The Role of Exosomes in Regenerative Medicine

Douglas Spiel, MD

Special Thanks Duncan Ross Ph.D. Andrew Laudisi MS

Conflict of Interest Statement: I, Douglas J. Spiel, am the Chief Medical Officer for Kimera Labs, Medical Director for an interventional pain private practice in New Jersey (Doug Spiel MD PA), and the Medical Director of Regenerative Solutions of NJ.

1.0 INTRODUCTION

Regenerative therapy (and those who practice in this space) is a microcosm of medicine focused on recapitulating the conditions of our youth in order to restore, rather than repair, morphological and physiological aberrancies as they appear. As a medical society, it is far more attractive to offer our patients the hope of regeneration in order to afford them the ability to restore a younger version of themselves rather than just repairing their injuries. Whether rehabilitating a disc, a joint, a nerve or the entire individual the dream has always been to live longer and better.

Mesenchymal Stem Cells (MSCs), named by Arnold Caplan nearly 30 years ago, were originally termed MSCs secondary to their innate ability to replicate while maintaining the attribute of multi-potential lineage. In short, these simple cells maintain the ability to form numerous types of mesodermal tissues (1). Their capacity to form bone, cartilage, fat, skin and other tissues in vitro led to the fallacious belief that they could be transplanted and would engraft in vivo to form these same elements.

Over the past few decades, we have seen thousands of clinics throughout the US, set up shop, with the hope of utilizing these same cells in directed protocols to tackle a myriad of medical conditions ranging from aesthetics to neurological diseases. Zealots of bone marrow, fat and allogeneic cells from umbilical cord and Wharton’s jelly, continue to argue over the benefits of their respective cell-based tissue products (Table 1). But what is the mechanism of action?

Table 1. Stem cell-based choices in regenerative medicine.

Dr. Caplan has released a paper, “Mesenchymal stem cells; time to change the name!” outlining the research of many other prominent scientists. In this treatise, Dr. Caplan eloquently points to these cells as “medicinal signaling cells”, capable of releasing paracrine effectors which thereby influence the body via immunomodulatory and trophic mechanisms (2). These bioactive factors essentially upregulate resident stem cells, which reside throughout all the tissues of our bodies, and affect the phenotypic and physiological expression of our immune system. So if these bioactive factors are what will be the honey, then why is so much attention paid to the bees? There are a few simple and some not so simple answers tied to this question. The first and most important answer lies in the fact that the old textbooks remain on the shelves and those who have read them continue to preach their teachings. Equally important is the fact that once we are trained as physicians we hold our initial teachings as dictum. Additionally, most of us reading this book are interventional pain physicians. We enjoy doing interventional cases. Bone marrow aspirations and

lipoaspirations are within the scopes of our training and are part of our armamentarium. Finally,

and not to be minimized is the fact that alternative therapeutic options have only recently become available on the marketplace.

2.0 WHAT ARE EXTRACELLULAR VESICLES ?

So what are exosomes and microvesicles? As paracrine effectors, they have a role in signaling by transferring their contents from one cell to another. We have discussed cells, specifically mesenchymal stem cells. Once we are born, all of our MSCs are termed adult. As adult MSCs these cells are too far down the cell cycle to directly transdifferentiate into other cell types. Only fetal or embryonal stem cells along with induced pluripotent stem cells retain the level of stemness to form different tissues. Currently in the US, ethical and moral considerations “along with cancer risks” preclude the use of these cells in clinical medicine. Adult MSCs cause changes via paracrine messengers (2). These messengers are termed exosomes and microvesicles, collectively known as extracellular vesicles (EVs).

Exosomes, the smaller of these two vesicles, measure 40 to 100 nm and are lipid membrane packets formed by a two-step budding process. Formed by inward budding of membranous vesicles in a multi-vesicular body, they fuse with the plasma membrane to release these ultra-tiny vesicles. Microvesicles refer to somewhat larger packets of a few hundred nanometers formed by budding directly from the plasma membrane. Both exosomes and microvesicles contain transmembrane proteins from their parent cells, which are important in regulating uptake by other cells (3). By conserving these transmembrane proteins it has been shown that uptake is facilitated by other cells to a much greater degree than if the cargo was simply released into the extracellular environment. Exosomes and microvesicles are not exclusive to stem cells and are released by many cells throughout the body. Immune cells, cancer cells and aging cells all secrete different vesicles which contain vastly different cargos of information. This information includes messenger RNAs, micro RNAs, and various proteins. The intrinsic durability of the extracellular vesicle membranes makes them uniquely durable and naturally biocompatible. Additionally, the wide spectrum of

proteins and messenger RNA contained within these EVs allows for a vastly greater capacity of information compared with single molecule messengers like hormones , growth factors and cytokines. Finally, the transmembrane protein receptors allow EVs to traffic or home to areas of injury and inflammation while facilitating uptake by numerous cells (4).

EVs are important in autocrine signaling (local between same cells), paracrine signaling (local between different cells) and endocrine signaling (between distant cells). EVs have been found in all bodily fluids. EV cargos are specific to each type of cell, while cells grown in different environments will also modify their production of EV contents. Commercially, at present, research grade purified EV solutions are only available from placental tissues whose MSCs secrete a cargo rich in growth and immunomodulatory substances. Of course, any resident stem cells who traffic to the areas of concern, will then secrete EVs specific to themselves and will be modified by their own local extracellular microenvironment. Much of the difficulty in bringing purified EVs to market is related to the scalability and standardization of the product. Similarly, concentrating the product to a physiologic level necessary to effect change has also proven to be a large obstacle for many companies and continues to be a significant barrier of entry into the marketplace. Variances of 0.1 to 2 mg of exosomes are isolated from cell numbers of up to 60 million MSCs (4). The current commercial product quotes 15 mg per standard five mL vial and also produces a higher concentration product containing 8 mg in a one mL dosing. A handful of companies are currently developing exosome products – some very specialized – and no doubt others will soon join the race. The Bioinformant, in their commercial publication, The Market for Stem Cell Exosomes lists multiple companies pursuing production of EVs (5, 6).

3.0 BENEFITS OF EXTRACELLULAR VESICLES

Similar to MSCs, EVs as secretory products have been shown to travel via local diffusion; deliver proteins, micro RNA and messenger RNA; and home (7, 8, 9). Unlike MSCs, EVs demonstrate a number of advantages distinct from their parent cells. They can travel systemically without the risk of clumping (as is seen with large peripheral intravascular doses of MSCs). As much smaller particles, EVs do not demonstrate a first pass effect into the lungs when administered intravascularly. EVs can cross the blood-brain barrier easily without utilizing mannitol (3). While allogeneic MSCs may be perceived as foreign by the innate and adaptive immune system and quickly whisked away, EVs are able to evade the immune response. EVs from healthy stromal cells do not contain DNA, so that there is no risk of malignant transformation. Alternatively, autologous MSCs are of the same age as the donor patient and are therefore limited by the inherent age of the individual. Older cells are less robust in the production of growth factors, micro RNA and messenger RNA and are frequently limited in total number. Finally, stem cell harvesting requires time and expertise whereas EVs provide an out of the freezer solution via easy storage, administration, and controllable dosing.

4.0 KEY THERAPEUTIC EFFECTS OF MSC EVS

Microvesicles and exosomes of mesenchymal stem cells provide very attractive therapeutic benefits. At the very core of these are their trophic (regenerative) and immunomodulatory capabilities which dictate their indications. A discussion of the trophic effects of EVs requires an understanding of the resident stem cells they act upon. Tissue-resident stem cells lie quiescently within niches throughout our bodies. This population of cells are partially undifferentiated and once activated can proliferate and migrate to sites of injury where they acquire a mature phenotype in order to facilitate repair and remodeling. The balance of progenitor cell quiescence and activation is a hallmark of a functional niche and is regulated by internal and external signals. Known niches are seen in the central nervous system, skeletal muscles, liver, skin, kidney, heart, lung, and joints (10, 11). In the joints alone, a myriad of cells have been described and include: chondrocyte progenitor cells (CPCs), cartilage-derived stem/progenitor cells (CSPCs), synovium resident multipotent progenitor cells, osteoblast/osteoclast resident MSCs within the subchondral bone, and chondrogenic cells within the infrapatellar fat pad (12).

Many of the immunomodulatory effects of MSC EVs are related to the influences upon phenotypic expression of certain cells. Macrophages and microglia (macrophages of the central nervous system) demonstrate two distinct appearances. M1 macrophages are pro-inflammatory and secrete inflammatory cytokines whereas M2 macrophages secrete an anti-inflammatory milieu regarding their secretions. MSC EVs were shown to influence the conversion of M1 macrophages into M2 macrophages (13, 14, 15, 16). Similarly, T-cells are described as predominantly T Helper (TH) or T Regulatory (TReg) cells with TH cells further subdivided into TH1 cells which stimulate cytotoxic T cells and TH2 cells which stimulate B-cells. TH1 cells are markedly more inflammatory than TH2 cells. TReg cells also downregulate inflammation. MSC EVs are also known to convert

TH1 to TH2 cells and increase the amount of TReg cells (13, 14). These phenotypic changes along with the internal production of significant anti-inflammatory cytokines like IL-10, TGF-ß3, TIMP, TNFαRA and IL-1RA provide for the immunomodulatory and anti-inflammatory effects seen after MSC EV administration.

Many of the anti-fibrotic benefits of MSC EVs are attributable to several factors. They produce large amounts of TGFß3 which regulates cell adhesion and extracellular matrix formation. In scar repair they increased the ratio of Collagen Type III to Type I. Additionally, MSC EVs displayed inhibition of granulation tissue leading to fine reticular collagen with fewer fibroblasts (17). Finally, MSC exosomes prevent apoptosis (cell death) through numerous techniques, including the promotion of redox homeostasis and appropriate autophagy/mitophagy.

During inflammatory and ischemic conditions (e.g., cerebrovascular accident, myocardial infarction) cells lose ATP/NADH, experience oxidative stress (e.g., increased production of reactive oxygen and nitrogen species [ROS/RNS]) and subsequently die. Assays have shown that MSC exosomes contain all five enzymes in the ATP-generating stage of glycolysis: GAPDH, PGK, PGM, ENO, and PKM2 (18).

This anti-apoptotic effect is perhaps the most important beneficial effect of MSC EVs because it relates to the powerhouse of the cell – the mitochondria. Mitochondrial dysfunction with subsequent death is a leading cause of endothelial injury and a cytoprotective effect here could lead to eventual decreases in cardiovascular disease, stroke, and myocardial infarction. By improving mitochondrial fitness, restoring a normal morphology, and removing damaged mitochondria appropriately (mitophagy), MSC EVs are able to ameliorate the effects of oxidative stress imposed by severe inflammation and ischemia (19).

5.0 BIODISTRIBUTION AND TARGETING OF EXTRACELLULAR VESICLES

Few studies have evaluated the biodistribution of extracellular vesicles in murine models. A short biodistribution phase is followed by a longer elimination phase (20). Route of administration has been shown to influence EV biodistribution. Intravenously delivered EVs show rapid uptake by macrophages of the mononuclear phagocyte system, accumulating predominantly within the liver, spleen, and lungs. In the liver, significant uptake is accomplished by Kupffer cells, while alveolar macrophages predominate in the lungs. High splenic levels are attributed to circulating lymphocytes and macrophages which bind EVs and then traffic there. Most EVs are subsequently eliminated from organs and biofluids within 360 minutes indicating active uptake and degradation by different cell type with some elimination via hepatic and renal processing (21).

The route of administration certainly influences EV biodistribution. Comparing intraperitoneal (IP), subcutaneous (SQ), and intravenous (IV) routes of delivery – certain salient differences are worth noting as they may play a role in establishing tailored protocols. In murine studies, EV administration by IV had the highest accumulations in the liver and spleen when compared to IP and SQ routes. The pancreas had a higher portion of accumulated EVs via IP followed closely by SQ injection, however overall rates of EV accumulation was highest in IP and IV administration when compared to SQ (22). Footpad administration resulted in highest localization into the lymph nodes while intranasal delivery to the cribriform plate yielded the highest brain delivery via peripheral injection. Finally, it has been noted that periocular injections of EVs reached the neurosensory retina, while intrathecal delivery allows for optimal CNS penetration (21). This is significant because when whole cells are delivered, either via autologous or allogenic sources, the first pass effect leaves the majority of the cells lodged within the lungs.

Different cell sources also play an important role in EV migration. While it has been proven that immune cells preferentially traffic to the spleen, so too do dendritic cell EVs likewise end up there. It is likely that the EVs maintain many of the surface receptor ligands and binding proteins of the parent cells. So important are these ligands that even species origin does not affect homing qualities (22); however, it is possible to affect targeting by changing certain membrane-bound protein ligands. RVG, a ligand that binds acetylcholine receptors, allows for a twofold greater accumulation in brain with increased levels in muscle and heart as well when attached to EVs (22). As for tumors, their leaky vasculature allows for permeation and retention, so that nanoparticles like exosomes and microvesicles will deposit there within sixty minutes if given intravenously. Additional modifications would no doubt provide even more exceptional targeting into tumors and other tissues (20).

6.0 PARABIOSIS – WHAT CAN BE LEARNED?

Parabiosis is defined as the procedure of joining two animals so that they share each other’s blood circulations. Heterochronic parabiosis occurs when two different aged animals are connected. Over the years it has been shown that factors from young animals were able to elegantly activate molecular signaling in the older counterparts to increase tissue regeneration within hepatic, muscle, and neural elements (23). Since the hallmark of aging is the decline of regenerative properties linked to impaired function of stem and progenitor cells this has sparked the launch of a number of companies to try to reproduce these results in humans. Alkahest in California has set up clinics to treat Alzheimer’s with plasma from 20-year-old human donors while Ambrosia, also based in California, sells plasma to all comers. But what is in the plasma? There are no appreciable stem cells present, but we should realize that because most stem cells are pericytes (cells sitting atop capillaries)(24) we may assume that plasma is a dilute fluid of extracellular vesicles. So, over time there is a relative rejuvenation of the older paired animal as the trophic and immunomodulatory mediators bathe the tissues and resident stem cells. But what of the younger animal? Much less attention has been paid in the literature to the less lucky of the two animals. In fact, new emerging evidence points to a senescence-associated secretory phenotype (SASP) which has a negative effect on stem cell niches. Hayflick defined a term in the 1960’s as “replicative senescence.” This was attributed to repeated cell divisions ultimately decreasing telomere length so that cells eventually could not divide. A newer term “premature senescence” relates to exogenous stressors affecting cells with normal telomere length. Senescence associated extracellular vesicles cause migration of phagocytic cells, induce inflammation, disrupt tissue architecture, and enhance malignant transformation and fibrosis. It has also been hypothesized that SASPs lead to impaired autophagy (i.e., appropriate death of senescent cells)

contributing to the pathogenesis of age-related diseases. Inflammatory cytokines seen within these SASPs are also associated with inflammatory and metabolic disorders (Table 2). And while “young blood” rejuvenated the older stem cell niches, SASPs decrease functionality of the niches thereby impairing tissue maintenance and repair, and they may even spread premature senescence to bystander cells (25).

Since the 1500’s when Ponce de Leon travelled the world looking for the fountain of youth, scientists have arduously sought for the singular factor responsible to stop the aging process. For those who have studied parabiosis, the answer may be attributed to a protein, GDF-11 (25). Many have asserted that this protein is responsible for the rejuvenation process. Perhaps it serves as an interesting footnote that the mRNA that codes for GDF-11 has been assayed within the exosomes of neonatal placental MSCs (Table 3).

Table 2. Key immune and growth factors present in MSC exosomes.

BMP5 GDF15 OPG G-CSF SCF TGFß3

VEGF ICAM-1 IL-1RA IL-6 IL-10

MCP-1
MIP-1 PDGF-BB TIMP1 & TIMP2 HGF

GDNF BDNF FGF TNFR1

Stimulates Bone Growth
Regulates inflammation, apoptosis, cell repair, and growth
Stimulates Bone Growth/Blocks Osteoclast Precursor Formation
Stimulates Bone Marrow to Procedure Granulocytes and Stem Cells
Responsible for Stem Cell and Melanocyte Growth
Most Important Anti-Inflammatory Protein. Converts Inflammatory T Cells into Anti- Inflammatory Regulatory T Cells.
Stimulates Formation of Blood Vessels
Binds Inflammatory Ligands on White Cells
Binds and Sequesters the Inflammatory Cytokine IL-1
Responsible for Macrophage Activation
Anti-Inflammatory Cytokine responsible for Immunomodulation and Regulatory T Cell Conversion
Recruits Mononuclear Cells to Treatment Area
Also known as CC1-4, Recruits Mononuclear Cells to the Treatment Area
Growth Factor Used to Stimulate Healing in Soft and Hard Tissues
Blocks Cartilage and Extracellular Matrix Degradation, Important for Cartilage Repair Involved in Organ Regeneration and Wound Healing
Promotes Survival of Neurons
Supports Survival of Neurons and Encourage Growth
Potent Growth Factors Affecting Many Cells
Binds and Inactivates the Inflammatory cytokine TNF-α

Table 3. Some key mRNA present in MSC exosomes.

7.0 HOW LONG SHOULD MSC EXOSOMES PERSIST IN VIVO?
In a study in 2016, forty patients with stage 3 and 4 chronic kidney disease were randomized into 2 groups. One group was given intravenous and intra-arterial exosomes, while the second group served as a control. Although renal functions improved significantly in the treatment arm as seen by increases in GFR and decreases in serum creatinine and BUN, what was most striking was the chronicity of benefit seen in anti-inflammatory markers TGF-ß and IL-10, and the pro-inflammatory TNF-α. The anti-inflammatory TGF-ß1 and IL-10 peaked at 12 weeks and persisted above baseline throughout the 52 week follow up while TNF-α showed a trough level at 12 weeks and persisted below baseline at 52 weeks. A rather remarkable persistence given the fact that only two injections were given initially one week apart. Given the rather transient traceability of EVs in vivo, and the limited half-life of proteins, the chronicity of these findings reinforces the

importance of the mRNA they bear (26).

8.0 COULD MSC EXOSOMES HELP FIGHT TYPE II DIABETES MELLITUS?

By the age of 65 it is estimated that 50% of the US population will suffer from Type II Diabetes Mellitus or impaired glucose tolerance (27). This disease is intimately associated with many of the severe afflictions suffered by seniors during their final decades of life. Cardiovascular disease, stroke, and myocardial infarction share Diabetes Mellitus as a prognosticating factor. Could MSC exosomes help fight or even prevent Type II Diabetes Mellitus and decrease the risk of these and other serious age-related diseases?

To this end, it has been proven that MSC exosomes promote a systemic anti-inflammatory milieu which persists for many months. Decreasing inflammation also enables insulin to bind its receptors with greater affinity thereby increasing its relative action. Additionally, the anti- apoptotic effects of exosomes decrease the death rate of B-Cells in the pancreas and increase production of insulin (28). Finally, the peripheral effects are a little more intricate and must be appreciated in steps. As we age, we develop sarcopenia. Sarcopenia is defined as loss of muscle tissue. Sarcopenia is marked by capillary rarefaction (27), which is a systemic loss of capillary volume. There two synchronous processes cause a relative decrease in our metabolism secondary to reduced muscle mass along with a concomitant difficulty for insulin and glucose to traffic to the muscle cells. Exosomes promote capillarization through VEGF and pro-angiogenic miRNAs (27, 29, 30). By increasing capillary surface area and through proliferating factors from the “young blood,” satellite cells (muscle resident stem cells) are activated and more muscle tissue is formed (30). This serves to increase the overall metabolism as well as enable delivery of insulin and glucose intracellularly – thereby effectively lowering postprandial glucose levels along with HgbA1C over time.

WHAT TYPES OF MEDICAL CONDITIONS MIGHT BE AIDED BY MSC EXOSOMES?
Musculoskeletal – Joints, discs, muscles, bones, ligaments, tendons
Neurodegenerative – MS, Parkinson’s, Alzheimer’s, Huntington’s, ALS, Cerebellar Ataxia CNS Injury/Trauma – CVA, CTE, TBI, SCI, Transverse Myelitis, Cerebellar Ataxia Burns/Scars/Ulcers

Heart Disease – MI, Angina, CHF
Lung Disease – COPD, Pulmonary Fibrosis, Interstitial Lung Disease Liver Disease
Kidney Disease
Inflammatory Bowel Disease – UC, Crohn’s
Alopecia
Neuropathy/CIDP
Erectile Dysfunction
Urinary Incontinence
Peripheral Vascular Disease
Cerebral Palsy/Seizure Disorders/Autism
Numerous Aesthetic Applications
Depression/Bipolar Disorder
Drug Addiction
Type II Diabetes Mellitus
Infertility
Aging

10.0 HOW CAN MSC EXTRACELLULAR VESICLES BE DELIVERED?

The tiny size of EVs allows for easy injection based therapies. Alone, these miniature powerhouses can be delivered through needles as diminutive as 30 gauge. Direct delivery is recommended intravenously, intrathecally, and intranasally. When injecting into other areas of the body, it is often prudent to utilize a scaffold to limit traffic out of the injection site. Common autologous scaffolds like PRP or PRFM serve the dual purpose of cell retention and cell migration as well, without significantly elevating the cost of the procedure. Combining EVs with PRP/PRFM utilizing a 22 gauge needle will allow time to inject without compromising safety. Some procedures do not require image guidance and can be accomplished in office. More invasive procedures like intradiscal injections, cervical intrathecal injections (31), and deep perispinal injections are best performed utilizing X-Ray guidance – while joint, tendinous, perineural, and other musculoskeletal indications are well suited for ultrasound guidance. Many courses around the country are available in order to help hone the skills necessary for image-guided therapy, and additional books will serve the injectionist well for easy on-site reference. Unlike bone marrow aspiration/concentration and mini-lipoaspiration with concentration (with or without enzyme degradation) an additional one or two hours do not have to be allotted for each procedure.