. This work was published in Restorative Neurology and Neuroscience, 2015 DOI: 10.3233/RNN-1140488.
Stem cells have the capacity to heal damaged tissues and replace death cells. However, harvesting and employing the right stem cell for the right job, at the right dose, and under the right conditions has proven to be a difficult puzzle to solve.
In particular, the damaged heart has proven rather difficult to heal with stem cells. Many different clinical trials have administered stem cells by direct injection, intracoronary delivery in coronary vessels, or injection into the surface of the heart. These studies have examined the efficacy of stem cells from fat, bone marrow, the heart itself, and other sources. The upshot of these studies is that some strategies work and others do not, but even those that work only work modestly well.
The biggest obstacle is overcoming the hostile environment into which stem cells are introduced when they are administered into the heart after a heart attack. The post-heart attack heart suffers from lots of inflammation, low oxygen concentrations, and the pervasive presence of dangerous molecules. Several laboratories have discovered that preconditioning stem cells by growing them in low-oxygen culture conditions can increase their survival in the post-heart attack heart as can genetically engineering cells to resist increased levels of cell stress. Now, a research team from Ohio State University has designed a different strategy to beat the hostility of the post-heart attack heart.
After a heart attack, oxygen-deprived tissues die and various chemical messengers instruct damaged cells to die. However, data from a host of clinical trials strongly suggests that this dangerous time is the best time to introduce stem cells into the heart. Thus, this window of opportunity is the “best of times and the worst of times” for cell therapy.
As it turns out, about 30% of all mammalian protein-encoding genes are regulated by small molecules called microRNAs (miRNAs). MiRNAs are single-stranded RNA molecules approximately 22 nucleotides in length that bind to messenger RNAs and regulate their translation into protein or half-life. Research has shown that miRNAs have substantial potential as a therapeutic target for the treatment of many diseases, including cardiovascular disease. A good deal of research in laboratory animals and in cultured heart cells that altered expression of miRNAs such as miR-1, miR-133, miR-21 and miR-208 contribute to the development of heart disease. The laboratory of Mahmood Khan, a scientist at the Davis Heart and Lung Research Institute at The Ohio State University Wexner Medical Center, has focused on miR-133a which seems to play a role in slowing fibrosis and cardiac remodeling. Importantly, levels of miR-133a are reduced in the heart tissues of patients who have suffered a heart attack.
Khan and his group predicted that increasing the levels of miR-133a in stem cells as they are cultured might preprogram the cells to survive in the hostile environment of the post-heart attack heart.
Khan’s team began by bioengineering a molecule to induce mesenchymal stem cells (MSCs) to produce miRNA-133a. When transplanted into an animal model of cardiac ischemia, the pre-treated MSCs showed improved survival over non-treated MSCs. These pre-treated MSCs also did a better job at decreasing the global damage to the heart, and increasing the thickness of the left ventricle, the main pumping chamber of the heart.
“We found that the pre-treated MSCs did a better job at decreasing the global damage to the heart, along with improvement in the left-ventricular wall thickness compared to the untreated MSCs,” said Dr. Angelos. “MSCs are a commonly used cell type in current heart failure studies, so our findings are definitely relevant to that work.”
The results of these experiments were published in the March issue of the Journal of Cardiovascular Pharmacology.
While trying to increase cell survival, Khan and his colleagues also addressed the problems surviving stem cells face in the heart – how to help them function along existing heart tissue without getting in the way or fouling things up.
To date, most stem cells are grown in flat culture plates and either injected directly into the heart muscle (typically on the periphery of scar tissue), or infused into the heart via an artery. While most of these stem cells either die or diffuse throughout the body, successfully transplanted stem cells sometimes inadvertently disrupt heart function.
“The heart is a constantly moving, connected matrix of muscle fibers working together to make the heart pump in sync,” said Dr. Angelo’s, an emergency medicine researcher and collaborator of Dr Khan. “Transplanted stem cells may not align with native tissue, potentially disrupting or attenuating signals that keep a steady heartbeat. There’s evidence that this could contribute to arrhythmias.”
To create a more secure environment that allows implanted stem cells successfully engraft into the heart, Drs. Khan and Angelos have used a biodegradeable nanofiber “patch” seeded with human inducible pluripotent stem cells derived cardiomyocytes (hiPSC-CMs). Khan and Angelos chose hiPSC-CMs because these cells are patient derived and can be used to model the heart disease of patients and for autologous stem cell transplantation in patients with failing hearts.
Both aligned nanofiber patch and standard culture plate were seeded with hiPSC-CMs. Both sets of cultures heart muscle cells were compared for calcium signaling (a measure of proper heart muscle function) and synchronous beating. Within two weeks, both stem cell cultures were spontaneously beating like a miniature heart, but the linear grain of the nanofiber formed an aligned pattern of cells that looked and functioned like a healthy heart tissue.
“The cardiomyocytes cultured on a flat plate are scattered and disorganized. Cardiomyocytes grown on the nanofiber scaffolding look more like healthy heart cells, beat more strongly and in greater synchronicity than cells from the flat plate,” said Dr. Khan. “Next, we hope to use what we’ve learned from this study to develop a thicker, multi-layer patch that could help restore thin and weakened heart walls.”
Drs. Khan and Angelos see great potential future clinical applications for their nanofiber bandage. This treatment could potentially bandage the damaged heart muscle of heart patients with the nanofiber cardiac patch. Also, it is possible that they could someday combine the microRNA pre-treatment technique and the patch to give stem cells a survival boost along with a protective structure to improve outcomes.
Khan, his co-workers and his collaborators published this work on May 19 in PLoS ONE.
Tissue engineers from the Universities of Liverpool and Bristol have invented a novel tissue “scaffold” technology that might one day enable the growth large organs in the laboratory.
According to data generated by these experiments, it is possible to combine cells with a special scaffold to produce living tissues in the laboratory. Hopefully, such organs can then be implanted into patients who need to have a diseased body part replaced. To this point, growing large organs in the laboratory has been impossible because growing larger structures in the laboratory limits the delivery of oxygen supply to the cells in the center of the organ. Therefore, growing tissues in the laboratory has been restricted to small structures that are readily served by the diffusion of oxygen.
In the experiments conducted by the University of Liverpool and Bristol teams, cartilage tissue engineering was employed as a model system for testing strategies for overcoming the oxygen limitation problem.
They manufacture a new class of artificial membrane binding proteins that attached to stem cells. Then they attached to these cell surface proteins the oxygen-carrying protein, myoglobin, before they used the cells to engineer cartilage. Since myoglobin is an oxygen-storage molecule, it will bind oxygen and provide a reservoir of oxygen for cells that cells can access when the oxygen in the scaffold drops to dangerously low levels.
Professor Anthony Hollander, Head of the University of Liverpool’s Institute of Integrative Biology, said: “We have already shown that stem cells can help create parts of the body that can be successfully transplanted into patients, but we have now found a way of making their success even better. Growing large organs remains a huge challenge but with this technology we have overcome one of the major hurdles. Creating larger pieces of cartilage gives us a possible way of repairing some of the worst damage to human joint tissue, such as the debilitating changes seen in hip or knee osteoarthritis or the severe injuries caused by major trauma, for example in road traffic accidents or war injuries.”
These results could expand the possibilities in tissue engineering, not only in cartilage, but also for other tissues such as cardiac muscle or bone. This new methodology in which a normal protein is converted into a membrane binding protein to which helpful molecules can be attached, is likely to pave the way for the development of a wide range of new biotechnologies.
Dr Adam Perriman, from the University of Bristol, added: “From our preliminary experiments, we found that we could produce these artificial membrane binding proteins and paint the cells without affecting their biological function. However, we were surprised to discover that we could deliver the necessary quantity to the cells to supplement their oxygen requirements. It’s like supplying each cell with its own scuba tank, which it can use to breathe from when there is not enough oxygen in the local environment.”
Previous work by Hollander’s group includes the development of a method of creating cartilage cells from stem cells. This method helped make the first successful transplant of a tissue-engineered trachea, which utilized the patient’s own stem cells, possible.
This work appeared in the paper, “Artificial membrane-binding proteins stimulate oxygenation of stem cells during engineering of large cartilage tissue,” which was published in Nature Communications.
Paul Tesar from Case Western Reserve in Cleveland. Ohio and his colleagues have discovered that two different drugs, miconazole and clobetasol, can reverse the symptoms of multiple sclerosis in laboratory animals. Furthermore, these drugs do so by stimulating the animals’ own native stem cell population that insulates nerves.
Multiple sclerosis (MS) is a member of the “demyelinating disorders.” The cause of MS remains unknown, but all of our available evidence strongly suggests that MS is an autoimmune disease in which the body’s immune system attacks its own tissues. In MS the immune system attacks and destroys myelin — the fatty substance that coats and protects nerve fibers in the brain and spinal cord. We can compare myelin to the insulation that surrounds electrical wires. When myelin is damaged, the nerve impulses that travel along that nerve may be slowed or blocked.
The myelin sheath is made by cells known as “oligodendrocytes,” and oligodendrocytes are derived from a stem cell population known as OPCs, which stands for oligodendrocyte progenitor cells. If this stem cell population could be stimulated, then perhaps the damaged myelin sheath could be repaired and the symptoms of MS ameliorated.
In a paper that appeared in the journal Nature (522, 2015 216-220), Tesar and the members of his research team, and his collaborators used a pluripotent mouse stem cell line and differentiated them into OPCs. Thyroid hormone is a known inducer of OPC differentiation. Therefore, Tesar and others screened a battery of drugs to determine if any of these compounds could induce OPC differentiation as cell as thyroid hormone. From this screen using cultured OPCs, two drugs, the antifungal drug miconazole and clobetasol, a corticosteroid of the glucocorticoid class, proved to do a better job of inducing OPC differentiation than thyroid hormone.
Was this an experimental artifact? Tesar and others devised an ingenious assay to measure the effectiveness of these two drugs. They used brain slices from fetal mice that were taken from animals whose brains had yet to synthesize myelin and applied OPCs to these slices with and without the drugs. With OPCs, no myelin was made because the OPCs did not receive any signal to differentiate into mature oligodendrocytes and synthesize myelin. However in the presence of either miconazole or clobetasol, the OPCs differentiated and successfully myelinated the brain slices.
Experiments in tissue culture are a great start, but do they demonstrate a biological reality within a live animal? To answer this question, Tesar and his crew injected laboratory mice with purified myelin. The immune systems of these mice generated a robust immune response against myelin that eroded the myelin sheath from their nerves. This condition mimics human MS and is called experimental autoimmune encephalitis, and it is an excellent model system for studying MS. When mice with experimental autoimmune encephalitis (EAE) were treated with either miconazole or clobetasol, the EAE mice showed a remarkable reversal of symptoms and a solid attenuation of demyelination. Tissue samples established that these reversals were due to increased OPC activity.
When the mechanisms of these drugs were examined in detail, it became clear that the two drugs worked through distinct biochemical mechanisms. Miconazole, for example, activated the mitogen-activated protein kinase (MAPK) pathway, but clobetasol worked through the glucocorticoid receptor signaling pathway. Both of these signaling pathways converge, however, to increase OPC differentiation.
Both miconazole and clobetasol are only approved for topical administration. However, the fact that these drugs can cross the blood-brain barrier and effect changes in the brain is very exciting. Furthermore, this work establishes the template for screening new compounds that might be efficacious in human patients.
In the meantime, human patients might benefit from a clinical trial that determines if the symptoms and neural damage caused by MS can be reversed by the administration of these drugs or derivatives of these drugs.
What if doctors could turn cancer cells into healthy cells? It would change everything about how we treat cancer. Researchers may have discovered a way to do that in colorectal cancer.
What if we could turn the clock back on cancer cells and return them to their healthy status? A new study in animals might have accomplished exactly that.
A research team from the Memorial Sloan Kettering Cancer Center has reactivated a defective gene in mice with colorectal cancer. This gene, adenomatous polyposis coli, or Apc, is commonly defective in colorectal cancer cells. Approximately 90 percent of colorectal tumors have a loss-of-function mutation of this gene.
At the onset of this research project, The Sloan Kettering group suppressed the expression of the Apc gene in mice. The Apc gene encodes a protein that regulates an important cell signaling pathway known as the Wnt signal pathway. Suppression of Apc activates the Wnt signaling pathway, which helps cancer cells grow and survive.
Afterwards, they reactivated the Apc gene, which returned Wnt signaling to its normal levels and the cancerous tumors stopped growing, and normal intestinal function was restored in four days. By two weeks after Apc was reactivated, the tumors were gone and there were no lingering signs of no signs of cancer relapse during the six-month follow-up.
The same approach turned out to be effective in mice with colorectal cancer tumors that result from activating mutations in the Kras gene and loss-of-function mutations in the p53 gene. In humans, about half of colorectal tumors have these mutations
This study was published in the prestigious international journal, Cell, by Scott Lowe and his colleagues. “Treatment regimens for advanced colorectal cancer involve combination chemotherapies that are toxic and largely ineffective, yet have remained the backbone of therapy over the last decade,” said Lowe.
Apc reactivation might very well be the way to improved treatment for colorectal cancer. It is doubtful it will be helpful in other types of cancer, but in the future, it might become so. “The concept of identifying tumor-specific driving mutations is a major focus of many laboratories around the world,” said Lukas Dow, Ph.D., of Weill Cornell Medical College, who is the first author of this study.
“If we can define which types of mutations and changes are the critical events driving tumor growth, we will be better equipped to identify the most appropriate treatments for individual cancers,” said Dow.
Colorectal cancer begins in the colon or rectum, and it remains the second-most prevalent cause of cancer death in developed countries.
According to the Surveillance, Epidemiology, and End Results Program, in 2012, there were 1,168,929 people living with colon and rectal cancer in the United States.
Estimates postulate that there will be 132,700 new cases of colorectal cancer in the United States in 2015, and about 49,700 people will lose their lives to this disease. Worldwide, colorectal cancer is the cause of approximately 700,000 deaths each year.
Internist and gastroenterologist Dr. Frank Malkin expressed optimism regarding genetic research into colorectal cancer. He said in an interview with the medical news service, Healthline: “They’ve identified a suppressor gene that can turn a tumor on and off. It can suppress the cancer and destroy it rapidly. That’s very promising.”
Cancers are normally treated with a combination of surgery, chemotherapy, and radiation. These rather harsh treatments can take a lasting toll. Easier and more effective treatments could change the lives of cancer patients.
Michelle Gordon, D.O., FACOS, FACS, finds it encouraging. “If this treatment is to be believed, all current modalities will be obsolete.”
However, Malkin and Gordon both cautioned that it is simply too early to bring this strategy to the clinic to treat human patients.
“There are so many unknowns when taking a mouse model to humans,” Gordon told Healthline. “This may be the foundational step that will lead to curing most colorectal cancers. This study can provide hope to future generations of colorectal cancer [patients], but I believe a cure is decades away.”
Researchers know Apc mutations initiate colorectal cancer, but they are unsure if Apc mutations are involved in promoting tumor growth after the cancer has developed.
The next step in this work will examine the ability of Apc reactivation to affect tumors that have spread or metastasized to distant locations in the body. Lowe and his colleagues are also hard at work to determine precisely how Apc works. That will help scientists develop safe treatments that change cancer cells into normal cells. Such a drug could make colorectal cancer treatment easier, faster, and safer.
How this research will impact other types of cancer remains unclear. “Cure rates for colorectal cancers are better than they used to be, especially when treated in the early stages,” said Malkin. Nevertheless, it is still far better to stop tumors before they start.
According to Malkin, the number of colon cancer cases has dropped dramatically since routine colonoscopy screening began. A colonoscopy allows doctors to find and remove polyps before they turn cancerous. Malkin also looks forward to genetic research that will identify those at greater risk for colorectal cancers.
“Right now, we’re using colonoscopy to screen people over 50, most who don’t have the genetic predisposition and will never get colorectal cancer,” he said. “We don’t yet have the genetic studies that would help us identify high-risk patients so we don’t have to screen everyone.”
I must admit that I remain skeptical as to whether or not this will work. The reasons for my skepticism lie in the fact that tumor cells in the colon are the result of a series of mutations in cells that cause the cells to overgrow and eventually become invasive. Colorectal carcinoma cells have mutations in several genes and not just Apc. Apc reactivation worked in these mice because this was the only gene affected in these animals. In a cancerous human colon, the cancer cells have a variety of mutations. Kurt Vogelstein’s work at Johns Hopkins has shown this in great detail. If Lowe could demonstrate the efficacy of his treatment in mice with humanized immune systems that have been infected with human colorectal carcinoma cells, then I will believe that this technique could work in human patients. For now, I remain skeptical.
Temple University stem cell researcher Raj Kishore, who serves as the Director of the Stem Cell Therapy Program at the Center for Translational Medicine at Temple University School of Medicine (TUSM), and his colleagues have used exosomes from stem cells to induce tissue repair in the damaged heart. The results of this fascinating research made the cover of the June 19, 2015 edition of the leading cardiovascular research journal, Circulation Research.
“If your goal is to protect the heart, this is a pretty important finding,” Dr. Kishore said. “You can robustly the heart’s ability to repair itself without using the stem cells themselves. Our work shows a unique way to regenerate the heart using secreted vesicles from embryonic stem cells.” Kishore’s group is in the early stages of characterizing the molecules in these exosomes that are responsible for inducing and potentiating tissue repair.
The heart beats throughout the lifetime of an individual. Despite its apparent constancy, the heart possess little to no ability to repair itself. When heart muscle is damaged in a heart attack, the heart is unable to replace the dead tissue and grow new contracting heart muscle. Instead, after a heart attack, it compensates for lost pumping ability by enlarging, a phenomenon known as ”remodeling.” .Remodeling, however, come with a high price, since the heart grows beyond the ability of the sparse cardiac circulatory system to properly convey blood to the enlarged heart muscle. Consequently, heart contraction weakens, leading to a condition known as congestive heart failure, which contributes to, or causes one in nine deaths in the United States. Heart disease is our nation’s leading killer.
Given the fact that heart disease is the result of the death of heart muscle cells, this condition seems to tailor-made for stem cell therapy. A variety of animal experiments with stem cells from bone marrow, muscle, fat, or embryos have shown that stem cells can regenerate heart muscle. However, the regeneration of the heart is much more complicated than was originally thought. For example, injecting damaged hearts with stem cells turned out to be a rather ineffective strategy because the heart, after a heart attack, is a very hostile place for newly infused cells. Dr. Kishore noted, “People know if they inject hundreds of stem cells into an organ, you’re going to be very lucky to find two of them the next day. They die. It’s as though you’re putting them into the fire and the fire burns them.”
Dr. Kishore has used a very different approach for regenerative medicine. Over 10 years ago, cancer researchers discovered tiny sacks excreted by cells that they called “exosomes.” These exosomes were thought to be involved with waste disposal, but later work showed that they were more mini-messengers, carry telegrams between cells. Exosomes proved to be one way a primary tumor communicated with distant metastases. Researchers later discovered that nearly all cell types excrete exosomes. Dr. Kishore and his team began to study the exosomes of stem cells to determine if these small vesicles could solve the heart-repair problem.
In 2011, Kishore’s team published the first paper to ever examine stem cell exosomes and heart repair. This paper established Kishore and his research team as a pioneer in exosome research and in the use of exosomes in the treatment of heart disease. A year after that paper, there were a total of 52 papers published on exosomes, but today there are 7,519 papers reporting on exosome research. Among those studies, only 13 or 14 have examined exosomes in heart disease. This new paper by Dr. Kishore’s team marks its third contribution to the science of exosomes and heart repair.
In the current study, Kishore and others used a mouse model of heart attack. Also involved in the research are Dr. Kishore’s colleagues from Temple’s Center for Translational Medicine, the Cardiovascular Research Center, and the Department of Pharmacology, as well as researchers from the Feinberg Cardiovascular Research Institute at Northwestern University in Chicago.
In this study, after suffering a heart attack, the mice received exosomes from either embryonic stem cells or exosomes from fibroblasts. Mice that received the fibroblast-derived exosomes served as the control group. The results were unmistakable. Mice that received exosomes from embryonic stem cells showed significantly improved heart function after a heart attack compared to the control group. More heart muscle cells in these mice survived after the heart attack, and their hearts also exhibited less scar tissue. Fewer heart cells committed suicide — a process known as programmed cell death, or apoptosis. Also, hearts from mice treated with embryonic stem cell-derived exosomes showed greater capillary development around the areas of injury. The increased density of blood vessels improved circulation and oxygen supply to the heart muscle. Further, there was a marked increase in endogenous cardiac progenitor cells, which is the hearts own internal stem cell population. These cells survived and created new heart cells. The heartbeat was more powerful in the experimental group compared to the control group, and the kind of unhealthy enlargement that compensates for tissue damage was minimized.
Vishore’s group also tested the effect of one of the most abundant gene-regulating molecules (microRNAs) found in the stem cell exosome; a microRNA called miR-294. When purified miR-294 alone was introduced to cardiac stem cells in the laboratory, it mimicked many of the effects seen when the entire exosome was delivered. “To a large extent, this micro-RNA alone can recapitulate the activity of the exosome,” Dr. Kishore said. “But we can never say it is responsible for all of the response because embryonic stem cell exosomes have many other microRNAs.”
Future research will examine both exosome therapy and the use of specific microRNAs for heart repair in large-animal models of heart attack with a view to eventually testing these components in human patients in clinical trials.
“Our work shows that the best way to regenerate the heart is to augment the self-repair capabilities and increase the heart’s own capacity to heal,” Dr. Kishore said. “This way, we’re avoiding risks associated with teratoma formation and other potential complications of using full stem cells. It’s an exciting development in the field of heart disease.”
The laboratory of Massimiliono Gnecchi at the Fondazione IRCCS Policlinico San Matteo in Pavia, Italy has used the products of amniotic mesenchymal stem cells to treat heart attacks in laboratory rodents. The results are rather interesting.
In a paper published in the May 2015 edition of the journal Stem Cells Translational Medicine, Gnecchi and his colleagues grew human amniotic mesenchymal stem cells derived from amniotic membrane (hAMCs) in cell culture.
These cells were isolated from amniotic membrane donated by mothers who were undergoing Caesarian sections. The membranes were removed, and grown in standard culture media under standard conditions. Once the cells grew out, they were collected and grow in a medium known as DMEM (Dulbecco’s modified Eagle Medium). After the cells had grown for 36 hours, they culture medium was filtered, concentrated, and readied for use.
The first experiments included the use of this conditioned culture medium to treat H9c2 embryonic heart muscle cells with in culture and then expose the heart muscle cells to low oxygen conditions. Normally, low oxygen conditions kill heart muscle cells. However, the cells pre-treated with conditioned medium from hAMCs showed much more robust survival in low-oxygen conditions. This shows that molecules secreted by hAMCs had promote the survival of heart muscle cells.
Next, Gnecchi and his team used their conditioned medium to treat laboratory rats that had suffered heart attacks. Some of the rats were treated with conditioned culture medium from cultured skin cells and others with sterile saline. The culture medium was injected directly into the heart muscle. The rats treated with conditioned medium from hAMCs showed far less cell death than the other rats. The rats treated with the hAMC-treated culture medium also had vastly denser concentrations of new blood vessels.
It is well-known that mesenchymal stem cells from many sources are filled with small vesicles known as exosomes that are loaded with healing molecules. Mesenchymal stem cells release these exosomes when they home to damaged tissues. The culture medium from the hAMCs were almost certainly filled with exosomes. The molecules released by these cells helped promote heart muscle cell survival in the oxygen-depleted heart, and induced the recruited large numbers of EPCs (endothelial progenitor cells), which established large numbers of new blood vessels. These new blood vessels gave oxygen to formerly depleted heart tissue and promoted heart healing. The size of the heart scar was smaller in the rats treated with hAMC-conditioned medium.
Unfortunately there were no measurement of cardiac function so we are not told if this treatment affected ejection fraction, or other physiological parameters. Nevertheless, this paper does show that exosomes from hAMCs do promote the production of blood vessels and cell survival.
Data from a Phase 2 clinical trial is creating quite a stir in cardiology circles. According to the findings of this study, the single administration of a gene on a non-viral-derived plasmid improves cardiac structure, function, serum biomarkers and clinical status in patients with severe ischemic heart failure one year after treatment.
The results from the final 12-months of the Phase 2 STOP-HF clinical trial for the JVS-100 treatment were presented at the European Society of Cardiology – Heart Failure 2015 meeting by the developer of this technology: Juventas Therapeutics Inc. The founder of Juventas, Marc Penn, M.D., Ph.D., FACC, is also the medical officer and director of Cardiovascular Research and Cardiovascular Medicine Fellowship at Summa Health in Akron, Ohio. Dr. Penn presented the results of this randomized, double-blind, placebo-controlled STOP-HF trial, which included treatments on 93 patients at 16 different clinical centers in the United States.
“The results from STOP-HF demonstrate that a single administration of 30 mg of JVS-100 has the potential to improve cardiac function, structure, serum biomarkers and clinical status in a population with advanced chronic heart failure who are symptomatic and present with poor cardiac function,” stated Dr. Penn. “These findings combined with our deep understanding of SDF-1 biology will guide future clinical trials in which we plan to prospectively study the patient population that demonstrated the most pronounced response to JVS-100. In addition, we will further our understanding of JVS-100 by determining if a second administration of drug may enhance benefits beyond those we observed with a single administration.”
These study. some patients received a 30 mg dose of JVS-100 while others received a placebo. Patients who received JVS-100 showed definite improvements 12 months after treatment. The cardiac function and heart structure of the patients who received JVS-100 were far better than those who had received the placebo. JVS-100-treated patients showed a changed in left ventricle ejection fraction of 3.5% relative to placebo, and left ventricular end-systolic volume of 8.5 ml over placebo. When patients were asked to walk for six minutes, the JVS-100-treated patients were better than patients who had received the placebo. Likewise, when patients were given the Minnesota Living with Heart Failure Questionnaire, the JVS-100-treat patients had a better score than those who had received the placebo. Also, there were no unanticipated serious adverse events related to the drug reported for the study.
JVS-100 is a non-viral DNA plasmid gene therapy. Plasmids are small circles of DNA that are relatively easy to manipulate, grow and propagate in bacterial cells. In the case of the JV-100 treatment, the plasmid encodes a protein called stromal cell-derived factor 1 (SDF-1). SDF-1 is a naturally occurring signaling protein that recruits stem cells from bone marrow to the site of SDF-1 expression. SDF-1, therefore, acts as a stem cell recruitment factor that summons stem cells to the places where they are needed.
When JV-100 is delivered directly to a site of tissue injury, it induces the expression of SDF-1 protein into the local environment for a period of approximately three weeks. SDF-1 secretion creates a homing signal that recruits the body’s own stem cells to the site of injury to induce tissue repair and regeneration.
Juventas is developing JVS-100 into a treatment of advanced chronic cardiovascular disease, including heart failure and late stage peripheral artery disease.
These improvements in heart function are relatively modest. Therefore, it is difficult to get too excited about these results. Also, Alexey Bersenev, a umbilical cord stem cell researcher, noted that the primary end points (or goalposts) for this trial were not met, and that makes this an unsuccessful trial. Despite this bad news, JV-100 does seem to be safe, and the theory seems sound, even if the results are more than a little underwhelming.
A research group from the Florida campus of The Scripps Research Institute (TSRI) has identified a new therapeutic approach that could promote the development of new bone-forming cells in patients suffering from bone loss.
The study was published in the journal Nature Communications, and it focused on a protein called PPARγ, which is a master regulator of fat, and the impact of this molecule on the fate of mesenchymal stem cells derived from bone marrow. Since these mesenchymal stem cells can differentiate into several different cell types, including fat, connective tissues, bone and cartilage. Consequently mesenchymal stem cells have a number of potentially important therapeutic applications.
A partial loss of PPARγ in a genetically modified mouse model led to increased bone formation. Could the use of drugs to inhibit PPARγ and potentially mimic that effect? This group combined a variety of structural biology approaches and then tried to design drugs that could fit PPARγ. This type of strategy is called “rational design,” and this yielded a new compound that could repress the biological activity of PPARγ.
The new drug, SR2595 (SR=Scripps Research), when applied to mesenchymal stem cells, significantly increased bone cell or osteoblast formation, a cell type known to form bone.
“These findings demonstrate for the first time a new therapeutic application for drugs targeting PPARy, which has been the focus of efforts to develop insulin sensitizers to treat type 2 diabetes,” said Patrick Griffin, chair of the Department of Molecular Therapeutics and director of the Translational Research Institute at Scripps Florida. “We have already demonstrated SR2595 has suitable properties for testing in mice; the next step is to perform an in-depth analysis of the drug’s efficacy in animal models of bone loss, aging, obesity and diabetes.”
In addition to identifying a new, potential therapeutic use for bone loss, this study may have even broader implications.
“Because PPARG is so closely related to several proteins with known roles in disease, we can potentially apply these structural insights to design new compounds for a variety of therapeutic applications,” said David P. Marciano, first author of the study, a recent graduate of TSRI’s PhD program and former member of the Griffin lab. “In addition, we now better understand how natural molecules in our bodies regulate metabolic and bone homeostasis, and how unwanted changes can underlie the pathogenesis of a disease.” Marciano will focus on this subject in his postdoctoral work in the Department of Genetics at Stanford University.
Research at Case Western Reserve, in collaboration with scientists from UT Southwestern Medical Center has identified yet another stem cell-activating drug. In animal models, this drug has helped mice regrow damaged liver, colon, and bone marrow tissue. The experimental drug examined in these experiments might open new possibilities for regenerative medicine. If clinical trials show that this drug therapy works in humans, it might save the lives of critically ill people with liver or colon disease or even some cancers.
This study was published in the journal Science. Even this work is exciting, this research is in the early stages and more work is necessary for the drug can be tested in people.
“We are very excited,” said co-author Sanford Markowitz, professor of cancer genetics at Case Western Reserve’s School of Medicine. “We have developed a drug that acts like a vitamin for tissue stem cells, stimulating their ability to repair tissues more quickly,” he added. “The drug heals damage in multiple tissues, which suggests to us that it may have applications in treating many diseases.”
This new drug is called SW033291. SW033291 works by inhibiting an enzyme with the formidable name of 15-hydroxyprostaglandin dehydrogenase, which is mercifully shortened to 15-PGDH. This enzyme degrades regulatory molecules called “prostaglandins.” One of these prostaglandins, known as prostaglandin E2, stimulates stem cell growth and differentiation. Inhibition of 15-PGDH increases the concentrations of prostaglandin E2 and stimulates the growth of tissue stem cells, which promotes healing.
Markowitz and his colleagues first showed that SW033291 inactivated 15-PGDH in a test tube. When they fed the drug to cells, it also inhibited 15-PGDH. Finally, they gave the drug to lab animals and showed that even in a living body, SW033291 inhibited 15-PGDH.
Does the drug augment healing? To determine this, Markowitz and others subjected mice to lethal doses of radiation, followed by a partial bone marrow transplant. Some of the mice were given SW033291 plus the bone marrow transplant while others received only the transplant. The mice that received SW033291 survived, while the others died.
In other studies, mice that had lost large amounts of blood were given SW033291, and mice given SW033291 recovered normal blood counts six days faster than mice that did not get the treatment.
Mice with an inflammatory disease called ulcerative colitis were given SW033291 and the drug “healed virtually all the ulcers in the animals’ colons and prevented colitis symptoms,” said the study’s authors.
“In mice where two-thirds of their livers had been removed surgically, SW033291 accelerated regrowth of new liver nearly twice as fast as normally happens without medication.” Additionally, SW033291 produced no adverse side effects.
Researchers who were not involved with the work said the study showed promise, but urged a heavy dose of caution. For example, Dusko Illic, a stem cell expert at Kings College London, said: “The drug seems to be too good to be true. We would have to be sure that nothing else was wrong with any organ in the body,” because if there were cancer cells present, the treatment would likely cause tumor cells to grow along with other tissue.
However, Ilaria Bellantuono, an expert in stem cell science and skeletal ageing at the University of Sheffield, said a key part of the drug’s promise could be in helping cancer patients, if it is proven safe. The “treatment has the potential of boosting patents’ resilience and improving their response to cancer treatment,” said Bellantuono. “This study is a proof of concept in mice and more experimental work is needed to verify the long-term safety of such an approach but it surely shows promise.”
The author of this study said that the first people to receive the experimental treatment in clinical trials would likely be patients who are receiving bone marrow transplants, have ulcerative colitis, or are undergoing liver surgery.
We normally think of bone as a very static tissue that does not change very much. However bone is actually a very dynamic tissue is constantly being remodeled in response to the needs of the organism. Bone remodeling is mediated by two different types of cells: osteoblasts that build bone and osteoclasts that resorb bone. Osteoblasts are derived from mesenchymal stem cells in the stroma of the bone marrow. The differentiation of mesenchymal stem cells into osteoblasts is mediated by molecules made by bone cells when bone is damaged. Osteoclasts come from pre-osteoclast cells that are monocyte-derived cells that fuse into multinucleate osteoclasts in response to the death of osteocytes (bone cells).
In healthy bone, osteocytes secrete a molecule called sclerostin, which prevents any new bone deposition. A break in bone causes the death of osteocytes near the site of the break, and the nearby osteocytes stop secreting sclerostin and start producing growth factors, nitric oxide and prostaglandins.
The lining cells of the bone marrow cavity detach and fuse with blood vessels. The mesenchymal stromal cells, under influence from IL-1, become pre-osteoblasts, and they start to secrete M-CSF, which prepares the pre-osteoclasts to fuse and become multinucleate osteoclasts. Pre-osteoclasts then express a molecule called RANKL, which binds to the RANK receptor on the surface of pre-osteoclasts and this induces them to fuse, and become mature osteoclasts. The osteoclasts secrete acid and cathepsin K to dissolve the damaged bone. The osteoclasts stop eating bone when the pre-osteoblasts mature into full-fledged osteoblasts that stop making RANKL and start making OPG, which binds to RANK, but does not activate it. Without this stimulation, the osteoclasts die. Then the osteoblasts divide, fill the cavity made by the now-deceased osteoclasts, and remake the bone. Some of the osteoblasts become entrapped in the bone matrix and become osteocytes. The bone takes several months to remineralize and 3-4 years to completely remineralize. See here for a video of this.
If there is a relative increase in bone resportion relative to bone deposition, the result is fragile, poorly mineralized bones, and this condition is known as osteoporosis. Decreased bone mass and bone strength causes an increased incidence of bone fractures, which often leads to further disability and early mortality. Bone healing is also impaired.
To treat osteoporosis, clinicians usually prescribe anti-resorptive agents that exert their effect by decreasing the rate of bone resorption. This strategy, however, has drawbacks, since as noted above, bone deposition relies on bone resorption. Inhibition of bone resorption also inhibits bone deposition, and bone tends to remain static and heal poorly.
A new paper has examined osteoporosis from the perspective of osteoblasts. It has been well established that in osteoblasts function is diminished in osteoporotic patients. Therefore increase osteoblast function is of chief interest. Work from the laboratories of Jihua Chen and Yan Jin from the Fourth Medical University has shown that a miniature RNA molecule called miR-26a plays a critical role in modulating bone formation during osteoporosis. Chen and Jin and others discovered that miR-26a treatment of mesenchymal stem cells effectively improved the osteogenic differentiation capability of these mesenchymal stem cells. In these experiments, they isolated mesenchymal stem cells from female mice that had their ovaries removed. Such mice are prone to undergo osteoporosis because they lack the hormone estrogen that stimulates osteoblast function. When these stem cells were treated with MiR-26a, they increased their bone-making capacities by in culture and when injected into live mice.
Further work showed that MiR-26a directly targets a gene called Tob1. Tob1 negatively regulates the BMP/Smad signaling pathway, and MiR-26a binds to the rear mRNA (3′-untranslated region) of Tob1, and prevents Tob1 translation.
These findings indicate that miR-26a is a potentially promising therapeutic candidate to enhance bone formation in order to treat osteoporosis and to promote bone regeneration in osteoporotic fracture healing.
Chiharo Akazawa from the Tokyo Medical and Dental University and his colleagues have tested two types of mesenchymal stem cells from human patients for their ability to make bone, cartilage, or fat. Their tests illustrated what has been shown several time before; mesenchymal stem cells tend to differentiate into the tissues that most closely resemble their tissue of origin.
Akazawa and his colleagues previously discovered a way to effectively isolated mesenchymal stem cells from bone marrow, which is no small feat because mesenchymal stem cells (MSCs) are a minority of the cells in bone marrow (Mabuchi and others (2013), Stem Cell Reports 1: 152-165). In a recent paper in the journal PLoS ONE, Akazawa and others used this technique to isolate MSCs from bone marrow and from synovial membrane – the fluid-filled sac that encases joints. In large joints, this synovium is large and called a “bursa.”.
In culture, the bone marrow-derived MSCs from several different human donors showed a marked tendency to form bone, but they did not make good cartilage or fat. The synovial MSCs, on the other hand, did not do so well at making bone, but made very good fat and cartilage. These differentiation trends were observed in MSCs culture for several different human donors. All cells were collected during arthroscopic surgery.
Since the synovial membrane of patients suffering from osteoarthritis undergoes, increased cell division, it is possible that the number of stem cells also increases. Alternatively, using MSCs from healthy donors who do not have arthritis may be even more preferable. Nevertheless, MSCs from synovial membrane show excellent cartilage-making potential and they may be a suitable source of cell for cartilage regeneration.
A new stem cell-based therapy has shown some very promising results. This therapy was designed to treat a rare and debilitating skin condition that affects children, for which no cure currently exists. This cell-based therapy provided pain relief and reduced the severity of the skin condition for patients who participated in the clinical trial.
The clinical trial was led by scientists at King’s College London, who collaborated with researchers from the Great Ormond Street Hospital (GOSH). They recruited 10 children afflicted with a disease called recessive dystrophic epidermolysis bullosa (RDEB).
RDEB is a painful skin disease in which very minor skin injury leads to blisters and wounds that tend to heal very slowly or not at all. The skin of RDEB patients is quite fragile and it tends to scar, develops contractures, and is also prone to life-threatening skin cancers.
This clinical trial, known as the EBSTEM trial, is a The Phase I/II trial whose results were published early online in the Journal of Investigative Dermatology. This study was designed to test the safety of infusions of stem cells and to determine if this treatment could help diminish the severity of the disease and improve quality of life for these patients.
During the first six months of the trial, participants were given three infusions of bone marrow- derived mesenchymal stromal cells from unrelated donors. Mesenchymal stem cells (MSCs) have been shown to home to wounded tissue and mediate wound healing in several previous studies. Although these infused stem cells do not survive permanently, they may still deliver therapeutic benefits.
The treated children were then monitored for a year after these cell infusions. Several different clinical tests failed to reveal any serious adverse effects in patients as a result of the stem cell treatment. When the pain levels of patients were measured, patients consistently reported lower pain levels after the treatment than before the treatment. Also the severity of their disease was also reported to have lessened following the stem cell infusions. Parents of these children reported better wound healing in their children and they also showed less skin redness and fewer blisters.
Overall, the outcomes of the trial are promising. However, this is an unblinded study of participants and may, therefore, contain positive biases in the way the information is reported. In interviews with families, participants reported a range of benefits from sleeping better, to the parents being able to return to work part-time because their children required less intense care. In fact, one family was actually able to plan their first vacation together.
Thus, further work is required to better understand the mechanisms that helped patients improve. Did the stem cells trigger the production of a growth factors and immune system regulators? Did these secreted compounds stimulate wound healing and reduce inflammation in the skin? Or did the presence of the cells somehow improve skin quality? Further studies are also required to confirm the efficacy of the treatment and establish the optimal dose of cells to give RDEB patients.
Ying-jian Zhud and Mu-jun Luan from the Shanghai Jiao Tong University in Shanghai, China teamed up to examine a new way to regenerate the bladder.
Several different synthetic and natural biomaterials have been pretty widely used in tissue regeneration experiments, particularly in the regeneration of the urinary bladder. The vast majority of this work has been done in rat model systems, which are fairly good animals to model bladder pathology and regeneration.
To date, the attempted reconstructive procedures don’t seem to work all that well, and this is due to the lack of appropriate scaffolding upon which cells can attach, grow and spread to form the new bladder tissue. Any scaffolding material for the bladder has to provide a waterproof barrier and it has to be able to support several different cell types. While this might not sound difficult on paper, it is in fact rather difficult. Some biomaterials might be well tolerated by the body, but cannot be fashioned into the shape of the organ. Others might support the growth of cells quite well, but are not tolerated by the body.
Zhud and Luan addressed these issues by turning to two different compounds that would compose a two-layered structure. Such a two-layered structure would support the cell types of the bladder. The outside layer was composed of silk fibroin, which is very moldable and usually well tolerated by cells. The inner layer consisted of a natural, acellular matrix (or BAMG for bladder acellular matrix graft). They used this two-layered structure to regenerate an injured bladder in rats.
First of all, it was clear that this material was relatively easy to make and it also could be nicely molded and sewn into the existing bladder. Tissue stains showed something even more interesting: the bilayer scaffold promoted the growth and recruitment of smooth muscles, blood vessels, and even nerves in a time-dependent manner. So by 12 weeks after implantation, bladders reconstructed with the bilayered matrix displayed superior structural and functional properties without significant local tissue responses or systemic toxicity.
Thus, the silk/BAMG scaffold could potentially be a promising scaffold for bladder regeneration. It shows good tissue compatibility, and allows the growth of cells on it. More work is required to take this to the next step, and the scaffold will undoubtedly undergo some changes. But this work represents a terrific start to what might be a superior scaffold for bladder regeneration.
Mesenchymal stem cells and multipotent adult progenitor cells (MAPCs) have received a good deal of discussion by scientists as agent for solid organ transplant recipients. Why? Because these cells, with their ability to suppress unwanted immune responses might be able to reduce the need for drugs that suppress the immune system, which have extensive side effects.
The study under discussion today is the clinical course of the first patient of the phase I, dose-escalation safety and feasibility study, MiSOT-I (Mesenchymal Stem Cells in Solid Organ Transplantation Phase I).
The patient received a living-related liver graft, each patient was given one intraportal injection (injection into the portal vein) and one intravenous infusion of third-party MAPC in combination with a low-dose of an anti-tissue-rejection drug.
The results so far are still coming in, but it seems that the administration of the cells is easy and is technically feasible. How well did the patients tolerate them? Quite well it turns out. There was no evidence of acute toxicity associated with infusions of the MAPCs. Also, there was some indication that the patient’s white blood cells were less reactive to foreign substances. However, it is difficult to make definitive statements about the efficacy of this treatment at this time.
Recruitment and follow-up of participants in the MiSOT-I trial continue, and completion of the study is currently projected for autumn 2016.
The moving pictures of American soldiers who lost limbs while serving their country come across our computer screens with some regularity. However, while we celebrate the courage of these young men and women, we should also be amazed at the technological advances that provide artificial limbs for these soldiers. What if, we could grow replacement limbs in culture? Is this science fiction? Maybe not.
The photo above comes from work done in the laboratory of Harald Ott who is at the Massachusetts General Hospital in Boston has succeeded in growing rodent forelimbs in the laboratory. “We’re focusing on the forearm and hand to use it as a model system and proof of principle,” said Ott. “But the techniques would apply equally to legs, arms and other extremities.”
“This is science fiction coming to life,” says Daniel Weiss at the University of Vermont College of Medicine in Burlington, who works on lung regeneration. “It’s a very exciting development, but the challenge will be to create a functioning limb.”
Modern amputees are often fitted with prosthetic limbs that have an excellent cosmetic look, but these artificial limbs don’t function as well as real limbs. Bionic replacement limbs that work well are now being made, but they look quite unnatural. Hand transplants have also been successful, but these surgeries are extremely expensive, and the recipient needs lifelong immunosuppressive drugs to prevent their body rejecting the transplanted hand.
Tissue engineered “biolimbs” would get round many of these obstacles as it only contains cells from the recipient and would, therefore, avoid the need for immunosuppression. Biolimbs would also look and behave naturally.
“This is the first attempt to make a biolimb, and I’m not aware of any other technology able to generate a composite tissue of this complexity,” says Ott.
To grow rat forelimbs in the laboratory, the so-called “decel/recel” technique was used. This same technique was previously been used to build hearts, lungs and kidneys in the lab. In fact, simpler organs such as windpipes and voice box tissue have been built and transplanted into people with varying levels of success, but not without controversy.
Decel stands for decellularization is the first step. In the decel step, organs from dead donors are treated with detergents that strips the soft tissue and leaves just the “scaffold” of the organ, which consists mainly from the inert protein collagen. This retains all the intricate architecture of the original organ. In the case of the rat forearm, these collagen structures include blood vessels, tendons, muscles and bones.
The second step, the recel step, recellularizes the flesh of the organ by seeding the scaffold with the relevant cells extracted from the recipient. This scaffold is then nourished in a bioreactor, which enables the new tissue to grow and colonize the scaffold. Because none of the donor’s soft tissue remains, this bioengineered limb, or biolimb, will not be recognized as foreign and rejected by the recipient’s immune system.
As tissue engineered organs go, the forearm is much more difficult to grow that a windpipe. It has a far greater number of cell types that need to be grown. Ott began by suspending the decellularized forelimb in a bioreactor, and then plumbing the collagen artery into an artificial circulatory system to provide nutrients, oxygen and electrical stimulation to the limb. Next, Ott and his colleagues injected human endothelial cells into the collagen structures of blood vessels to recolonize the surfaces of blood vessels. This was important, because, according to Ott, this made the blood vessels more robust and prevented them from rupturing as fluids circulated through them.
But would the limb’s muscles work? In order to work, the muscles must be connected to motor nerves from the central nervous system. To try this out, Ott’s team used electrical pulses to activate the muscles and found that the rat’s paw could clench and unclench. This experiment “showed we could flex and extend the hand,” says Ott. They also attached the biolimbs to anaesthetized healthy rats and saw that blood from the rat circulated in the new limb. However, they didn’t test for muscle movement or rejection.
While they have decellularized around 100 rat forelimbs, recellularizing at least half of them, there is still a great deal of work to do, said Ott. First they need to seed the limb with bone, cartilage and other cells to see whether these structures can be grown in the biolimb. Then they must demonstrate that a nervous system will develop in these cells. Results of hand transplants have shown the re-enervation occurs by means of the recipient’s nerve tissue growing into the transplanted hand and penetrating it. These growing nerves then make connections with the appropriate muscles. Thus, Ott believes that this would enable the recipients of a transplanted biolimb to control of their new organ. However, whether this also works in regenerated limbs remains to be seen.
Ott and his colleagues have also shown that forearms from nonhuman primates can be successfully decellularized. His team has begun recolonizing the primate scaffolds with human cells that line blood vessels, which is the first step towards human-scale biolimb development. They have also started experiments using human myoblasts in rats instead of the mouse myoblasts. Considerable work is needed to perfect this technology and it will be at least a decade before the first biolimbs are ready for human testing, says Ott, which is probably an optimistic estimate.
“It’s a notable step forward, and based on sound science, but there are some technical challenges that Harald’s group has to tackle,” says Steve Badylak of the University of Pittsburgh in Pennsylvania, who has used grafts built on scaffolds made from pig muscle to rebuild damaged leg muscles in 13 people. “Of these, the circulation is probably the biggest challenge, and making sure even the tiniest capillaries are successfully lined with endothelial cells so that they don’t collapse and cause clots,” he says. “But this is really an engineering approach, taking known fundamental principles of biology and applying them as an engineer would.”
Others are more critical. “For a complex organ like the hand, there are so many tissues and compartments that this definitely will not be a feasible protocol,” says Oskar Aszmann of the Medical University of Vienna in Austria, inventor of a bionic hand that people can control through their own thoughts. “Also, the hand must be innervated by thousands of nerves to have meaningful function, and that is at this point an insurmountable problem. So although this is a worthy endeavor, it must at this stage remain in the academic arena, not as a clinical scenario.”
In humans, Ott envisages organ donation schemes being extended to include transplantation of biolimbs. Cells for regenerating blood vessels could come from minor vessels supplied by the recipient, while muscle cells could come from biopsies from large muscles, such as in the thigh. “If you took about 5 grams, the size of a finger, you could grow it into human skeletal myoblasts,” he says.
With 1.5 million amputees in the US alone, this regeneration work is important, says Ott. “At present, if you lose an arm, a leg or soft tissue as part of cancer treatment or burns, you have very limited options.”
What if you could take a pill to induce regeneration in your wounded body? Amphibians can lose a leg or tail and readily regenerate it spontaneously. Mammals, unfortunately, generally form scars over the injury site during the process of wound repair. However, a strain of mouse known as the MRL mouse strain is an exception because these mice have the ability to spontaneously regenerate and heal, and this animal is a model system for regeneration.
Ellen Heber Katz and her colleagues at the Wistar Institute in Philadelphia, Pennsylvania have examined the MRL mouse in some detail and have discovered that a protein called hypoxia-inducible factor 1α (HIF-1α) plays a central role in the regeneration ability of adult MRL mice. The HIF-1α protein is usually degraded when normal levels of oxygen are available. Degradation of HIF-1α is mediated by enzymes called PHDs, which stands for prolyl hydroxylases. The presence of oxygen provides the substrate for PHDs to modify HIF-1α so that the cell sees it as a protein that is marked for degradation. MRL mice seem to have a rather stable form of HIF-1α, which stimulates healing.
Heber Katz and her co-workers designed a drug that would inhibit PHDs and stabilize HIF-1α. Next they took their drug (1,4-dihydrophenonthrolin-4-one-3-carboxylic acid or 1,4-DPCA) and encased it in hydrogel that would slowly release it over a course of 4 to 10 days. Heber Katz and her fellow researchers then injected this drug-laced hydrogel beneath the skin of Swiss Webster mice, which do not show an ability to spontaneously regenerate. They discovered that their hydrogel increased stable expression of HIF-1α protein over a 5 day period. Then, when they subjected these mice to skin wounds by punching a hole in their ears, the Swiss-Webster mice showed regenerative wound healing. No stem cells were injected, but this drug-laced hydrogel increased the regenerative ability of these animals.
Thus, increased expression of the HIF-1α protein seems to provide a starting point for future studies on regeneration in mammals. This work in preliminary, but think of it – taking a pill or getting an injection of some hydrogel that increases your body’s healing ability many fold. Of course, this is far in the future, but the possibilities are remarkable.
Stem cell-based therapies usually require the differentiation of stem cells into various cell types that are used for regenerative therapies. Such a strategy requires that the differentiated cells be purified from the rest of the cells. Typically, cell surface proteins are used as the means to distinguish cell types. Unfortunately, many undesired cell types may also share the same cell surface receptors, which will badly compromise the efficiency of cell purification.
Hirohide Saito from the Center for iPS Cell Research and Application (CiRA) at Kyoto University has designed a new way to isolate differentiated cells using microRNAs. This technique appears to be better than using cell surface proteins and it may revolutionize stem cell science.
Readers of this blog will recognize the term induced pluripotent stem cells or iPSCs, but for newer readers, I will provide a brief explanation of these cells. Induced pluripotent stem cells are made from mature, adult cells by means of genetic engineering and cell culture techniques. When the expression of four different genes (Oct4, Klf4, Sox2 and c-Myc) is forced in adult cells, a fraction of the cells de-differentiate and become like young, embryonic cells. When these cells are cultured ion special culture systems, they will aggregate and grow into an iPSC cell line. These cells have many, though not all, the features of embryonic stem cells, and they can, theoretically, differentiate into any adult cell type.
iPSCs are so popular in medical research because they are derived from a patient’s own body and they can be differentiate into any cell type. However, the protocols that are normally used to differentiate iPSCs lead to a mixed population of cells that are very heterogeneous, and the desired cell type has to be isolated from this mixture. Normally, antibodies that bind to surface receptors unique to the desired cell type are used for this purpose but in many cases such purification strategies are inefficient and the cell yield is rather poor. Also, these cell purification techniques have a tendency to damage cells.
New RNA-based procedures designed at CiRA may avoid these problems. Hirohide Saito and his colleagues designed tiny RNA molecules (microRNAs or miRNAs) that are designed to detect and sort live cells not by surface receptors, but by miRNAs. MicoRNAs are better markers of cell types and can improve purity levels. These “miRNA switches” as they are called, consist of synthetic mRNA sequences that include a recognition sequence for miRNA and an open reading frame (ORF) that codes a desired gene, such as a regulatory protein that emits fluorescence or promotes cell death. If the miRNA recognition sequence binds to miRNA expressed in the desired cells, the expression of the regulatory protein is suppressed, which helps distinguish one cell type from others that do not contain the miRNA and express the protein.
Senior Lecturer Yoshinori Yoshida, a heart muscle specialist who works with Professor Saito, immediately saw the potential of this technology. Dr. Yoshida has been studying how iPS cells can be used to combat cardiac diseases, but he has been stifled by unsatisfactory cell purification protocols. Heart muscle cells (cardiomyocytes) are especially difficult to purify because they do not possess unique cell surface proteins. So Professor Saito and Dr. Yoshida put their heads together to test the effectiveness of miRNA switches for isolating differentiated heart muscle cells from iPSCs.
First, they went to established heart muscle cell lines (which, by the way, are a colossal pain in the neck to deal with). They used these cells to define the miRNAs that are unique to cardiomyocytes. Then they designed several miRNA switches that contained sequences complementary to these miRNAs. After constructing the miRNA switches, they used them to isolated differentiated heart muscle cells from iPSCs.
The results were remarkable. Dr. Yoshida saw far better purification than he ever seen with standard methods. Furthermore, because this technology is RNA-based, it does not integrate into the genome and cause mutations. This could potentially make the cells eligible for clinical application.
Yoshida sees this tool as remarkably simple and something that can be used by stem cell researchers studying any organ. “It is just synthesizing RNA and transfecting them. It is not difficult,” he said. To prove this point, he and Saito used their miRNA switches to purify liver cells and pancreatic cells from iPSCs. This is significant, because neither of these cell types possess unique cell surface markers, but miRNA switches wre able to effectively purify them.
Intriguingly, the performance of different miRNA switches varied with the stages of cell development. This suggests that strategic selection of miRNAs could separate heart muscle cells that are at different developmental stages, which could also lead to even more homogeneous cell pools and potentially better cell therapy outcomes.
Saito believes that with further development, miRNA switches will be applicable to all cell types at all cell stages. “We want to make an active miRNA dictionary for each cell type, so that if we want to isolate this kind of cell type, we know how to use this kind of switch,” he said.
Fortunately, skeletal muscles have a high potential for regeneration, unlike other organs. When injured, muscle stem cells, known as satellite cells and located between the individual muscle fibers, rapidly begin to proliferate and subsequently replace the damaged muscles cells. New research from researchers from the Max Planck Institute for Heart and Lung Research in Bad Nauheim, Germany, have shown that a protein called Prmt5 plays a key role in regulating the activity of muscle satellite cells. These data gave rise to new studies that would like to examine the impact of Prmt5 in muscle disorders.
Satellite cells in skeletal muscles are small, spherical stem cells in between the individual muscles fibers. Normally, these cells remain almost completely inactive, but when a muscle is immediately begin to proliferate and heal the injury by replacing damaged muscles fibers.
When satellite cells react to an injury, they undergo a transition from their inactive state to one of increased activity. This transition must be finely balanced because uncontrolled proliferation of satellite cells in healthy muscle tissue increases the risk of tumor formation. Conversely, muscle regeneration is impeded if the satellite cells are not activated fast enough when muscles are injured.
Now a research team headed by Thomas Braun from the Max Planck Institute for Heart and Lung Research in Bad Nauheim has now identified a gene that plays a decisive role in regulating the activity of satellite cells. Braun and his colleagues isolated muscle satellite cells from laboratory mice and identified 120 genes that are instrumental for the function of these cells.
Next, they switched off one of these genes, Prmt5, in the satellite cells of adult mice. “In healthy mice, switching off Prmt5 in the satellite cells had no effect on the muscles. But when the mice had a muscle injury, the results were completely different”, says Ting Zhang, the study’s lead author. No signs of regeneration were observed in Prmt5-deficient mice, but the muscles of control mice that had an active Prmt5 gene healed normally. “Instead of growing new muscle tissue, the mice without Prmt5 eventually developed clear signs of fibrosis”.
Braun and others further examined how Prmt5 regulates muscle regeneration. In mice without Prmt5, the number of satellite cells was noticeably reduced. Prmt5 seems to regulate proliferation activity of satellite cells. Furthermore, these results indicated that Prmt5 also prevents satellite cells from dying prematurely and plays a key role in transforming them into functional muscle fibers.
Braun and his colleagues hope their study will help them gain a better understanding of muscle disorders in humans. “The loss of muscle tissue in the absence of Prmt5 shows clear parallels to degenerative muscle disorders such as Duchenne muscular dystrophy”, says Johnny Kim, a member of Braun’s working group. In fact, the Bad Nauheim team now hopes that in the future, mice lacking the Prmt5 gene can serve as models for this particular disorder. “But we also want to study the etiological effects of Prmt5 regarding the genesis of muscular hypertrophies and certain tumor types,” Kim adds.