A single layer of tightly spaced cells, known as the endothelium, rests on the posterior side of the cornea. This endothelium regulates the stroma’s relative dehydration, which is essential for corneal clarity. Cell therapy is an innovative method being used to repair various corneal abnormalities. Mesenchymal stem cells (MSCs) are now one of the most significant types of stem cells scientists have studied. This study aimed to evaluate the role of MSCs for corneal endothelial regeneration.

A systematic review was performed by searching for articles from reputable databases with many study-type references, including PubMed, Cochrane Library, Science Direct, and Google Scholar, up to January 2024. The resulting data were displayed using the 2020 PRISMA flowchart and evaluated using the PRISMA 2020 checklist. Most of the included studies were in vivo and used topical application and anterior chamber injection as the administration routes.

Based on the findings of this review, MSCs increased corneal endothelial cell density, improved the defect area and corneal transparency, facilitated endothelial cell regeneration and wound healing, and decreased neovascularization and corneal pro-inflammatory cytokines as compared to controls.

Administration of MSCs into the anterior chamber could increase regeneration and proliferation of corneal endothelial tissue.

Keywords: Anterior chamber, corneal endothelial cell density, corneal endothelial regeneration, in vivo, mesenchymal stem cells

The cornea is the clear aperture that allows light to enter the eye. In adults, the diameter and thickness of this avascular tissue measure 10–12 mm and 500–600 μm, respectively, with a light-refractive index of 1.38. The outer layer of the cornea consists of stratified corneal epithelial cells. A monolayer of densely packed, hexagon-shaped corneal endothelial cells (CECs) lines the innermost layer of the cornea. The CECs reside in contact with the stroma on the Descemet membrane.¹

On the posterior side of the cornea, a single layer of tightly spaced cells called the endothelium regulates the stroma’s relative dehydration, which is essential for corneal clarity.² A corneal endothelium with adequate cell density maintains normal corneal moisture and transparency by acting as both a passive barrier and an active ion transporter. Endothelial cell density diminishes with age steadily and linearly. However, more rapid reduction in cell density may occur in response to surgical or accidental trauma, corneal transplantation, and illnesses, including endothelial dystrophy, glaucoma, and diabetes.³

Corneal endothelial decompensation occurs when the density of endothelial cells in the cornea drops below a critical level.⁴ The only known treatment is to transplant a donor cornea with a healthy endothelium.⁵ Human CECs are not physically capable of regenerating in vivo. Cell migration and expansion in the defect area facilitate endothelial wound repair.⁶ On the other hand, ex vivo studies have revealed that the periphery of the corneal endothelium has a greater capacity for in vitro regeneration than the center.⁷

One of the leading causes of blindness worldwide is corneal damage. Corneal transplantation is a way to treat corneal damage. However, approximately 12.7 million people globally are in need of corneal transplants.⁸ Among the difficulties and obstacles associated with corneal transplantation are its high cost, the risk of transplant rejection, the scarcity of donors, as well as legal and cultural concerns.⁹ Cell therapy is an innovative method for repairing various corneal abnormalities. One of the newer cell therapies is related to stem cells. Tissue engineering uses adult and embryonic stem cells to repair damaged organs and tissues because of their capacity for both differentiation and reproduction. Mesenchymal stem cells (MSCs) are now one of the most significant types of stem cells studied by scientists.¹⁰ This study aimed to evaluate the role of MSCs in corneal endothelial regeneration.

For the purposes of this review, “corneal endothelium” refers to the monolayer of hexagon-shaped cells that line the posterior surface of the cornea and divides the aqueous humor of the anterior chamber from the corneal stroma. This review included all animal model studies that used an MSC-related intervention in corneal endothelial tissue.

Each study considered for inclusion had to assess the effects of MSCs on injured rabbit corneal endothelium using a controlled methodology. Therefore, this review included only studies where MSC-related therapy was the main element of the intervention being assessed. The inclusion criteria were as follows: (1) in vivo animal studies and human trials, including both small animals (mice, rats, rabbits, and hamsters) and large animals (pigs, dogs, and horses), standardized according to the appropriate animal laboratory protocols; (2) studies using models of damaged corneal endothelium; (3) interventions involving human-derived stem cells injected or transplanted into specified or defective sites; and (4) studies published in the last 10 years (2013–2023) in internationally recognized journals. The exclusion criteria were: (1) in vitro animal studies; (2) interventions using only non-human-derived stem cells; and (3) studies using mixed interventions with additional treatments such as specified growth factors.

Objective and self-reported outcome measures (Table 1) were retrieved from studies that met the inclusion requirements.

Table 1 Outcome Measures.

The authors used the Population, Intervention, Comparison, Outcome, Studies (PICOS) criteria listed in Table 2 to identify relevant articles. All data taken from systematic searches were recorded in the 2020 PRISMA flowchart and evaluated using the PRISMA 2020 checklist.

Table 2 PICOS Strategies.

The data used in this review were secondary data obtained from included studies. Literature searches were performed using controlled vocabulary based on the PICOS framework to get general search terms (keywords). After selecting keywords and their synonyms using Medical Subject Headings (MeSH), searches of research journals were performed using advanced search, bibliographic searching, and Boolean operators (AND, OR, and NOT) on keywords arranged according to the research topic. The keywords and Boolean operators used were: (“mesenchymal stem cells” OR “mesenchymal stem cell” OR “mesenchymal”) AND (“corneal endothelial cell” OR “corneal regeneration”) AND (“animal model”).

Searches were performed for all dates up to January 2024 using the following reputable databases: PubMed, Cochrane Library, Science Direct, and Google Scholar. All relevant studies that met the inclusion criteria, but not the exclusion criteria, were used in this review.

Following a full-text analysis, the following data were collected from the included articles: title, author details, publication year, evaluated outcome(s), kind of animal model, animal weight, age, and sex, dosage administered, duration of research, mode of administration, and primary findings.

A synthesis matrix was used to collate all findings, together with each article’s weaknesses and strengths, enabling presentation of the data in a structured manner. After reviewing the data, descriptions and conclusions could be made regarding the similarities and differences among the findings. The research results were then reviewed again with a supervisor and examiner to ensure more precise, accurate, consistent, and unambiguous analyses and presentation of the data.¹¹

Risk of bias and quality of the animal studies were evaluated using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) Risk of Bias instrument. The SYRCLE technique is a modified version of the Risk of Bias tool, made available by the Cochrane Collaboration, and intended to translate risk assessments from human research to animal studies. The SYRCLE tool comprises 10 items that are linked to biases in reporting, attrition, detection, performance, and selection. Overall bias is divided into low risk of bias, unclear risk of bias, and high risk of bias based on assessor assessments in five domains.¹² The risk of bias for each study was thoroughly evaluated by two researchers. In case of disagreements among the researchers, an impartial third researcher made the final decision. Relevant studies were eliminated from the study if the estimated risk of bias was high.

Data analysis was performed by systematically integrating and describing all data to obtain conclusions. The data consisted of study characteristics (name of primary author, year of publication, and research location), participant characteristics (animal type, age, and weight), and pre-determined outcomes. All data from the analysis are presented in Table 3.

Table 3 Characteristics of the Studies.

The literature search found a total of 1,493 studies relevant to this review. Referring to the PRISMA flowchart (Figure 1), 1,294 records were excluded prior to screening in line with the inclusion and exclusion criteria specified by the tools in each database. A total of 141 studies were disqualified after reviewing the title and abstract, 14 studies could not be retrieved, and 31 studies were excluded due to unclear stem cell intervention, unclear corneal endothelial model, and non-in vivo animal studies.

A total of 13 studies were included in this review.¹³–²⁵ Based on the SYRCLE Risk of Bias instrument, all 13 studies had a low risk of bias.

Of the 13 included studies, 10 were in vivo studies, and three were combined in vivo and in vitro studies. No human trials were retrieved via the literature search process. Of the 13 studies, 3 were from Spain; 2 each were from China, South Korea, and USA; and 1 each from India, Italy, Malaysia, and Turkey. Most administration routes included in the studies were topical application and anterior chamber injection. The duration of the studies varied from 48 hours to several months. Table 3 provides an overview of the included studies.

The studies reported that MSCs increased CEC density, improved the defect area and corneal transparency, facilitated endothelial cell regeneration and wound healing, and decreased neovascularization and corneal pro-inflammatory cytokines compared to the control. The study outcomes are provided in Table 4.

Table 4 Study Outcomes.

This literature review has shown that MSCs have the potential to regenerate CECs. Depending on their origin, stem cells may be classified as adult or embryonic stem cells and have the ability to develop into MSCs.⁸ They interact with both innate and adaptive immune cells and are essential for regulating immune responses via paracrine signaling.²⁶ The role of MSCs vary, depending on their source: both bone marrow and adipose MSCs exhibit the phenotypic markers CD13, CD73, CD90, CD105, and STRO-1; however, their CD34, CD49d, CD54, and CD106 expression patterns are distinct. Compared to MSCs from birth-associated tissues, MSCs obtained from adult tissues such as bone marrow have lower proliferation, engraftment ability, and differential potential.⁸ In addition, MSCs can also have molecular derivatives, namely exosomes that function in paracrine signaling between cells directly. Exosomes play an important role in intercellular communication, which affects the cellular environment; in the context of corneal therapy, exosomes help regulate tissue inflammatory responses, modulate cytokine and chemokine balance, and improve epithelial wound healing.²⁷

Because MSCs can control immune responses, they are more effective as therapeutics and less likely to be rejected in both the in vitro and in vivo environments. Believed to be immune-privileged cells, the cell surfaces of MSCs have fewer major histocompatibility complex class II molecules and co-stimulatory molecules (CD80, CD86, and CD40). Mesenchymal stem cells influence the innate immune system in vitro by inhibiting the cytotoxicity of natural killer cells and developing and activating dendritic cells. Additionally, they impede the maturation of B and T cells and their ability to proliferate and secrete cytokines, thereby suppressing adaptive immunological responses.²⁸ In addition, scientists are investigating in-cell treatment as a potential substitute for corneal transplantation, which is sometimes hindered by a lack of donors. It has been shown that corneal endothelium transplantation is a feasible alternative since rho-associated protein kinase (ROCK) can renew and repair CECs.⁸

The rho protein is an essential regulator of skeletal structure. It is hypothesized that ROCK signaling inhibition may influence cell adhesion characteristics, and CEC transplantation may be used as a therapeutic intervention in regenerative medicine. Corneal transparency was enhanced in a rabbit with endothelial failure by transplanting CECs with a ROCK inhibitor. This approach may be used in human clinical trials despite using an animal model for this work.¹⁰ Using Descement membrane biomimetic microphotography, human MSCs may be induced to develop into corneal endothelial-like cells. The ZO-1 and Na/K-ATPase proteins and COL81, COL8A2, and PITX2 genes are exclusive to the endothelium and may all be expressed by MSCs.²⁹

Mesenchymal stem cells derived from the lining of the umbilical cord and bone marrow can differentiate into keratocyte-like cells and may be able to reinstate transparency to the corneal stroma. Injecting human umbilical cord lining MSCs into the corneal stroma improved the aberrant collagen structure, restored corneal thickness, and enhanced corneal transparency. Furthermore, there was little chance of rejection since the injected cells reduced the inflammatory cytokine levels.³⁰

A limitation of this review is that it only included English-language literature; hence, some of the unincluded studies may have had skewed data. Also, since use of MSCs is a contentious strategy, no studies could be located that used human models.

Although MSC therapy holds promising potential in treating corneal tissue-related diseases, several challenges must be addressed before widespread clinical application can be achieved. First, there is currently no standardized protocol for isolating and characterizing MSCs across studies, making it difficult to conduct valid comparative analyses of MSCs treatment efficacy. Therefore, future research should focus on establishing standards for MSCs isolation and characterization, including determining effective administration methods, such as intrastromal, subconjunctival, and topical approaches.

Second, the challenge of maintaining the survival of transplanted MSCs in corneal tissue remains significant. While several studies reviewed here reported favorable long-term outcomes, the results varied considerably. In consequence, future research should also focus on strategies to enhance long-term MSCs survival, such as genetic modification or preconditioning.

Third, in vivo efficacy measurements of MSCs, through to clinical trials, are still limited. This may be due to resource constraints and limited patient availability, which could impact the findings on the safety and efficacy profiles of the therapy. Nonetheless, these challenges are likely surmountable, allowing the continued development of MSC therapy to show increasingly promising results.

In conclusion, the administration of MSCs both topically and by anterior chamber injection could increase regeneration and proliferation of corneal endothelial tissue in animal models based on histological and molecular findings (cytokine and growth factors). Further research is needed to evaluate the application of human-derived stem cells in both animal and human populations.

The authors would like to thank all relevant parties for the opportunity and facilities provided to carry out the research so that this review could be completed.