miRNAs from mesenchymal-stem-cell-derived extracellular vesicles: Emerging players in regenerative medicine and disease therapy

Abstract

Mesenchymal stem cells (MSCs) hold immense promise in regenerative medicine due to their unique capabilities, including differentiation into various cell types and paracrine signaling. Among their secretions, extracellular vesicles (EVs) play a pivotal role in mediating therapeutic effects through their bioactive cargo, including microRNAs (miRNAs). These miRNAs are key regulators of gene expression, modulating critical biological processes such as inflammation, immune response, and tissue repair. MSC-derived EVs have demonstrated significant therapeutic potential across diverse diseases, including cardiovascular, neurological, autoimmune, and oncological conditions. This review explores the biogenesis, isolation, and characterization of MSC-EVs, with a focus on the functional roles of miRNAs in disease modulation. The therapeutic prospects of MSC-EV miRNAs as potential biomarkers, drug delivery agents, and modulators of pathophysiological pathways are discussed, providing insights into future translational research and clinical applications.

Graphical abstract

Mesenchymal-stem-cell-derived extracellular vesicles (MSC-EVs) carry microRNAs (miRNAs) that regulate inflammation, tissue repair, and immune responses. In this review, Jafarinia and colleagues highlight the therapeutic potential of MSC-EV miRNAs in cardiovascular, neurological, autoimmune, and oncological diseases, offering a promising cell-free approach for regenerative medicine and disease modulation.

Introduction

Mesenchymal stem cells (MSCs) are a type of adult stem cell found in various tissues, including bone marrow, umbilical cord (UC), placental tissue, adipose tissue, and menstrual blood. These cells possess the ability to differentiate into multiple cell types. Extensive clinical studies have focused on the therapeutic role of MSCs in the protection and regeneration of tissues by replacing injured cells.¹ MSCs have quickly become a focus of interest in clinical research for their multifaceted biological roles. The initially emphasized capability of multilineage differentiation into different cell types was followed by a growing number of studies focused on their immunomodulatory, anti-inflammatory, and neuroprotective roles, along with the promotion of tissue repair.² These promising features have sparked extensive research into their potential therapeutic applications in the treatment of various diseases, including neurological disorders,^3,4 liver diseases,⁵ cardiovascular diseases,⁶ skin diseases,⁷ lung diseases,⁸ and renal injuries.⁹ It was initially thought that MSCs migrate through injured tissues, thus assuming the types of injured tissues by cellular replacement after injury. Further research threw light on this direct cell replacement mechanism as minor compared to what had been expected. Most of the administered intravenous MSCs were trapped in the lungs, while only a few reached the targeted site of injury. Moreover, the immune rejection, genetic instability, and malignant potential of cell therapy added further confusion. Indeed, studies have initiated a paradigm shift by indicating that most therapeutic effects of MSCs occur through a paracrine mechanism rather than through direct cell-to-cell interaction. The conditioned medium of MSC cultures, which contains the secreted factors, has been shown in studies to provide similar therapeutic benefits compared to the cells. This observation also highlights that extracellular vesicles (EVs) are critical mediators of paracrine signaling by MSCs.^10,11,12 In more detail, recent literature suggests that the immunomodulatory effect of MSCs is predominantly mediated by EVs.¹³

MSC-derived EVs have taken part in immunoregulation through the secretion of various cytokines and growth factors, such as interleukin (IL)-10, IL-6, transforming growth factor (TGF)-β1, and hepatocyte growth factor (HGF).¹⁴ In addition to proteins, MSC-EVs also contain microRNAs (miRNAs), a class of non-coding RNAs that regulate gene expression post-transcriptionally.¹⁵ Several miRNAs, such as miR-23b, miR-125b, miR-451, miR-223, miR-31, miR-24, miR-214, and miR-122, have been identified in MSC-EVs and are involved in various biological processes, including epigenetic regulation, immunoregulation, organism development, tumorigenesis, and tumor progression.^16,17 Considering that MSC-EVs have been extensively studied in various animal models, it is postulated that the major mechanisms of their action are through their cargo, comprising cytokines and miRNAs.¹⁰ miRNAs within MSC-EVs, essential for the therapeutic activity of MSC-EVs, could target genes associated with immune response, inflammation, and tissue repair. Since MSC-EVs are among the promising therapeutic methods with minimal potential to cause side effects, and miRNAs are one of the important constituents of MSC-EVs, this review discusses the therapeutic potential of MSC-EVs, focusing on miRNA functions, biogenesis, and characterization, and their roles in modulating various diseases.

Therefore, this review aims to provide an exhaustive overview of existing knowledge regarding the biogenesis, isolation, and characterization of MSC-derived EVs, particularly their miRNA cargo. Particular emphasis is given to the explanation of therapeutic mechanisms of MSC-EV miRNAs, their disease-modulating roles, and their translational promise in various clinical settings. Disease-specific applications covered in this review include cardiovascular, neurological, oncological, autoimmune, hepatic, and renal disorders, with both preclinical evidence and potential for clinical translation being underscored.

Mesenchymal stem cells

MSCs are a type of stromal cell capable of self-renewal and can differentiate into multiple cell lineages.^10,18 Due to their easy accessibility, these versatile cells can be obtained from various tissues, including umbilical cord, adipose tissue, bone marrow, and dental pulp.¹⁹ Although they comprise approximately 0.001%–0.01% of nucleated cells from adipose tissue or bone marrow,²⁰ their proliferative and differentiating abilities make them an attractive tool in regenerative medicine. The International Society for Cellular Therapy (ISCT) has defined stringent minimum criteria to define MSCs. These include attachment, differentiation potential, and surface marker expression. MSCs should be able to attach to plastic surfaces when cultured. Differentiation potential suggests that MSCs should be able to differentiate into at least more than one cell type, including, but not limited to, fat, bone, and cartilage. MSCs must also express specific surface markers, including CD73, CD105, and CD90, while lacking expression of CD14, CD45, CD19, and HLA-DR.²¹ These markers help distinguish MSCs from other cell types. Even though MSCs represent a somewhat heterogeneous cell population due to variability related to tissue source and donor specificity, they have been reliably shown to produce anti-inflammatory paracrine activity. However, their inflammatory phenotype can be dynamically modulated by the surrounding microenvironment.^22,23

Extracellular vesicles

EVs are minute, membrane-enclosed particles released by many cell types. These vesicles range from 30 to 1,000 nm in size and may be secreted by virtually all cell types, including blood platelets, dendritic cells, T and B lymphocytes, adipocytes, mast cells, neurons, tumor cells, stem cells, and epithelial and endothelial cells.^24,25,26,27 They are found in various bodily fluids, including urine, blood serum, amniotic fluid, saliva, cerebrospinal fluid, breast milk, and nasal secretions. EVs can be categorized into three main types based on their size and cellular origin: (1) exosomes: these small vesicles, in the size range of 30–150 nm in diameter, are formed from the inward budding of late endosomes; (2) microvesicles are larger vesicles measuring between 100 and 1,000 nm in diameter, which are shed from the plasma membrane; and (3) apoptotic bodies: larger vesicles, ranging in size from 1,000 to 4,000 nm, originate from cells dying due to programmed cell death.^24,28,29,30

However, since there are no specific markers available for exosomes and microvesicles, and current methodologies allow separation by size and density rather than by origin, the International Society for Extracellular Vesicles (ISEV) has agreed to name all experimentally isolated vesicles, collectively, EVs.¹⁰ EVs contain a wide array of biological materials, including proteins, lipids, and RNAs. They bear a wide variety of membrane proteins on their surface, including those involved in immune response (major histocompatibility complex [MHC] class I and II); protein synthesis (EEF1A1 and EEF2); stress response (Hsp60, Hsp70, Hsp90, and Hspa8); and cell adhesion such as integrins, tetraspanins (CD9, CD63, CD81, and CD82), membrane fusion (flotillin and annexin), and intracellular trafficking (TSG101 and Alix).^25,31,32 They also express some of the surface markers that are common with MSCs, like CD73, CD44, CD90, and CD29.^33,34 They also contain a multitude of lipids, including ceramide, cholesterol, sphingomyelin, ganglioside GD3, and phosphatidylserine. In addition, they are enriched with a wide variety of both coding and non-coding RNA molecules.^35,36,37,38 Whereas EVs play some important biological functions in hemostasis and the spread of biomolecules to other adjacent cells and tissues.³⁹

Mechanisms and selectivity of miRNA incorporation into EVs

miRNAs are 19-24-nucleotide non-coding RNAs that modulate the expression of 30% of all mammalian protein-coding genes and are involved in a variety of biological processes such as proliferation and differentiation, apoptosis, and immune cell activity.^40,41

Evidence suggests that EVs, especially MSC-derived exosomes, contain numerous miRNAs that regulate gene expression post-transcriptionally. Many miRNAs in MSC-EVs contribute to pathological and physiological processes such as epigenetic regulation, immune regulation, organismal growth, and tumorigenesis and progression.^16,17

Packaging of miRNA content in MSC-EVs occurs selectively and through a specific mechanism. During EV biogenesis, specific miRNAs are sorted and enriched based on their interaction with RNA-binding proteins or affinity for tetraspanin proteins on the EV membrane. This targeted packaging allows for the delivery of specific miRNAs to the recipient cell via EVs, where they can mediate the effects of their parent cell and transfer cellular function to another cell.^42,43,44

The regulation of the secretion mechanism of miRNAs is through a ceramide-dependent pathway in which ceramide is synthesized by neutral sphingomyelinase 2 (nSMASe2) and controls the secretion of EVs and subsequent secretion of miRNAs, which is a completely interdependent and direct process. That is, inhibition of nSMASe2 reduces ceramide synthesis and subsequently reduces the secretion of EVs and miRNAs, and increased expression of nSMASe2 is associated with increased ceramide synthesis and increased secretion of EVs and miRNAs.⁴⁵ Since the mechanisms of miRNA sorting are not well understood, miRNA loading into EVs may depend on factors including the GGAG motif, GGCU motif, and miRNA-related proteins such as Ago2, Alix, MEx3c, hnRNPA2B1, SYNCRIP, YBX-1, MVP, and La protein.⁴⁶ To further demonstrate the various proteins and miRNA motifs that are important for the selective sorting of miRNAs into extracellular vesicles, Table 1 summarizes the particular RNA-binding proteins, motifs, and corresponding miRNAs implicated in the regulation of miRNA loading into EVs.

Table 1.

List of proteins and miRNA motifs involved in the regulation of miRNA sorting into EVs

Article	Proteins/Motifs	miRNAs	Description
Lee et al.47	caveolin-1 (cav-1)	miR-17, miR-93	caveolin-1 enhances the O-GlcNAcylation of hnRNPA2B1 through phosphorylation, promoting its binding to specific miRNAs such as miR-17 and miR-93, thereby facilitating their incorporation into microvesicles.
Beltri et al.46	GGAG motif	miR-198, miR-601	the GGAG motif in miRNAs is recognized by the nuclear ribonucleoprotein hnRNPA2B1, which is involved in the packaging of miRNAs into EVs. Mutations or changes in hnRNPA2B1 sumoylation can alter the loading process.
Santangelo et al.48	GGCU motif	miR-3470a, miR-194-2-3p	the GGCU motif in miRNAs is recognized by SYNCRIP, which facilitates the sorting of miRNAs into EVs. Loss of SYNCRIP disrupts this sorting, while adding the GGCU motif to poorly exported miRNAs enhances their sorting into EVs.
Gibbings et al. McKenzie et al. 49,50	Argonaute 2 (Ago2)	let-7a, miR-100, miR-320a	Ago2 binds to miRNAs to form the RNA-induced silencing complex (RISC). This complex is involved in miRNA sorting to EVs. Ago2’s activity can also regulate the stability and packaging of miRNAs into EVs.
Zietzer et al.51	heterogeneous nuclear ribonucleoprotein U (hnRNPU)	miR-30c-5p	hnRNPU binds to miR-30c-5p in the nucleus, stabilizing it and enhancing its nuclear expression. Downregulation of hnRNPU leads to increased cytoplasmic levels of miR-30c-5p, facilitating its export into large EVs.
Chang et al.52	heterogeneous nuclear ribonucleoprotein C1 (hnRNPC1)	miR-30d	hnRNPC1 associates with miR-30d and EVs in endometrial epithelial cells; reducing hnRNPC1 lowers miRNA levels in EVs.
Groot et al.53	Y-box binding protein 1 (YBX-1)	miR-223, miR-133	YBX-1 regulates miRNA sorting by binding to miRNAs like miR-133. Overexpression of YBX-1 increases the presence of miR-133 in EVs, whereas silencing YBX-1 reduces it.
Intine et al. Diaz et al. 54,55	La protein	miR-122	La protein binds miR-122 with high affinity and shuttles it between the nucleus and cytoplasm. It is important for the stability and sorting of miR-122 into EVs.
Groot et al. Teng et al. 53,56	Major vault protein (MVP)	miR-193a	MVP is involved in the sorting of miR-193a into EVs. MVP knockout increases cytosolic miR-193a but decreases its levels in exosomes. MVP overexpression leads to reduced cytosolic miR-193a and increased exosomal levels.
Lu et al.57	Mex-3 RNA-binding family member C (MEX3C)	miR-451a	MEX3C is an RNA-binding protein involved in miRNA sorting to EVs. It interacts with Ago2 and affects miR-451a sorting. Knockdown of MEX3C reduces miR-451a levels in exosomes.

Therapeutic potential of MSC-derived EV miRNAs in disease modulation

miRNAs from MSC-EV have gained attention as potential therapeutic entities for disease modulation because of their capability to modulate gene expression, decrease inflammation, and augment tissue regeneration.²³ These EVs can transport miRNAs acting on recipient cells, providing benefits compared to whole-cell therapies, like lower immunogenicity and risk of tumorigenesis. They have shown promise for clinical use in treating diseases, such as cardiovascular, neurological, cancer, autoimmune, liver, and renal diseases¹⁰ (Figure 1; Table 2).

Figure 1.

Therapeutic applications of MSC-derived EV miRNAs in various diseases

The figure summarizes the broad therapeutic potential of MSC-EV miRNAs across multiple disease categories, demonstrating their ability to modulate key pathological processes through specific miRNA-mediated mechanisms MSC-EV: mesenchymal-stem cell-derived extracellular vesicle.

Table 2.

MSC-EV miRNAs and their therapeutic applications across different pathologies

Article	Disease model	Cell source of EV	miRNAs	Key results
Zhao et al.58	Myocardial ischemia/reperfusion (I/R)	BM-MSC	miR-182	modified the polarization of M1 macrophages to M2 macrophages
Yang et al.59	atherosclerosis	HUC-MSC	miR-145	•downregulate JAM-A •inhibit migration in vitro •reduce atherosclerotic plaque in vivo
Zhu et al.60	myocardial infarction	BM-MSC	miR-125b-5p	•cardiomyocyte restoration •alterations in p53 and Bcl-2 homologous antagonist killer (BAK) 1 protein expression levels
Yang et al.61	aneurysm	BM-MSC	miR-144-5p	•suppresses vascular endothelial injury through PTEN
Pu et al.62	heart failure	BM-MSC	miR-30e	•decline in myocardial injury, apoptosis, and fibrosis •downregulation of lectin-type oxidized LDL receptor 1 (LOX1)
Du et al.63	peripheral arterial diseases (PADs)	BM-MSC	miR-210-3p miR-126	•promote angiogenesis and improve outcomes in PAD
Zhang et al.64	pulmonary hypertension (PH)	BM-MSC	miR-483	•attenuate inflammation and fibrosis
Geng et al.65	stroke	Adipose-derived MSC	miR-126	•promote cell proliferation •enhance the expression of von Willebrand factor and doublecortin (a neuroblast marker) •improve functional recovery and decrease neuronal cell death
Li et al.66	spinal cord injury (SCI)	BM-MSC	miR-133b	•axonal growth •decrease lesion sizes and protection of neurons •downregulate the neuron-degenerating molecule RhoA
Nakano et al.67	cognitive impairment	BM-MSC	miR-146a	•reduce NF-κB levels and an anti-inflammatory response •promote synaptogenesis
Xu et al.68	traumatic brain injury (TBI)	BM-MSC	miR-216a-5p	•prevent cell death •promote neurogenesis through neurogenin-2 inhibition
Yan et al.69	glioblastoma	BM-MSC	miR-512-5p	•slows glioblastoma growth
Mi et al.70	rheumatoid arthritis	HUC-MSCs	miRNA-451a	•target ATF2 •reduce RA cell growth and spread, •improve joint inflammation
Chen et al.71	autoimmune hepatitis (AIH)	BM-MSC	miR-223	•reduce levels of ALT and AST. Reduce inflammation (TNF-α, IL-17A, and IL-1β)
Fujii et al.72	GVHD	BM-MSC	miR-125a-3p	•suppress the proliferation and differentiation of effector T cells
Lou et al.73	liver fibrosis	ADSCs	miR-122	•reduce of the expression of genes such as cyclin G1 and prolyl-4-hydroxylase α inducing collagen synthesis in hepatic stellate cells
Gu et al.74	acute renal ischemia reperfusion injury	HWJ-MSCs	miR-30	•inhibit mitochondrial fission in tubular epithelial cells •ameliorate acute renal IRI
Venkat et al.75	diabetic stroke	HUC-BCs	miR-126	•promoted angiogenesis and neuroprotection
Su et al.76	insulin resistance (aging-related)	BM-MSCs	miR-29b-3p	•miR-29b-3p ameliorated insulin resistance via SIRT1 pathway modulation
dos Santos et al.77	acute lung injury (ALI)	MSCs	miR-21-5p	•MSC-EV-derived miR-21-5p reduced apoptosis and inflammation by targeting PTEN and PDCD4, thereby suppressing pro-apoptotic pathways and NF-κB signaling
Wei et al.78	Alzheimer disease	MSCs	miR-223	•MSC-exosomal miR-223 reduced neuronal apoptosis via PTEN-PI3K/Akt signaling
Tao et al.79	osteoarthritis (cartilage repair)	HS-MSCs	miR-140-5p	•enhanced chondrocyte proliferation & migration, preserved ECM secretion, prevented OA progression in vivo

BM-MSC, bone marrow mesenchymal stem cell; HUC-MSC, human umbilical cord mesenchymal stem cell; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ADSCs, adipose-derived mesenchymal stem cells;IGFR-1, insulin-like growth factor receptor 1; HWJ-MSCs, human Wharton Jelly mesenchymal stromal cells; IRI, ischemia reperfusion injury; HUC-BCs, human umbilical cord blood cells; HS-MSCs, human synovial mesenchymal stem cells.

Cardiovascular disease

Atherosclerosis

Atherosclerosis is a vascular inflammatory disease originating from endothelial injury.⁸⁰ Regarding causes, researchers have extensively described the origins of atherosclerosis because this complex condition involves numerous factors, such as endothelial cell (EC) injury, vascular smooth muscle cell (VSMC) aggregation, macrophage infiltration, inflammation, and disorders in lipid metabolism.^81,82 Atherosclerosis is initiated by endothelial dysfunction, facilitating the subendothelial accumulation of oxidized low-density lipoproteins (ox-LDL) and their uptake by macrophages to form foam cells. These foam cells—and activated vascular smooth muscle cells (VSMCs)—release proinflammatory cytokines and growth factors, promoting plaque formation and vascular inflammation. Regulatory T cells (Tregs) play a counterbalancing anti-atherogenic role, but their numbers decline during disease progression, enabling unchecked inflammation and plaque development.⁸³

Research demonstrated that EVs carrying miRNA-155 could induce atherosclerosis and impair endothelial cells, while MSC-derived exosomes could modulate the inflammatory response of atherosclerotic plaques, representing a future potential way for preventing this disease.⁸⁴ Several miRNAs, such as miR-21-5p, miR-146a, miR-30a, and let-7, secreted by MSCs in EVs, have been reported to regulate macrophage function.⁸⁵ Research by Yang et al. demonstrated that delivering extracts from the human umbilical cord-MSCs (hUC-MSCs) to produce miR-145 could reduce the migration of human umbilical vein endothelial cells (HUVEC) by targeting junctional adhesion molecule (JAM)-A. This subsequently resulted in a reduction in the formation of atherosclerotic plaques.⁵⁹ Research showed that EVs carrying miR-125 b-5p from bone marrow stem cells (BMSCs) could induce smooth muscle cell (SMC) apoptosis, decrease inflammation, reduce blood lipid levels, and inhibit plaque formation by targeting mitogen-activated protein 4 kinase 4 (Map4k4).⁸⁶ Another study found that EVs from BMSCs containing miR-21a-5p could delay the progression of atherosclerosis by inducing a favorable switch in macrophage polarization and lessening their infiltration within the vessel wall. This was realized through targeting some signaling pathways involving Kruppel-like factor 6 (KLF6) and extracellular signal-regulated kinase ½ (ERK1/2).⁸⁷ Besides, another study showed that EVs from adipose-derived stem cells (ADSCs) could suppress miR-324-5p expression in an injury model and then protect endothelial cells against atherosclerosis.⁸⁸ Moreover, EVs derived from BM-MSCs containing miR-133 can reduce the expression of genes involved in cholesterol efflux from macrophages, including ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1). This disruption in cholesterol efflux inhibits the formation of foam cells.⁸⁹ EVs from BM-MSCs containing small interfering RNA (siRNA) si-LOC100129516 can increase cholesterol efflux from foam cells and reduce the accumulation of intracellular lipids, thus slowing down the process of atherosclerosis through the peroxisome-proliferator-activated receptor γ/liver X receptor α/ATP binding cassette subfamily A member 1 (PPARγ/LXRα/ABCA1) pathway.⁹⁰

In summary, MSC-derived EVs and their miRNAs offer promising therapeutic potential for atherosclerosis by modulating inflammation, cholesterol metabolism, and plaque formation, paving the way for innovative treatments.

Myocardial infarction

An MI occurs when there is a blockage within a coronary artery, which results in an inadequate oxygen supply to the myocardial tissue. This hypoxia leads to irreversible damage and necrosis of cardiac muscle cells.⁹¹ MI arises from acute ischemia leading to cardiomyocyte death, which triggers a biphasic immune response: an early inflammatory phase marked by neutrophil infiltration and pro-inflammatory M1 macrophages, followed by a reparative phase dominated by anti-inflammatory M2 macrophages, fibrosis, and scar formation mediated by cardiac fibroblasts. Tachycardia also induces natriuretic peptides (e.g., atrial natriuretic peptide [ANP] and brain natriuretic peptide [BNP]) with anti-fibrotic and anti-apoptotic effects on infarcted myocardium.⁹²

Transplantation of MSCs into the infarcted heart can improve cardiac function. This therapeutic strategy can be further optimized by genetic modification of MSCs to overexpress miR-1.⁹³ Such modification increases the survival of MSCs and their differentiation into heart muscle cells. Significantly, combination therapy in sequence with EVs and then BM-MSCs achieved greater efficacy in reducing scar size and improving cardiac function in rats with acute myocardial infarction (AMI) compared to the single use of EVs or BM-MSCs alone.⁹⁴ Zhu et al. investigated the effect of hypoxia on BM-MSCs. Data showed that EVs secreted by hypoxic BMSC-enriched in miR-125b-5p facilitate cardiomyocyte restoration in the MI. Such protection was associated with reduced apoptosis in cardiomyocytes, which the authors claimed was due to alterations in p53 and Bcl-2 homologous antagonist killer (BAK) 1 protein expression levels.⁶⁰ Furthermore, researchers have found that treating BM-MSCs from mice with hypoxia enhances the cardiac damage repair activity of their EVs.⁹⁵ This enhanced reparative capability is due to the activity of miR-210 and neutral sphingomyelinase 2 carried by the EVs. Sun et al. showed that long non-coding RNA (lncRNA)-urothelial cancer-associated 1 (UCA1), when delivered via EVs secreted by hypoxic cells, exerts cardioprotection against AMI. The mechanisms underlying this protection depend on lncRNA-UCA1 targeting and inhibiting miR-873-5p. Thus, lncRNA-UCA1 inhibits miR-873-5p from repressing its target gene, XIAP. The latter effect involves the activation of AMPK and higher expression levels of Bcl-2, which decreases apoptosis.⁹⁶ miR-25-3p derived from MSCs decreases MI through targeting pro-apoptotic proteins and inhibition of enhancer of zeste homolog 2 (EZH2) activity. Both in vitro and in vivo cardioprotection happens by inhibition of EZH2 or overexpression of miR-25-3p in cardiomyocytes.⁹⁷ Yet another study showed that the treatment of TXL significantly enhanced the capacity of MSCs in cardiac myocyte regeneration, while at the same time suppressing inflammation and apoptosis. Further, it was found that this was mediated through EVs containing miR-146a-5p, which targets particularly the IL-1-receptor-associated kinase 1 (IRAK-1)/nuclear factor κB (NF-κB) p65 signaling pathway. It suppresses the cardiac muscle cell repair mediated by the EVs secreted from TXL-preconditioned MSCs, reduces the area of infarction, and finally improves left ventricular ejection fraction.⁹⁸ One mechanism by which MSC transplantation improved outcomes post-MI was through the induction of autophagy in cardiomyocytes.⁹⁹ These effects were mediated, at least in part, by EVs secreted by MSCs containing miR-125b-5p. More recently, human MSCs (hMSCs) have been shown to secrete EVs containing the lncRNA KLF3-AS1.¹⁰⁰ This lncRNA acts to inhibit the activity of the Sirt1 protein, which itself inhibits pyroptosis. KLF3-AS1 acts by directly targeting miR-138-5p and inhibiting it. KLF3-AS1 efficiently impedes the initiation and process of MI.

MSC-EVs from hemin-pretreated MSCs have been demonstrated to potentially reduce aging by inhibiting the high-mobility group protein B1 (HMGB1)/ERK pathway, increasing the delivery of miR-183-5p into cardiomyocytes.¹⁰¹ Huang et al. used atorvastatin-pretreated MSCs, and the obtained results showed better functional recovery in rats with AMI. The increase in rat heart function has been related to enhanced angiogenesis. More specifically, they identified key contributions for lncRNA H19 in modulating miR-675 and, more crucially, the activity of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule 1 (ICAM-1). MSC-derived EVs enriched with specific miRNAs may offer a promising treatment for AMI.¹⁰²

In summary, MSC-derived EVs enriched with specific miRNAs and lncRNAs demonstrate promising therapeutic potential for MI by reducing apoptosis, enhancing cardiac repair, and promoting angiogenesis.

Aneurysm

An aneurysm represents a serious vascular disorder featuring a local, permanent dilation of an artery.¹⁰³ They are mostly asymptomatic, of complex etiology, and occur mostly in the aorta, cerebral arteries, and visceral arteries.¹⁰⁴ Aneurysm arises from chronic aortic wall inflammation, VSMC apoptosis/dysfunction, and extracellular matrix degradation (e.g., elastin breakdown by proteases), weakening the media and predisposing to dilation and rupture.^105,106

Now, it can be noted that BMSCs are rapidly becoming potential therapeutic tools for the management of vascular system diseases. miR-144-5p and miR-23b-3p display extraordinary promise in lessening intracranial aneurysm (IA). miR-144-5p suppresses vascular endothelial injury through phosphatase and tensin homolog (PTEN).⁶¹ However, miR-23 b-3p inhibits the PI3K/Akt/NF-κB pathway by repressing KLF5, thereby inhibiting the development of IA.¹⁰⁷ On the other hand, some studies have treated abdominal aortic aneurysms with ADSCs. ADSCs-derived miR-17-5p mediates the progression and inflammatory response of abdominal aortic aneurysm (AAA) by regulating the thioredoxin-interacting protein (TXNIP)-NLR family pyrin domain containing 3 (NLRP3) pathway.¹⁰⁸

In summary, MSC-derived miRNAs show potential in aneurysm management by targeting pathways involved in vascular injury and inflammation, offering a promising therapeutic approach for these complex vascular disorders.

Heart failure

Heart failure (HF) is a parameter reflecting impaired cardiac function, based on insufficiency in blood flow to the peripheral tissues, which, in turn, cannot meet their demand for blood and oxygen due to reduced delivery.^109,110 Heart failure reflects ventricular remodeling with myocyte loss/hypertrophy and interstitial fibrosis, maintained by neurohormonal activation (renin-angiotensin-aldosterone and sympathetic systems) and systemic inflammation, which together worsen loading conditions, calcium handling, and energetics.^111,112

According to a suggestion by Matsumoto et al.,¹¹³ patients who had an AMI and developed HF within a year demonstrated much higher levels of some p53-responsive miRNAs, such as 34a, 192, and 194, in EVs 18 days after AMI. This suggests that these EV-derived miRNAs can be utilized for prognosis in HF. Moreover, these miRNAs can also be predictive markers of left ventricular remodeling after AMI. Pu et al. illustrated that miR-30e-overexpressed BMSCs-EVs contributed to a decline in myocardial injury, apoptosis, and fibrosis, through the downregulation of lectin-type oxidized LDL receptor 1 (LOX1) and subsequent inhibition of the NF-κB p65/Caspase-9 pathway.⁶² The other angle of cardiac angiogenesis induced through miR-1246 in HUC-MSCs-EVs, inhibiting PRSS23, may block the Snail/α-SMA pathway.¹¹⁴ Similar dysregulation in miRNA profiling was also noted in serum EVs from HF patients. High levels of miRNA-21 and miRNA-92¹¹⁵ and low levels of miRNA-425 and miRNA-744¹¹⁶ were detected in such patients compared with controls. Most of these miRNAs commonly implicated in fibrosis may serve as biomarkers for the diagnosis of hypertrophic cardiac diseases and as promising targets for therapy against the development of HF.¹¹⁷ In summary, exosomal membrane vesicles isolated from in vitro experiments derived from healthy donors’ cardiomyocytes induce cell proliferation and decreased apoptosis in neonatal rat cardiomyocytes. On the contrary, EVs isolated from HF patients exert an opposite effect, probably linked to their lower levels of miRNA-21-5p. A decrease in miRNA-21-5p could be responsible for the activation of PTEN and downregulation of Akt phosphorylation in cardiomyocytes.¹¹⁸ These results confirm that MSC-derived EVs may become a new therapeutic strategy in HF.

In summary, MSC-derived EVs and their miRNAs hold potential as both diagnostic biomarkers and therapeutic agents for HF, aiding in reducing fibrosis, promoting angiogenesis, and improving cardiac function.

Peripheral arterial diseases

PADs primarily involve peripheral arteries, which reduce blood flow to the extremities. The most common pathological process associated with PAD is atherosclerosis, or the hardening and narrowing of arteries, which impairs blood flow to the lower and, in some cases, upper limbs.¹¹⁹ This may present symptoms of claudication that can substantially affect the patient’s quality of life.¹²⁰ PAD is driven by atherothrombotic narrowing of limb arteries from endothelial injury, lipid deposition, and inflammation; downstream ischemia induces skeletal-muscle and nerve changes that feed back to worsen perfusion and symptoms.^121,122

Recent studies have demonstrated that MSCs-EVs may be a promising method for the treatment of PAD by inducing angiogenesis.^123,124 EVs from BM-MSCs rich in VEGF and miR-210-3p enhance the formation of blood vessels in ischemic limbs, as well as the upregulation of VEGFR1 and VEGFR2 in endothelial cells.¹²⁵

Du et al.⁶³ further showed that MSC-derived EVs could be engineered to have superior angiogenic capability with the addition of a nitric-oxide-releasing polymer. The approach yielded higher levels of VEGF and miRNA-126 cargo, demonstrating promising angiogenesis in a murine model of hindlimb ischemia. Furthermore, EVs secreted by CD34⁺ cells are capable of transporting miRNA-126-3p into target cells, downregulating the Sprouty-related Ena/Vasp homology 1 (EVH1) domain, and upregulating genes responsible for angiogenesis.¹²⁶

In summary, MSC-derived EVs, enriched with pro-angiogenic factors such as VEGF and miRNAs like miR-210-3p and miR-126, offer significant potential for promoting angiogenesis and improving outcomes in PAD.

Pulmonary hypertension

Pulmonary hypertension (PH) is a multifactorial disease characterized by pulmonary blood vessel remodeling and hypertrophy of the right ventricle, leading to its dysfunction.^80,81 PH features endothelial dysfunction and fibroproliferative remodeling of distal pulmonary arteries (endothelial cells, smooth muscle cells, and fibroblasts), leading to vasoconstriction, plexiform lesions, and right-heart strain; dysregulated endothelial transcriptional programs (e.g., SOX17/RUNX1) and inflammation are key drivers.^127,128

More importantly, intravenous administration of MSC-derived EVs increased the expression of miR-204 in the lungs, leading to reduced right ventricular systolic pressure in an experimental model of hypoxia-induced PH.^129,130

EVs from mice with PH or patients with idiopathic pulmonary arterial hypertension expressed higher levels of some miRNAs, such as miR-19b, miR-20a, miR-20b, and miR-145. By contrast, MSC-derived EVs were rich in anti-proliferative and anti-inflammatory miRNAs, including miR-34a, miR-122, miR-124, and miR-127. These data underlined the role of miRNAs in the pathogenesis and the prevention of PH.¹³¹ Besides, MSC-derived EVs may contain miRNAs with a protective role, including, but not limited to, miR-483 and miR-21-5p.^64,132 Overexpression of miR-483 in endothelial cells is reported to induce a protective effect by promoting the attenuation of inflammation and fibrosis, leading eventually to alleviated PH in rats.⁶⁴ MSC-derived EVs are documented to attenuate lung injury related to ischemia and subsequent reperfusion by transporting anti-apoptotic miR-21-5p into target cells.¹³²

In summary, MSC-derived exosomes, enriched with protective and anti-inflammatory miRNAs such as miR-204, miR-21-5p, and miR-483 show potential in alleviating PH by reducing vascular remodeling, inflammation, and fibrosis.

Ischemia/reperfusion

I/R injury is a serious complication that predisposes to cardiovascular disorders and represents one of the leading causes of death in conditions including heart attack, stroke, and organ failure. The blockage of blood flow to a tissue precipitates a cascade of oxygen and nutrient deprivation, thus inducing cell injury. While restoration of blood flow is essential, it may itself provoke further injury that may progress to irreversible tissue injury and organ dysfunction.^133,134 Myocardial I/R injury may induce cardiomyocyte apoptosis and necrosis, thus influencing the therapeutic outcomes of cardiac disease.¹³⁵ Reperfusion paradoxically exacerbates ischemic damage via bursts of reactive oxygen species, calcium overload, mitochondrial permeability transitions, and innate immune activation, propagating local and remote organ injury.^136,137

Recently, many studies have established the protective functions of MSC-derived EVs in myocardial I/R injury. For example, exosomal miR-143-3p derived from MSCs repressed myocardial cell death by targeting the CHK2-Beclin 1 pathway.¹³⁸ Besides, ADSC-derived exosomal miR-22-3p has been suggested to lower infarct size and inhibit apoptosis to facilitate neuronal survival after ischemic brain injury.¹³⁹ Moreover, MSC-derived exosomal miR-182-5p inhibits I/R-induced inflammation, apoptosis, and injury of cardiomyocytes by targeting gasdermin D (GSDMD) inhibition.¹⁴⁰ In addition, EVs secreted from ADSCs overexpressing miR-221/222 target p53 upregulated modulator of apoptosis (PUMA) and ETS-1, which prevents cardiac I/R injury-mediated apoptosis and hypertrophy. Thus, this may provide a future therapeutic approach.¹⁴¹ Zhang et al. also demonstrated that ADSC-derived exosomal miR-22-3p exerts protection against ischemic injury by reducing infarct size while inhibiting neuronal apoptosis and promoting neuronal viability. All these data indicate that MSC-derived exosomes could be a hopeful therapeutic method in ischemic brain injury treatment.¹³⁹

In summary, MSC-derived EVs, carrying miRNAs such as miR-143-3p, miR-22-3p, and miR-182-5p, show therapeutic promise in mitigating I/R injury by reducing inflammation, apoptosis, and tissue damage while promoting cell survival and repair.

Aside from cardiovascular health, MSC-EVs also show significant promise in the central nervous system. With their capability to cross the blood-brain barrier (BBB) and transport bioactive miRNAs, they are touted as potential therapeutic agents for a range of neurological conditions.

Neurological diseases

The MSC-derived EVs are among the most widely studied EVs in animal models. According to studies, their cargo is what constitutes the possible therapeutic effect of these cells, mainly through cytokines and miRNAs.²¹ A major advantage of MSC-EVs in neurological disorders is their ability to cross the BBB. The exact mechanism of crossing the BBB is unknown, but it is hypothesized that EVs may utilize similar strategies as viruses crossing the BBB¹⁴² (Figure 2).

Figure 2.

Potential applications of MSC-derived EV miRNAs in neurological disease therapy

In this diagram, the neuroprotective and regenerative actions of miRNAs derived from MSC-EVs were illustrated in neurological disorders. miRNAs contained within EVs pass through the BBB and play a role in regulating neurogenesis, angiogenesis, inflammation suppression, and neural repair. BBB, blood-brain barrier, MSC-EV, mesenchymal-stem-cell-derived extracellular vesicle.

Stroke

Arterial occlusion precipitates energy failure, excitotoxicity, ionic imbalance, and oxidative stress, followed by BBB disruption and sterile inflammation involving microglia, astrocytes, and infiltrating leukocytes that expand the infarct.¹⁴³

Preclinical studies have already documented the capability of MSC-EVs in augmenting the higher order of neurological-function-implicated events such as neurogenesis, angiogenesis, and neural plasticity in animal stroke models.^144,145,146 Further, in animal models of post-stroke cognitive impairment, MSC-EVs induce favorable M2 polarization of microglia/macrophages.¹⁴⁷ In a cortical injury model, the administration of MSC-EVs improved motor function and microglia-mediated neuroinflammation.¹⁴⁸ The study of Pan et al. is the first to show that EV-mediated transfer of miR-132 exerts its inhibition against brain injury through inhibition of blood vessel oxidative stress or suppression of BBB dysfunction. Thus, MSC-EVs-miR-132-3p has also been reported to decrease reactive oxygen species (ROS) production, apoptosis of neurons, and disruption of the integrity of tight junctions in vitro. Its target site identification includes RAS P21 protein activator 1 (RASA1) signaling as its direct target. Here, miR-132-3p/RASA1 axis governs the process of angiogenesis. MiR-132-3p has been shown to induce vascularization in culture as well as enhance the tight junction in cultured ECs by downregulating RASA1 and hence activating the Ras/PI3K/Akt signaling pathway. This pathway has been reported to govern EC proliferation and apoptosis.¹⁴⁹ Recently, Moon et al. have documented the role of MSC-EVs on angiogenesis by their cargo of miRNAs. Moon et al. showed that miR-210 specifically derived from MSC-EVs plays an important role in the stimulation of angiogenesis in a rat stroke model. The angiogenic function of miR-210 was investigated by tubular formation assay using HUVECs. The efficacy of the miR-210 group on HUVEC tube formation was the highest among them. Furthermore, miR-210 has been shown to downregulate the expression of Ephrin A3 (EFNA3) in HUVECs. They also discovered that miR-184 and miR-210 represented the top two most highly upregulated miRNAs during neurogenesis and angiogenesis following stroke in a rat model. To further validate the role of these two miRNAs, their pre-miRNAs were transfected into human neural progenitor (ReN) cells and HUVECs. Proliferation assays demonstrated that miR-184-transfected ReN cells enhanced proliferation. Western blotting also showed that expression of Numb-like was downregulated in miR-184-transfected ReN cells.¹⁵⁰

The miR-126 from the EVs derived from the MSCs of the adipose tissues has been reported to contribute significantly to neurogenesis. Geng et al. reported reduced plasma and brain tissue levels of miR-126 in both rat models with acute ischemic stroke and affected individuals. To investigate the function of exosomal miR-126, miR-126 expression in the EVs was overexpressed and knocked down by miR-126⁺ and miR-126⁻ EVs, respectively. It was observed that the delivery of ADSC-derived miR-126⁺ EVs promoted cell proliferation, enhanced the expression of von Willebrand factor and doublecortin (a neuroblast marker), improved functional recovery, and decreased neuronal cell death compared to miR-126⁻ and control EVs.⁶⁵

Yang et al. demonstrated that rabies virus glycoprotein (RVG) fused with Lamp2b-engineered EVs efficiently delivers miR-124 to the infarct site, which promotes neurogenesis through enhanced differentiation of cortical neural progenitors into neural lineage cells after ischemic injury.¹⁵¹ Further, miR-138-5p from BM-MSC-EVs was shown to promote proliferation and inhibit apoptosis and expression of inflammatory factors in astrocytes in an animal model of ischemic stroke.¹⁵² Research has identified that miR-126-3p secreted by ADSC-EVs reduces neuronal cell death and promotes cell proliferation in a rat stroke model. Besides, exosomal miR-126 has been proven to suppress microglial activation and inflammatory factor expression both in vitro and in vivo.⁶⁵ Jiang et al. investigated the role of ADSC-derived EVs rich in miR-30d-5p in acute ischemic stroke (AIS). They pointed out that exosomal miR-30d-5p alleviates cerebral injury by inducing M2 macrophage polarization and repressing autophagy.¹⁵³ For example, one study by Xin et al. demonstrated that MSC-EV-mediated delivery of miR-133b promotes neural plasticity and functional recovery in animal models of stroke. They found that MSC-EVs promote the transfer of miR-133b to astrocytes and neurons, modulating gene expression to promote neurite remodeling and functional recovery after stroke. miR-133b may target connective tissue growth factor (CTGF), thus downregulating its expression to facilitate axonal growth from the site of injury.¹⁴⁴ miR-17-92, originally derived from MSC-EVs, promotes neuroplasticity and functional recovery in animal models of stroke. The mechanism of this recovery was further associated with enhanced oligodendrogenesis, neurogenesis, and neurite remodeling by miR-17-92. The target of this miRNA is the PTEN gene. Downstream processes to PTEN include neurite remodeling, cell proliferation, and differentiation through the PI3K/Akt/mTOR signaling pathway. miR-17-92 targets PTEN to reduce its expression. This, in turn, increases the phosphorylation of Akt and mammalian target of rapamycin (mTOR) due to PTEN inhibition. Activated mTOR phosphorylates glycogen synthase kinase 3β (GSK-3β). GSK-3β is known to inhibit axonal outgrowth. Thus, its inactivation due to phosphorylation mediated by miR-17-92 promotes axonal growth in neurons.¹⁵⁴

In summary, MSC-derived EVs enriched with miRNAs such as miR-132, miR-210, and miR-126 show significant potential in stroke therapy by enhancing neurogenesis, angiogenesis, and neural plasticity while reducing inflammation and neuronal apoptosis, thereby improving functional recovery.

Spinal cord injury

Spinal cord injury (SCI) comprises a primary contusion/laceration followed by secondary injury—ischemia, excitotoxicity, inflammation, demyelination, and glial-scar formation—with microglia/macrophages, reactive astrocytes, and oligodendrocyte loss driving persistent neurological deficits.^155,156

Li et al. demonstrated that EVs from miR-544-overexpressing BM-MSCs exert a therapeutic effect on the treatment of SCI in rats. This type of EV promotes the survival of nerve cells, lessens inflammation, and has an effect on the functional recovery of the hind limbs. This would, therefore, point toward a probable therapeutic application of the EVs obtained from overexpression of miR-544 on BM-MSCs as one possible candidate to treat SCI in cases involving attenuating neurological deficits, inhibition of inflammation, and the facilitation of repair mechanisms.¹⁵⁷ In another work by Li et al., it was recorded that MSC-derived miR-133b-enriched EVs facilitate functional recovery after SCI by participating in axonal growth, apart from decreasing lesion sizes and protection of neurons, downregulation of the neuron-degenerating molecule RhoA, and induction of pivotal pathways involved in neural outgrowth: ERK1/2, signal transducers and activators of transcription 3 (STAT3), and cyclic AMP-responsive element-binding protein (CREB) signaling. These findings define miR-133b-based EVs as new candidates against SCI.⁶⁶ Previously, Zou et al. demonstrated that one of the highly upregulated miRNAs in MSC-derived EVs participated in miR-21-5p treatment for SCI. Suppression of miR-21-5p in MSC-EVs may further deteriorate functional recovery and trigger cell death through a mechanism related to SCI rats. The favorable function of miR-21-5p mainly exerts its role by targeting the proapoptotic gene FasL to downregulate its expression. This indicated that the miR-21-5p-loaded MSC-EVs can be a promising therapeutic approach for SCI.¹⁵⁸ Liu et al. showed that hypoxic preconditioned MSC-derived EVs loaded with miR-216a-5p significantly enhanced functional recovery after SCI by shifting the microglial polarization pattern from M1 to the M2 phenotype in the SCI animal model through modulation of Toll-like receptor (TLR) 4 signaling and inhibition of inflammatory-associated signaling pathways, including TLR4/NF-kB/PI3K/AKT.¹⁵⁹ Wang et al. showed that UC-MSC-derived EVs induce functional recovery in the spinal cords through the increased levels of miR-199a-3p and miR-145-5p, which target Casitas B-lineage lymphoma (Cbl) and Cblb to stabilize tropomyosin receptor kinase A (TrkA), thereby activating a downstream cascade critical to neuronal survival and regrowth processes. Thus, these EVs are of great promise as therapeutic entities in SCI by bringing about neuronal differentiation and having a neuroprotective role in it.¹⁶⁰ Yu et al. investigated the role of EVs containing miR-29b expressed from BM-MSCs following SCI. They showed that treatment of these EVs in SCI rats significantly improved locomotor function and reduced neurological loss. This study showed that the exosomal miR-29b elevated the expression of proteins associated with neuronal regeneration, including neurofilament (NF) 200, growth-associated protein 43 (GAP-43), and glial fibrillary acidic protein (GFAP). These findings reflect using miR-29 b-loaded EVs in promoting nerve cell repair and functional recovery following SCI.¹⁶¹

In conclusion, MSC-derived EVs enriched with specific miRNAs show promising potential for treating SCI by promoting nerve cell survival, reducing inflammation, and aiding functional recovery. These findings suggest that exosomal therapies could become a novel approach for enhancing repair mechanisms in SCI.

Cognitive impairment

Alzheimer disease (AD) is a major neurological disorder characterized by cognitive decline. A key feature of AD is the accumulation of beta-amyloid peptide (Aβ) in the brain due to an imbalance between its production and clearance. Neprilysin (NEP), an enzyme that breaks down Aβ, is highly expressed in MSCs.¹⁶² AD progression is fueled by chronic glial activation (microglia/astrocytes) in response to amyloid-β and tau pathology, generating ROS, nitric oxide, and pro-inflammatory cytokines that impair clearance mechanisms and exacerbate synaptic/neuronal loss.¹⁶³

According to our discussion above, miRNAs, as one component of MSC-EVs, are capable of playing an enormous role in the improvement process of neurocognitive diseases like AD. Jahangard et al. found that EVs from rat BM-MSCs, carrying miR-29, were able to exert a protective effect against Aβ-induced cognitive impairment in an AD rat model. This neuroprotective effect was most likely due to the downregulation of neuron navigator 3 (NAV3) and Bcl-2-interacting mediator of cell death (BIM), two target genes of miR-29b.¹⁶⁴ Wei et al. demonstrated that MSC-derived EVs harboring miR-223 protected neuronal cells against programmed cell death by targeting the PTEN-PI3K/Akt signaling pathway. Their findings pointed toward a potentially active therapeutic approach in the treatment of AD.⁷⁸ Cui et al. demonstrated that EVs from hypoxia-preconditioned BM-MSCs carrying miR-21 reduced synaptic dysfunction and modulated inflammatory responses, hence improving learning and memory capabilities in amyloid precursor protein/presenilin 1 (APP/PS1) mice. In particular, these infused EVs enhanced spatial learning and memory by reducing Aβ accumulation, microglial transformation into dendritic cells, enhancing the expression of synaptic proteins synapsin 1 and postsynaptic density 95 (PSD-95), upregulating anti-inflammatory factors, inhibiting astrocyte and microglial activation, downregulating pro-inflammatory factors, reducing phosphorylation of STAT3, and suppressing NF-κB activation. Cui et al. researched and stated that one of the potential mechanisms for the anti-inflammatory role of EV treatment is miR-21. They further established the efficacy of miR-21 in restoring memory impairments among APP/PS1 mice.¹⁶⁵ Besides AD, Kubota et al. showed that MSC-EVs overexpressing miR-146a attenuated astroglial inflammation in a rat model induced with streptozotocin, consequently restoring cognitive functionality. The authors explored the anti-inflammatory role of miR-146a in astrocytes by overexpressing miR-146a in astrocytes. The overexpression resulted in reduced tumor necrosis factor alpha (TNF-α), IL-1-receptor-associated kinase 1, and NF-κB in the diabetes model.¹⁶⁶ In one study, Nakano et al. showed that BM-MSC-derived EVs containing miR-146a were incorporated into astrocytes, which resulted in a reduction of NF-κB levels and an anti-inflammatory response. Given the role of astrocytes in synaptogenesis, restoration of astrocyte function by miR-146a may further contribute to the promotion of synaptogenesis and thus lead to improved cognitive outcomes.⁶⁷

In conclusion, MSC-derived EVs, particularly those enriched with specific miRNAs like miR-29, miR-223, miR-21, and miR-146a, demonstrate promising therapeutic potential for cognitive impairment in diseases such as AD. These EVs help reduce Aβ accumulation, modulate inflammatory responses, and promote neuroprotection, offering a potential strategy for improving cognitive function and slowing disease progression.

Subarachnoid and intracerebral hemorrhage

After aneurysmal subarachnoid and intracerebral hemorrhage (SAH), early brain injury includes abrupt intracranial pressure surges, BBB disruption, oxidative stress, and neuroinflammation, followed by delayed cerebral vasospasm and microvascular dysfunction—together causing ischemia and poor outcomes.¹⁶⁷

The effects of miRNAs secreted by MSC-EVs during SAH are less well understood using animal models. Zhang et al. demonstrated that MSC-derived EVs loaded with miR-21 may target transient receptor potential cation channel subfamily M member 7 (TRPM7) and protect neurons from injury after intracerebral hemorrhage (ICH). MiR-21 inhibits the NF-κB pathway, reducing inflammation and promoting neuronal survival. Histological analysis showed a smaller hematoma size in the MSC-miR-21 group. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining revealed a reduction of cell apoptosis in the brain treated with miR-21-MSCs.¹⁶⁸ Lai et al. demonstrated that intravenous administration of EVs carrying miR-193b-3p could reduce neuroinflammation in the context of early brain injury after SAH in mice. This miRNA suppresses histone deacetylase 3 (HDAC3) expression and leads to increased acetylation of NF-κB p65. Administration of miR-193b-3p has been shown to reduce brain edema, neurodegeneration, neurological deficit, BBB damage, and inflammatory cytokine expression after SAH in mice. More specifically, EVs with miR-193b-3p decreased IL-1β, IL-6, TNF-α, and caspase-3 expressions while increasing Bcl-2 expression.¹⁶⁹ Gao et al. demonstrated that MSC-EV-derived miR-21-5p exerts a protective effect on neurons from apoptosis and alleviates SAH-induced cognitive impairment in an animal model. They further suggested that the PTEN/Akt signaling pathway might be a target of miR-21-5p in neurons to help mitigate early brain injury after SAH.¹⁷⁰

In conclusion, MSC-derived EVs with miRNAs like miR-21 and miR-193b-3p show promise in protecting neurons, reducing inflammation, and alleviating cognitive impairments in SAH. These EVs offer the potential for therapeutic intervention in these conditions.

Traumatic brain injury

Traumatic brain injury (TBI) is one of the most common causes of death and long-term disability around the world.¹⁷¹ TBI initiates primary mechanical damage and widespread diffuse axonal injury, then secondary cascades of neuroinflammation, oxidative stress, and BBB compromise; microglia/astrocytes and peripheral immune cells orchestrate chronic degeneration and circuit dysfunction.^172,173

Besides others, miRNAs represent one of the key factors underlying the therapeutic efficacy of MSC-derived EVs. Among active players, miR-216a-5p has been identified to play an active role in the therapeutic effect mediated by MSC-EVs, able to prevent neuronal cell death and promote neurogenesis. They identified neurogenin-2 as a target of miR-216a-5p and postulated that this miRNA might prevent cell death and promote neurogenesis through neurogenin-2 inhibition.⁶⁸

In conclusion, miR-216a-5p in MSC-derived EVs shows potential in treating TBI by preventing neuronal cell death and promoting neurogenesis through the inhibition of neurogenin-2. This highlights its therapeutic promise for TBI.

Besides their neuroprotective and regenerative properties, MSC-EVs have been increasingly studied in the field of oncology. Their miRNA cargo can modulate tumor growth, metastasis, and the tumor microenvironment, providing new insights into cancer treatment.

Cancer

Role of miRNAs in tumor suppression and anti-metastasis

Regarding the expression of miRNA in EVs (EV-miRNA) in MSCs, it should be noted that the expression level of EV-miRNA in healthy individuals is different from that of patients with malignant tumors, and the tumor can be manipulated by targeting EV-miRNA. Also, the expression level of miRNA in different cell types is generally upregulated and downregulated,^{69,174,175,176,177,178,179,180,181,182,183,184} which is related to factors such as tumor prognosis,^{176,178,181,185} tumor grading,¹⁷⁶ TNM (tumor, node, metastasis) staging,^174,175 and degree of differentiation.¹⁷⁵ Recent studies generally consider three strategies for regulating miRNA expression in tumors, including inactivation of oncogenic miRNAs, activation of tumor suppressor miRNAs, and restoration of drug sensitivity.¹⁸⁶ Studies conducted in early 2013 indicated that BMSC-derived microvesicle (MV) has an inhibitory effect on cell-cycle progression, thereby inducing apoptosis and necrosis in hepatocellular carcinoma, Kaposi sarcoma, and ovarian tumors.¹⁸⁷ These studies also suggest that miRNAs in ADSC-EVs are responsible for antitumor activity. In other words, ADSC-derived EVs have a similar effect on ovarian cancer cells that is mediated by RNAs rather than proteins in the EVs.¹⁸⁸

miRNAs in MSC-EVs play a role in regulating key pathways of cancer and tumors, such as progression, proliferation, invasion, angiogenesis, dominance of the immune response, and manipulation of tumor chemoresistance, and miRNAs in MSC-EVs play a role in the development of precancerous lesions.^189,190 Studies conducted by Xu et al. indicated that BMSC-EVs in acute myeloid leukemia (AML) inhibit cell proliferation and cell-cycle progression and increase apoptosis.¹⁹¹

Key antitumor miRNAs in MSC-EV and their targets in cancer pathways

As previously mentioned, tumor proliferation is one of the key pathways regulated in cancer, and aberrant miRNAs can suppress this process. Some of the most important miRNAs involved are listed below.

A study by Xu et al. found that BMSC-EVs hindered growth and induced death in AML cells. Removing miR-124-5p from BMSCs eliminated this effect, suggesting miR-124-5p’s role in EVs’ impact on AML.¹⁹¹ According to the research conducted by Yan et al., exosomal miR-512-5p from BMSC slows glioblastoma growth.⁶⁹ miR144 in BMSC-EVs reduces cyclin E1 (CCNE1) and CCNE2 levels, slowing non-small cell lung cancer (NSCLC) growth.¹⁹² MSC-EVs contained miR-100, affecting the miR-100/mTOR/miR-143 axis and impacting colorectal cancer cell (CRC) growth, and miR-143 was found in higher amounts in MSC-EVs.¹⁹³ Lin et al. found that human bone marrow stromal cell (hBMSC)-derived exosomal miR-484 blocked the Wnt/MAPK pathway, reduced pancreatic cancer cell growth, increased cell death in vitro, and lowered tumor size and weight in vivo.¹⁹⁴ Exosomal miR-124-3p from hBMSCs lowered the levels of cellular myelocytomatosis oncogene (cMYC) by reducing nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), and had similar effects on diffuse large B cell lymphoma.¹⁹⁵ In another study, it has been observed that hMSC-EVs with miR-23 b-5p reverse tripartite motif-containing 14 (TRIM14) promote AML cell growth via PI3K/AKT pathway.¹⁹⁶ According to research conducted by Xu et al., MSC-EVs with miR-133b slow glioma cell growth by blocking the Wnt-catenin pathway through enhancer of EZH2 inhibition,¹⁹⁷ and also Khazaei-Poul et al.’s studies indicated that miR-3182 from human umbilical cord MSCs (hUCMSCs) affects triple-negative breast cancer (TNBC) cells.¹⁷⁷ Li et al. discovered that CD90^low ADSCs and EVs help slow down tumor growth in humoral mice. They do this by reducing the growth of tumor cells and increasing the death of those cells. This effect became stronger when they added miR-16-5p mimic to the CD90^low ADSC EVs.¹⁹⁸ Another study has demonstrated that let-7f in hMSC-EVs is upregulated by tumor cells through the hypoxia-inducible factor 1α (HIF-1α) pathway under hypoxic conditions and leads to decreased tumor cell proliferation and invasion, and MSCs treated with inflammatory factors and chemokine stromal-cell-derived factor 1 alpha (SDF-1α) have antitumor effects.¹⁹⁹

Key anti-tumor miRNAs and inhibition of invasion and metastasis

According to research conducted by Wang et al., it has been stated that let-7i derived from BMSC-EVs decreased the expression of FXYD domain-containing ion transport regulator 3 (FXYD3), a sodium-potassium ATPase regulator, and decreased cell migration and invasion in lung cancer cells.²⁰⁰ Also, Zhang et al. showed that targeting the transformer-2 protein homolog beta (TRA2B) with miR-206 delivery by BMSC-EVs into osteosarcoma cells reduces cell migration and invasion.²⁰¹ According to He et al.’s study, hUCMSC-EVs with miR-375 suppresses esophageal squamous cell carcinoma invasion and migration by downregulating the expression of ENAH and the expression levels of EMT-related proteins (E-cadherin, N-cadherin, and Snail).²⁰² miR-205 via EVs secreted by hBMSCs inhibits invasion and migration by inhibition of Rhophilin Rho GTPase binding protein 2 (RHPN2) in prostate cancer cells.²⁰³ Xu et al. showed that when BMSC-EVs overexpress miR-16-5p, CRC progression is reduced by integrin alpha-2 (ITGA2) downregulation.²⁰⁴ miRNAs in MSC-EVs help change the extracellular matrix, which helps tumor cells invade; for instance, as stated by Xu et al. and Yang et al., miR-331-3p from BMSC-EVs lowers DNA methyltransferase 3a (DNMT3A) in cervical cancer cells, impacting LIM zinc finger domain containing 2 (LIMS2) expression and how it interacts with proteins.^205,206 Based on Yang et al.’s study, miR-16-5p inhibits EMT, a process that is activated during tumor pathogenesis and causes the phenotype of epithelial cells to change to mesenchymal cells and reduces NF-κB signaling pathway, reduces EPHA1 expression, and prevents cancer progression.^207,208 MiR-34a-5p in MSC-EVs suppresses CRC cell growth and the EMT process by affecting c-MYC binding to DNA methyltransferase 3a (DNMT3A) and epigenetic regulation of PTEN.¹⁸³ Relative to Jahangiri et al.’s article, miR-100 and miR-143 suppress CRC cell invasion and migration and the EMT process.¹⁹³ miR-381 in ADSC-EVs reduces TNBC cell survival, movement, and invasion.²⁰⁹ MiR-34c in MSC-EVs suppresses nasopharyngeal carcinoma tumors with β-catenin inhibition.²¹⁰

Key anti-tumor miRNAs and inhibition of invasion and modulation of distant metastasis

MiRNAs in MSC-EVs can affect how tumors spread. To give an instance from Zhang et al.’s article, EVs that are high in miR-101 from ADSCs lowered the spread of osteosarcoma cells.²¹¹ Chen M. et al. stated that miR-655-3p in hUCMSC-EVs suppresses esophageal squamous cell carcinoma (ESCC) liver metastasis by inactivating HIF-1α via the LMO4/HDAC2 axis.²¹² On the other hand, according to another study, miR-139-5p derived from BMSC-EVs inhibits the tumorigenesis and metastasis of bladder cancer cells by downregulation of kinesin family member 3A (KIF3A) and activation of p21.²¹³ miR-1827 carried by hUCMSC-EVs impairs CRC liver metastasis.²¹⁴

Key miRNAs in the negative modulation of the immune system

Since M2 macrophage polarization of tumor-associated macrophages (TAMs) is considered an important component of the tumor microenvironment (TME), it is considered an immunosuppressive site for tumor progression.²¹⁵ MiR-744-5p in MSC-EVs inhibits glioma progression by reducing M2 macrophage polarization through decreasing MAPK signaling activity and also downregulating TGFB1.²¹⁶

Key miRNAs in the angiogenesis process

miR-29a-3p in MSC-Exos and establishment of a VEGF-induced microvascular cycle reduce angiogenesis mimicry in glioma cells by overexpression.²¹⁷ Additionally, miR-100-5p and miR-1246 inhibit endothelial cell proliferation and migration and induce apoptosis in oral squamous cell carcinoma.²¹⁸

In conclusion, MSC-derived exosomes carrying specific miRNAs play crucial roles in tumor suppression, anti-metastasis, and immunomodulation. These miRNAs can inhibit tumor growth, cell invasion, and metastasis by targeting key pathways involved in cancer progression.

Aside from cancer biology, MSC-EVs have key functions in immune regulation. Their ability to regulate inflammatory pathways and immune cell function renders them useful in the management of autoimmune and inflammatory conditions.

Autoimmune diseases

Autoimmune diseases are chronic disorders in which loss of immune tolerance leads to immune-mediated damage against self-tissues. These conditions include rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), multiple sclerosis (MS), type 1 diabetes (T1D), and others. Recent studies suggest that MSC-EV-derived miRNAs play crucial roles in regulating immune pathways underlying both innate and adaptive immunity in these diseases.²¹⁹

Rheumatoid arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by persistent inflammation of the synovial membrane, leading to the deterioration of articular cartilage and adjacent skeletal structures.²²⁰ RA is characterized by synovial hyperplasia, persistent inflammation, and joint destruction driven by interactions among fibroblast-like synoviocytes (FLS), macrophage-like synoviocytes (MLS), endothelial cells, and infiltrating immune cells. These cell types promote synovial hyperplasia, pannus formation, angiogenesis, and production of proinflammatory cytokines (e.g., TNF-α, IL-1, and IL-6), matrix-degrading enzymes (e.g., matrix metalloproteinases [MMPs]), and osteoclast activation—culminating in cartilage erosion and bone damage.^221,222

miR-146a and miR-155 are the first miRNAs to be abnormally expressed in RA and were previously reported as important regulators of inflammation. Recent studies have shown that MSC-EVs containing miR-155 modulate the phenotype and function of immune cells, such as monocytes and dendritic cells, and influence the inflammatory microenvironment in RA.²²³ According to Tavasolian et al.’s studies on transduced MSC-EVs on the immune response of RA miR-146a/miR-155 in collagen-induced arthritis (CIA) mice, in addition to significant changes in the level of Treg cells that led to an anti-inflammatory response and suppression of RA, it increased the expression of IL-6, IL-17, and retinoic-acid-receptor-related orphan receptor-gamma-t (RORγt) genes that are associated with a pro-inflammatory response. They also reported that treatment with MSC-EVs containing miR146a increased the expression of forkhead box P3 (FOXP3), TGF-β, and IL-10 genes in CIA mice.²²⁴ The apparent contradiction between the induction of miR-146a expression at high levels in RA fibroblast-like synoviocytes (RAFLSs) by lipopolysaccharide and IL-1β and the negative regulatory effect of miR-146a on inflammation in immune cells is due to the lack of comprehensive data on the role of miR146a in fibroblast-like synoviocytes (FLS); thus its negative regulatory role may not be due to fibroblasts.^225,226 MSC-derived EVs carrying miR-223-3p modulated the inflammatory microenvironment by targeting genes involved in the activation and recruitment of immune cells, such as chemokine ligand 2 (CCL2) and myocyte enhancer factor 2C (MEF2C).²²⁷ MSC-EV-transported miR-132 regulate immune responses and inflammation in RA by targeting genes producing pro-inflammatory cytokines IL-6 and IL-17 and suppressing T cell activation and differentiation.²²⁸ In vitro, EVs derived from BM-MSCs increased miR-34a expression by targeting cyclin I and activating the ATM/ATR/p53 signaling pathway, reducing RA inflammation and inhibiting RAFLS proliferation and resistance to cell death.²²⁹ As a result, decreased miR-132 expression promoted RA progression. Studies have also shown that BMSCs-EVs and miR-378a-5p play a role in modulating the interferon regulatory factor 1 (IRF1)/STAT1 axis. IRF1 and STAT1 are transcription factors that play a role in regulating immune responses and inflammatory processes. Abnormal activation of these two transcription factors contributes to the progression of RA. BMSC-EVs carrying miR-378a-5p prevent the progression of RA by reducing the STAT1 and IRF1 expression of IRF1 in human synovial microvascular endothelial cells (HSMECs), as well as increasing proliferation, migration, and angiogenesis in HSMECs and inactivating the IRF1/STAT1 axis, and can be considered as a potential treatment for RA.²³⁰ The transfer of miR-146a by MSC-EVs to recipient cells, such as synovial fibroblasts and macrophages, suppresses pro-inflammatory signaling pathways such as NF-κB by targeting genes involved in inflammation, such as TNF-receptor-associated factor 6 (TRAF6) and IL-1-receptor-associated kinase- 1 (IRAK-1).²³¹ Transfer of miR-1246 by MSC-EVs reduces pro-inflammatory cytokine production and inhibits immune cell activation in RA by targeting genes involved in immune cell activation and cytokine signaling, such as IL-6 and STAT3.²³²

In addition to targeting genes involved in the NF-κB signaling pathway, such as inhibitory kappa B kinase beta (IKKβ) and STAT3, which reduces the production of pro-inflammatory cytokines, miR-124a transfer by MSC-EVs promotes the survival and proliferation of FLS, reduces the expression of PI3K-AKT-NF-κB signaling, and inhibits the inflammatory factors TNF-α and IL-6.²³³ Transfer of miR-146b by MSC-EVs (known to regulate the immune response in RA) by targeting genes involved in TLR signaling, such as IRAK1 and TRAF6, results in the reduction of pro-inflammatory signaling pathways and modulation of immune cell activation.²³¹

Research by Ma and others showed that chondrogenic EVs from BMSCs can lower inflammatory substances in RAFLSs. These EVs contain miR-205-5p, which helps reduce inflammation by affecting murine double minute 2 (mdm2). This process reduced inflammation and joint damage in mice with an inflammatory condition.²³⁴ A study by Mi et al. found that EVs from HUC-MSCs with miRNA-451a targeted ATF2, reducing RA cell growth and spread and improving joint inflammation and imaging results in rats with CIA.⁷⁰

MSC exosomes with miR-1505p, called Exo150, can help reduce joint damage. Researchers explored how genetically changed MSCs with the miR-1505p gene affect protein MMP 14 (MMP14) and VEGF related to RA. They found higher MMP14 and VEGF levels in RA patients and lower miR1505p levels. Exo150 treatment reduced harmful protein levels and cell movement, indicating potential as a future treatment.²³⁵

In conclusion, MSC-EVs and their miRNA cargo represent a novel and promising approach for RA therapy. By modulating key inflammatory pathways and immune cell activity, MSC-EVs have the potential to provide targeted treatment for RA, offering a more effective and personalized therapeutic strategy.

Autoimmune hepatitis

Autoimmune hepatitis (AIH) is a form of liver inflammation that predominantly occurs in women. Its diagnosis is based on three key features: distinctive liver cell damage (interface hepatitis) seen under a microscope, elevated liver enzyme levels (aspartate and alanine aminotransferase levels, AST and ALT) in blood tests, and the presence of specific autoantibodies and high immunoglobulin G in the serum.²³⁶ miRNAs derived from MSC-EVs can potentially ameliorate AIH. Chen et al. demonstrated that BMSC-EVs, as well as EVs loaded with miR-223, enhanced liver function in a mouse model of AIH. They supported this using lower levels of liver enzymes, ALT and AST, lower inflammation (TNF-α, IL-17A, and IL-1β), better liver morphology (structure), and lower lymphocyte infiltration. More importantly, EVs with a higher content of miR-223 increased the therapeutic benefit. Both miR-223 and miR-223-3p are miRNAs with deep anti-inflammatory and liver-protective roles. Although both of them work to avoid liver damage, they are of different natures. miR-223 will generally protect the liver by inhibiting inflammation and cell death. It does this mainly through the inhibition of NLRP3 and caspase-1, the core proteins for these roles.⁷¹ miR-223-3p, on the other hand, has shown remarkable protective roles in AIH only. It decreases liver damage by reducing inflammatory cell infiltration and tipping the immune response toward a regulatory phenotype through enhanced Treg cells and reduced Th17 cells, reducing pro-inflammatory cytokines (IL-1β, IL-6, and IL-17), and enhancing the anti-inflammatory cytokine IL-10. These effects are being mediated at least in part through the downregulation of the signaling molecule STAT3. While both miRNAs have actions on inflammation, miR-223-3p has a greater impact on the immune system and cytokine regulation in AIH. Significantly, the effects of MSCs-exosomal miR-223-3p were equivalent to and even better than prednisolone and azathioprine and better than MSC-EVs. No apparent adverse events were observed for any of the groups.²³⁷ In summary, miR-223 and miR-223-3p are precious miRNAs with enormous potential for the protection of the liver. More studies are needed to delineate their exact functions and modes of action in different liver disorders.

Inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic condition involving systemic inflammation primarily targeting the digestive tract, and its main types are Crohn disease and ulcerative colitis.²³⁸ miRNAs from MSC-EVs may offer a therapeutic strategy for IBD. hUC-derived EVs can decrease macrophage counts in colon tissue and decrease the expression of inducible nitric oxide synthase in IBD mice.²³⁹ Studies by Zhao et al. have shown that these MSC-EVs are capable of modulating macrophage function from a pro-inflammatory (M1) to an anti-inflammatory phenotype (M2).⁵⁸ Further studies have shown that one of how these EVs bring about this change is through delivering miR-182, a microRNA that targets and downregulates TLR4, a molecule central to the inflammatory response. BMSC-EVs containing miR-146a significantly relieved colitis in the rat model. MSC-EV-miR-146a therapy markedly suppressed colon inflammation, as revealed by reduced colon shortening, decreased inflammatory cell infiltrations, and reduced ulcers. Mechanistically, the exosomal miR-146a suppressed the NF-κB signaling pathway as evidenced by suppression of TRAF6 and IRAK1, inhibition of phosphorylation of NF-κB p65 and IκBα, reduction in the levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β, and elevation in the level of anti-inflammatory cytokine IL-10. All these findings strongly suggest that the anti-inflammatory function of MSC-EV-miR-146a in colitis is via suppression of the NF-κB signaling pathway.²⁴⁰

Systemic sclerosis

Systemic sclerosis (SSc) is a chronic autoimmune disorder of unknown cause, primarily characterized by excessive scarring (fibrosis) that affects the skin and lungs and vascular dysfunction that leads to tissue ischemia and damage to organs like the kidneys and lungs.²⁴¹ miRNAs from MSC-EVs may attenuate the progression of systemic sclerosis. In SSc mice, BMSC transplantation restored osteopenia. BMSCs that overexpressed miR-151-5p suppressed the IL4α/mTOR pathway. MSC-EVs that carried miR-151-5p induced bone formation, decreased fat deposition, normalized disease parameters (autoantibodies and renal function), and suppressed Th2 cell activity. These results indicate that MSC-EVs that carry miR-151-5p could be an effective therapeutic agent for SSc by preventing disease progression and alleviating its symptoms.²⁴²

Asthma

Asthma is a chronic airway disease marked by immune cell infiltration and activation, including dendritic cells, eosinophils, and mast cells, which causes symptoms like coughing, wheezing, and shortness of breath.²⁴³ miRNAs from MSC-EVs could modulate the chronic inflammation and immune cell activation in asthma, offering a potential therapeutic strategy to alleviate airway symptoms. Zhuansun et al. demonstrated that BMSCs-EVs enhance the expression of miR-1470 in CD4⁺ T cells derived from asthma patients. The elevated expression levels of miR-1470 enhance the differentiation of CD4⁺ T cells into Tregs. This process is through the direct targeting of c-Jun by miR-1470, indirectly leading to the overexpression of P27KIP1, a cell-cycle inhibitor. Notably, the inhibition of P27KIP1 leads to the dampening of elevated levels of Treg cells, thereby validating its essentiality in this pathway. In essence, MSC-exosomal miR-1470 is responsible for immune control in asthma by increasing the yield of Treg cells, with the net effect being suppression of overactive inflammation.²⁴⁴

Graft-versus-host disease

Graft-versus-host disease (GVHD) is a condition where donor immune cells attack the host’s body, primarily targeting the skin, liver, and gastrointestinal tract.²⁴⁵ miRNAs from MSC-EVs may mitigate GVHD by modulating the donor immune response. Fujii et al. demonstrated that miR-125a-3p has a role in alleviating GVHD in mouse models. In the acute GVHD experiments, miR-125a-3p suppressed the proliferation and differentiation of effector T cells but preserved the cell number of naive T cells. As a result, it enhanced the survival duration of GVHD mice and reduced pathological injury in organs. Further studies are needed to establish the exact function of miR-125a-3p in GVHD.⁷²

Type 1 diabetes mellitus

Type 1 diabetes mellitus (T1DM) is a complex autoimmune disease where the immune system mistakenly attacks and destroys insulin-producing beta cells in the pancreas, leading to a permanent loss of insulin production and chronic high blood sugar (hyperglycemia).²⁴⁶ miRNAs from MSC-EVs could potentially halt the autoimmune destruction of pancreatic β cells in T1DM. A study by Chen et al. demonstrated that exosomal miR-21, derived from hUC-MSCs, shielded pancreatic β cells from death under low-oxygen conditions. This protection was achieved by blocking p38 MAPK signaling, reducing endoplasmic reticulum stress, and enhancing overall cell survival.²⁴⁷ To enhance therapeutic efficacy, novel strategies are engineering the cargo of MSC-EVs; for instance, enriching exosomes with miR-146a has shown superior results in treating diabetic peripheral neuropathy by improving neurovascular function and suppressing immune responses beyond the capabilities of natural exosomes.¹⁰¹

In conclusion, MSC-EV-miRNAs represent a promising therapeutic avenue for managing autoimmune diseases by modulating immune responses and reducing inflammation. The precise roles of miRNAs such as miR-223, miR-146a, and miR-151-5p in targeting key inflammatory pathways highlight their potential to alleviate disease symptoms and prevent progression. While preclinical studies have demonstrated their efficacy in conditions like AIH, IBD, SSc, asthma, colitis, and GVHD, further research is essential to fully elucidate their mechanisms and optimize their application in clinical settings.

The medicinal prospects of MSC-EVs go even further to organ-specific disease, including liver disease, where their miRNAs help in the decrease of fibrosis, inflammation, and tissue regeneration.

Liver diseases

MSCs improve the progression of various liver diseases, such as fibrosis and cirrhosis, in animal models.⁵ Furthermore, according to studies, similar results can be achieved by using MSC-conditioned medium (MSC-CM) instead of parent cells.^5,248,249 MSC-EVs improve carbon tetrachloride (CCl4)-induced liver fibrosis in a mouse model of liver fibrosis.¹⁰ According to research by Li et al., transplantation of hUCMSC into CCl4-induced fibrotic liver is associated with a reduction in superficial fibrous capsular and hepatic inflammation and collagen deposition, as well as a decrease in collagen types I and III, which ultimately leads to liver tissue softening, and TGF-β1 and recovery of AST activity. hUCMSC-EVs protect liver cells by inhibiting epithelial transition to EMT.²⁵⁰ According to studies conducted by Hyun et al., MSCs exert their therapeutic effects through EVs and their components, especially miRNAs. For example, suppression of miR-125b activation from EVs derived from CP-MSCs by transplantation of chorionic-plate-derived MSCs (CP-MSCs) into a mouse model of CCl4-induced liver fibrosis.²⁵¹

In addition, according to studies conducted by Lou et al. on hematoma cells, the reduction of CCl4-induced liver fibrosis is mediated by the reduction of the expression of genes such as cyclin G1, insulin-like growth factor receptor 1, and prolyl-4-hydroxylase α (inducing collagen synthesis in hepatic stellate cells) by overexpression of miR-122 in hepatic stellate cells carried by MSC-EVs.⁷³ Another study also showed that CCl4-induced liver fibrosis was reduced through activation of autophagy by overexpression of miRNA-181-5p.²⁵² According to studies by Chen et al., in a mouse model of experimental autoimmune hepatitis, they found that liver damage was reduced by transferring miR-223 from MSC-EVs, resulting in a decrease in NLRP3 and caspase-1 in vitro.⁷¹

In summary, MSC-EVs show great promise in treating liver diseases like fibrosis, cirrhosis, and autoimmune hepatitis. By delivering miRNAs such as miR-122, miR-181-5p, and miR-223, MSC-EVs reduce inflammation, fibrosis, and apoptosis while promoting liver regeneration. These findings highlight MSC-EVs as a potent, non-cellular alternative to stem cell therapy for liver repair.

Similar regenerative and anti-inflammatory processes have also been documented in kidney disease, where MSC-EVs ensure renal recovery and functional restoration through miRNA-mediated targeting.

Renal diseases

Reduction of ischemia-reperfusion-induced kidney injury by transfer of miRNAs to renal resident cells was first demonstrated by Cantaluppi et al. In a mouse model of glycerol-induced acute kidney injury, intravenous administration of MSC-EVs resulted in improvement in functional and morphological properties, but because MSCs with global downregulation of miRNAs via Drosha knockdown have lower miRNA content, intravenous administration of MSC-EVs with global downregulation of miRNAs is not effective.^253,254,255

Another study was conducted by Lindoso et al. to investigate the mechanism of MSC-EVs on miRNA upregulation in proximal renal tubular epithelial cells.²⁵⁶ According to studies by Wang et al., overexpression of miRNA-let7c by MSCs and their transfer via EVs to renal tubular epithelial cells in a mouse model of unilateral ureteral obstruction attenuates renal fibrosis in vitro by upregulating miRNA-let7c and targeting TGF-β receptor 1.²⁵⁷ Another study in a rat model of acute renal ischemia-reperfusion injury showed that treatment with MSC-EVs restored miR-30 levels and inhibited mitochondrial fission in tubular epithelial cells. Treatment with miR-30 antagomirs also abolished the protective effect of MSC-EVs.⁷⁴

In summary, MSC-EVs aid renal repair by delivering miRNAs to kidney cells. They improve kidney function, reduce fibrosis via miRNA-let7c, and protect tubular cells by restoring miR-30 levels, highlighting their therapeutic potential in kidney injuries.

Current challenges and future perspectives

The therapeutic use of MSC-derived EVs faces several challenges that need to be addressed for successful clinical translation. One major challenge lies in the standardization of EV isolation and characterization methods. Current techniques lack consistency, leading to variability in EV quality, quantity, and efficacy. Furthermore, the mechanisms underlying miRNA packaging and selective targeting remain poorly understood, necessitating further research to optimize therapeutic cargo.

Another limitation is the scalability of MSC-EV production, which is critical for meeting clinical demands. Advances in bioreactor technologies and scalable culture systems are essential for large-scale production. Additionally, while MSC-EVs show promise in preclinical studies, there is a pressing need for comprehensive clinical trials to validate their safety, efficacy, and long-term effects in humans.

Future perspectives include engineering MSC-EVs to enhance their specificity and potency. For instance, bioengineering approaches can be employed to enrich EVs with specific miRNAs or surface molecules to target diseased tissues more effectively. Moreover, the integration of MSC-EVs with advanced drug delivery systems could revolutionize their application in precision medicine.

As research progresses, the regulatory landscape must evolve to establish clear guidelines for MSC-EV therapies. This includes defining standards for manufacturing, quality control, and clinical testing. Collaborative efforts between academia, industry, and regulatory bodies are crucial to accelerate the development of MSC-EV-based therapeutics.

Despite promising preclinical results, translating MSC-derived EV miRNAs into clinical applications remains plagued by several critical issues. To date, the number of clinical trials is relatively small, and most studies are primarily aimed at establishing safety and feasibility rather than efficacy. Some critical safety concerns relating to off-target effects, unintended gene regulation, and pro-tumorigenic activity are also important areas of consideration for susceptible patients. Their biodistribution after administration remains poorly comprehended, and their tissue-targeted action with evasion of premature reticuloendothelial system clearance is an active challenge. Determining optimal dosing regimens is equally complex, as therapeutic effects may vary depending on disease type, severity, and patient-specific factors. Immune responses to EVs delivered exogenously, albeit overall weaker than to whole-cell therapy, are a pivotal concern in patients with autoimmune or inflammatory conditions. From the regulatory perspective, the lack of universalized EV protocols for isolation, characterization, and potency testing is still an obstacle to approval. Overcoming these issues will require coordinated efforts among scientists, clinicians, and regulators, along with the establishment of robust, multicenter clinical trials that can stand witness to both efficacy and safety across different populations of patients.

Conclusion

In conclusion, MSC-EVs and their miRNA content are revolutionizing regenerative medicine and disease modulation. MSC-EVs can selectively package and transport miRNAs, allowing them to modulate gene expression that regulates inflammation, immune, and tissue repair processes in a wide variety of diseases, including, but not limited to, cardiovascular, neurological, autoimmune, and oncological diseases. Thus, MSC-EVs have several advantages over whole-cell therapies, including decreased immunogenicity and tumorigenesis risk and preserved therapeutic effect. Growing evidence from preclinical studies indicates that they have the potential to modulate pathological processes like atherosclerosis, myocardial infarction, and immune dysregulation using miRNA-mediated mechanisms. These results highlight the promise of MSC-EV miRNAs as new biomarkers, drug delivery systems, and therapeutic candidates. Efforts to optimize EV isolation, improve miRNA loading specificity, and standardize clinical therapies through rigorous clinical trials need to be the focus of future research to translate these potential biological tools for use in an effective, safe, and standardized manner. This outline raises awareness of the distinct contribution of MSC-EV miRNAs in pathology and opens new avenues for developing novel translational applications that would ultimately transform treatment paradigms.

Besides, the therapeutic potential of MSC-derived EV miRNAs holds tremendous promise for clinical translation, potentially advancing from preclinical models to standardized, regulatory-approved therapies. Their built-in versatility also holds out the promise of entry into personalized medicine, with EV-miRNA profiles tailored to a patient’s individual genetic and disease-specific characteristics. Furthermore, the integration of MSC-EV-based therapies with existing therapeutic strategies, such as pharmacotherapeutics or cell therapies, may enhance therapeutic efficacy while minimizing adverse effects, paving the way for next-generation regenerative medicine strategies.

Acknowledgments

The authors gratefully acknowledge our respective institutions for their support and provision of research facilities.

Author contributions

Y.S. and F.B., conceptualization, investigation, and writing—original draft preparation. M.J., writing—original draft preparation, writing—reviewing and editing, and supervision. F.N., writing—reviewing and editing and supervision. S.S., conceptualization and investigation. All authors read and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the writing process

During the preparation of this work, the authors used ChatGPT to improve the readability and language of the manuscript/edit grammar and style. After using this tool, the authors carefully reviewed and edited the content as needed and took full responsibility for the final version of the manuscript.