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.”