Regenerative medicine has the potential to assist the spontaneous regeneration of damaged tissues and organs, as well as restore normal function to birth defects. Thus, regenerative medicine strategies are increasingly being improved using materials and new generative cells to contribute to regeneration, as well as various combinations of these (1). Cell therapy, a regenerative medicine that meets this need, has become an attractive option to induce in situ tissue regeneration through host cell transplantation and stem cell transplantation. In particular, stem cells derived from fetuses or adults are attracting more attention than general cells in terms of their clinical application ability because they have self-renewal, multipotent or pluripotent properties as undifferentiated cells. Stem cells can be classified into three categories: embryonic stem cells (ESCs), pluripotent stem cells (PSCs), and mesenchymal stem cells (MSCs) (2). Among them, MSCs as tissue-specific stem cells, which do not have ethical problems compared to ESCs, and show less tumorigenicity compared to PSCs, are widely used in tissue damage repair and regenerative medicine.
Although stem cell transplantation has various beneficial effects, abnormal cell differentiation and low engraftment rate at damaged sites are their limitations. Considering that the general consensus of the regenerative mechanism following stem cell transplantation is the paracrine effect through the release of growth factors and cytokines, research to solve the limitations should be continued (3). 1) Nutritional imbalance with the transplant site when culturing stem cells at the laboratory level, 2) the need for hundreds of millions of stem cells, 3) the generation of a large number of stem cells that cannot be delivered to the transplant site, 4) the presence of carcinogenic potential are the problem of stem cell therapeutics that it must be a necessary and sufficient condition for clinical application in regenerative medicine (4-8). In the ischemic heart, it was reported that 1% of cells at 24 hours and less than 0.44% at 4th day after MSC transplantation were survived (9, 10). Furthermore, it has been reported that most of the administered stem cells did not penetrate the blood-brain barrier (BBB) and exert a therapeutic effect due to the limited characteristics of the BBB in the ischemic brain (11).
Stem cell-free-based therapies that can ameliorate the shortcomings of cell therapies are emerging as safe and effective treatment options compared to conventional cell therapies. MSC secretome secreted by paracrine effect, i.e. culture medium, protein, RNA, and lipid, can be a strategy for cell-free therapy, which contributes to physiological or pathological improvement of tissue damage. The MSC secretome effect is manifested by secreting extracellular vesicles (EVs), which are biochemical components according to large amounts of activation signals, rather than single growth factors or cytokines. EVs play a role in delivering functional genetic material to other cells, and it transmit various information surrounding it to neighboring cells (microenvironment), thereby activating cell-to-cell interaction as a mediator of cell communication (12). In particular, it has been known that microRNA (miRNA), a short RNA molecule that regulates several cellular processes through post-transcriptional gene silencing, communicates between neighboring cells, however it has reported recently that miRNAs have been commonly or artificially delivered by inducing multiplexed target into the microenvironment via EVs (13, 14).
In this review, we mainly focus on the potential regenerative medicine by which stem cell-free-based therapies attenuate cardiovascular and brain tissue damage. We also discuss specific strategies of miRNA-enriched EV therapeutics for multiplexed targeting.
EVs are cellular derivatives with a size of 40-1000 nm composed of adhesion molecules and soluble mediators. It has become clear that they exchange cellular biological information and deliver contents using soluble mediators including growth factors, cytokines and small molecules as small extracellular membrane fragments containing various types of cellular information and physiological properties. EVs are mainly classified into exosomes, microvesicles (MVs), microparticles, ectosomes, oncosomes, apoptotic bodies, and many other names according to their size and biogenesis. Proposed through Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines for the field in 2014 in the International Society for Extracellular Vesicles (ISEV), it was promulgated to be collectively referred to as EV (12, 13, 15).
EVs are largely divided into exosomes, MVs and apoptotic bodies according to their biogenesis, release pathway, size, content, and function. Although the specific protein markers that distinguish EVs have not been established, each protein profile is different. Exosomes are released to the outside of the cell in the form of microvesicular endosomes, and are extracellular release materials of 40-150 nm that mainly contain CD63 (CD81/CD9). MV is a plasma membrane formed by the budding of the cell plasma membrane outward, and is a subunit of EV with a typical diameter of 100-1000 mm. They express predominantly annexin A, a cytoskeletal component such as actin and microtubules. Apoptotic bodies have a diameter of about 50-5000 nm and are a programmed cell death phenomenon, and are released into the extracellular space in the form of active amphisome and passive cell death by hydrostatic pressure. In general, it has double-stranded DNA or histones as markers (16, 17). The diameter of stem cells is about 15 μm, whereas the capillary is less than about 8 μm and becomes clogged when intravenous cell transplantation is performed. However, since EVs containing exosomes and MVs have a diameter of 40-1000 nm, they have the advantage of penetrating to the periphery (Fig. 1).
Since the formation and transport of exosomes are regulated by the endosomal sorting complexes required for transport (ESCRT) signaling pathway, signal-regulating proteins such as Alix, TSG101, HSC70, and HSP90β are found in exosomes. Also known as exosome marker proteins, tetraspanin family membrane proteins (CD63, CD51 and CD9) are their components (17, 18). Although tetraspanin proteins were originally thought to be specific markers of exosomes, these proteins have been recently identified in MVs and apoptotic bodies (16, 18). MVs contain cytoskeletal proteins, integrin family proteins of adhesion function, and proteins responsible for translational modification. Specifically, glycan-binding protein responsible for interaction with other cells and Annexin A1 and A2, an abundant membrane-associated protein classified as an exosomal constituents, is recognized as a novel content of MV. Apoptotic bodies contain Annexin A5 protein that specifically binds to the negative curvature-specific lipid phosphatidylserine, and since it contains some organelles, proteins related to the nucleus, mitochondria, or endoplasmic reticulum can be observed (histones, heat shock protein, and glucose-regulated protein) (18, 19).
Cell-derived EVs have already been studied to contain many physiologically active substances (20, 21). It has been reported that certain substances showing potential therapeutic efficacy are contained through proteomic profiling synthesized in EVs. However, for induction of cell-derived EVs containing a lot of tissue-specific or target-specific substances, extrinsic substances must be expressed at high concentrations and constant levels in EVs. In the context of regenerative medicine, RNA modification of cell-derived EVs can participate in genetic communication and transfer genetic information to target cells, while simultaneously altering target gene regulatory networks to accelerate disease recovery.
Among many types of RNAs, mRNAs contain new protein sources and information necessary for tissue regeneration. mRNAs of less than about 1 kb in length can be included and delivered to EVs (22). Cuesta
Unlike mRNA, transfecting miRNAs into EVs has the advantage of being able to simultaneously target a variety of cellular signals. miRNA is a short noncoding and single-stranded RNA molecule that regulates the expression of protein-coding genes by promoting degradation and interfering with translation based on the mRNA-miRNA complementary sequence (26, 27). Although miRNAs cannot inhibit 100% protein coding like siRNA, they can cover about 60% of inhibition with a similar mechanism, and have the advantage of achieving efficient transformation due to their small size and low weight (28). The reason that miRNAs play a key function from the point of view of regenerative medicine for disease treatment is that miRNAs released from EVs internalized in donor cells can access neighboring genes and affect their expression with influencing cell-cell communication as cell-free therapy (29). To our knowledge, this function implies direct or indirect influence on neighboring cells or tissue microenvironment like stem cell paracrine effect at the transplanted site.
In addition, there is an increasing number of experimental studies demonstrating the relevance of direct miRNA delivery using EVs in various physiological environments. For example, in neuronal to astrocyte signaling, EV-mediated miRNA-124a modulated glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1), by using SOD G93A mice, an end-stage animal model of amyotrophic lateral sclerosis (ALS). It was recognized that miRNA-124a controls perisynaptic function through the characterization of neuron-astrocytic communication (33). Furthermore, Salvucci
An antisense oligonucleotides (ASO) construct antagonizing oncogenic miR-125b was induced in red blood cells (RBCs), and the potential for treatment of leukemia cells through the RBC-EVs was presented. Because RBCs lacking nuclear and mitochondrial DNA were treated with ASO-miRNA-125b, a safe and scalable platform without the risk of horizontal gene transfer was demonstrated (Table 1) (35).
To our knowledge, two approaches can be used to generate EVs containing miRNAs that induce multiplexed targeting. The first is to induce overexpression by directly injecting miRNA into cells. The easiest and simplest method for loading miRNAs is to incubate them with miRNA-secreting cells. Migration to intracellular vesicles by concentration gradient can be a natural integration process that does not pose any threat to cells. However, this strategy has the disadvantage of not being able to predict the concentration of miRNAs that diffuse into EVs in cells, and low efficiency due to miRNA instability.
A method for stably inducing miRNA into EVs is the design of chemical transfection. As mentioned earlier, stem cells are basically loaded with paracrine molecules, and EVs inside the cells can be the best biogenesis plant and delivery tool that can over-produce and deliver desired miRNAs. It is to transduce miRNA itself or a specific plasmid/virus-based construct designed so that miRNA can be activated in the cell, and induce it to be expressed ectopic, and naturally package it in EVs (36, 37).
Physical force can be used to induce transient micropores in EV membranes or membrane recombination sites to promote miRNA incorporation. Sonication and electroporation are examples of methods that researchers can actively utilize. Ultrasound has been focused on methods that can shape solid images using sound waves. However, it is possible to apply a mechanical shear force that can increase the temporary membrane permeability by using low physical energy. Lamichhane
Second, it is a direct method to isolate EVs from stem cells, concentrate them, and transfer miRNAs. Zhang
A study that examines the improvement in regenerative medicine by introducing a stem cell-mediated miRNA into EVs present inside the cell has been recently published. This chapter represents to examine the case of multiplexed targeting of various signals related to cell/tissue improvement by stem cell-derived EVs with increased miRNA used in heart and brain diseases (Table 2).
Coronary artery diseases, a representative cardiovascular disease, are caused by atherosclerosis and are known to cause arrhythmias and myocardial infarction. In order to maintain the homeostasis of various cells (cardiac cells, fibroblasts, lymphocytes, mast cells, and macrophages) present in the cardiovascular system and the difficulties in cardiac regeneration and repair (14), so stem cell-derived EV therapy is essential, especially the special treatment of EV-derived miRNAs functioned as multiplexed targeting.
As with cardiovascular disease, stem cell-derived EV-derived miRNA research that performs multiplexed targeting in CNS disease has not been conducted to a large extent. In order to control Rett syndrome (RTT), one of the neurodevelopmental disorders, Pan
Therapeutic research for the regenerative medicine with stem cells has made a lot of progress. Researches for stem cell-derived EV are also currently in progress, however it is necessary to secure therapeutic efficacy and safety in vivo. In the case of stem cell-derived EVs with increased specific miRNAs, it is expected that it will be a powerful cell-derived therapeutic agent that can overcome the limitations of existing studies and has the advantages of stem cells and multiplexed targeting of miRNAs.
This work was supported by a 2-Year Research Grant of Pusan National University.
The authors have no conflicting interests.
A summary of enhancing information for cell-derived extracellular vesicles
|Types of RNAs||Target||Cell source of EVs||Effects||Reference|
|mRNA||TGF-β1||Human pulmonary artery smooth muscle cells||Vascular remodeling||23|
|GDNF||Human adipose mesenchymal stem cells||Angiogenesis||24|
|Decrease of renal fibrosis|
|GLO-1||Adipose derived stem cells||Angiogenesis||25|
|Improvement of T2DM|
|miRNA||miRNA-133a||Adult cardiac progenitor cells||Indirect EV treatment||30|
|Improvement of cardiac function|
|miRNA-126||Endothelial cells||Indirect EV treatment||31|
|Atheroprotective laminar shear stress|
|Improvement of ALS|
|miRNA-155||Adipose tissue macrophage||Glucose tolerance||34|
|ASO-miRNA-125b||Red blood cells||Treatment of leukemia cells||35|
A summary of stem cell-derived EV therapies mediating multiplexed targeting by miRNAs
|Ischemic heart||miRNA-210||Ptp1b||Human adipose-derived stem cells||Regulation of apoptosis and angiogenesis||14|
|miRNA-133a||Bmf||Cardiac progenitor cells (SCA-1＋Lin−)||Anti-apoptosis||30|
|Bim||Improvement of cardiac function|
|miRNA-25-3p||Fasl||Bone marrow mesenchymal stem cells||Cardioprotection||45|
|Heart||miRNA-199a||22 genes included Rb1||Mesenchymal stem cells||Cell death/proliferation||46|
|Lkb1||Cell cycle regulation (Target Prediction)|
|Rett syndrome||miRNA-21-5p||EphA4||Urine-derived stem cells||Facilitation of early nerve formation||47|
|Spinal cord injury||miRNA-126||Spred1||Mesenchymal stem cells||Angiogenesis||50|