Repairing Nerves Using Exosomes to Hijack Cell-Cell Communication

Biomedical engineers from Tufts University have discovered a new protocol that can induce mesenchymal stem cells (MSCs) derived from bone marrow to differentiate into neuron-like cells by treating them with exosomes from cultured cells.

PC12 cells are neuron-like progenitor cells derived from rats that can be successfully grown in culture. The Tufts team, led by Qiaobing Xu, found that exosomes extracted from cultured PC12 cells at various stages of differentiation could drive MSCs to differentiate into neuron-like cells.

Exosomes are very small, hollow particles that a wide range of cells types secrete. These tiny vehicles contain proteins, RNA, and other small molecules, and serve as a vehicle for communication between cells. In the nervous system, exosomes guide the direction of nerve growth, and they control nerve connection and direct peripheral nerve regeneration.

Xu and his team showed that these exosomes contain microRNAs (miRNAs), which a small RNA molecules that regulate gene expression and are known to play a role in neuronal differentiation. They hypothesized that these miRNAs activate neuron-specific genes in the MSCs that receive them and this is the reason these cells begin their journey towards differentiating into neurons.

“In combination with synthetic nanoparticles, we may ultimately be able to use these identified miRNAs or proteins to make synthetic exosomes, thereby avoiding the need to use any kind of neural progenitor cell line to induce neuron growth,” said Xu.

This work was published in PLoS ONE 2015; 10(8): e135111 DOI: 10.1371/journal.pone.0135111.

Exosomes Work As Well As Stem Cells to Heal Stroke Damage

A German research team at the University of Duisburg-Essen has published a study in the latest issue of STEM CELLS Translational Medicine that shows tiny membrane-enclosed structures that travel between cells work as well as adult stem cells to help the brain recover from a stroke.

Extracellular vesicles (EVs), which are small, membrane-enclosed structures that pass between cells, which are also referred to as exosomes, were given to one group of stroke-impaired mice and adult stem cells from bone marrow to another. After monitoring these mice for four weeks, both groups experienced the same degree of neurological repair. Besides promoting brain recovery in the mice, the EVs also down-regulated the post-stroke immune responses and provided long-term neurological protection.

This study could lead to a new clinical treatment for ischemic strokes, since exosomes carry far fewer risks than adult stem cell transplants, according to the co-leaders of this research, neurologist Thorsten Doeppner, and Bernd Giebel, a transfusion medicine specialist.

“We predict that with stringent proof-of-concept strategies, it might be possible to translate this therapy from rodents to humans, since EVs are better suited to clinical use than stem cell transplants,” said Doeppner and Giebel.

Scientists think that EVs carry biological signals between cells and direct a wide range of processes. Exosomes are under a good deal of scientific investigation for the role they could play in cancer, infectious diseases, and neurological disorders.

Other studies have shown that exosome administration can be beneficial after a stroke, but the Duisburg-Essen study is the first to supply evidence through a side-by-side analysis that they act as a key agent in repairing the brain.

“The fact that intravenous EV delivery alone was enough to protect the post-stroke brain and help it recover highlights the clinical potential of EVs in future stroke treatment,” Doeppner and Giebel said.

This study included contributions from ten different researchers from Duisburg-Essen’s Department of Neurology and Institute for Transfusion Medicine. The study was supported by the university, Volkswagen Foundation and German Research Council.

“The current research, combined with the previous demonstration that EVs are well tolerated in men, suggests the potential for using this treatment in conjunction with clot-busting therapies for treatment of stroke,” said Anthony Atala, M.D., editor of STEM CELLS Translational Medicine and Director of the Wake Forest Institute for Regenerative Medicine.

Treating a Damaged Liver: Bone Marrow CD45 Cells are Superior to Mesenchymal Stem Cells

Scarring of the liver, otherwise known as “liver fibrosis” usually results when the liver is constantly assaulted by inflammation. Conventional treatments for liver fibrosis are usually not very effective. Therefore, mesenchymal stem cells (MSCs) is an attractive alternative due to their ability to suppress inflammation. Unfortunately, transplanted MSCs tend to show poor survival in the scarred liver, and they have an additional tendency to stimulate the formation of new scar tissue. These characteristics have bred skepticism among many investigators.

New work by Asok Mukhopadhyay and his colleagues from New Delhi, India has compared bone marrow (BM)-derived cells with MSCs as a treatment for liver fibrosis. They used CCl4 to induce liver fibrosis in laboratory mice. Then they treated liver-damaged mice with either BM-CD45 cells or fat-based MSCs.

Liver tests and tissue samples of both sets of mice clearly showed that the BM-CD45 cells did a much job attenuating liver scarring than did the fat derived MSCs. Interestingly, the anti-scarring capacity of the BM-CD45 cells was compromised by the presence of MSCs.

Why did the BM-CD45 cells do a better job? The bone marrow cells expressed rather high level expressions of matrix metalloproteinases. These enzymes chopped through scar tissue and also suppressed the hepatic stellate cells, which are responsible for making the scar tissue in liver. Apparently, the BM-CD45 cells induced the die off of the stellate cells. MSCs, however, released two growth factors (TGFβ and IGF-1) that are known to activate hepatic stellate cells, and promote the formation of scar tissue. As an added bonus, transplantation of CD45 cells led to functional improvement of the damaged liver, and this functional improvement seems to the result of improved liver repair and regeneration.  Thus transplanted MSCs were pro-scarring while transplanted BM-CD45 cells were pro-regeneration, at least in the liver.

To summarize the results of these experiments, BM-derived CD45 cells appear to be a superior candidate for the treatment of liver fibrosis. The structural and functional improvement of CCl4-damaged livers was substantially better in animals that received transplants of BM-CD45 than those who received fat-derived MSCs.

Pancreatic Cancer Stem Cells Could Be “Suffocated” by an Anti-Diabetic Drug

A new study by researchers from Queen Mary University of London’s Barts Cancer Institute and the Spanish National Cancer Research Centre (CNIO) in Madrid shows that pancreatic cancer stem cells (PancSCs) are very dependent on oxygen-based metabolism, and can be “suffocated” with a drug that is already in use to treat diabetes.

Cancer cells typically rely on glycolysis; a metabolic pathway that degrades glucose without using oxygen. However, it turns out that not all cancer cells are alike when it comes to their metabolism.

PancSCs use another metabolic process called oxidative phosphorylation or OXPHOS to completely oxidize glucose all the way to carbon dioxide and water. OXPHOS uses large quantities of oxygen and occurs in the mitochondria. However, this is precisely the process that is inhibited by the anti-diabetic drug, metformin.

Ever crafty, some PancSCs manage to adapt to such a treatment by varying their metabolism, and this leads to a recurrence of the cancer. However, this English/Spanish team thinks that they have discovered a way to prevent such resistance and compel all PancSCs to keep using OXPHOS. This new discovery might open the door to new treatments that stop cancer stem cells using oxygen and prevent cancers from returning after conventional treatments. A clinical trial is planned for later next year.

Dr Patricia Sancho, first author of the research paper, said: “We might be able to exploit this reliance on oxygen by targeting the stem cells with drugs that are already available, killing the cancer by cutting off its energy supply. In the long-term, this could mean that pancreatic cancer patients have more treatment options available to them, including a reduced risk of recurrence following surgery and other treatments.”

PancSCs become resistant to metformin by suppressing a protein called MYC and increasing the activity of a protein called PGC-1α. However, this resistance mechanism of PancSCs can be abolished if a drug called menadione is given. Menadione increases the amount of reactive oxygen species in mitochondria. Additionally, resistance to metformin can be prevented or even reversed if the MYC protein is inhibited by genetic or pharmacological means. Therefore, the specific metabolic features of pancreatic Cancer Stem Cells are amendable to therapeutic intervention and can provide the basis for developing more effective therapies to combat this lethal cancer.

Pancreatic cancer is still one of the most difficult cancer types to treat. It rarely causes symptoms early on and does not usually trigger diagnosis until its later and more advanced stages. Unfortunately, many patients do not live longer than a year after being diagnosed. These cancers are also becoming more common due to obesity, which increases the patient’s risk for metabolic syndrome and diabetes, which are pancreatic cancer risk factors. Limited treatment options and a failure to improve survival rates mean that finding new treatment strategies is a priority.

PancSCs could be an important but as yet overlooked piece of this puzzle, since they compose only a small proportion of the tumor. PancSCs also have the potential to make new tumors, even if all the other cells are killed, and are prone to spreading around the body (metastasis). Therefore killing these PancSCs is a better way to treat such dangerous cancers.

Closing the Door on the STAP Episode

Last year, a group of Japanese researchers, led by scientists from the high-regarded RIKEN Center for Developmental Biology, reported a break-through in stem cell technology. Their so-called STAP or stimulus-triggered acquisition of pluripotency cells could be derived from mature, adult cells by exposing those cells to stressful conditions. Even though the papers that reported these advances were published in the prestigious journal Nature, immediately, people found problems in the papers that could not be easily resolved. Several laboratories tried to replicate the STAP results, with no success. The papers were eventually retracted and an internal investigation by the RIKEN Center also suggested that foul play might have been at work. Amidst all this, a question that hung in the air was this, “Was there something to the original discoveries but it was overstated?”

That question has now been definitely answered in the negative, thus closing the door for good on this whole sordid affair. Two papers were published on 23 September in the journal Nature, which was the same journal that published the original, ill-fated papers early last year that showed that STAP cells should be called NE (never existed) cells.

The original STAP papers were published in January 2014 by a team led by researchers at the RIKEN Center for Developmental Biology (CDB) in Kobe, Japan, in collaboration with scientists from Harvard Medical School in Boston, Massachusetts. These two papers claimed that embryonic-like stem cells could be produced by exposing adult body cells to stress, such as acidic conditions or physical pressure. These papers dubbed their technology “stimulus-triggered acquisition of pluripotency,” or STAP. Unfortunately, other scientists quickly discovered problems with data in the research. These problems then generated an investigation, and these papers were eventually retracted.

The paper retraction, however, did not answer the nagging questions as to whether or not the STAP procedure might have worked, and where the pluripotent stem cells labelled STAP in the RIKEN laboratory came from.

Such questions were addressed by seven teams in four countries who tried to replicate the procedure under various conditions (De Los Angeles, A. et al. Nature (2015). These teams collaborated to generate 133 attempts to produce STAP cells, and all of these attempts failed. One of these teams was led by researchers at Harvard Medical School who had worked with one of the original STAP co-authors. In this laboratory, cells were engineered to express a fluorescent protein when a gene related to pluripotency was expressed. When cells were exposed to stressful conditions, they did find some fluorescence, which suggested that pluripotency genes were expressed when cells were subjected to such conditions. However further testing showed this result to be an artifact since cells can naturally emit light; a phenomenon known as autofluorescence. Six other groups also observed autofluorescence in stressed cells, but no convincing evidence of STAP conversion.

A group of RIKEN researchers that did not include any authors of the original STAP papers analyzed the genomes of purported STAP cell lines that had be derived at the CDB. These scientists discovered multiple instances of contradictory data that probably resulted from contamination of purported STAP cells by other known cell types. The RIKEN group’s analyses showed that all remaining purported STAP stem cell lines, for example, were genetically identical to embryonic stem cell lines that already existed in the laboratory.

Additionally the “chimeric” mice that were reportedly produced by injecting STAP cells into the embryo of a developing mouse were found to have been produced by injecting pre-existing embryonic cell lines, rather than STAP cells, into the embryo. The production of chimeric mouse embryos is an experiment that definitively shows that particular cells are truly pluripotent.

Cell contamination also explains one of the most puzzling features of the original work, and that has to do with why the alleged STAP cells were reported to be capable of forming placental tissue, which is something that embryonic stem cells are not able to do (De Los Angeles, A. et al. Nature 525, 469–478 (2015)). These most recent analyses show that mixtures of trophoblast stem cells (which form the placenta in a developing embryo) were mixed with embryonic stem cells and that this mixture was used in the mouse chimeric experiments, leading to the production of mouse placental and embryonic tissue.

Stem-cell scientist Rudolf Jaenisch of the Massachusetts of Technology in Cambridge, who was part of the replication efforts, originally suggested in April 2014 to Nature’s news team that contamination was the reason for the results in the STAP papers. Unfortunately, he did not have evidence at the time for his hypothesis, but this most recent work has vindicated Jaenisch’s hypothesis.

A lingering question is how these embryonic stem cells and trophoblast stem cells came to replace purported STAP cells when the chimeric mouse experiments were performed. So-called cross-examination, which is the accidental contamination of one cell culture by another type of cell, is a well-known problem in cell culture experiments and biological research that depends on cultured cells. However, to properly explain the results in the original STAP papers, multiple independent contamination events must be invoked. “It is very difficult to reconcile the data with simple contamination or careless mislabeling,” says stem-cell scientist George Daley at Harvard Medical School. Unfortunately, requests for clarifying comments from corresponding authors of the original papers went unanswered.

In a review article published in Nature, Daley, Jaenisch argue that all new reports of new types of pluripotency should be subjected to rigorous “forensic” analysis that examines the genomes of the cells under consideration before publication. According to the authors, besides the failed STAP papers, “numerous groups are reporting ever more nuanced states of pluripotency.” In particular, the article focuses on genomic analyses, which are enabled by advances in sequencing technology, that will help evaluate such cell types.

Daley says that these experiments bring some well-desired closure to the STAP. He ended, however, with a warning to scientists who are looking for ways to reprogram cells to an embryonic-like state: “We will all be a tad more cautious in evaluating such claims.”

Laboratory-Grown Kidneys Work in Laboratory Animals

A Jikei University School of Medicine research team based in Tokyo, Japan, led by Takashi Yokoo, in collaboration with scientists from Meiji University and St. Marianna University School of Medicine in Kawasaki, Keio University School of Medicine in Tokyo, and the School of Veterinary Medicine at Kitasato University in Towada, has shown that mini kidneys grown in vitro from human stem cells can be effectively connected to the excretory systems of rats and pigs.

This is not the first time that research groups have successfully grown mini kidneys in the laboratory. However, connecting these laboratory-grown organs to a laboratory animal’s excretory system constitutes a major technical challenge. The Jikei University team used an approach that employs a step-wise peristaltic ureter or SWPU to connect its lab-grown mini kidneys to the ureter of the transplant animal.

Previous attempts to use laboratory-grown kidneys in laboratory animals have failed because while the transplanted kidneys made urine, they were unable to pass that urine to the animal’s bladder and the kidneys swelled up and failed. Yokoo and his collaborators and colleagues used a stem cell method to make their mini kidneys, as others have in the past.  However, he and his team grew more than just the kidney for the host animal; that also grew a drainage tube, known as a ureter, as well, in addition to a  bladder to collect and store the urine.

Yokoo and others used laboratory rats as the incubators for their growing tissue.  When they connected the new kidney and its tubular systems to the animal’s existing bladder, the system worked.  Urine passed from the transplanted kidney into the transplanted bladder and then into the rat bladder.  The transplant was still working well when they checked eight weeks later.  Then Yokoo and others repeated their procedure in pigs, which are larger mammals than rats and better model systems for human beings.  Fortunately, they achieved the same results.

Although this technology is still years away from clinical trials with human patients, this work provides a paradigm for making organs in the laboratory that will work in sick people.  In the United Kingdom alone, more than 6,000 people are waiting for a kidney.  Because of a shortage of kidney donors, fewer than 3,000 transplants are carried out each year, and more than 350 people die each year waiting for a transplant.  Growing new kidneys using human stem cells could solve this problem.

“To our knowledge, this is the first report showing that the SWPU system may resolve two important problems in the generation of kidneys from stem cells: construction of a urine excretion pathway and continued growth of the newly generated kidney,” Yokoo and others wrote in their paper, which was published in the Proceedings of the National Academy of Sciences, USA, which was communicated to the journal by National Academy of Science member R. Michael Roberts from the University of Missouri-Columbia.

Stem cell expert Prof Chris Mason from University College London, said: “This is an interesting step forward. The science looks strong and they have good data in animals.  But that’s not to say this will work in humans. We are still years off that. It’s very much mechanistic. It moves us closer to understanding how the plumbing might work.  At least with kidneys, we can dialyse patients for a while so there would be time to grow kidneys if that becomes possible.”

Human Muscle Satellite Cells Isolated and Characterized

A research group from the University of California, San Francisco have isolated and characterized human muscle stem cells. In addition, they have established that these stem cells can robustly replicate and repair damaged muscles when they are grafted onto an injured site. These remarkable findings might open the door to potential treatments for patients with severe muscle injuries, paralysis or genetic diseases that adversely affect skeletal muscles (e.g., muscular dystrophy).

Jason Pomerantz, MD is an assistant professor of plastic and reconstructive surgery at UCSF, and served as the managing author of this work. “We’ve shown definitively that these are bona-fide stem cells that can self-renew, proliferate and respond to injury,” said Pomerantz.

Badly damaged muscles can suffer terrible depletion of their native populations of stem cells or even obliteration of the stem cell niches and populations. Since such muscles have lost the very things that can heal them, these muscles will not be able to heal the damage they have sustained. This very fact represents a terrible hurdle for physicians who specialize in patients who have been crippled by muscle injury and paralysis. One of the worse cases is those conditions that cause damage or paralysis in the critical small muscles of the face, hand and eye, according to Pomerantz.

When muscles are badly damaged, they can lose the native populations of stem cells that are needed to heal. This has posed a major roadblock for treating patients crippled by muscle injury and paralysis, particularly in the critical small muscles of the face, hand and eye, Pomerantz said.

Fortunately, there have been remarkable surgical advances in restoring nerves in damaged muscles. Unfortunately, if the healing process takes too long, the stem cell pool is exhausted and the regenerative capacity is attenuated and eventually. Such injured muscles fail to connect to the nerve tissue and without accompanying motor and sensory nerves, skeletal muscles then to degenerate.

“This is partly why we haven’t had major progress in treating these patients in 30 years,” Pomerantz said. “We know we can get the axons there, but we need the stem cells for there to be recovery.”

A group of stem cells called “satellite cells” line the borders of muscle fibers and, in mice, can function as stem cells and contribute to muscle growth and repair. Until now, however, it wasn’t clear whether human satellite cells worked the same way. It was also terribly unclear how to isolate muscle satellite cells from human tissue samples or even adapt them to help treat patients with muscle damage.

Muscle satellite cells in section

Pomerantz and colleagues tackled this problem used muscle tissue from surgical biopsies of muscles of the head, trunk and leg. Then they used antibody staining to show that human satellite cells can be identified by the expression of the transcription factor PAX7 in combination with the cell-surface proteins CD56 and CD29. Pomerantz and his colleagues use this molecular signature to isolate populations of human satellite cells from these patient biopsies. Then they grafted these satellite cells into mice with damaged muscles whose own muscle stem-cell populations had been depleted. Five weeks after the transplantation, these human cells had successfully integrated into the mouse muscles and divided to produce families of daughter stem cells; effectively replenishing the stem cell niche and repairing the damaged muscle tissue.

This characterization of human muscle stem cells and the ability to transplant them into injured muscles has varied and wide-ranging implications for patients who are presently suffering from muscle paralysis, whose damaged muscles have lost the ability to regenerate. Additionally, protocols that allow us to isolate and manipulate human stem cells also may have applications for understanding why our muscles lose their regenerative capacity during normal aging or in the case of genetic diseases such as muscular dystrophy.

“This gives us hope that we will be able to extract healthy stem cells from other muscles in the patient’s body and transplant them at the site of injury,” Pomerantz said. “If replenishing a healthy muscle stem cell pool facilitates reinnervation and recovery, it would be a significant leap forward.”

These findings appeared the Sept. 8 edition in the open access Cell Press journal, Stem Cell Reports.