Discover the Regenerative Potential of Exosomes

By: Jason Sanders, MD

The following arti cle is not endorsed and/or supported by The American Academy of Anti -Aging Medicine. The purposes of this publicati on do not imply endorsement and/or support of any author, company or theme related to this arti cle.

Regenerative medicine is an innovative, medical subspecialty that strives to treat or prevent injury and disease by naturally repairing, restoring and regenerating damaged or diseased organs and tissues.  is  eld has exploded in recent years to meet the needs of patients with both complex and common medical problems. Some regenerative medical therapies aim to slow or stop degenerative or pathophysiologic processes that ultimately present themselves as symptomatic conditions. Other regenerative therapies activate the body’s native repair system by in uencing the behavior of somatic and progenitor cells to stop degenerating and start regenerating. Stem cell therapy is decades-old regenerative medical technology that has shown the ability to supercharge the body’s ability to repair itself and to combat disease and injury. Recent scienti c evidence indicates that the core mechanism of stem cell therapy, which has been very successful in the treatment of di erent medical conditions, lies in exosomes.

Stem cell therapy most commonly employs the mesenchymal stem cell – a natural precursor cell involved in the development of connective tissues that has the capacity to transform into other cell types with more speci c functions. Mesenchymal stem cells also have the ability to support normal growth, development and tissue repair. For regenerative medicine applications, health care providers have used both young mesenchymal stem cells, which may originate from perinatal tissue such as the umbilical cord, and adult mesenchymal stem cells, which may originate from bone marrow or fat. Originally, scientists and physicians believed that these stem cells exerted their e ects by engrafting and di erentiating into di erent cell types to replace diseased or damaged tissues. More recent evidence clearly demonstrates that these stem cells do not engraft, but work by a paracrine mechanism, meaning that they in uence the behavior of other cells within the same tissue environment. In the case of allogeneic cells, those harvested from another person, these cells survive less than 3 days after transplantation. In the case of autologous cells, those harvested from tissues in the same person, these cells survive for about 7 days. In either case, the function of these cells during that time is to produce and release exosomes.  e nature of these exosomes depends on the parent cell type and the tissue environment, but these exosomes mediate the paracrine e ect of the stem cells.

So, what are exosomes? Exosomes are sophisticated, nano-scale, biologic signals that are contained within extracellular vesicles.

 ese membrane-enclosed capsules ranging in size from about 20 to 200 nanometers are  lled with cellular signaling information. Virtually every type of cell produces exosomes as a means of intercellular communication, so exosomes are the essence of the message of the parent cell.  at message may tell other cells to reduce in ammation, repair damaged tissues or even reverse aging.

Proteins constitute one part of the message of exosomes.  e most familiar type of proteins contained within exosomes may be referred to as growth factors.  ese proteins trigger cellular signaling pathways that initiate speci c behaviors in the target cells that internalize the exosomes. Some of the proteins contained within exosomes are growth factor receptors, which may cause growth factors to in uence the behavior or the target cells, in di erent ways than the growth factor would alone. Binding proteins, also contained within exosomes, may have a similar e ect. Enzymes contained within exosomes may augment normal enzymatic pathways or restore lost or de cient enzymatic processes exogenously. Finally, immune modulators are signaling proteins that in uence in ammation and the activity of the immune system.

Messenger RNA comprises another important part of the message contained within exosomes. mRNA is the blueprint for protein production, and proteins govern the structure and function of cells.  e mRNA contained within exosomes induces target cells to translate numerous copies of each of the proteins that correspond to each exosomal mRNA. In this way, exosomes can directly hijack the natural protein production machinery of the target cells. Each strand of mRNA remains viable for about 30 days, which may contribute, in part, to the sustained e ect exosomes exert on target cells.  e persistent e ects of exosomes that extend beyond that 30 days may result from the e ect of the exosomes on the signaling of the target cells. Cells have the capacity to selectively load exosomes with speci c contents, and if the parent cell exosomes in uence the loading of the target cell exosomes, the target cell exosomes will perpetuate the message of the parent cells, as they are internalized by secondary or tertiary target cells.  ese cascading e ects may allow the original exosomes to cause lasting e ects on cells and tissues that persist much longer than the original contents of the exosomes. recently begun to understand.  ese short, non-coding strands of RNA bind to complementary strands of mRNA. When microRNA binds to mRNA, it has two e ects. One, microRNA obstructs the binding of ribosomes to mRNA, preventing the translation of speci c mRNA.  e other e ect results from micro RNA destabilizing the mRNA strands, causing them to degrade more quickly.  e combined e ects of microRNA on endogenous mRNA act as an o -switch for speci c protein production, making micro RNA just as important, if not more important, a signaling mediator as the proteins and mRNA contained within the exosomes.

All of the contents of exosomes are contained within a lipid bilayer membrane very similar to the parent cell membrane.  e exosome membrane, while similar to the parent cell membrane, is not identical, as the membrane proteins and the phospholipid composition may vary.  ese variances may have some signi cance related to exosome homing or internalization.  e primary function of the exosome membrane is to protect the contents of the exosome from degradation by circulating proteases or RNAses.  e exosome membrane also serves to facilitate internalization of the exosomes into the target cells.

Perinatal MSC exosomes contain the proteins, mRNA and microRNA packaged by mesenchymal stem cells, the youngest progenitor cells of the connective tissue lineage.  ese cells naturally support the development of connective tissues like skin, hair, bone, muscle and cartilage. Mesenchymal stem cells also possess bene cial biologic properties that could have applications in regenerative medicine, and these biologic e ects are mediated by the exosomes that the MSCs produce.  e most important property of perinatal MSC exosomes is that they are produced by very young stem cells, less than a day old, that have the greatest regenerative potential.

Numerous studies have demonstrated that perinatal MSC exosomes have multiple innate biologic properties that could have profound e ects in regenerative medicine.  ese exosomes reduce in ammation, which is now understood to be a core mechanism of many diseases and traumatic processes. Excessive activity of immune cells also plays a role in many autoimmune and degenerative conditions, and perinatal MSC exosomes have demonstrated e cacy in modulating the activity of immune cells.  ese exosomes also promote the development of connective tissues and

blood vessels. Perinatal MSC exosomes have also demonstrated e cacy in preserving the viability of damaged cells, which has the e ect of preventing apoptosis and necrosis. In severely damaged tissues that do not survive, these exosomes reduce the extent of  brosis and scarring in the healing tissues. Perinatal MSC exosomes have also shown suppression of tumor growth in pre-clinical models that have been studied. It is important to note that these biologic properties may be related to one or more of the proteins, as well as the micro RNAs contained within these exosomes.

One of the most important points to understand, when considering the biologic properties of perinatal MSC exosomes, is that their e ects are not the result of any single protein, mRNA or micro RNA that is contained within the exosomes. Instead, the biologic properties of these exosomes result from the combined e ects of hundreds of proteins, mRNAs and micro RNAs acting in concert on multiple signaling pathways and having direct e ects within the target cell.  e composite e ect of these exosomal signaling factors sometimes causes predictable e ects based on the exosome constituents. At other times exosomes may exhibit paradoxical e ects that cannot be easily explained by the well understood biologic activity of their individual contents. In the case of perinatal MSC exosomes, they appear to have positive net biologic e ects across the models of injury and disease that have been studied in vitro and in vivo.

One explanation for the bene cial biologic properties of perinatal MSC exosomes exert may be related to their role in nature.  e natural function of perinatal MSC exosomes is to support the development of the fetus. A central function in this regard is to promote the rapid, controlled cell proliferation that underlies growth.  ese exosomes also play a role in suppressing in ammation in utero to allow the pregnancy to be carried to term, and modulating the maternal immune system to prevent rejection of the fetus. Supporting the development of connective tissues and their blood supply are also important roles of perinatal MSC exosomes. Finally, these exosomes participate in the suppression of oncogenesis by controlling the growth of the rapidly dividing cells.

 e biologic properties of perinatal MSC exosomes di er from those of adult MSC exosomes.  e closest analog to mesenchymal stem cells in the adult are progenitor cells that may be harvested from bone marrow or fat.  ese adipose-derived or bone marrow-derived stromal cells behave di erently and produce exosomes with di erent contents than perinatal MSCs because of the age-associated decline in the numbers and level of function of these senescent progenitor cells. For example, at birth there are about 1 in 10,000 MSCs in bone marrow, while at age 20, there are about 1 in 100,000 MSCs in bone marrow.  ese senescent progenitor cells also express di erent proteins and miRNA than perinatal MSCs. One familiar example of this loss of protein expression may be illustrated by the reduction in lactase expression in adults, which results in lactose intolerance in many adults. Another clinically relevant example of the loss of protein expression in adult cells is the reduced expression of tumor suppressor proteins, which explains why many cancers are frequently diagnosed later in life. Finally, recent cataloguing of micro RNA present in circulating microvesicles indicates that the levels of speci c micro RNAs change with aging or in speci c disease states.

Tissue-derived exosomes also di er from isolated, perinatal MSC exosomes for several reasons, one of which is that sampling tissues for exosomes yields a multitude of di erent exosomes produced by di erent cell types, which di er signi cantly in their contents and corresponding biologic properties. MSC exosomes constitute only a very small fraction of the population of exosomes and other biologic material in both adult and perinatal tissues. In order to concentrate MSC exosomes from composite perinatal tissue, di erent methods may be employed. One such method is sterile  ltration, which can remove cells and other larger particulate material from the samples, but unfortunately leaves nano-particulate debris with similar size and density to the exosomes along with the entire fraction of exosomes produced by di erent cell types. One example of particulate contamination that may persist after this type of  ltration may be ABO blood type incompatibility antibodies, which could cause lifethreatening complications, if unknowingly administered along with exosomes. Another method for MSC exosome isolation is antibody-coated bead extraction, which uses antibodies that bind to MSC exosome membrane proteins to separate these exosomes from the other biologic material in tissues. Unfortunately, the antibody remains attached to the exosome after this separation, creating the potential to trigger an immune response. Compared with both of these methods, isolated MSC exosomes can be much more safely and e ectively harvested from isolated MSC cultures, which virtually eliminates the potential for contamination with particulate biologic material and infectious agents.

 e results of nano-tracking analysis demonstrate a clear di erence between the exosomes harvested from isolated MSC cultures, illustrated above, and exosomes derived from processing tissue samples. Nano-tracking analysis can determine the size and number of nano-particles re ecting light in a sample. In the upper row, the size distribution shows a single peak indicating a consistent population of nano-particles, as opposed to the multiple peaks in the lower row, which indicate signi cant particulate contamination. Also, the scatter plot in the upper right shows dense clustering of particles by size indicating the consistency of the sample of isolated MSC exosomes. In the lower right, there is signi cant scattering of the results representing a very inconsistent sample consistent with particulate biologic contamination.

Optimal methods for isolation of MSC exosomes begin with rigorous screening of tissue donors, and subsequently, the donated tissues that exceeds the standards required by the American Academy of Tissue Banks. After successfully screening the donors and tissues, isolated mesenchymal stem cells may be harvested and cultured on chemically de ned synthetic growth media, which contains no animal or human products. Over a period of about 4 weeks, the cultured MSCs will produce signi cant numbers of exosomes and release them into the growth media.  is conditioned media can be sterile-  ltered to remove the cells and any material that is not the same size and density as an exosome. Unlike processed, composite tissue samples, conditioned media produced by isolated MSC cultures does not contain any biologic nano-particulates that might contaminate the exosomes fraction after  ltration process. Despite the intrinsic purity of exosomes isolated in this fashion, the product of  ltration should still undergo sterility and endotoxin testing to con rm the absence of any infectious agents. Specialized protein and RNA sequencing identi es the speci c contents of the exosome sample. Finally, STORM super resolution  uorescence microscopy is able to con rm that these protein and RNA contents are contained within MSC exosomes.

STORM super resolution  uorescence microscopy is able to quantify single molecule  uorescence and identify individual proteins on the surface of exosomes. CD 9, 63 and 81 are membrane proteins that are characteristic of mesenchymal stem cells. Detection of these proteins by STORM microscopy in a con guration that resembles the size and three-dimensional structure of the exosomes, as illustrated on the lower right, allows direct visualization of the exosomes and con rms that these exosomes originated from mesenchymal stem cells. Unlike nano-tracking analysis, which can only detect size and number without identifying nano-particles, STORM microscopy can also directly count the MSC exosomes in a sample and can visualize exosome movement and internalization into cells in real time.

YOU MAY BE WONDERING WHY ANY OF THIS MATTERS…

Consider why the science and clinical potential of exosomes might matter to a cancer patient. If physicians could use advanced exosome characterization modalities to detect abnormal exosomes produced by cancer cells long before the cancer could be detected by physical exam or traditional diagnostic tests, treatment could begin sooner with potentially better outcomes. Perhaps physicians could utilize the innate perinatal MSC exosome property of tumor suppression to slow or stop the growth of tumors, or prevent the development of cancer before it begins.  e intrinsic biologic properties of perinatal MSC exosomes may also provide some bene t in the preservation of normal tissues damaged by the chemotherapy or radiation commonly used in the treatment of cancer. Ultimately targeted exosome-based therapies could be developed for destruction of speci c cancer cells without the collateral damage to normal tissues. All of these exosome-based diagnostic and therapeutic modalities could eventually help to improve the survival of cancer patients.

Consider the impact that therapeutic MSC exosomes could have on the life of a spinal cord injury patient. Currently, the only real tool in the armamentarium for an acute spinal cord injury patient is high dose corticosteroids, and this is part of the initial treatment protocol for spinal cord injury in trauma centers.  e objective of this type of treatment is to reduce the in ammation, which is what causes the swelling and ischemia that lead to neuronal cell death and motor and sensory de cits below the level of the direct mechanical injury.

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