Tiny, But Mighty. The Power Of Mesenchymal Stem Cell-Derived Extracellular Vesicles: A Review
A literature review on the future potential of MSC-EVs for regenerative medicine therapeutics
For most of history, people died from the common cold. Before antibiotics, bloodletting was a tactic used to remove infections and other serious illnesses. Doctors prescribed cocaine and heroin to cure coughs, headaches, and sore throats in children. Surgeons cut up human brains to cure mental illnesses and neurological diseases. A smoothie of human flesh, blood, bones was given to sick people to cure epilepsy and improve health and well-being [1, 2].
Crazy, right?!?
Medicine has come a long way since this time, but we are still fighting a losing battle against some of the largest medical problems in the world. Cancer, ageing, chronic, and genetic diseases are still winning against our current medical tactics.
Our current weapons used for battle are cutting out organs from sick people and replacing them with a brain-dead person’s organs, then stuffing them full of anti-rejection medications for the rest of their lives. Flooding a cancer patient’s body with poison to remove bad cells while simultaneously killing the good cells. Sending an entire army of drugs into the body, but only having around 10% reach the target area.
We’ve evolved from lobotomies and bloodletting, but today’s treatments are still in need of an upgrade. With emerging technologies that are being researched and tested today, the next evolution of medicine is here.
One technology that we should all keep on our radars is regenerative medicine. Mesenchymal stem cell-derived extracellular vesicles, or MSC-EVs. Long and confusing name, I know, but it’s pretty simple. MSC-EVs are derived from mesenchymal stem cells (MSCs) and are nanosized particles produced by every single cell in our body. In the past few years, research on MSC-EVs and their benefits have been increasing. In this literature review, I will be diving into how we go from MSCs to MSC-EVs, their therapeutic applications for a range of diseases, their limitations, and the future of this technology.
Table of Contents
- The Origins of MSC-EVs
∘ The Parent of MSC-EVs: Mesenchymal Stem Cells
∘ Extracellular Vesicles
∘ Isolating the MSC-EVs - The Therapeutic Benefits and Applications of MSC-EVs
∘ MSC-EVs For Cardiovascular Diseases
∘ MSC-EVs For Lung Diseases
∘ MSC-EVs For Kidney Diseases
∘ MSC-EVs For Neurological and Neurodegenerative Diseases
∘ MSC-EVs as A Delivery Mechanism - Current Limitations To Clinical Applications
- Conclusion
- References
The Origins of MSC-EVs
Before we dive into the nitty-gritty of MSC-EVs, their properties, and how they work, we have to understand what regenerative medicine is. Regenerative medicine is an emerging technology that focuses on replacing or regenerating human cells, tissues, and organs to restore or replace lost function. The function of these cells, tissues, and organs can be a result of disease, ageing, and trauma [3]. Regenerative medicine is a multidisciplinary field that encompasses subfields like tissue engineering, gene therapy and stem cells [4]. Now that we have a basic understanding of regenerative medicine let’s dive into how it’s connected to MSC-EVs.
The Parent of MSC-EVs: Mesenchymal Stem Cells
MSC-EVs are derived from mesenchymal stem cells (MSCs), which are multipotent stem cells that can be found in almost all tissues, but large quantities can be found in bone marrow, adipose tissue (fat tissue), placenta, and cord blood [5,6,7].
Multipotent stem cells mean that they can turn into a limited lineage of cells, and MSCs can differentiate into cartilage cells (chondrocytes), bone cells (osteoblasts), fat cells (adipocytes), neurons, and muscle cells (myocytes) [8]. They express the surface markers CD90, CD105, and CD73 without expressing CD14, CD34, and CD45 [9]. MSCs for tissue engineering and regeneration are looking promising to researchers because of their unique qualities. MSCs have a large capacity for self-renewal and differentiation. In addition, they have no significant histocompatibility complexes, which means that they do not trigger an immune response, and they can even secrete factors like cytokines and growth factors [10, 11].
However, there are limitations to whole cell MSC therapies that make in vivo administration challenging. Firstly, MSCs are present in multiple tissues, but the overall quantity in the body is scarce, and extraction of these stem cells requires invasive and painful procedures. Because cell therapies need hundreds of millions of MSCs for each treatment, in vitro cell expansion is necessary to reach the required number, which can take about ten weeks, time a patient may not have [12].
Secondly, many studies have shown that <1% of MSCs survive for more than one week after systemic administration, and many cells do not reach the desired location [12]. The MSC implantation time is too short to produce a large-scale impact, meaning that treatments typically do not work.
Finally, each of the stem cells is slightly different, creating a heterogeneous population. Heterogeneity of MSCs is determined by factors such as donor or patient’s age, health status, genetics, gender, tissue sources, cell populations, culture conditions, cell isolation techniques, and cryoprotective and thawing protocols [13]. Because of the heterogeneous population of the cells, it can be hard to predict the benefit of the treatment because every treatment will be different.
Several studies have attributed at least part of the regenerative potential of MSC therapies to their secretome [5,14,15]. The stem cell secretome is the biological molecules and factors that are secreted into the extracellular space. The secretome includes soluble proteins, free nucleic acids, lipids, and extracellular vesicles (EVs) [12]. Extracellular vesicles play a large part in the paracrine regenerative actions of MSCs as they communicate between the stem cells and other cells by transferring information and regulatory genes for cell-free therapy [16].
Extracellular Vesicles
EVs are a part of the stem cell secretome, but in addition to being the middle man between cells, they also play an important role in cell-to-cell communication, immune responses, homeostasis, inflammation, angiogenesis, and more [5]. The EVs are produced by all cells in the body and can be derived from all types of bodily fluids like blood, urine, breast milk, bronchoalveolar lavage fluid, amniotic fluid, pleural effusions, and more [6, 16]. There are two main subclasses of EVs: exosomes and microvesicles.
Exosomes
Exosomes are the most numerous type of EVs. Their size ranges from 40–100 nm in diameter and has a density of 1.13–1.19g [5]. Exosomes can be isolated through centrifugation at 100 000 x g [5]. The biogenesis of exosomes occurs via the endocytosis-exocytosis pathway, where early endosomes formed by endocytosis mature into late endosomes [17, 18]. Multivesicular bodies (MVBs) or intraluminal vesicles (ILVs) fuse to the cell membrane and then release the exosomes into the extracellular environment [17]. Once formed by the cell, exosomes can enter recipient cells via three pathways. Exosomes can enter through endocytic uptake, a direct fusion of vesicles to the cell membrane, or they can transmit their contents through a lipid-ligand receptor [5].
Microvesicles
Microvesicles are bigger than exosomes but smaller than apoptotic bodies, and they range from 100–1000 nm in diameter and 1.04–1.07 g in density [5]. They can be isolated by ultracentrifugation. The biogenesis of microvesicles is through the external budding of the plasma membrane. Microvesicles interact with recipient cells by specific receptor-ligand interactions [5]. A ligand and receptor are like a lock and key where the ligand is a key, and the receptor is a lock.
Isolating the MSC-EVs
There is no current gold standard for isolating the EVs from the stem cells, but centrifugation and ultracentrifugation is the most commonly used method for isolating the EVs. An ultracentrifuge is a machine that uses centrifugal force to spin fluids at speeds up to 1,000,000 g to separate different-sized molecules [19]. Ultracentrifugation is known for generating EVs of higher specificity because different sized EVs can be isolated by varying spin speeds and duration. The procedure for separating the MSCs and the EVs varies depending on the MSC tissue source. However, ultracentrifugation has drawbacks as it has a low yield, and the centrifugal force could cause aggregation and potentially alter function [16].
Another commonly used method for EV isolation is manufactured precipitation isolation kits. They can quickly isolate EVs at a much higher recovery rate than ultracentrifugation but have potentially more impurities, which is why most researchers will prefer to use centrifugation [16].
Another method that is increasing in popularity is the tangential flow filtration (TFF) system. TFF uses crossflow filtration and transmembrane permeation of fluids to create a purified EV population. The flow rate is adjusted to eliminate sedimentation stress and allows large quantities of media to be filtered. TFF improved the yield of small EVs by 27-fold compared to ultracentrifugation [16].
The Therapeutic Benefits and Applications of MSC-EVs
MSC-EVs have multiple therapeutic benefits because of their biological cargo. Because they are derived from MSCs and are a part of the secretome, they contain a load of lipids, proteins, and nucleic acids like messenger RNA (mRNA), microRNA (miRNA), and other non-coding RNAs [5]. This cargo can increase cell proliferation, angiogenesis, cell function, and regeneration. It can also decrease inflammation, cell apoptosis, oxidative stress, and fibrosis [6].
In addition to their therapeutic cargo, MSC-EVs also offer advantages in patient safety. They are less likely to elicit an immune response, cannot directly form tumours, and are biocompatible since they are derived from the patient or a donor [6, 17]. These factors make MSC-EVs applicable to a variety of diseases and clinical applications.
MSC-EVs For Cardiovascular Diseases
17.9 million people each year die from cardiovascular diseases such as myocardial infarctions (MIs), cardiomyopathy, coronary artery disease, making it the leading cause of death [20]. The heart is an essential organ of our bodies that never stops beating, so when ageing, trauma, or disease damages it, it can lead to dire consequences.
A study done by Lai et al. [5] proved that MSC-EVs possess cardioprotective qualities, meaning that they can protect the heart from injury and disease through their miRNAs. These cardioprotective qualities can increase cardioprotective by reducing the expression of the p53 upregulated modulator of apoptosis (PUMA). In addition, Feng et al. [21] have shown that MSC-EVs containing miR-22 can downregulate methyl CpG binding protein 2 (Mecp2) in an acute myocardial infarction (AMI) mouse model. Yu et al. [22] showed that MSC-EVs containing miR-221 displayed anti-apoptotic effects in an in vitro ischemic heart injury model. Multiple studies with animal models have proved that MSC-EVs can reduce cardiac apoptosis, fibrosis, inflammation, and a reduced MI size by 60% in a porcine model [6]. They can also increase angiogenesis and restore cardiac function in the heart through their mRNA and miRNAs.
Delivery of the MSC-EVs to the heart is simple. They can be systematically administered into the body via an IV. Once they reach the heart, they are internalized by the target cells via endocytosis. Endocytosis is where cells take in substances from outside of the cell by encasing them in a vesicle.
MSC-EVs For Lung Diseases
In addition to cardiovascular diseases, MSC-EVs can be used to treat lung diseases like chronic obstructive pulmonary disorder (COPD), asthma, acute respiratory distress syndrome (ARDS), bronchopulmonary dysplasia, and more [23].
COPD is a chronic inflammatory lung disease that causes obstructed airflow from the lungs [24,25]. It is caused by lung irritants that can damage the lungs and airways, the most prominent example being cigarette smoke. COPD causes difficulty breathing, coughing, mucus production and wheezing [25]. The disease causes the natural elasticity of the bronchial tubes and air sacs to over-expand and loses their elasticity, which leaves air trapped in your lungs when you exhale. When you have COPD, you are more likely to have respiratory infections. MSC-EVs can significantly improve lung function, elevate oxygen saturation in the lungs, increase the quality of life, and reduce inflammation in COPD patients [23,26]. EVs can also be used as a drug delivery mechanism to deliver drugs directly into the lungs of COPD patients [27].
ARDS is a life-threatening lung injury with a 30–40% morbidity rate [26]. It allows fluid to leak into the alveolar sacs in the lungs. The alveolar sacs are where the lungs and the blood exchange oxygen and carbon dioxide while we breathe. ARDS is caused by sepsis, severe pneumonia, smoke inhalation, and near-drowning injuries. Even a respiratory virus like COVID-19 can lead to pneumonia and ARDS in severe cases [28]. MSC-EVs can potentially be a treatment for ARDS because they can improve alveolar fluid clearance, trigger tissue repair, decrease inflammatory cells in the lungs, reduce oxidative stress and cell apoptosis. Furthermore, EVs can decrease the influx of inflammatory cells into the lungs by 36% and neutrophils by 73%, decreasing the inflammatory response [6].
MSC-EVs For Kidney Diseases
Acute Kidney Injury (AKI) is a sudden episode of failure or severe damage to the kidneys that causes a build-up of waste products in your blood [29]. The kidneys are an essential organ because they filter about 200 quarts of fluid in our bodies every 24 hours and remove waste products, drugs, balance the body’s fluids, and more [30]. AKI can affect other organs such as the brain, heart, and lungs and is a significant cause of morbidity and mortality among hospitalized patients. Renal ischemia/reperfusion injury (IRI) is a common cause of AKI. IRI results from a sudden impairment of blood flow to the kidney and is associated with harsh inflammatory and oxidative stress responses, disturbing organ function [31].
In AKI, the administration of MSCs has shown in animal models that systematic and localized treatment resulted in the amelioration of AKI. One of the most important approaches to treating AKI is to control the inflammatory and immune response. MSC-EVs can modulate T-cells as well as innate immune cell functions [6]. In a study, both MSC and MSC-EV treated mice had similar benefits of reduced fibrosis and interstitial lymphocyte infiltration and reduced tubular atrophy [6]. Both the MSCs and MSC-EVs had similar benefits, and because of the EV’s safety benefits, it makes them the better option for treating AKI without sacrificing the efficacy of MSCs.
Results suggest that MSC-EVs work because of the activation of the proliferative pathway in tubular cells that occurs via the transfer of specific miRNAs and mRNAs through the MSC-EVs. These RNAs were able to influence cell cycle entry and progression and regulate anti-apoptotic pathways, which helped restore the damage in the kidney [32]. With the introduction of MSC-EVs into animal models, they increased the expression of anti-apoptotic genes such as Bcl-cl, BCl2, and BIRC8 while decreasing apoptosis genes like caspase 1 and 9 and lymphotoxin alpha [33].
In addition to enhancing proliferation and reducing apoptosis, Zou et al. [34] observed through a rat model of IRI that EVs also improve renal function, increase angiogenesis, and decrease apoptosis [34,35]. MSC-EVs also have anti-oxidate properties ad can reduce oxidative stress in renal injuries like IRI and AKI.
MSC-EVs For Neurological and Neurodegenerative Diseases
For neurological diseases like strokes and traumatic brain injuries (TBIs) and neurodegenerative diseases like Alzheimer’s and Parkinson’s Disease, there is no cure, and treatment is limited. These diseases are among the leading causes of death and disability worldwide [36]. MSC-EVs have neuroprotective qualities and are also biocompatible, and because of this, they can cross the blood-brain barrier (BBB).
A stroke occurs when a part of your brain is deprived of oxygen and nutrients because the blood supply is interrupted or reduced [37,38]. Strokes can lead to paralysis, difficulty talking or swallowing, memory loss or cognitive decline, emotional problems, pain, behavioural changes, and brain death [38]. Through a rat stroke model, Xin et al. demonstrated that the intravenous administration of MSC-EVs improved neurological recovery and neurovascular plasticity [39, 40, 41]. In addition, the MSC-EVs enhanced neurite remodelling and improved functional recovery through the transfer of miR-133b [40]. Similarly, Doeppner et al.’s [42] study found that MSC-EVs used to treat a stroke in a mouse model induced long-term neuroprotection, improved neurological recovery, increased neurogenesis and angiogenesis, and adjusted post-ischemic immune responses. It also showed that MSC-EVs improved brain function and promoted long-lasting cognitive functions.
Traumatic brain injury (TBIs) is an injury that affects how the brain works and is a substantial cause of death and disability in the US [43]. There were 61,000 TBI-related deaths in 2019 in the US [43]. TBIs can be caused by a penetrating injury or bump, blow, or jolt to the head. People that get TBIs may only face health problems for a few days or weeks, but in severe cases for the rest of their lives. MSC-EVs have also been tested in several studies to treat TBIs. The results showed that in a TBI mouse model, MSC-EVs increase angiogenesis and neurogenesis, reduce neuroinflammation, newly generated endothelial cells, and formed immature and mature neurons to improve functional recovery [44]. In another TBI mouse model, treatment inhibited neuroinflammation and salvaged spatial learning impairments [45].
Neurodegenerative diseases are conditions of the brain and/or spinal cord, where a progressive loss of brain cells or brain cell function causes problems with memory, thinking, and movement [46]. The most common neurodegenerative diseases are Alzheimer’s Disease (AD) and Parkinson’s Disease (PD). Ageing is the primary risk factor for most neurodegenerative diseases and is the 7th leading cause of death worldwide [36,47]. Katsuda et al. [48] used adipose-derived MSC-EVs that possessed large amounts of neprilysin to treat AD in animal models. Neprilysin is the most prominent enzyme that degrades β-amyloid peptides in the brain. β-amyloid plaque buildup is thought to be the underlying cause of why the brain cells in patients with AD die [49]. Results showed that there was a reduction of secreted and intracellular β-amyloid peptide levels. A study done by Jarmalavičiūtė et al. [50] used dental pulp MSC-EVs to treat PD. They used these MSC-EVs on dopaminergic neurons in a 3D culture, and the results showed that the neurons were rescued from 6-hydroxy-dopamine-induced apoptosis by the MSC-EVs.
MSC-EVs as A Delivery Mechanism
Not only can MSC-EVs repair and regenerate damaged tissues and organs via their natural biological cargo, but they be used for gene editing, drug delivery, and nanomedicine applications. EVs are natural transporters as they already participate in intracellular communication. They can reach a wide range of tissues following systemic administration, including the central nervous system [6]. One of the barriers to synthetic nanobots for drug delivery is because they are toxic to the body. They are easily recognized by the immune system and most of them cannot cross the BBB. MSC-EVs can overcome all of these barriers because they are biocompatible and do not trigger the immune system, and because of this, they can cross the BBB [6].
MSC-EVs naturally protect their cargo from degradation and can be loaded with hydrophilic and lipophilic drugs. The EVs can be filled with gene editors like CRISPR-Cas9 and cancer therapeutics. The EVs can be loaded with the cargo after they have been isolated or during EV biogenesis [6]. To help better reach the target cells, the MSC-EVs can be surface engineered with a ligand-receptor pair.
Current Limitations To Clinical Applications
MSC-EVs have gained significant interest for their use as cell-free regenerative therapies. However, there are limitations to clinical application and scalability, making it difficult for MSC-EVs to become a reality for human use.
Although the risk is low, there is still potential for tumorigenesis and other potential adverse effects of MSC-EVs. EVs cannot directly form cancerous tumours like MSCs, and they can still indirectly cause abnormal growth of tumours or accelerate the progression of pre-existing cancers. In addition, some studies have found that MSC-EVs can modulate the tumour microenvironment, creating a niche for cancer cell metastasis [6]. These risks are very low, but they are possible, and because of this, more research and testing are to prevent this.
Scalability is the other main limitation to the clinical usage of MSC-EVs. Firstly, the cell population of the EVs is heterogeneous, and because of this, each treatment will vary slightly and make it difficult to achieve the intended benefit. Secondly, the use of ultracentrifugation as an EV isolation technique requires a labour-intensive process. The high requirement of MSC-EVs varies depending on the treatment, but the low output from the ultracentrifugation makes scale-up more difficult because it will require a large amount of MSCs. In addition, the process of EV isolation could also compromise its integrity and functionality, making MSC-EV therapies more expensive and harder to scale [6, 16].
Conclusion
MSC-EVs and the broader field of regenerative medicine have gained significant interest in the scientific community because of their regenerative capabilities. They present a safer, more efficient cell-free therapy option than traditional stem cell therapies and could possibly become the preferred option for regenerative medicine. MSCs can be easily derived from multiple tissue sources and can be readily isolated and filled with biologically active cargo. The cargo can exert therapeutic benefits like regenerating function and damage and increasing beneficial processes like angiogenesis while decreasing detrimental processes like fibrosis and apoptosis.
Before MSC-EVs reach human trials and clinical usage, they must overcome their clinical limitations for EV isolation and clinical scalability. Next step efforts should be directed towards creating a standardized procedure for isolating and manufacturing the EVs and more research into preventing the adverse reactions and improve therapies.
It’s time for our weapons against the world’s biggest medical problems need an upgrade. The next evolution of medicine is regenerative medicine and MSC-EVs. The next evolution of medicine will shift us from ineffective drugs and delivery, organ transplants, and millions of people dying from curable diseases. MSC-EVs could bring more effective and powerful therapies that regenerate damage to the market.
References:
There were a lot of references so I have compiled them onto a Notion document where you can view them.
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