Mesenchymal stem cell-derived microRNAs: Key immunomodulators to prevent ocular tissue degeneration

Abstract

MicroRNAs (miRNAs) are small non-coding RNAs of 20-22 nucleotides in length. They have been identified as major regulators in the secretome of mesenchymal stem cells (MSCs) including adipose tissue, bone marrow, Wharton’s jelly, and dental pulp. These MSCs and their secretome with specific miRNAs are known modulators of the immune response, angiogenesis, inflammation, and apoptosis. In this review, the application of MSC-derived miRNAs in treating several ocular conditions including dry eye, glaucoma, and retinal degenerative diseases has been compiled. In addition, the emerging role of MSC-derived extracellular vesicles carrying miRNAs as a major cargo, regulating the target cells in the human eye has been reviewed. Finally, the bioengineering of nanovesicles with specific MSC-derived miRNAs as novel drug therapy has been discussed.

Key Words: Mesenchymal stem cells; MicroRNA; Extracellular vesicles; Ocular diseases; Exosomes

Core Tip: Mesenchymal stem cells (MSCs) are emerging as potent therapeutic agents for ocular degeneration due to their regenerative and immunomodulatory properties. A key mechanism of action is through extracellular vesicles, which deliver bioactive cargoes, particularly microRNAs (miRNAs), to target cells. This review highlights the role of MSC-derived miRNAs in modulating immune responses and promoting repair in ocular diseases such as corneal injury, dry eye, diabetic retinopathy, age-related macular degeneration, retinitis pigmentosa, and glaucoma. It also explores the potential of engineering extracellular vesicles enriched with specific MSC-derived miRNAs as innovative, cell-free therapeutic strategies.

INTRODUCTION

Mesenchymal stem cells (MSCs) are a heterogenous population of adult, fibroblast-like multi-potent cells characterized by two principal properties: Self-renewal and differentiation. MSCs can differentiate into various lineages including mesodermal (adipocytes, osteocytes, and chondrocytes), ectodermal (neurocytes), and endodermal lineage (hepatocytes)[1,2]. MSCs can be obtained from various tissues and organs including bone marrow, adipose tissue, Wharton’s jelly, peripheral blood, umbilical cord, placenta, amniotic fluid, and dental pulp[1,3]. MSCs have demonstrated much promise in recent years for graft-vs-host disease (GVHD)[4,5], multiple sclerosis[6], spinal cord injury[7], cardiac conditions[8], and Crohn’s disease[9,10] due to their exceptional anti-inflammatory, immunoregulatory, and regenerative capabilities. The intravenous infusion of MSCs has also been proposed as a treatment for coronavirus disease 2019 to suppress inflammatory storms in the lungs[11]. Their capacity to secrete bioactive components is a major advantage in regenerative medicine[12]. Despite the initial model of MSCs homing into injury sites and differentiating into multiple cell types, paracrine signaling has emerged as a critical player. Recent studies draw attention to the diverse spectrum of bioactive elements released by MSCs into the medium, or formed as encapsulated MSC extracellular vesicles (MSC-EVs): Apoptotic bodies, microvesicles, and exosomes/small EVs (sEVs)[13,14]. MSC-exosomes have been shown to act as regulators of MSC paracrine protection through their role in delivering microRNAs (miRNAs or miRs), mRNAs, and proteins to the targeting cells[13]. MiRNAs are a significant component of the contents of exosomes and play an essential role in the biological efficacy of the exosomal contents[15]. MiRNAs were shown to be enriched in exosomes relative to their parent cells, and thus showed selective incorporation to meet a dynamic, rapid response to biological need[16,17].

MiRNAs are noncoding RNAs with the length of about 22 nucleotides, and they negatively regulate the expression of target genes. As a significant component of MSCs, miRNAs reportedly play an important role in tissue regeneration[18,19]. MiRNAs are key in regulating gene expression, inflammation, wound healing, and immune function[20], and are widely reported to mediate the therapeutic value of MSC-sEVs in various diseases[21].

As aforementioned, miRNAs are able to regulate the immune response[22]. MSC-derived miRNAs (MSC-miRNAs) can stimulate Toll-like receptor-mediated intracellular signaling cascades in immune cells that infiltrate tumors, leading to greater synthesis of inflammatory and antitumorigenic cytokines[23]. Most importantly, the regulatory role of miRNAs is highly context-dependent. Endogenous miRNAs regulate transcription within cells and play a role in the physiopathological functions of homologous cell types[24]. By contrast, exosomal miRNAs remain intact after being delivered into cells and possess the ability to serve as mediators of a wider array of recipient cells[25]. Differences of miRNAs between extracellular vesicles (EVs) and their parental MSCs have been shown by numerous studies. For example, one study observed that miR-15 and miR-21 expression was significantly increased in MSCs compared to their EVs[26]. Baglio et al[27] showed miR-136-3p, miR-34c-5p, miR-34a-5p and miR-15a-5p to be expressed more in MSCs compared to their EVs, and miR-4485 to be enriched in MSC-EVs. This review comprehensively summarizes the current knowledge on MSC-miRNAs, their underlying molecular mechanisms, and their potential applications in treating ocular degenerative diseases.

To date, a total of 1566 clinical trials have utilizing MSCs for various disease conditions, reflecting the broad therapeutic potential of MSC-based interventions. Of these, 40 clinical trials specifically focus on treating ocular disorders, underscoring growing interest in their application for eye diseases. In parallel, MSC-derived exosomes have emerged as a promising cell-free therapeutic modality, with 50 reported clinical trials investigating their use across diverse medical conditions. Notably, four of these trials are related to ocular indications: Two targeting dry eye disease (DED), one addressing macular hole, and another focusing on retinitis pigmentosa (RP) (https://clinicaltrials.gov). These clinical efforts highlight the expanding role of MSCs and their derivatives, such as exosomes and miRNAs, in the management of ocular degenerative diseases.

IMMUNOMODULATORY EFFECT OF MSCS

Human MSCs express minimal major histocompatibility complex class I but lack all co-stimulatory molecules including cluster of differentiation 80 (CD80), CD86, and CD40 according to Krampera et al[28]. Moreover, MSCs fail to induce proliferation of allogeneic or xenogeneic lymphocytes[29]. MSCs drive macrophage cell populations to shift into anti-inflammatory M2 phenotype cells, which enhances tissue repair together with regeneration[30]. MSCs have dual immune regulatory capabilities through their ability to stop inflammation while activating anti-inflammatory responses. The immunomodulation process consists of several steps; from monitoring the inflammatory response, to MSC migration into the area of lesion, to MSC activation when there is a need, and pathogen removal[31]. MSCs, as well as the supernatant from their culture, stimulate macrophage migration in vitro, and increase their in vivo mobilization to the healing tissue area, accelerating its renewal process[32]. MSCs suppress detrimental immune responses in the eyes and alleviate ongoing inflammation in ocular tissues by modulating the phenotype and function of all immune cells that play pathogenic roles in the development and progression of inflammatory and autoimmune eye diseases (monocytes/macrophages, dendritic cells, natural killer, natural killer T cells, and T and B lymphocytes)[33]. Additionally, MSCs possess potent anti-angiogenic properties, which can be beneficial in the treatment of neovascularization-associated inflammatory eye diseases, such as diabetic retinopathy (DR) and age-related macular degeneration (AMD)[34]. Thus, “immunomodulatory strategies” appear promising to control immune cell functions across disease stages. The research shows MSCs utilize paracrine signaling as their primary method for immunomodulation by releasing exosomes according to Liao and Hsu[35]. The exosomes derived from adipose tissue derived MSCs (ADMSC-Exos) direct CD4+ T lymphocytes toward regulatory cell differentiation through their encapsulation of miR-23a-3p that controls transforming growth factor beta (TGF-β) receptor 2 at the post-transcriptional level[36]. Bolandi et al[37] reported that ADMSC-Exo containing miR-10a directs the transition of uncommitted CD4+ T cells toward becoming both T helper type 2 (Th2) cells and regulatory T cells (Tregs). Forkhead box P3-positive expression together with TGF-β pathway regulation enable ADMSC-Exo miR-10a to control the differentiation of Tregs and Th17 cells. MSCs demonstrate strong paracrine properties, enabling them to produce several trophic factors that work towards controlling inflammation while reducing tissue fibrosis and cell death in the target environment. Thus, the combined action of immunomodulatory effects makes MSC-EV-miRNAs valuable therapeutic candidates for treating ocular degenerative conditions.

CORNEAL DISEASES

The human cornea functions as the transparent avascular tissue of the eye, which both refracts and transmits light toward the retina through the lens. The cornea protects eyes against harmful radiation and toxic dangers, and gives both optical precision and mechanical strength to the eyes. Epithelial cells in the cornea protect against infections through tight intercellular junctions, which also generate fresh basal layer cells to sustain optical surface smoothness. Disruption of this protective layer results in conditions that enable eye infections, which, together with stromal ulceration and perforation, lead to scarring and a decrease in visual acuity[38,39]. Medical treatment of deep corneal epithelial defects represents an ongoing therapeutic challenge. Conventional surgical approaches become necessary when existing medical treatments fail to heal defects or ulcers which persist for longer than 3 weeks. Such severe cases exhaust the limbal stem cell population and diminish its repair capabilities, leading to limbal stem cell deficiency according to McCulley et al[40] and Inatomi et al[41]. MSCs represent an accessible non-immunogenic stem cell source for treating persistent epithelial defects of corneal surface disorders with therapeutic potential in corneal epithelial regeneration[42]. Corneal scarring/fibrosis represents an overactivated healing process of the corneal stroma. Corneal blindness occurs due to development of persistent scarring from chemical exposure or mechanical trauma or infectious keratitis, which ends in the loss of corneal transparency[43]. Visual impairment resulting from corneal scarring constitutes a main reason for corneal transplant surgeries[44]. Multiple new research approaches for modelling corneal fibrosis/scarring are under development[45].

Research suggests that MSCs can reduce inflammation and promote the restoration of corneal transparency following ocular injuries[46-48]. Interestingly, while MSCs promote angiogenesis in other tissues, they exhibit anti-angiogenic effects in the cornea, making them particularly suited for corneal therapy[49]. The majority of MSCs are found in the corneal stroma, indicating that their therapeutic effects are likely mediated via a paracrine mechanism. This involves the release of soluble factors, including EVs and exosomes, which carry bioactive molecules such as miRNAs[50-52]. Multiple studies have highlighted the potential of MSC-sEVs in promoting corneal epithelial healing. Liu et al[53] demonstrated that human umbilical cord MSC (HUMSC)-sEVs significantly enhanced the proliferation and migration of human corneal epithelial cells via the repression of phosphatase and tensin homolog (PTEN) and activation of downstream protein kinase B phosphorylation. Subconjunctival injection of HUMSCs was found to facilitate corneal epithelial wound healing. These sEVs were particularly rich in miR-21, which appears to function as a key physiological and pathological regulator. sEVs derived from miR-21 knockdown HUMSCs showed diminished effects on human corneal epithelial cell proliferation and migration, suggesting that the regenerative function of HUMSC-sEVs is partly dependent onmiR-21. In addition, miR-143-3p, miR-3168, miR-21-5p, miR-10a-5p, miR-150-5p, and miR-1910-5p are significantly upregulated in corneal epithelial stem cells[54], indicating their potential role in maintaining epithelial homeostasis and repair.

ADMSC-Exos also exhibit potent epithelial healing properties. Ryu et al[55] reported that exosomal miRNAs such aslet-7a-5p, miR-23a-3p, miR-29b-3p, miR-302-3p, and miR-1246 play critical roles in promoting wound healing by regulating pluripotency, inhibiting epithelial-mesenchymal transition, and preventing cellular senescence. Moreover, ADMSC-derived EVs (ADMSC-EVs) enriched with miR-24-3p accelerate healing of corneal epithelial defects and enhance cell migration and maturation, while simultaneously reducing fibrosis and inflammatory cytokines such as CD163 and matrix metalloproteinase-9 in both in vitro and in vivo models of alkali-induced corneal injury[56].

MSC-EVs have also shown significant promise in modulating corneal stromal responses, particularly by inhibiting fibrosis and extracellular matrix (ECM) remodeling. Shen et al[57] demonstrated that ADMSC-EVs carrying miR-19a suppress homeodomain-interacting protein kinase 2 expression, thereby inhibiting fetal bovine serum-induced differentiation of rabbit corneal keratocytes into myofibroblasts via modulation of p53 and Smad3 signaling pathways. This downregulation of homeodomain-interacting protein kinase 2 further leads to the inhibition of TGF-β/Smad3 signaling, reduced pro-fibrotic markers, and ECM degradation, thus preserving stromal integrity. Altug et al[58] explored the therapeutic effects of human limbal MSC-EVs in fibrosis regulation. Their study found that miR-155 and miR-29 contained in these EVs negatively regulated collagen matrix synthesis, thus reducing fibrosis without impacting myofibroblast formation. In a recent study, Liu et al[59] used bone marrow derived MSC (BMSC) exosomes as carriers to deliver miR-29b in corneal injury models. MiR-29b effectively modulated multiple pro-inflammatory genes and enhanced autophagy by inhibiting the mechanistic target of rapamycin signaling pathway. This not only reduced the inflammatory response but also promoted autophagic flux and tissue repair, emphasizing the therapeutic potential of miR-29b-loaded MSC-exosomes in controlling corneal inflammation and fibrosis (Table 1).

Table 1 Effects of mesenchymal stem cell-derived microRNAs in corneal diseases.

Type of MSC	MiRNA	Target	Expression pattern	Function	Ref.
Human umbilical cord-MSC	MiR-21	PTEN	Upregulated	Enhanced corneal epithelial cell proliferation, migration, and wound healing, both in vitro and in vivo	Liu et al[53]
Human limbal MSC	MiR-155, miR-29	TGF-β1/β3	Upregulated	Negatively regulated corneal fibrosis by inhibiting collagen matrix synthesis instead of inhibiting myofibroblast formation	Altug et al[58]
Mouse bone marrow-MSC	MiR-29b-3p	PI3K	Upregulated	Improved the therapeutic effect via activated autophagy and inhibited corneal inflammation and fibrosis	Liu et al[59]
Rabbit adipose derived MSC	MiR-19	HIPK2	Upregulated	Suppressed fetal bovine serum induced differentiation of rabbit corneal keratocytes into myofibroblasts by inhibiting HIPK2 expression	Shen et al[57]
Rabbit adipose-derived MSC	MiR-24-3p	-	Upregulated	Enhanced healing of corneal epithelial defect, cell migration, and maturation while inhibiting fibrosis and reducing the levels of inflammatory cytokines (CD163, and MMP9) in both in vitro and in vivo	Sun et al[56]
Human adipose-derived MSC	Let-7a-5p, miR-23a-3p, miR-29b-3p, miR-302-3p, and miR-1246	-	Upregulated	Improved the wound healing and migration of corneal epithelial cells by inducing a shift in the cell cycle, and inhibiting senescence and autophagy pathways	Ryu et al[55]

HIPK2: Homeodomain-interacting protein kinase 2; miRNA: MicroRNA; MMP9: Matrix metalloproteinase-9; MSC: Mesenchymal stem cell; PI3K: Phosphatidylinositol 3-kinase; PTEN: Phosphatase and tensin homolog; TGF-1: Transforming growth factor beta 1.

DED

Dry eye is a chronic progressive disease characterized by decreased tear production, inducing ocular discomfort and/or visual dysfunction. The prevalence of DED widely varies between 5.5% to 33.7% globally[60], and it is characterized by an unstable tear film or unbalanced ocular surface microenvironment. DED pathogenesis is complex and may entail inflammation and immune modulation within the meibomian glands, lacrimal gland, and/or the conjunctival goblet cells, which generate different components of the tear film[61]. The aim of MSCs for dry eye treatment is regenerating new tissue to repair the damage of cornea and conjunctiva resulting from inflammation, and to restore tear film stability. MSCs and their secretome are considered as potentially novel therapeutic agents due to their capacity to release a high quantity of different immunomodulatory factors that can effectively modulate pathological immune responses in the eyes, and diminish systemic inflammation without any major adverse effects.

Weng et al[62] treated DED associated with chronic GVHD using intravenously administered MSCs and reported that MSCs are a safe and potentially effective therapy. Similarly, Zhou et al[63] injected MSC-exosomes into mice with chronic GVHD-associated dry eye, and into patients with refractory dry eye. Their findings demonstrated that MSC-exosomes alleviated dry eye symptoms, with the therapeutic effect attributed to miR-204 contained within the exosomes.

Møller-Hansen et al[64] administered allogeneic ADMSCs directly to the lacrimal glands of five patients with aqueous-deficient dry eye, resulting in increased tear secretion and decreased tear film osmolarity after treatment. Complementing these findings, Wang et al[65] explored the therapeutic potential of HUMSC-EVs in a mouse model of DED, observing significant amelioration of symptoms, enhanced tear secretion, and preservation of corneal integrity.

Several studies have highlighted the anti-inflammatory effects of MSC-miRNAs in DED. In a rabbit model, Li et al[66] demonstrated that HUMSC-sEVs alleviated DED by promoting M2 macrophage polarization and increasing Treg populations, primarily driven by miR-100-5p. Wang et al[65] attributed the therapeutic efficacy of HUMSC-EVs to their suppression of inflammation and restoration of ocular surface homeostasis, mediated by miR-125b, let-7b, and miR-6873 targeting the 1 (IL-1) receptor-associated kinase 1/TGF--activated kinase 1/nuclear factor kappa B (NF-κB) signaling pathway, which led to the downregulation of pro-inflammatory cytokines such as IL-4, IL-8, IL-10, IL-13, IL-17, and tumor necrosis factor alpha (TNF-α). Similarly, Zhao et al[67] showed that BMSC-derived exosomes enriched with miR-21-5p alleviated DED symptoms by inhibiting the Toll-like receptor 4 (TLR4)/myeloid differentiation primary response 88 (MyD88)/NF-κB signaling pathway, thereby rebalancing the Treg/Th17 ratio and reducing pro-inflammatory cytokines. Furthermore, Wang et al[68] reported that miR-223-3p delivered via exosomes from mouse ADMSCs significantly improved ocular surface integrity and reduced inflammation in benzalkonium chloride- and scopolamine-induced DED mouse models by targeting and down regulating F-box and WD repeat domain-containing 7 (Table 2).

Table 2 Effects of mesenchymal stem cell-derived microRNAs in dry eye disease.

Type of MSC	MiRNA	Target	Expression pattern	Function	Ref.
Human umbilical cord-MSC	MiR-204	IL-6R	Upregulated	Downregulated IL-6/IL-6R/STAT3 signaling responsible for macrophage phenotypes and alleviated dry eye (prospective clinical trial)	Zhou et al[63]
Human umbilical cord-MSC	MiR-100-5p	mTOR	Upregulated	Attenuated by skewed macrophages into a M2 phenotype and increased Treg proportion	Li et al[66]
Human umbilical cord-MSC	MiR-125b, let-7b, miR-6873	IRAK1	Upregulated	Inhibited ocular surface inflammation by multitargeting the IRAK1/TAB2/NF-κB signaling pathway	Wang et al[65]
Rat bone marrow-MSC	MiR-21-5p	TLR4	Upregulated	Decreased production of inflammatory and promoted synthesis of immunosuppressive cytokines and enhanced Tregs-driven suppression of inflammatory Th17 cells	Zhao et al[67]
Mouse adipose-derived MSC	MiR-223-3p	Fbxw7	Upregulated	Alleviated ocular surface damage and decreased pro-inflammatory cytokines or chemokines by targeting Fbxw7	Wang et al[68]

Fbxw7: F-box and WD repeat domain containing 7; IL: Interleukin; IL-6R: Interleukin 6 receptor; IRAK1: Interleukin 1 receptor-associated kinase 1; miRNA: MicroRNA; MSC: Mesenchymal stem cell; mTOR: Mechanistic target of rapamycin; NF-κB: Nuclear factor kappa B; STAT3: Signal transducer and activator of transcription 3; TAB2: TGF-beta activated kinase 1; Th17: T helper type 17; TLR4: Toll-like receptor 4; Treg: Regulatory T cell.

A clinical trial (NCT04213248), which aims to evaluate the efficacy of umbilical cord derived MSC-exosomes treatment in relieving DED-induced symptoms in patients with ocular GVHD, is in the recruitment stage. In the study protocol, patients with ocular GVHD will be treated first with artificial tears for 14 days (for standardization and stabilization of ocular surfaces), and then treated with umbilical cord derived MSC-exosomes eyedrop (10 μg/drop) four times per day for 2 weeks. A second clinical trial (NCT06543667) that is currently recruiting patients will evaluate therapeutic capacity of limbal stem cell-derived exosomes in attenuating DED-related symptoms in patients who responded suboptimally to conventional tear drop-based treatment. Patients with moderate or severe DED will be treated with artificial tears for 2 weeks, followed by administration of limbal stem cell-derived exosomes (10 μg/drop; 0.15 mL/single eye/one time), four times per day for 3 months. Since MSC-exosomes also contain proinflammatory and proapoptotic proteins or nucleic acids, the safety profile of all MSC-exosomes based eye drops needs to be carefully examined in clinical trials before they could be recommended as novel immunoregulatory agents for treatment of severe DED.

RETINAL DEGENERATIVE DISEASES

Retinal degenerative diseases represent major causes of blindness, which encompasses RP, AMD, DR, and glaucoma. Age-related pathological changes and genetic predisposition, along with environmental triggers, cause progressive cell function decline leading to the loss of retinal cells. Three key aspects allow MSCs to be considered as a relevant therapeutic tool for retinal degeneration and retinal repair: First, tissue repair through paracrine signaling, specifically through secretion of neurotropic factors that induce the repair of neuro-retinal cells; second, MSCs have immunomodulatory properties[69] to reduce the pro-inflammatory microenvironment that is present in retinal degenerative diseases; and third, their ability to secrete anti-angiogenic factors to counteract the pro-angiogenesis involved in the etiology of some ocular diseases[70].

DR

As a manifestation of diabetic pathology, DR involves damage to the optic vessels, precipitating a significant decline in visual acuity. Chemically-induced streptozotocin (STZ) is one of the most popular diabetic models that has been routinely used in basic studies and therapeutic drug experiments. This model exhibits rapid onset of hyperglycemia within a few days after STZ injection in rats[71]. The therapeutic effects of MSC transplants against STZ-induced DR in rats were attributed to cytokine regulation, which led to a reduction in retinal tissue damage, prevention of cell death, control of irregular blood vessel formation, and regulation of inflammatory processes[72,73].

ADMSC-EVs have demonstrated therapeutic potential in DR by delivering key miRNAs such asmiR-192 and miR-222, which mediate anti-inflammatory, anti-angiogenic, and tissue repair effects. Safwat et al[74] examined the ameliorative effects of ADMSC-Exo on retinal degeneration and demonstrated the significance of miR-222 on the repair of retinal tissue. The decrease of miR-222 was related to the progression of the severe retinal damage in the retinal tissue of STZ-induced diabetic rats. Gu et al[75] demonstrated that ADMSC-EVs enriched with miR-192 exerted protective effects against DR by downregulating integrin alpha 1. Integrin alpha 1 is implicated in ECM remodeling and retinal vascular dysfunction, contributing to DR pathogenesis. This study found that miR-192-loaded EVs effectively reduced retinal inflammation by lowering the levels of TNF-α, IL-6, IL-1β, vascular endothelial growth factor, monocyte chemoattractant protein-1, CD68, vascular endothelial growth factor A, CD31, and Ki67; reducing oxidative stress; and minimizing vascular leakage, thereby preserving retinal function.

In addition to ADMSC-EVs, HUMSC-EVs have also shown promising therapeutic potential in DR through the delivery of specific miRNAs miR-17-3p, miR-126, miR-5068, miR-10228, miR-30c-5p, and miR-18b, which collectively target inflammatory, oxidative, apoptotic, and angiogenic pathways to preserve retinal structure and function. Zhang et al[76] demonstrated that diabetic rats receiving intravitreal injections of HUMSC-exosomes overexpressing miR-126 exhibited significant reductions in retinal levels of inflammatory markers, including caspase-1, IL-1β, and IL-18, compared to untreated diabetic controls. MiR-126 contributed to the downregulation of high mobility group box 1 and its downstream molecules involved in inflammation. MiR-126-exosomes decreased high mobility group box 1 expression and inhibited activation of NF-κB/p65, which is a crucial mediator in modulating the expression of inflammatory factors, and is likely the mechanism behind the amelioration of the high glucose-induced inflammatory response. Li et al[77] reported that exosomal miRNA-17-3p from HUMSCs effectively alleviated retinal damage in DR by targeting signal transducer and activator of transcription 1 (STAT1). STAT 1 is a key regulator of inflammatory pathways, and its inhibition by miR-17-3p led to reduced pro-inflammatory cytokine expression and oxidative stress, thereby mitigating retinal cell apoptosis and vascular dysfunction. Xu et al[78] identified the significant upregulation of miR-18b in HUMSC-sEVs, which led to a significant reduction in retinal vascular leakage and thickness compared to untreated DR rats. Human retinal microvascular endothelial cells exposed to high glucose conditions showed increased inflammation and apoptosis. However, treatment with HUMSC-sEVs led to a marked decrease in pro-inflammatory cytokine levels and apoptotic cell counts, suggesting a protective role of the sEVs under diabetic conditions through miR-18b. Bioinformatics predictions and subsequent validations revealed that miR-18b targets mitogen-activated protein kinase kinase kinase 1, leading to the inhibition of NF-κB p65 phosphorylation, thereby alleviating DR symptoms. He et al[79] showed that intravitreal injection of HUMSC-EVs significantly improved retinal vascular integrity, reduced inflammation, and suppressed oxidative stress in a diabetic rat model through miR-30c-5p. The authors demonstrated that miR-30c-5p-enriched HUMSC-EVs significantly downregulated pro-inflammatory cytokines (TNF-α, IL-1β, and IL-6) and oxidative stress markers while increasing the expression of antioxidant enzymes such as superoxide dismutase. Furthermore, target gene analysis identified that miR-30c-5p directly inhibited the expression of thioredoxin-interacting protein, a key regulator of oxidative stress and inflammation in DR pathology. Suppression of thioredoxin-interacting protein led to reduced nucleotide-binding oligomerization domain-, leucine-rich repeat-, and pyrin domain-containing receptor 3 inflammasome activation, thereby decreasing retinal cell apoptosis and preserving retinal function.

Sun et al[80] explored the therapeutic potential of engineered HUMSC-sEVs enriched with miR-5068 and miR-10228 in treating DR by modulating the hypoxia-inducible factor 1 alpha/enhancer of zeste homolog 2/proliferator-activated receptor gamma coactivator 1 alpha pathway. In vivo experiments using a diabetic mouse model revealed that intravitreal administration of HUMSC-sEVs significantly reduced retinal vascular leakage, preserved retinal thickness, while improving retinal function compared to untreated diabetic mice. Molecular studies revealed that HUMSC-sEV treatment upregulated hypoxia-inducible factor-1α, which in turn enhanced the expression of proliferator-activated receptor gamma coactivator 1 alpha, a key regulator of mitochondrial function, while suppressing enhancer of zeste homolog 2, a known epigenetic modulator of oxidative stress. This regulation helped restore mitochondrial homeostasis, thereby reducing retinal oxidative stress and protecting against DR-induced damage in mouse models. The functional inhibition of miR-5068 and miR-10228 nullified the retinal therapeutic effects of HUMSC-sEVs, thus confirming that HUMSC-sEVs alleviate DR mainly through the delivery of miR-5068 and miR-10228. Compared to natural MSC-sEVs, HUMSC-sEVs engineered with miR-5068 and miR-10228 demonstrated heightened therapeutic capabilities in mitigating retinal injury.

BMSCs have also emerged as a potent source of therapeutic EVs. Notably, BMSC-derived EVs enriched with miR-486-3p have shown significant promise in alleviating DR through the repression of the TLR4/NF-κB signaling pathway. In high glucose-treated Muller cells, miR-486-3p expression was significantly reduced, whereas TLR4 and NF-κB levels were elevated. Overexpression of miR-486-3p or silencing of TLR4 Led to decreased oxidative stress markers, reduced inflammatory cytokine production, and lower apoptosis rates. Additionally, cell proliferation rates improved under these conditions. By binding to TLR4 mRNA, miR-486-3p suppressed its expression, leading to inhibition of the downstream NF-κB signaling pathway. This suppression resulted in reduced inflammatory responses and cellular apoptosis, thereby offering protective effects against DR progression[81] (Table 3).

Table 3 Effects of mesenchymal stem cell-derived microRNAs in diabetic retinopathy.

Type of MSC	MiRNA	Target	Expression pattern	Function	Ref.
ADMSC	MiR-192	ITGA1	Upregulated	Negatively regulated ITGA1 and ameliorated diabetic retinal damage	Gu et al[75]
Rabbit ADMSCs	MiR-222	STAT5	Upregulated	Induced repair of diabetic retinal degeneration	Safwat et al[74]
Human BMSC	MiR-486-3p	TLR4	Upregulated	Inhibited oxidative stress, inflammation and apoptosis, and promoted proliferation of high glucose-treated Muller cells by downregulating TLR4; through TLR4/NF-κB axis repression	Li et al[81]
Human umbilical cord-MSC	MiR-18b	MAP3K1	Upregulated	Reduced vascular leakage and inflammation by targeting MAP3K1, thereby inhibited NF-κB p65 phosphorylation	Xu et al[78]
Human umbilical cord-MSC	MiR-17-3p	STAT1	Upregulated	Decreased contents of inflammatory factors and VEGF, alleviated oxidative injury, and inhibited retinal cell apoptosis through inhibiting STAT1	Li et al[77]
Human umbilical cord-MSC	MiR-30c-5p	PLCG1	Upregulated	Inhibited oxidative stress and inflammation in high glucose-treated retinal endothelial cells via the PLCG1/PKC/NF-κB axis	He et al[79]
Human umbilical cord-MSC	MiR-5068, miR-10228	HIF-1α	Upregulated	Exhibited enhanced retinal therapeutic efficiency by regulating HIF-1α/EZH2/PGC-1α pathway	Sun et al[80]

ADMSC: Adipose tissue-derived mesenchymal stem cell; EZH2: Enhancer of zeste homolog 2; HIF: Hypoxia-inducible factor; ITGA1: Integrin alpha 1; MAP3K1: Mitogen-activated protein kinase kinase kinase 1; miRNA: MicroRNA; MSC: Mesenchymal stem cell; NF-κB: Nuclear factor kappa B; PGC-1α: Proliferator-activated receptor gamma coactivator 1 alpha PKC: Protein kinase C; PLCG1: Phospholipase C gamma 1; STAT5: Signal transducer and activator of transcription 5; TLR4: Toll-like receptor 4; VEGF: Vascular endothelial growth factor.

AMD AND RP

AMD represents a progressive disease that affects the neuronal retina along with retinal pigment epithelium (RPE) and choriocapillaris complex specifically within older adults in developed nations[82]. Neovascular AMD-induced subretinal fibrosis persists as the leading reason for severe vision damage worldwide. The pathogenesis of subretinal fibrosis connects to epithelial-mesenchymal transition in RPE cells[83]. Li et al[84] investigated the efficacy of miR-27b derived from HUMSC-exosomes in a mouse model of laser-induced subretinal fibrosis. The results demonstrated that miR-27b inhibited homeobox C6, suppressing the epithelial-mesenchymal transition process triggered by TGF-β2 and attenuating subretinal fibrosis (Table 4).

Table 4 Effect of mesenchymal stem cell-derived microRNAs in age-related macular degeneration and glaucoma.

Type of MSC	MiRNA	Target	Expression pattern	Function	Ref.
Mouse bone marrow-MSC	MiR-21a-5p	PDCD4	Upregulated	Promoted RGC survival	Su et al[100]
Rat bone marrow-MSCs	MiR-21	PDCD4	Upregulated	Maintained photoreceptor survival by targeting PDCD4 to inhibit apoptosis	Deng et al[87]
TNF-α-stimulated gingival MSC	MiR-21-5p	PDCD4	Upregulated	Increased retinal cell viability and neuroinflammation in vitro, prevented the apoptosis and inflammation of retina	Yu et al[101]
Human bone marrow-MSC	MiR-144-5p, miR-126-5p, and miR-100-5p	-	Upregulated	Increased neuroprotection and functional preservation of RGC	Mead et al[98]
Human bone marrow-MSC	MiR-17–5p, miR-30c-2 and miR-92a; miR-92a, miR-292 and miR-182	PTEN	Upregulated	Protected RGCs and their axons from degeneration and dysfunction through PTEN knockdown	Mead and Tomarev[99]
Human umbilical cord-MSC	MiR-27-b	HOXC6	Upregulated	Inhibited TGF-β-induced epithelial-mesenchymal transition by targeting HOXC6 and attenuated subretinal fibrosis	Li et al[89]
Human umbilical cord-MSC	MiR-146a	Nr43a	Upregulated	Significantly decreased the expression of LPS-induced pro inflammatory cytokines	Zhang et al[88]
Human umbilical cord-MSC	MiR-22-3p	MAPK312	Upregulated	Ameliorated NMDA-induced RGC injury through the inhibition of MAPK signaling pathway-mediated apoptosis	Yu et al[102]

HOXC6: Homeobox C6; LPS: Lipopolysaccharide; MAPK: Mitogen-activated protein kinase; miRNA: MicroRNA; MSC: Mesenchymal stem cell; NMDA: N-methyl-D-aspartic acid; PDCD4: Programmed cell death 4; PTEN: Phosphatase and tensin homolog; RGC: Retinal ganglion cell; TGF: Transforming growth factor; TNF: Tumor necrosis factor.

RP represents progressive hereditary degenerative disorder which creates permanent vision damage through RPE dysfunction and photoreceptor apoptosis[85]. The condition typically leads to progressive visual-field deterioration, along with night blindness and pigmentary retinopathy, which produces abnormal electroretinogram readings[86]. Deng et al[87] demonstrated that MSC transplantation conferred significant protection to photoreceptors in retinal degeneration, primarily through the release of exosomal miR-21. Their study revealed that miR-21 exerts its neuroprotective effects by inhibiting apoptosis and reducing oxidative stress in degenerating retinal cells. Zhang et al[88] investigated the therapeutic potential of HUMSC-EVs in a mouse model of RP, focusing on their anti-inflammatory effects mediated through the miR-146a-Nr4a3 axis. Their study revealed that MSC-EVs inhibited the NF-κB pathway and modulated cytokine expression, upregulating anti-inflammatory and downregulating pro-inflammatory cytokines through miR-146a. This aligns with previous findings where MSC-derived exosomal miRNAs have been implicated in retinal repair by modulating inflammatory and survival pathways. Notably, earlier studies reported the role of MSC-derived exosomes in mitigating neurodegeneration through miR-21-mediated suppression of pro-apoptotic genes[89].

GLAUCOMA

Glaucoma stands as the leading cause of worldwide blindness because it causes gradual retinal ganglion cell (RGC) and axonal deterioration followed by optic nerve damage[90]. An increase in aqueous humor production or a disturbance in its outflow system leads to elevated intraocular pressure which remains the essential factor in glaucoma-mediated RGC apoptosis[91]. The widespread adoption of MSCs as new therapeutic agents in glaucoma cell-based therapy stems from their functional capabilities according to Sun et al[92], Daliri et al[93], and Harrell et al[94]. In glaucomatous eyes, MSCs generate neurotrophins that encourage both the survival and regenerative capacity of injured RGCs[95]. MSCs restore RGCs through the combination of creating functional RGC-like cells and boosting residential retinal stem cells in mature RGCs[96]. The maintenance of trabecular meshwork integrity along with altered trabecular meshwork cell function by MSCs allows intraocular pressure reduction in glaucomatous eyes[97].

The therapeutic effects of MSC-exosomes function primarily through mechanisms that rely on miRNA regulation. Argonaute 2 protein knockdown has been shown to reduce BMSC-exosome-induced effects since this protein plays an essential role in miRNA-mediated processes[98]. RNA sequencing indicated that BMSC-exosomes produced more than forty unique miRNAs compared to fibroblast-derived exosomes. MiR-144-5p, miR-126-5p, miR-100-5P, miR-17-92, miR-21, and miR146a were identified as the primary therapeutic miRNAs for RGC regeneration in glaucoma[98,99]. Interestingly, the miR-17-92 and miR-21 regulated the expression of PTEN, which is a critical inhibitor of RGC axonal growth and survival, and miR-146a controlled the expression of epidermal growth factor receptor which inhibits axon regeneration[99]. Later, they expressed combinations of six candidate miRNAs (miR-26a, miR-17, miR-30c-2, miR92a, miR-292, and miR-182) using self-complimentary adeno-associated virus-2 in the RGCs of rats that underwent optic nerve crush injury. PTEN, a confirmed target of many of the delivered miRNA was qualitatively reduced and coincided with the therapeutic effects observed.

Su et al[100] and Yu et al[101] explored the role of miR-21a-5p in mediating MSC-induced neuroprotection in a rat model of acute glaucoma. The study demonstrated that miR-21a-5p played a critical role in MSC-induced RGC survival by targeting programmed cell death 4 (PDCD4), a pro-apoptotic gene. Inhibiting miR-21a-5p reversed the neuroprotective effects of MSCs. The study identified the miR-21a-5p/PDCD4 axis as a key regulator of RGC survival, providing mechanistic insights into MSC-miRNA functions in glaucomatous neurodegeneration. Yu et al[102] modified MSC by transfecting them with a lentivirus carrying miR-22 and then collected their sEVs. They found that treatment with miR-22-3p-enriched sEVs led to a significant reduction in RGC apoptosis in N-methyl-D-aspartic acid-induced RGC injury through caspase dependent pathway. This neuroprotective effect was associated with modulation of the mitogen-activated protein kinase pathway, a critical signaling cascade involved in cell survival and apoptosis.

MiR-21

MiR-21 is a commonly found and functionally prominent miRNA in different ocular degenerative conditions, and it is always involved in tissue repair, modulation of the immune system, neuroprotection. Remarkably, miR-21 targeting PTEN occurs in corneal injury[89] and glaucomatous retinal damage[100], both of which involve similar cascades that rely on PTEN inhibition to sustain cell survival, cell growth, and axon regeneration. MiR-21-5p has an anti-inflammatory effect in DED by intervening in the TLR4/MyD88/NF-κB signaling pathway to restore the balance between Tregs and Th17, and limit damage caused by cytokines[67]. During retinal degeneration, miR-21 also appears to play a neuroprotective role by reducing apoptosis and oxidative damage of photoreceptors. In glaucoma miR-21 helps in the survival of RGC by targeting PTEN, and in certain models, PDCD4 (a pro-apoptotic gene) through the miR-21a-5p/PDCD4 axis. These results suggest that although miR-21 is always protective and regenerative, its molecular mechanism of action and associated signaling differs based on the disease and the type of affected cell. Such versatility highlights the necessity to characterize miRNA action on a disease-specific level when formulating miRNA-based therapies using MSC-EVs. Figure 1 summarizes the key MSC-miRNAs, their validated targets, and associated signaling pathways implicated in ocular degenerative diseases.

Figure 1 Summary of mesenchymal stem cells-derived microRNAs, their targets, and signaling pathways involved in ocular diseases. AKT: Protein kinase B; HIF: Hypoxia-inducible factor; HIPK2: Homeodomain-interacting protein kinase 2; HOXC6: Homeobox C6; IL: Interleukin; IRAK1: Interleukin 1-receptor associated kinase 1; MAPK: Mitogen-activated protein kinase; mTOR: Mechanistic target of rapamycin; NF-кB: Nuclear factor kappa B; PI3K: Phosphatidylinositol 3-kinase; PLCG1: Phospholipase C gamma 1; PTEN: Phosphatase and tensin homolog; STAT3: Signal transducer and activator of transcription 3; TGF: Transforming growth factor; TLR4: Toll-like receptor 4.

Bioengineering of nanovesicles with MSC-miRNA

Nano-therapeutic properties of EVs are now increasingly being explored for regenerative medicine because these particles possess natural capabilities for cellular communication and tissue regenerating functions. Multiple barriers including restricted output quantities, along with differences in biological function, and separation and purification obstacles have prevented these nanovesicles from reaching clinical practice. Current research dedicated to MSC-EV therapeutic enhancement targets two main aspects: Molecular content optimization, and surface modification of these particles[103,104]. Along with the display of tissue-specific peptides, molecular content optimization includes both overexpression of angiogenic miRNAs and silencing inflammatory messenger RNAs[105], and injecting drug compounds into EVs[105,106]. Surface modification of EVs with active nucleic acid encapsulation in cancer therapies created a basis for adopting this technique in regenerative medicine applications.

MSC-EVs engineered to deliver pro-angiogenic miRNAs and growth factors contribute greatly to wound angiogenesis through their action of triggering endothelial cell migration and proliferation, while simultaneously boosting angiogenic growth factor production and activating regeneration signaling pathways within the wound tissue. MSC-EVs can transport particular miRNAs including miR-126 and miR-210, or miR-132[107-110] because these miRNAs demonstrate recognized proangiogenic properties and tissue regenerative abilities.

Aging-induced vascular dysfunction treated with miR-675-enriched EVs inside silk fibroin hydrogels in mouse demonstrated effective blood perfusion in ischemic hindlimbs together with diminished proinflammatory molecule expression[111]. ADMSC-EVs enriched in miR122 were successfully deployed for hepatocellular carcinoma chemotherapy[112]. The lentiviral transduction process enable modifications to miR-let7c expression levels in MSCs, which produce EVs containing high concentrations of miR-let7c. Evidence has shown that these modified EVs use miR-let7c to improve transport to the kidneys where they reduce renal fibrosis[113]. Professional engineering of EVs allows nucleic acid delivery at a cellular level through preconditioning and exogenous transfection to enhance natural miRNA content in EVs.

Cell preconditioning has also been validated as a method of enriching key miRNAs. Next-generation sequencing revealed that EVs originating from human neural stem cells cultured under hypoxic conditions increase levels of 53 miRNAs and decrease levels of 26 miRNAs[114]. EV-based therapies will lead to better efficacy through specific MSC-miRNA engineering approaches in the manufacturing process of EVs. Recent research developments create more specific therapeutic regenerative solutions, which help improve present constraints while expanding potential clinical usage of EVs.

Although MSC-derived nanovesicles are promising tools in ocular therapeutics, a number of issues have to be addressed to entertain clinical translation. The majority of unmodified EVs are distributed in the liver, spleen, and kidneys, and very few reach target tissue like the retina or brain[115]. Pharmacokinetic predictions are further complicated by interspecies differences, because in non-human primates studies, prolonged circulation times have been found compared to rodent models[116]. To address these shortcomings, attempts to bioengineer EVs have been established to increase their targeting and half-life. Genetic engineering may present ligands, homing peptides, or immune checkpoint proteins (e.g., CD47, programmed death-ligand 1) on the surface of EVs complementing tissue-specific delivery and avoiding immune clearance[117,118]. It has also been illustrated that the PEGylation of EVs can inhibit their rapid clearance[119]. Therapeutic precision can be enhanced with innovations like light-responsive release systems and targeting through the lysosome-associated membrane protein 2B platform[120,121]. Microfluidics allows scalable, reproducible synthesis of multifunctional nanovesicles of specific size and encapsulation properties, and with tunable surface characteristics. These platforms are optimal when co-delivering miRNAs and other therapeutics, and achieved greater batch consistency[122]. Nevertheless, issues of safety do occur; especially the cytotoxicity of positively charged nanocarriers which are used to load miRNAs. A pH-responsive coating, and surface charge-shifting are potential approaches to limit toxicity while enhancing biocompatibility[123]. Taken together, the combination of targeting ligands, immune-evasive coating and improved microfluidics shows the obvious road to clinically relevant MSC-derived nanovesicle therapies. Still, interspecies pharmacokinetic variability, safety over months/years, and scalable good manufacturing practice-grade production have to be dealt with in the future to reach the full translational potential.

FUTURE PERSPECTIVES AND CLINICAL TRANSLATION CHALLENGES

MSC-EV-based therapies are only possible with miRNA-loaded vesicles when the latter can be consistently loaded with the required RNA[124]. In general, more loading efficiency can be achieved with electroporation and chemical transfection compared to co-incubation[125]. Nevertheless, these techniques still require additional research to optimize them, to achieve a better balance of loading and the integrity of the vesicles[126]. It will involve streamlining factors like the amount of miRNA, the intensity of the electric field used in electroporation, and the kind of transfection agents to be used[127]. The use of these vesicles and associated tightly bound proteins provides an environment that shields the miRNAs against degradation during protracted storage and transportation, including its delivery to the cancer tissues[128]. Stabilizing agents and improved storage conditions are also under investigation to enhance the stability of miRNA carried by MSC-EVs[129]. Chemical modifications to enhance stability and delivery are also under scrutiny in MSC-EVs[130]. Polyethene glycol-based surface modification of MSC-EVs has the potential to enhance their survival in the blood by preventing opsonization and clearance by the immune system[131]. Additionally, it is also possible to enhance the on-demand release ability of miRNAs within the tumor microenvironment by introducing pH-sensitive or enzyme-cleavable linkers in this MSC-EV structure[132].

The clinical possibility of miRNA diagnostics encounters the challenge of high variation in circulating miRNA expression levels, which could make the interpretations difficult[133-135]. Another aspect is the limited availability of standard protocols[136] and low sensitivity and specificity of existing techniques when analyzing heterogeneous samples[137]. Although EVs have recently undergone major breakthroughs in the field, continued work in the areas of standardization, production, governing regulations, and integration technologies are necessary to transfer the full potential into clinical practices[138]. Uniformity of EV isolation procedures is a critical objective to the discipline, because it will ensure more reliable characterization of vesicle-associated miRNAs and lead to their greater applicability in the field of diagnosis. On another note, miRNA detection is a fast-developing discipline with profound potential in the discovery of biomarkers, disease diagnostics, and therapy monitoring. Although next-generation sequencing, microarray analysis, quantitative PCR, in situ hybridization, biosensors, digital droplet PCR, and NanoString each have their own benefits, none is regarded as superior to the other. The choice of the most appropriate detection platform will depend on the sensitivity, specificity, cost and throughput. Future directions must aim at the improvement of current methods and the creation of uniform guidelines to enable cross-study comparisons and clinical translation.

CONCLUSION

Among adult stem cells, MSCs have revolutionized the field of ophthalmology due to their immunomodulatory and regenerative capability. These multipotent stem cells are non-immunogenic and they exert their action through paracrine effect. One of the major factors that mediate this process are the miRNAs. With emerging focus on MSC-derived exosomes, several clinical trials have been initiated as an alternate to MSC therapy for various conditions, including ocular diseases. Among the exosomal cargo, miRNAs have been extensively analyzed. The current focus is on bioengineering these exosomes with modified cargo or surface to exert the desired effect on target cells. This will enable the development of novel therapeutics with easy delivery of drugs for various ocular diseases.

ACKNOWLEDGEMENTS

The authors thank Dr. Beth Mills, UKRI Future Leaders Fellow, Translational Healthcare Technologies, Centre for Inflammation Research, Institute for Regeneration and Repair, University of Edinburgh, for the help in language editing and Lady Tata Memorial Trust, Mumbai, India for the Junior Research Fellowship (Sneha Nair).