Exosomes from Placental Mesenchymal Stem Cells Confer Plasticity to Fibroblasts


Journal of Cellular Biochemistry
DOI: 10.1002/jcb.25459

Cells have the ability to internalize small portion of their membranes into small vesicles called endosomes. Once inside the cell, these endosomes can then internalize bits of their own membranes to form a kind of vesicle-within-a-vesicle structure called a “multivesicular endosomes” or MVEs. Cells can load up these MVEs with various proteins and RNAs and lipids, and then secrete them into their environment. Once released, these former MVEs become known as “extracellular vesicles” or EVs, and the smallest of the EVs are known as exosomes.

Exosome_Basics

Neighboring cells can take up exosomes released by nearby or far-flung cells and the proteins and RNAs delivered by the exosomes can elicit profound changes in cell behavior. Therefore, exosomes act as agents of cell-cell communication. Detection of exosomes from cancer cells into blood, or other bodily fluids can provide diagnostic insights into the diagnosis of cancers. Also exosomes from stem cells can augment tissue healing and repair (see here).

Exosomes from mesenchymal stem cells (MSCs) are released into the culture medium when MSCs are grown in the laboratory. Exosome-containing culture medium is often referred to as “conditioned medium” and in this case it is conditioned medium from MSCs, which we will refer to as MSC-CM. MSC-CM has been reported to enhance wound healing in several different experiments.

In a new study by Motohiro Komaki and his colleagues at the Tokyo Medical and Dental University, in Tokyo, Japan has examined the effects of exosomes from human placenta MSCs in a controlled culture system to better characterize the biological roles of these exosomes.

In this study, placental MSCs were from donated human placenta from newborn babies by means of enzymatic digestion. Exosomes were prepared from these placental MSCs (PlaMSCs) by growing the cells in culture and then subjecting the conditioned culture medium to ultracentrifugation. Exosomes, fortunately, are relatively easy to isolate and can be frozen and stored quite effectively without loss of function.

These isolated PlaMSC exosomes were then given to cultured human dermal fibroblasts. Changes in gene expression were then ascertained by real-time reverse transcriptase PCR analysis (real-time PCR).

After incubating fibroblasts with exosomes from PlaMSCs, the expression of stemness-related genes, such as OCT4 and NANOG were significantly increased. These exosomes also stimulated the differentiation of these fibroblasts into bone cells and fat cells when the cells were subjected to bone and fat induction media. The fibroblasts expressed bone-specific and fat-specific genes and enzymes.

Thus, these experiments showed that exosomes from PlaMSCs increased the expression of the stemness genes OCT4 and NANOG in fibroblasts, which normally do not express these genes. Consequently, these exosomes influence ability of fibroblasts to differentiate in both bone and fat cells. This work demonstrates a new feature of MSCs and their exosomes, and suggests new possible clinical applications for MSC exosomes.

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.

Stem Cell-Based Exosomes Heal Hearts After a Heart Attack


A new paper in the journal Circulation Research by a research team from the Temple University School of Medicine (TUSM) has examined the use of tiny stem cell-based vesicles to help limit the damage caused by a heart attack. Even those these experiments were performed with laboratory mice, the result are very promising.

A heart attack tends to badly damage it, and since the heart has little innate ability to repair itself, it has to compensate by growing large and flabby, which can lead to congestive heart failure, Congestive heart failure is currently responsible for one in nine deaths in the United States.

The research team of Raj Kishore at the Temple University School of Medicine turned to exosomes to heal the heart. Exosomes are tiny sacks secreted by cells that act as messengers that pass messengers between cells in various parts of the body. While these extracellular vesicles are secreted by nearly all types of cell, exosomes from stem cells might be a useful tool in mitigating damage caused by heart attacks.

Exosomes
Exosomes

“If your goal is to protect the heart, this is a pretty important finding,” Dr. Kishore said. “You can robustly increase 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 also beginning to determine those members of this “work crew” within the vesicles may be responsible for the damage repair.

Previous studies have shown that injecting damaged hearts with stem cells increases heart function after a heart attack. However, the injected cells tend to not survive very long when placed in the damaged heart, and most of their benefits are due to molecules that the administered stem cells secrete. Pluripotent stem cells (embryonic stem cells, for example) run the risk of creating a tumor made up of a mass of cells of different tissue types, known as a teratoma. Therefore, Khan’s tram approached the problem from a slightly different angle by injecting only the exosomes made by stem cells. It was known that this would avoid the teratoma problem, and could have positive effects on damaged heart tissue.

Exosome poster

The study examined mice that had suffered heart attacks. These animals were split into two groups; one group received exosomes from mouse embryonic stem cells, and the other group were injected with fibroblast exosomes.

The results were extremely promising. The mice that had received stem cell-derived exosomes exhibited improved heart function, less scar tissue, lower levels of programmed cell death and better capillary development around the damaged area. There was also a higher presence of cardiac progenitor cells – the heart’s own stem cells – in the stem cell exosome-injected mice. Overall, the heartbeat of the mice was stronger than those in the control group, with less unhealthy enlargement of the organ.

Khan and others examined an abundant gene-regulating molecule (microRNA) from the stem cell-derived exosomes, known as miR-294. They introduced this microRNA into cultured cardiac stem cells. This microRNA recapitulated many of the positive effects of the stem cell exosomes that had been observed in the animal study.

Khan and his coworker plan to continue their research by studying the effects of individual microRNAs on damaged heart tissue.

“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,” says researcher Dr Mohsin Khan. “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.”

Temple University Lab Shows Exosomes from Stem Cells Heal Hearts After a Heart Attack


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.”

Culture Medium from Human Amniotic Membrane Mesenchymal Stem Cells Promotes Cell Survival and Blood Vessel Production in Damaged Rat Hearts


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.

Cardiac Stem Cells or their Exosomes Heal Heart Damage Caused by Duchenne Muscular Dystrophy


One of the research institutions that has been at the forefront of developing investigational stem cell treatments for heart attack patients is The Cedars-Sinai Heart Institute. Recently, a research team at Cedars-Sinai Heart Institute (CSHI) has injected cardiac stem cells into the hearts of laboratory mice afflicted with a rodent form of Duchenne muscular dystrophy. This disease can also adversely affect the heart, and these stem cell injections actually improved the heart function of these laboratory animals and resulted in greater survival rates for those mice. This work might provide the means to extend the lives and improve the quality of life of patients with this chronic muscle-wasting disease.

The CSHI team presented their results at the American Heart Association Scientific Sessions in Chicago. Their results clearly demonstrated that once laboratory mice with Duchenne muscular dystrophy were infused with cardiac stem cells, the animals showed progressive and significant improvements in heart function and increased exercise capacity.

Specifically, 78 lab mice that had been given laboratory-induced heart attacks were injected with their own cardiac stem cells, and over the next three months, these mice demonstrated improved pumping ability and exercise capacity in addition to a reduction in heart-specific inflammation. The CSHI team also discovered that the stem cells work indirectly, by secreting tiny vesicles called exosomes that are filled with molecules that induce tissue healing. When these exosomes were purified and administered alone, they reproduced the key benefits of the cardiac stem cells.

Apparently, this particular procedure could be ready for testing in human clinical studies as soon as next year.

Duchenne muscular dystrophy or DMD is a genetic disease that results from mutations in a gene found on the X chromosome in humans. DMD affects 1 in 3,600 boys and is a neuromuscular disease caused by abnormalities in a muscle protein called dystrophin.  Because dystrophin is an important structural protein for muscle that anchors muscle to other muscles and to the substratum, deficiencies for functional copies of the dystrophin protein cause progressive muscle wasting, destruction, and muscle weakness.

Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin (see Fig. 2A for details of dystrophin domains). αDG, α-dystroglycan; βDG, β-dystroglycan
Dystrophin acts as an important link between the internal cytoskeleton and the extracellular matrix. Neuronal nitric oxide synthase (nNOS) binds to α-syntrophin but also has a binding site in repeat 17 of the rod domain of dystrophin (see Fig. 2A for details of dystrophin domains). αDG, α-dystroglycan; βDG, β-dystroglycan.  See here

The majority of DMD patients lose their ability to walk by twelve years of age, although the severity of the disease varies from patient to patient. The average life expectancy is about 25, and the cause of death is usually heart failure. Dystrophin deficiency causes heart muscle weakness, and, ultimately, heart insufficiency, since the chronic weakness of the heart muscle prevents the heart from pumping enough blood to maintain a regular heart rhythm and provide for the needs of the rest of the body. Such a heart condition is called “cardiomyopathy.”

“Most research into treatments for Duchenne muscular dystrophy patients has focused on the skeletal muscle aspects of the disease, but more often than not, the cause of death has been the heart failure that affects Duchenne patients,” said Eduardo Marbán, MD, PhD, who is the director of the CSHI and the principal investigator of this particular study. “Currently, there is no treatment to address the loss of functional heart muscle in these patients.”

In 2009, Marbán and his team completed the world’s first procedure in which a patient’s own heart tissue was used to grow specialized heart stem cells. Stem cells from the heart were isolated, cultured, and then injected back into the patient’s heart in order to repair and regrow healthy heart muscle that had been injured by a heart attack. Results, Marbán and his colleagues published these results in The Lancet in 2012, and also demonstrated that one year after their patients had received the experimental stem cell treatment, they showed significant reductions in the size of the heart scar that had been produced by their heart attacks.

Earlier this year, CSHI researchers commenced a new clinical trial entitled “ALLSTAR,” which stands for Allogeneic Heart Stem Cells to Achieve Myocardial Regeneration (Clinical trial number NCT01458405). In this study, heart attack patients are given injections of stem cells from healthy donors, which should work better than the patient’s own stem cells, which were damaged by the heart attack.

CSHI has recently opened the nation’s first Regenerative Medicine Clinic, which is designed to match heart and vascular disease patients with the appropriate stem cell clinical trial being conducted at CSHI and other institutions.

“We are committed to thoroughly investigating whether stem cells could repair heart damage caused by Duchenne muscular dystrophy,” Marbán said.

The protocols for growing cardiac-derived stem cells were developed by Marbán when he was on the faculty of Johns Hopkins University. Johns Hopkins has filed for a patent on that intellectual property and has licensed it to Capricor, a company in which Cedars-Sinai and Marbán have a financial interest. Capricor is providing funds for the ALLSTAR clinical trial at Cedars-Sinai.

Regenerating Injured Kidneys with Exosomes from Human Umbilical Cord Mesenchymal Stem Cells


Zhou Y, Xu H, Xu W, Wang B, Wu H, Tao Y, Zhang B, Wang M, Mao F, Yan Y, Gao S, Gu H, Zhu W, Qian H: Exosomes released by human umbilical cord mesenchymal stem cells protect against cisplatin-induced renal oxidative stress and apoptosis in vivo and in vitro. Stem Cell Res Ther 2013, 4:34.

Ying Zhou and colleagues from Jiangsi University have provided helpful insights into how adult stem cell populations – in particular, mesenchymal stem cells (MSCs) isolated from human umbilical cord (hucMSCs) – are able to regulate tissue repair and regeneration. Adult stem cells, including MSCs from different sources, confer regenerative effects in animal models of disease and tissue injury. Many of these cells are also in phase I and II trials for limb ischemia, congestive heart failure, and acute myocardial infarction (Syed BA, Evans JB. Nat Rev Drug Discov 2013, 12:185–186).

Despite the documented healing capabilities of MSCs, in many cases, even though the implanted stem cells produce genuine, reproducible therapeutic effects, the presence of the transplanted stem cells in the regenerating tissue is not observed. These observations suggest that the predominant therapeutic effect of stem cells is conferred through the release of therapeutic factors. In fact, conditioned media from adult stem cell populations are able to improve ischemic damage to kidney and heart, which confirms the presence of factors released by stem cells in mediating tissue regeneration after injury (van Koppen A, et al., PLoS One 2012, 7:e38746; Timmers L, et al., Stem Cell Res 2007, 1:129–137). Additionally, the secretion of factors such as interleukin-10 (IL-10), indoleamine 2,3-dioxygenase (IDO), interleukin-1 receptor antagonist (IL-1Ra), transforming growth factor-beta 1 (TGF-β1), prostaglandin E2 (PGE2), and tumor necrosis factor-alpha-stimulated gene/protein 6 (TSG-6) has been implicated in conferring the anti-inflammatory effects of stem cells (Pittenger M: Cell Stem Cell 2009, 5:8–10). These observations cohere with the positive clinical effects of MSCs in treating Crohn’s disease and graft-versus-host disease (Caplan AI, Correa D. Cell Stem Cell 2011, 9:11–15).

Another stem cell population called muscle-derived stem/progenitor cells, which are related to MSCs, can also extend the life span of mice that have the equivalent of an aging disease called progeria. These muscle-derived stem/progenitor cells work through a paracrine mechanism (i.e. the release of locally acting substances from cells; see Lavasani M, et al., Nat Commun 2012, 3:608). However, it is unclear what factors released by functional stem cells are important for facilitating tissue regeneration after injury, disease, or aging and the precise mechanism through which these factors exert their effects. Recently, several groups have demonstrated the potent therapeutic activity of small vesicles called exosomes that are released by stem cells (Gatti S, et al., Nephrol Dial Transplant 2011, 26:1474–1483; Bruno S, et al., PLoS One 2012, 7:e33115; Lai RC, et al., Regen Med 2013, 8:197–209; Lee C, et al., Circulation 2012, 126:2601–2611; Li T, et al., Stem Cells Dev 2013, 22:845–854). Exosomes are a type of membrane vesicle with a diameter of 30 to 100 nm released by most cell types, including stem cells. They are formed by the inverse budding of the multivesicular bodies and are released from cells upon fusion of multivesicular bodies with the cell membrane (Stoorvogel W, et al., Traffic 2002, 3:321–330).

Exosomes are distinct from larger vesicles, termed ectosomes, which are released by shedding from the cell membrane. The protein content of exosomes depends on the cells that release them, but they tend to be enriched in certain molecules, including adhesion molecules, membrane trafficking molecules, cytoskeleton molecules, heat-shock proteins, cytoplasmic enzymes, and signal transduction proteins. Importantly, exosomes also contain functional mRNA and microRNA molecules. The role of exosomes in vivo is hypothesized to be for cell-to-cell communication, transferring proteins and RNAs between cells both locally and at a distance.

To examine the regenerative effects of MSCs derived from human umbilical cord, Zhou and colleagues used a rat model of acute kidney toxicity induced by treatment with the anti-cancer drug cisplatin. After treatment with cisplatin, rats show increases in blood urea nitrogen and creatinine levels (a sign of kidney dysfunction) and increases in apoptosis, necrosis, and oxidative stress in the kidney. If exosomes purified from hucMSCs, termed hucMSC-ex are injected underneath the renal capsule into the kidney, these indices of acute kidney injury decrease. In cell culture, huc-MSC-exs promote proliferation of rat renal tubular epithelial cells in culture. These results suggest that hucMSC-exs can reduce oxidative stress and programmed cell death, and promote proliferation. What is not clear is how these exosomes pull this off. Zhou and colleagues provide evidence that hucMSC-ex can reduce levels of the pro-death protein Bax and increase the pro-survival Bcl-2 protein levels in the kidney to increase cell survival and stimulate Erk1/2 to increase cell proliferation.

Another research group has reported roles for miRNAs and antioxidant proteins contained in stem cell-derived exosomes for repair of damaged renal and cardiac tissue (Cantaluppi V, et al., Kidney Int 2012, 82:412–427). In addition, MSC exosome-mediated delivery of glycolytic enzymes (the pathway that degrades sugar) to complement the ATP deficit in ischemic tissues was recently reported to play an important role in repairing the ischemic heart (Lai RC, et al., Stem Cell Res 2010, 4:214–222). Clearly, stem cell exosomes contain many factors, including proteins and microRNAs that can contribute to improving the pathology of damaged tissues.

The significance of the results of Zhou and colleagues and others is that stem cells may not need to be used clinically to treat diseased or injured tissue directly. Instead, exosomes released from the stem cells, which can be rapidly isolated by centrifugation, could be administered easily without the safety concerns of aberrant stem cell differentiation, transformation, or recognition by the immune system. Also, given that human umbilical cord exosomes are therapeutic in a rat model of acute kidney injury, it is likely that stem cell exosomes from a donor (allogeneic exosomes) would be effective in clinical studies without side effects.

These are fabulously interesting results, but Zhou and colleagues have also succeeded in raising several important questions. For example: What are the key pathways targeted by stem cell exosomes to regenerate injured renal and cardiac tissue? Are other tissues as susceptible to the therapeutic effects of stem cell exosomes? Do all stem cells release similar therapeutic vesicles, or do certain stem cells release vesicles targeting only specific tissue and regulate tissue-specific pathways? How can the therapeutic activity of stem cell exosomes be increased? What is the best source of therapeutic stem cell exosomes?

Despite these important remaining questions, the demonstration that hucMSCderived exosomes block oxidative stress, prevent cell death, and increase cell proliferation in the kidney makes stem cell-derived exosomes an attractive therapeutic alternative to stem cell transplantation.

See Dorronsoro and Robbins: Regenerating the injured kidney with human umbilical cord mesenchymal stem cell-derived exosomes. Stem Cell Research & Therapy 2013 4:39.

Recovery of the Brain After a Stroke


A stroke results when the brain suffers from “ischemia” or a lack of blood flow for an extended period of time. Blockage in the small vessels that feed blood to the brain can cause a trans-ischemic attack (TIA) or stroke. The lack of oxygen causes localized death of brain cells. The dying cells dump a whole gaggle of molecules into the spaces surrounding nearby brain cells, and these cell-derived molecules can actually poison surrounding cells, thus increasing the area that dies as a result of a stroke.

Stroke pathology

New work from by Henry Ford Hospital researchers in Detroit, Michigan suggests that some of the molecules released by brain cells during a stroke might actually help the brain heal after a stroke. Small RNA molecules or microRNAs that are packaged into lipid-bound vesicles in cells known as exosomes are released by stem cells after a stroke and seem to contribute to neurological recovery.

Exosomes are secreted vesicles that were first discovered nearly 30 years ago. They were, at first, considered little more than garbage cans whose job was to discard unwanted cellular components. However, once cell biologists began to study these little structures, evidence began to accumulate that these dumpsters also act as messengers that convey information to distant tissues. Exosomes contain cell-specific payloads of proteins, lipids, and genetic material that are transported to other cells, where they alter function and physiology.

Exosome_Basics

Therefore, it is little wonder that exosomes can also transport microRNAs. In this present study from the laboratory of Michael Chopp, rats were given experimentally induced strokes, and then the neurological recovery of the rats was examined at the molecular level.

Chopp and his colleagues first isolated mesenchymal stem cells (MSCs) from the bone marrow of their laboratory rats. Then they genetically engineered these MSCs to release exosomes laden with specific microRNAs; in particular miR-133b.

MicroRNAs are a class of post-transcriptional regulators. Since they are usually only about 22 base pairs in length, they are far too short to encode anything. microRNAs usually bind to complementary sequences in the 3’ untranslated region of messenger RNAs, and this binding silences the RNA, which simply means that the RNA cannot be recognized by ribosomes and will not be translated into protein, or that the RNA is degraded by special enzymes that target RNAs bound by microRNAs. Single microRNAs target hundreds genes at a time, and some 60% of all genes are regulated by microRNAs. MicroRNAs are abundantly present in all human cells. They are also highly conserved in organisms ranging from the unicellular algae Chlamydomonas reinhardtii to mitochondria in vertebrates, which suggest that they are a vital part of genetic regulation throughout the plant and animal kingdoms.

The Actions of Small Silencing RNAs (A) Messenger RNA cleavage specified by a miRNA or siRNA. Black arrowhead indicates site of cleavage. (B) Translational repression specified by miRNAs or siRNAs. (C) Transcriptional silencing, thought to be specified by heterochromatic siRNAs.
The Actions of Small Silencing RNAs
(A) Messenger RNA cleavage specified by a miRNA or siRNA. Black arrowhead indicates site of cleavage.
(B) Translational repression specified by miRNAs or siRNAs.
(C) Transcriptional silencing, thought to be specified by heterochromatic siRNAs.

The microRNA known as miR-133b has been shown to enhance the death of prostate cancer cells when they are delivered to them (see Patron JP, Fendler A, Bild M, Jung U, Müller H, et al. (2012) MiR-133b Targets Antiapoptotic Genes and Enhances Death Receptor-Induced Apoptosis. PLoS ONE 7(4): e35345. doi:10.1371/journal.pone.0035345). However, because different cell types show different responses to the same reagents, exposing brain cells to this microRNA after a stroke might elicit a very different response.

By raising or lowering the amount of miR-133b in MSCs, Chopp and his colleagues were able to determine the effects of miR-133b on brains cells after a stroke. Chopp and others injected their genetically engineered MSCs into the bloodstream of rats 24 hours after inducing a stroke in these animals. When the exosomes of the MSCs were enriched in miR-133b, the neurological recovery in the rats was amplified, but when injected MSCs were deprived of miR-133b, the neurological recovery was substantially less.

To measure neurological recovery, researchers separated the disabled rats into several groups and injected each groups with saline, nongenetically-engineered MSCs, MSCs with low levels of miR-133b, and MSCs with high levels of miR-133b. The rats were given behavioral tests 3, 7, and 14 days after treatment. These tests measured the gait of the animals on a grid to determine if the rats could walk on an unevenly spaced grid (foot-fault test). The second test determined how long it took the rats to remove a piece of adhesive tape that was stuck to their front paws.

in every test, the rats injected with miR-133b-enriched MSCs showed superior levels of neurological recovery. Autopsies of these same animals revealed that the rats treated with miR-133b-enriched MSCs had enhanced rewiring of the brain and axonal outgrowth. In the areas of the brain adversely affected by the stroke, the rats showed increased axonal plasticity and neurite remodeling.

Most stroke victims recover some ability to use their hands and other body parts on a voluntary basis, but almost half of all stroke victims are left with some weakness on one side of their body and many are permanently disabled by the stroke.

No treatment presently exists for improving or restoring this lost motor function in stroke patients, mainly because of mysteries about how the brain and nerves repair themselves.

Chopp said, “This study may have solved one of these mysteries by showing how certain stem cells play a role in the brain’s ability to heal itself to differing degrees after stroke or other trauma. Chopp also serves as the scientific director of the Henry Ford Neuroscience Institute.

Stem Cell-Derived Exosomes May Prevent Lung Disease in Babies


Now there’s a word you don’t see everyday: Exosome. What on earth is an exosome? They are small, membrane-enclosed vesicles that are released by cells. Exosomes contain proteins and nucleic acids, and they are have surfaces that are decorated with various types of proteins. Exosomes are 40-100 nm in diameter and there are several different cell types that are known to secrete exosomes. Cancer cells use exosomes to mold surrounding cell into structures that the cancer needs (see Huang et al., (2011). Cancer Lett 315, 28-37 and Cho et al., (2012). Int J Oncol. 40(1):130-138). Likewise, exosomes from mesenchymal stem cells seem to delivery proteins and RNA to heart muscle cells that help them heal after a heart attack (Lai et al., Regen. Med. (2011) 6(4), 481–492). Finally, there have been reports that exosomes can protect against tissue injury such as acute kidney damage (see Bruno S, et al. (2009). J. Am. Soc. Nephrol. 20, 1053–1067.

New work from Harvard University’s Boston Children’s Newborn Medicine division in Massachusetts suggests that exosomes from stem cells can protect the fragile lungs of premature babies from serious lung diseases and chronic lung injury. Mesenchymal stem cells (MSCs) can decrease inflammation under several different conditions. They seem to do so by secreting exosomes that hold inflammatory cells at bay. In a mouse model of lung inflammation, infused MSCs can quell inflammation, but the culture medium in which the MSCs were grown can do as good a job and decreasing inflammation as the whole cells. This suggest that the MSCs are making something that assuages inflammation (see Ionescu, L. et al., (2012). Am J Physiol Lung Cell Mol Physiol. doi: 10.​1152/​ajplung.​00144.​2011).

Babies born prematurely have to struggle to get sufficient oxygen into their small, incipient lungs. This causes these poor babies to suffer from chronic oxygen insufficiencies (hypoxia) and they usually need artificial respirators to breathe. The lungs of these babies are susceptible to inflammation and this can lead to chronic lung disease and poor lung function.

Lung inflammation also causes pulmonary hypertension, which simply means that the blood pressure in the artery that carries blood from the heart to the lungs (pulmonary artery) is abnormally high. This causes foaming in the lungs, which reduces gas exchange in the lungs, but the lungs also start to thicken and scar over and that also permanently decreases gas exchange.

Stella Kourembanas is the chair of Boston Medical’s Newborn Medicine Division, and she is heading up investigations into the efficacy of MSC-derived exosomes to stave off inflammation in the lungs of premature babies.

Kourembanas explained, “PH (pulmonary hypertension) is a complex disease fueled by diverse, intertwined cellular and molecular pathways. We have treatments that improve symptoms but no cure, largely because of this complexity. We need to be able to target more than one pathway at a time.”

In 2009, Kourembanas and her colleagues showed that injections of MSCs could prevent PH and chronic lung injury in a newborn mouse model of the disease (Bone marrow stromal cells attenuate lung injury in a murine model of neonatal chronic lung disease (Aslam M, et al., (2009). Am J Respir Crit Care Med. 180(11):1122-30). They also showed that they could achieve the same results by injecting the growth medium that had previously fed the cells.

According to Kourembanas, “We knew, then, that the significant anti-inflammatory and protective effects we saw had to e caused by something released by he MSCs.”

To further their search for factors that heal damaged lungs, Kourembanas and co-workers searched the growth medium of MSCs and they came upon exosomes. By purifying exosomes from the growth medium, Kourembanas’ team showed that the anti-inflammatory effects of the MSCs could be completely recapitulated by simply applying isolated exosomes to the lungs of newborn mice.

Kourembanas said, “We are working to figure out what exactly within the MSC-produced exosomes causes these anti-inflammatory and protective effects. But we know that these exosomes contain microRNAs as well as other nucleic acids. They also induce expression of specific microRNAs in the recipient lung.”

MicroRNAs a small RNA molecules that regulate gene expression. Thousands of microRNAs have been discovered and they are also found in many different biological organisms ranging from plants to worms, to fish, frogs and people.

Kourembanas noted that, “What we may be seeing is the effect of these microRNAs on the expression of multiple genes and the activity of multiple genes and the activity of multiple pathways within the lungs and the immune system all at once.”

Kourembanas hopes that exosome research will someday act alongside stem cell-based therapies. MSC-based exosomes could potentially be used as treatments for premature babies at risk of suffering from chronic lung disease and PH. Also, MSCs are not the only cells that make useful exosomes. Umbilical cord stem cells also secrete exosomes with therapeutic value. Even though many different types of stem cells are recognized by the immune system as foreign, exosomes are not, which gives them an added advantage as therapeutic agents. Kourembanas notes, “they could potentially be collected, banked and given like a drug without the risks of rejection or tumor development that can theoretically come with donor cell or stem cell transplantation.”