Extracellular vesicles for corneal regeneration: the new frontier

Corneal disease is a significant contributor of blindness. It is among the top five leading causes of blindness, affecting around 6 million people globally [1]. The cornea forms the transparent window at the front of the eye and is essential in transmitting light onto the retina for visual perception. The cornea is clear in health and comprises three cellular layers – the epithelium, stroma and endothelium. Disruption in any one of these three layers, by corneal haze or swelling (edema), results in loss of corneal clarity and blindness.

The mainstay of treatment for corneal disease is its replacement by transplantation [2]. Classic corneal transplantation involves the transplantation of all layers of the cornea (known as penetrating keratoplasty). Contemporary transplantation methods involve specific transplantation of different layers of the cornea, depending on which layer is affected. This is known as lamellar keratoplasty and includes endothelial keratoplasty for diseased corneal endothelium. Recently, cellular transplantation methods have been used for the cornea, including corneal epithelial stem cell transplantation for diseased corneal epithelium [3] and corneal endothelial cell transplantation for diseased corneal endothelium [4]. All the above transplantation techniques require the ready availability of healthy donor corneas, surgical training for corneal transplantation, and financial resources to undertake surgery. As a result, corneal transplantation remains out of reach for the majority of patients globally.

In view of the above limitations of corneal transplantation, there is a need to research alternative therapies for corneal pathology. Regenerative acellular corneal therapies that reduce the need for donor corneal tissue and corneal surgery would be of particular value. Recently, extracellular vesicles (ECVs) derived from specific corneal cells have garnered interest due their ability to promote corneal regeneration via paracrine effects [5]. In this editorial, we outline the research done to date.

1. Extracellular vesicles (ECVs)

ECVs are cell-derived nanovesicles composed of proteins (matrix proteins and receptors), metabolites, lipids and nucleic acids (small strand DNAs, mRNAs and micro-RNAs) bound by a lipid bilayer. They are classified into three main types based on size: apoptotic bodies (ranging from 1 to 5 μm), microvesicles (1–2 μm) and exosomes (40–120 nm) [6]. ECVs are potent mediators of cell-to-cell communication, cellular differentiation and proliferation. They carry cargo that can influence cell behaviors, impacting immune modulation, tissue repair and cellular regeneration. As such, they show great promise in providing a novel strategy for acellular regenerative therapies. It is thought that cell-derived ECVs could capitalise on the therapeutic benefits of stem-cell-based therapies with fewer biosafety concerns associated with the transplantation of living cells. ECVs have already shown promise in other fields, as biomarkers in cancer diagnosis and also potential therapeutic agents [7]. An important goal for corneal research is to understand the role of ECVs in the cornea and to explore their therapeutic potential.

2. ECVs in corneal disease

Corneal injury initiates a cascade of repair pathways. The healing response comprises cell proliferation, migration and transformation; the release of growth factors, cytokines and proteases; and the deposition of extracellular matrix proteins. This response can lead to scar formation and compromise corneal transparency. An example of this is during corneal injury, where interaction between the corneal epithelium and stroma plays an important role in the transformation of stromal keratocytes into myofibroblasts [8]. By delivering bioactive molecules such as proteins, mRNAs and microRNAs, ECVs are thought to be key mediators of this intercellular communication. The presence of ECVs between epithelial cells and the stroma of wounded rat corneas has been captured by transmission electron microscopy. Zieske et al. have demonstrated the ability of these rat corneal epithelial-derived ECVs to induce phenotypic changes in keratocytes to generate myofibroblasts [9]. Although the exact pathways of intercellular communication are to be determined, given their role in the transfer of genetic material and intercellular signalling, ECVs show great potential as mediators of corneal wound healing. They most likely do this by enabling cell migration (deposition of matrix components), cellular proliferation (by cell cycle progression) and communication between the epithelial cells and stromal keratocytes.

3. ECVs in corneal epithelial regeneration

Several corneal surface diseases impact corneal epithelial stem cells (known as limbal stem cells) and this results in the impairment of normal corneal epithelial renewal, resulting in pain and vision loss. Stem cell replacement by limbal stem cell (LSC) transplantation has been successfully used to treat diseases of the corneal epithelium [3]. In allogeneic LSC transplants, it has been shown that transplanted stem cells and their progeny cannot be identified after 9–12 months post surgery, despite continued normalization of corneal epithelial renewal [10]. It is hypothesized that transplanted LSCs promote normalization of the host corneal epithelium through paracrine effects, possibly via ECVs.

Ramos et al. have demonstrated the in vitro effects of corneal epithelial-derived ECVs on non corneal epithelial cells [11]. This resulted in molecular changes associated with a corneal epithelial cell type. This furthers our understanding of potential therapeutic targets in the treatment of corneal epithelial disease. In addition, mesenchymal stem cell derived ECVs have shown promise in corneal epithelial wound healing, by accelerating epithelial wound healing [12].

4. ECVs in corneal stromal scarring

Stromal scarring commonly occurs secondary to corneal repair pathways, and is the main pathological process in the corneal stroma that leads to vision loss. Funderburgh et al. have studied the therapeutic potential of corneal stromal stem cells for corneal scarring. They have shown that corneal stromal stem cells-based regeneration is in part mediated by ECVs. Testing this hypothesis, they found that topical application of CSCC-secreted ECVs reduced corneal inflammation and scarring in a mouse model of superficial stromal corneal wounding [13,14]. Such a cell-free approach would vastly simplify the ease of therapy as the treatment could be through topical application rather than surgery. It would also eliminate issues of immune rejection that is associated with corneal transplantation.

5. ECVs in corneal endothelial function & regeneration

The function of the corneal endothelium is to prevent excess fluid accumulation within the cornea. A dysfunction of the corneal endothelium causes corneal swelling (edema) and therefore loss of corneal transparency. Humans are born with a finite number of corneal endothelial cells, which decline in number over time [15]. Human corneal endothelial cells cannot proliferate in vivo. The corneal endothelium therefore has limited regenerative capacity, and normal repair occurs via stretching of existing endothelial cells. Apart from age, there are several pathologies that can accelerate the loss of corneal endothelial cells. The most common endothelial disease is Fuchs’ Endothelial Corneal Dystrophy (FECD) and is one of the main reasons for corneal endothelial keratoplasty. Due to the global shortage of donor corneas, it is vital to identify alternative strategies for corneal endothelial repair.

One important area of regenerative research for corneal endothelial disease is that of ECVs. In one study, ECVs derived from human corneal endothelial cells have been shown to inhibit the proliferation of endothelial cells [16]. Endothelial cells exposed to high ECV concentrations in this study started to appear like those seen in FECD. This led to the hypothesis that continuous increased release and uptake of ECVs under stress may accelerate endothelial cell loss leading to FECD. Investigating the content of these inhibitory ECVs has identified micro-RNAs (miRNAs) that are important in the proliferation of corneal endothelial cells. It has been proposed that inhibiting miR-195-5p induces the proliferation of human corneal endothelial cells in vitro and ex vivo and this could be of therapeutic benefit [17].

6. Challenges & future potential

For all their therapeutic potential in corneal regeneration, ECVs come with several challenges that need to be addressed prior to clinical application. Being a relatively new field of research, ECV isolation methods face limitations such as low yield, purity of ECVs and sample heterogeneity. This is particularly important given that the goal is clinical grade production by good manufacturing practice (GMP) standards. The regulatory approval of ECVs also requires clarity on whether they are classified as advanced therapeutic medicinal products or gene therapies (due to their miRNA content), although it can be argued they are neither. The International Society for Extracellular Vesicles has formulated some limited guidance for their clinical use.

A second consideration for clinical application is to determine the best mode of delivery, and a better understanding of the dose-response relationship. Topical eye drops activate the blink reflex which washes away administered doses within 30 s after instillation [18]. Alternative delivery methods, such as hydrogels have been investigated with good success for corneal delivery of ECVs [19]. For corneal endothelial regeneration, injection into the anterior chamber of the eye might be needed.

Finally, for a viable off-the-shelf product, optimal storage considerations are critical. There has been promising preliminary work on the lyophilization of ECVs, with one study demonstrating freeze-dried clinical grade ECVs being stored at -20°C for at least 2 months [20]. Further research is however needed prior to clinical application.

Over the past two decades, corneal stem cell research has paved the way for novel clinical therapeutics. The challenges however have been the requirement of specialized laboratories, high costs and complex logistics, and the need for new surgical skills. ECVs beckon a paradigm shift in corneal regenerative therapy toward safer and hopefully cheaper acellular therapies that can be delivered without the need for complex surgery. ECV therapy holds the hope to democratise regenerative corneal treatments for a much wider population. In addition, our experience from corneal stem cell therapies has shown that close collaboration across disciplines is needed to accelerate the path for ECVs into clinic.

Author contributions

K Jafari: writing of manuscript. AS-Safi: editing of manuscript draft. S Ahmad: concept development and writing/editing of manuscript.

Financial disclosure

This paper was not funded.

Competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.