Chapter

Stem Cells for Therapy of Eye Diseases: Current Status

Zala Lužnik Marzidovšek, Janina Simončič, Petra Schollmayer, Elvira Maličev, Primož Rožman and Marko Hawlina

Abstract

In recent years, the treatment of various ocular diseases using stem cells and stem cell-derived exosomes has rapidly evolved. In this chapter, we present the potential of different stem cells and their secreted extracellular vesicles for the treatment of ocular diseases based on a careful review of relevant pre-clinical and clinical studies. The regenerative and immunomodulatory capacity of stem cells is analyzed together with the complex role of extracellular vesicles in intercellular communication, regulation of inflammation, and tissue repair. In addition, the inevitable chal-

lenges in clinical translation and ethical considerations are presented. Thus, in this chapter, we highlight the importance of current advances in the field of stem cell- based therapy in ophthalmology, with a growing body of evidence confirming the potential of these therapeutic modalities not only to restore vision but also to inhibit the progression of various ocular diseases, promising a significant improvement in quality of patients’ life.

Keywords: ophthalmology, stem cell therapy, regenerative medicine, extracellular vesicles, clinical trials

1. Introduction

The human eye is one of our most important sensory organs, which allows us to see. The anatomy is complex and can be divided into anterior and posterior struc- tures (Figure 1). Anterior structures such as the cornea and conjunctiva are in direct contact with the external environment [1, 2]. The transparent cornea enables the transmission of light through the crystalline lens to the innermost light-sensitive neu- rosensory structures of the eye. The retina is accountable for the visual processing of light energy into an electrical signal that is transmitted via the optic nerve to the brain [1]. Several eye diseases and trauma can affect the normal function of these major ocular structures and can lead to progressive vision loss or blindness, severely affect- ing individual’s mobility, independence, and quality of life [3]. Some of the most common and debilitating eye diseases are severe ocular surface disease, glaucoma,

Figure 1.

Schematic cross-section of a human eye with an expanded view of the cornea (on the left) and retinal layers (on the right).

macular degeneration, autoimmune and genetic eye diseases [3], for which, there is currently available only symptomatic treatment.

Thus, extensive research effort is being put into the development of new preven- tive and causative therapeutic options such as advanced therapy medicinal products (ATMPs). ATMPs are medical products based on genes, tissues, or cells [4], including various stem cell (SC) and extracellular vesicle (EV) therapies [4, 5]. These new

SC-based therapies offer the possibility of permanently restoring or replacing previ- ously irreparable tissues or organs and have several well-described paracrine func- tions that can prevent or halt disease progression [5]. In recent years, the therapeutic potential of cell-free therapy using SC-secreted EVs has gained tremendous interest and considerable promise in regenerative medicine [6]. Due to its relative ease of accessibility, ocular immune privilege, and the accessibility of non-invasive diagnos- tic methods to follow-up and visualize eye structures after therapy (e.g., slit-lamp examination, optical coherence tomography, and in vivo confocal microscopy) pre- dispose the human eye to be a prime target for SC-based therapy development [7, 8].

As presented in this chapter, we provide an overview of the current clinical advances in various SC-based therapies for treating several ocular diseases. Although published clinical studies using the therapeutic potential of EV secreted by SCs

are currently still rare, with most reports in the pre-clinical stages, we addition- ally included some promising pre-clinical studies as this cell-free therapy may have several advantages over conventional SC therapies, as presented below.

2.  Stem cell biology and types

Stem cells are undifferentiated or unspecialized cells present in the embryonic, fetal, and adult human body. The SC-specific characteristics are self-renewal and their ability to differentiate into different cell types, enabling embryonic development and, in adult life, tissue regeneration and repair after injury [9]. They can be classified into two types depending on their source. Embryonic or adult (somatic) SCs [10].

Another classification is based on their potency; the varying ability of SCs to dif- ferentiate into specialized cell types; and classifies SCs into totipotent (e.g., a zygote), pluripotent (e.g., embryonic SCs and induced pluripotent SCs), multipotent (e.g., hematopoietic SCs), oligopotent (e.g., myeloid SCs), or unipotent SCs (e.g., corneal/ limbal epithelial SCs) [10]. Totipotent SCs can form embryonic and extra-embryonic tissues, whereas pluripotent SCs can form all germ layers but not extra-embryonic

structures such as the placenta [10]. Another example of pluripotent SCs is artificially produced induced pluripotent SCs (iPSCs), which can be produced by reprogram- ming adult cells into a pluripotent state [11].

The differentiation potential is a continuum, starting from completely pluripotent SCs such as embryonic SCs or iPSCs and ending on representatives of adult SCs with much narrower spectrums of differentiation (e.g., multi-, oligo-, or unipotent SCs) [10]. Adult SCs can be found in various tissues and organs (e.g., skeletal muscles, brain, bone marrow, dental pulp, liver, spinal cord, cornea, and fat tissue) in specific microenvironments called SC niches [9], usually being able to differentiate into a limited number of terminated cell types allowing the maintenance/repairing of adult body tissues and organs.

Among these, one of the most important adult SC sources is multipotent mes- enchymal stem cells (MSCs) that play a vital role in tissue repair and regeneration and can modulate immune responses via paracrine function [7, 12]. To date, they are the most commonly used SCs in clinical trials (https://clinicaltrials.gov/) and can be isolated from various adult as well as fetal tissues (such as placenta, Wharton’s jelly, and umbilical cord blood) [12]. Thus, the International Society for Cellular Therapy (ISCT) has proposed a set of minimal in vitro criteria to define MSCs as cells that

(a) can adhere to plastic, (b) express specific markers such as the cluster of dif-

ferentiation (CD) 73, CD90, CD105, and lack the expression of CD14, CD34, CD45, and human leucocyte antigen-DR (HLA-DR), and (c) differentiate into adipocyte, chondrocyte, and osteoblast cell types [13]. Adult MSCs are most commonly isolated from bone marrow and adipose tissues [7].

Another interesting SC source for the development of SC-based therapies is tissue- specific adults SC. In the human eye, adult SCs have been identified in various regions such as the cornea, trabecular meshwork, crystalline lens, iris, ciliary body, retina, choroid, sclera, conjunctiva, eyelid, lacrimal gland, and the orbital fat [14]. However, to date, only cultured corneal epithelial SC transplantation revolutionized the current treatment options for patients with advanced painful and vision-threatening ocular surface disease, such as total limbal stem cell deficiency (LSCD) [15], as described

in more detail below. A healthy corneal epithelium plays a vital role in the mainte- nance of corneal integrity and transparency, which is renewed by a small population of adult corneal epithelial SCs primarily found at the peripheral corneal area, the limbus (thus also named limbal epithelial SCs (LESC)) [2, 16]. Small populations of multipotent limbal mesenchymal or corneal stromal SCs have also been observed in the anterior limbal stroma [17] with increasing evidence suggesting a direct role of corneal stromal SCs in the provision of cells for corneal maintenance and regenera- tion [18]. Corneal stromal SCs could be used to regenerate the corneal stroma without a scar formation, thus preserving corneal transparency [19]. Although human corneal endothelium, the innermost corneal monolayer of neural crest-derived cells, has very low proliferative potential in vivo [20], recent in vitro studies also suggest the exis- tence of corneal endothelial SCs located in the transition zone between the peripheral corneal endothelium and the beginning of the non-filtering portion of the trabecular meshwork called the Schwalbe’s ring region, which might supply new cells for the corneal endothelium and trabecular meshwork [21]. Other eye-specific SC types were found also in the posterior segment of the eye in the retina [22, 23]. For example, Mueller cells display upon injury neural SC characteristics, capable of generating neurons and glial cells [22]. In addition, the retinal pigment epithelium also exhibits plasticity, with a subset of cells expressing SC features such as multipotency, poten- tially contributing to retinal repair [23].

3.  Stem cell-derived extracellular vesicles

Extracellular vesicles (EVs) are cell-derived lipid membrane vesicles of varying sizes that can be secreted by many cell types [24–26]. Depending on the size and their biogenesis, they can be classified as (1) exosomes (30–150 nm in diameter), released into the extracellular space by the intracellular budding of endosomes; (2) microves- icles (100–1000 nm in diameter), formed by budding of the cell membrane; and into apoptotic bodies (1000–5000 nm in diameter), compartmentalization of the cell during programmed cell death [24]. EV carries important bioactive molecules, such as cytokines, growth factors, signaling lipids, mRNAs, and regulatory miRNAs, that are involved in intracellular communication and several signaling cascades [27]. The specific EV cargo depends on the cell type of origin and can influence a wide variety of biological functions, such as cell proliferation, regeneration, migration, apoptosis, and immunoregulation [27].

By transferring mRNA or miRNA, EVs can influence new protein synthesis and modulate gene expression [27]. MSCs are known to secrete EVs, which are supposed to have similar paracrine therapeutic effects as the original SCs, thus could be safer to use clinically due to their cell-free nature and can also be administered in higher concentrations [28]. Moreover, by genetic engineering, the original MSCs could be modified, and new therapeutic factors could be introduced into their EVs, avoiding safety concerns [28]. However, although the therapeutic potential of EVs is promis- ing, the reproducibility, vesicle integrity, and maintenance of their biological activ- ity to ensure the final product homogeneity remains challenging [25]. Thus, new Minimal Information for Studies of Extracellular Vesicles (MISEV) 2023 guidelines have been published to update the experimental requirements for EVs definition and their functions [29].

4.   Clinical trials using stem cell therapy and stem cell-derived extracellular vesicles in various ocular disorders

The field of regenerative medicine using SC-based therapies holds immense promise for the treatment of debilitating ocular diseases. Based on encouraging results obtained from pre-clinical studies on animal models [30–38], several clinical trials utilizing various sources of SC therapies [6, 39–120] and a few studies using SC-derived EVs [121–125] emerged as novel approaches to address a wide range of ocular disorders. In this chapter, we focused on major pathologies with a large unmet medical need for new treatment strategies affecting the anterior segment (cornea), the retina (diabetic retinopathy, macular degeneration, and dystrophies), and the optic nerve (glaucoma).

4.1   Ocular surface and corneal regeneration

Corneal diseases represent the fifth leading cause of blindness worldwide, with approximately 4.5 million individuals being visually impaired due to loss of corneal clarity [126]. In case of treatable corneal blindness, the diseased or damaged corneal layers can be replaced by healthy donated corneal tissue (corneal transplantation

or corneal graft). Corneal transplantation remains the most common form of solid tissue transplantation [127] with about 180,000 transplants being performed annu- ally worldwide [128]. However, despite significant advances in surgery techniques

and tissue storage, there are still major issues related to the availability and quality of donated tissues and postoperative corneal transplant survival [126–128]. Moreover, if patient’s limbal area is severely compromised (e.g., LSCD disease), corneal transplan- tation is not curative in the long term as limbal epithelial SCs are absent in corneal grafts [129–131]. Thus, regenerative approaches using SC-based therapies provide a novel alternative treatment solution addressing severe corneal diseases such as LSCD, corneal stromal scarring, and endothelial dysfunction [132], as presented below.

• Cultured epithelial stem cell transplantation for limbal stem cell deficiency disease

Severe ocular surface damage may lead to complete vision loss due to ocular surface failure as a result from insufficient corneal epithelial renewal and stromal scaring, which is clinically referred to as LSCD [16, 133, 134]. LSCD disease develops due to loss or dysfunction of LESC, the ultimate source of corneal epithelium, and can result from various causes including trauma, chemical burns, autoimmune diseases, infections, and genetic disorders [16, 133, 134]. LSCD can be partial or total and can affect one or both eyes.

Management of patients with total LSCD requires SC transplantation for corneal epithelial restoration. In unilateral cases, an autologous limbal graft from the fellow healthy eye can be used, which carries a hypothetical risk of inducing LSCD to the donor eye [135]. In bilateral cases, donor limbal tissue is needed. In case of no avail- able or willing living related donor, the keratolimbal allograft (KLAL) transplantation can be performed, which uses cadaveric allogeneic limbal tissue as the source of corneal SCs and therefore allows for a larger SC supply [135]. However, the success rate of KLAL is limited by graft immune rejections that may develop despite intensive systemic immunosuppressive therapy in more than half of the cases [39]. As conven- tional surgical management of patients with total LSCD faces several challenges, a new approach using ex vivo cultured SC transplantation has been tried to be imple- mented into routine clinical practice since 1997 in many ophthalmological centers worldwide [40–73]. SCs are most commonly isolated and cultured from a small limbal biopsy of 1 to 2 mm² harvested from the peripheral corneal area (e.g., cultivated limbal epithelial transplantation (CLET)).

Autologous CLET was first documented by Pellegrini and colleagues in 1997

[53, 74] in two patients with LSCD caused by alkali burns and laid the foundation for several subsequent clinical studies. Until now, using several variants of autologous or allogeneic CLET technique, over 1250 eyes have been treated worldwide with promising clinical results including centers in Australia, Belgium, Germany, India,

Iran, Italy, Japan, Norway, Spain, Taiwan, Thailand, the United States, and the United Kingdom [73, 135]. Moreover, in 2015, the European Medicines Agency (EMA) approved “Holoclar” as the first SC-based therapy for LSCD therapy in the world

[75, 76], and in 2020, another autologous CLET product called “Nepic” was approved in Japan [77, 78]. In 2023, Jurkunas et al. published a case series of phase 1 clinical trial which enrolled five patients with unilateral LSCD using a new xenobiotic-, serum-, and antibiotic-free protocol for cultivated autologous limbal epithelial cell (CALEC) transplantation in the United States [73].

Long-term results of autologous CLET report improved corneal transparency and visual acuity, which is achieved in 50–86% [79], similar to conventional surgical approaches, but less invasive to the contralateral donor eye and with the ability to ex

vivo expand or amplify the SC pool pre-transplantation [29, 53]. Similarly, in bilateral

LSCD cases, allogeneic CLET reported complete re-epithelialization of corneas and significant visual improvements postoperatively [80]. In addition, two meta-analyses found no differences in success rates between autologous and allogeneic CLET

[81, 82]. The overall safety profile was favorable, with all side effects being amenable and transient to subsequent treatments [82, 83]; however, although allogeneic CLET is produced from small donor limbal biopsies with less immunogenic potential than the conventional KLAL transplantation (as fewer immune antigen-presenting cells are transplanted), systemic administration of immunosuppressive agents was needed to obtain good clinical results [81, 82].

Thus, other autologous SC sources such as oral mucosal cells or MSCs were sought for the ex vivo preparation of epithelial sheets in bilateral cases. Already in 2002, the first clinical study of autologous cultivated oral mucosal epithelial cell sheet transplanta- tion (COMET) showed successful corneal surface reconstruction in four patients with bilateral total LSCD [84, 85]. In 2021, the first COMET product was approved in Japan, called “Ocural” [86]. In a recent comparative analysis of long-term results of three epi-

thelial cell transplantation procedures comparing either CLET, COMET, or simple limbal epithelial transplantation, more than half of the patients achieved a stable ocular surface and visual acuity improvement up to 7 years postoperatively [74].

Another promising and potentially more sustainable SC source in ocular surface reconstruction is MSCs. Although MSCs have been widely studied in several animal disease models [127], to date, only a few research groups have reported clinical results with allogeneic bone marrow or autologous adipose-derived MSC transplantation

in restoring corneal epithelium in LSCD [6, 87–89]. Calonge et al. reported the first prospective, randomized, double-masked clinical trial to treat patients affected by LSCD with cultured allogeneic bone marrow derived-MSC transplantation (MSCT), which was compared to CLET [87, 90]. The corneal epithelial phenotype improved in patients treated with MSCT without significant differences to CLET, with no reported adverse events related to cell products [87, 90].

• Stem cell-based therapy in dry eye disease

Dry eye disease (DED) is a very common multifactorial disease characterized by a persistently unstable and/or deficient tear film causing discomfort and/or visual impair- ment, accompanied by variable degrees of ocular surface epitheliopathy, inflammation, and neurosensory abnormalities affecting millions of people [136]. Currently, there

are only a few reported clinical trials in which severe DED was treated with MSC- or MSC-derived EV-based therapy [121, 122]. In an open-label, prospective, phase I clinical trial, seven patients affected by aqueous-deficient DED were treated with a single dose of allogeneic adipose-derived SCs, which were administered directly into the lacrimal gland through transconjunctival injection. Improvement in clinical signs and symptoms without adverse events was seen. Another study successfully used allogeneic bone- marrow-derived MSCs administered by intravenous injection to treat Graft-versus-host disease (GVHD)–associated DED [121]. Zhou et al. reported successful topical use of EV obtained from human umbilical cord-derived MSCs as eye drops in a prospective clinical trial to treat GVHD-associated DED [122].

• Corneal stromal regeneration with stem cell-based therapy

The corneal stroma represents around 90% of corneal thickness and is com- posed of parallel collagen fibers (the extracellular matrix) and interspersed scarce

keratocytes [137]. The precise spacing of fibers is essential for stromal transparency [137]. Stromal keratocytes remain quiescent throughout life and are derived from the embryonic periocular mesenchyme that originates from the neural crest [137]. Corneal stromal SCs (CSSC), a distinct cell population, are located in the anterior stroma close to limbal niche and serve as a reservoir of progenitor cells that can differentiate into functional keratocytes, which are responsible for producing and organizing the extracellular matrix [137]. CSSCs respond rapidly to corneal injury and also play a vital role in preserving corneal avascularity and immune privilege [137]. They can be characterized as MSCs according to the in vitro characteristics and ISCT guidlines, however, with more specific differentiation potentials [137]. After injury or severe corneal infections, the accompanying inflammatory response and

fibrogenic growth factors activate keratocytes into fibroblasts and myofibroblasts ini- tiating a wound-healing response which ultimately results in the formation of stromal scars [137]. In addition, many corneal diseases such as dystrophies or ectatic corneal disorders affect physiologic corneal stromal anatomy and transparency [137].

Thus, to find an alternative to classical corneal transplantation, CSSCs are of particular interest in regenerative medicine, offering a new therapeutic approach for corneal stromal scar therapy (regeneration instead of transplantation, enhancing the tissue’s inherent but limited capacity to regenerate) [137].

CSSCs and MSCs have been widely studied in several animal disease models and were shown to restore corneal homeostasis and transparency. Although CSSCs might have greater differentiation potentials to differentiate into keratocytes as they are lineage committed compared to other MSCs sources [138], isolating CSSCs autologously is technically more demanding due to small amounts of obtained corneal tissues [6].

Previous studies also reported that extraocular sources of MSCs are capable to differenti- ate into keratocytes and produce new collagen in vitro and in vivo without inducing any inflammatory reaction [30] and can modulate pre-existing scars by stromal remodeling [31, 32]. For example, studies in rabbit LSCD models have shown that adipose-derived and dental pulp-derived MSCs improve corneal transparency, reduce inflammation, and promote re-epithelialization when transplanted to the cornea [33]. Similarly, intrastro- mal injections of umbilical cord-derived MSCs adopted the phenotype of corneal stro- mal keratocytes and improved corneal thickness and transparency [34]. Some research groups focused on iPSCs to generate healthy keratocytes in vitro [139].

However, to date, only Alió et al. [91–93] reported first promising results with autologous adipose-derived MSCs transplantation into a mid-stroma femtosecond laser-assisted lamellar pocket in patients with advanced keratoconus and poor visual function that were already candidates for corneal transplantation. Although they observed new collagen synthesis in the injected areas, it was quantitatively not enough to sufficiently restore corneal thickness [91–93]. Direct injection of MSCs might thus better provide a new treatment strategy for corneal scar modulation [6].

To treat severely thinned corneas, a better approach was to use decellularized corneal stroma as a carrier for MSCs [91]. During the 3-year follow-up, the authors noted no complications with a transparent cornea in all patients [94]. The authors also sug- gested that while SC-free therapy with MSC-derived EVs could potentially achieve a similar therapeutic effect, the in vivo production of extracellular matrix would prob- ably still require the implantation of cells [6]. In India, a clinical trial is assessing the efficacy and safety of human donor cornea derived allogeneic CSSCs delivered to the ocular surface embedded into a fibrin gel for the prevention and treatment of corneal scars in pathologies such as corneal burns and ulcers with promising preliminary results [6].

• Cultured human corneal endothelial cell transplantation

Corneal endothelium has very low proliferative potential in vivo and a gradual decline in endothelial cell density is observed during adulthood due to age-related cell death [140, 141]. In case of substantial endothelial cell loss or dysfunction, pathological corneal hydration (termed ‘edema’) occurs, leading to visual impair- ment [140, 141]. To date, the best treatment option is a corneal transplant [142]. Due to advancements in corneal transplantation surgery selective replacement of dysfunctional corneal endothelium enables fast visual rehabilitation in patients with corneal endothelial disease [142]. However, in many parts of the world, a shortage of donor corneas limits access to treatment, prompting a search for alternatives. One recent new strategy that can be performed only in patients with a healthy peripheral endothelium is central Descemetorhexis without endothelial transplantation, which necessitates migration of peripheral endothelial cells to cover the denuded stromal area. Thus, slower visual rehabilitation is expected [142]. Therefore, new clinical tri-

als evaluating cultured human corneal endothelial cells, which aim to ex vivo multiply

endothelial cells from a single donor, are studied [142].

Early attempts to culture human corneal endothelial cells began in the early 1980s with several published protocols [143]. As endothelial cells are growth arrested in vivo, it is extremely challenging to get them to proliferate in sufficient quantities

in vitro without undergoing endothelial-mesenchymal transition or premature

senescence [144]. In 2004, Amano and colleagues presented the potential of primary cultured human corneal endothelial cells isolated from explant cultures to adhere to human corneal stroma deprived of endothelial cells [145]. Refinements in protocols for in vitro endothelial cell expansion culminated in a pioneering clinical trial. In 2018, Kinoshita and colleagues achieved a significant milestone by restoring vision in patients with bullous keratopathy using cell injection with 1 × 10⁶ passaged cultured endothelial cells supplemented with a Rock inhibitor [95]. At 5 years follow-up, normal corneal endothelial function was reported in 10 of 11 eyes [96]. Currently, there are a few other clinical trials studying the safety and efficacy of cultured human corneal endothelial cells with limited reports in the published literature: one in El Salvadore (NCT05309135) using human cultured corneal endothelial cells and Rho kinase Y-27632 inhibitor injection; one in Singapore (NCT04319848) using a tissue engineered graft consisting of a biological carrier and tissue engineered cells; and one in the United States (NCT04894110) using technology integrating biocompat- ible magnetic nanoparticles with cultured endothelial cells; similarly one other study in Mexico also using cell injection approach with human corneal endothelial cells laden with magnetic particles; and the last in India using an ultrathin carrier [96].

In addition, pre-clinical studies explore alternative sources for corneal endothelial cell differentiation from human embryonic SCs and iPSCs [146] or MSCs-derived EV with cytoprotective properties to induce proliferation and survival of damaged human corneal endothelial cells [123].

4.2   Retinal degenerative disorders

The neural retina is composed of several layers of neurons interconnected by synapses. The outermost cell layer consists of photoreceptor cells, which are sup- ported by a layer of pigment epithelium cells. Photoreceptor cells are of two types, rods, and cones, and are essential components of the retina responsible for light perception. While rods function in low light conditions, cones are activated in bright

light, particularly in the macula, the central region of the retina crucial for high acuity and color vision [147]. The innermost retinal layers (closest to the vitreous body) are formed by the ganglion cells (ganglion cell layer) and their axons that form the nerve fiber layer of the optic nerve [147]. Retinal degenerative disorders represent a major global health problem due to permanent vision loss characterized by pathological degeneration or loss of various retinal cells such as photoreceptors, retinal ganglion cells (RGC), and/or retinal pigment epithelium (RPE) with an inability to regenerate in vivo [148]. Therefore, cell replacement therapy emerges as a promising new thera- peutic avenue for retinal degenerative disorders such as advanced retinitis pigmentosa (RP), age-related macular degeneration (AMD), and diabetic retinopathy (DR), as presented below.

• Diabetic retinopathy

Diabetic retinopathy, a complication of diabetes that causes damage to the blood vessels in the retina, is one of the leading causes of blindness in the developed world in working-age population [97] DR can be classified into non-proliferative (NPDR) and proliferative DR (PDR). Early stages are characterized by inflammation, microg- lial activation, loss of endothelial cells and pericytes, loss of neuronal cells, and dam- age to the retinal microvasculature integrity [97]. With the progression of the disease, macular edema can lead to vision loss, and retinal ischemia stimulates new vessel growth and vision-threatening PDR [97]. Current treatment options include laser photocoagulation of ischemic peripheral retinal regions, intravitreal anti-VEGF, and glucocorticoid therapy, all of which can minimize vision loss if conducted promptly, but may have adverse side effects [97]. Thus, SC-based therapy is a promising new therapeutic option to replace lost endothelial cells or pericytes in DR, which are important for the maintenance of the inner blood-retinal barrier and vascular func- tion, or to provide trophic factors and cytoprotective support to retinal neurons [97].

Several clinical trials (available at: https://www.clinicaltrials.gov) are currently underway to investigate the potential of SC-based therapies for DR. In one clinical study (NCT01736059), subjects with irreversible vision loss from retinal degenera- tive diseases or retinal vascular disease, including DR, are receiving intravitreal injections of autologous CD34+ bone marrow MSCs. The purpose of another trial (NCT03403283) is to determine if the reparative cells of blood vessels, called endo- thelial progenitor cells, are defective in people with diabetes. A third clinical trial (NCT03403699) is recruiting patients with diabetes and age-matched controls to generate iPSCs from the peripheral blood cells to be further used to generate meso- derm cells for injection into the vitreous cavity of diabetic rodents and primate eyes. In another recent pilot clinical trial, the safety and efficacy of intravenous autologous bone marrow-derived MSCs for the treatment of DR were evaluated [98]. Thirty-four eyes with non-proliferative or proliferative DR from 17 patients received one intra- venous infusion of 3 × 10⁶/kg cells. Treatment led to decreased fasting blood glucose and CRP levels, with improved visual acuity observed in the NPDR group [98]. No acute reactions or severe adverse events were reported, which suggests this treatment method is a safe and potentially effective option [98].

In addition, DR might also be improved after intravitreal injection of MSC exosomes, as demonstrated in a mouse model. In an animal model, Moisseiev et al. improved oxygen-induced retinopathy by the intravitreal injection of exosomes from human MSCs, as there was a suppression and amelioration of retinal ischemia and damage [35].

These trials thus represent significant steps toward understanding and potentially treating DR and related ocular conditions through innovative SC-based approaches.

• Retinal dystrophies

Retinal dystrophies encompass a range of degenerative diseases affecting the retina with high genetic and clinical heterogeneity including syndromic or non- syndromic cases [149]. Typical symptoms may include impaired color or night vision and tunnel vision [149]. Vision impairment usually increases with age and eventually can progress to complete blindness [149]. Retinal dystrophies can be categorized depending on the type of photoreceptor affected into rod-dominated diseases,

cone-dominated diseases, or generalized retinal degenerations involving both rod and cone photoreceptors [149]. One of the most common rod-cone retinal dystrophies

is retinitis pigmentosa (RP), with high genetic heterogeneity [149]. It manifests as progressive night blindness (loss of the ability to see in dim light), followed by loss of peripheral vision that slowly progresses toward the center, resulting in tunnel

vision [149]. Stargardt disease (STGD) is the most common cause of juvenile macular dystrophy [150]. It is a genetic disorder commonly inherited as an autosomal recessive trait that leads to the accumulation of lipofuscin in the retina, ultimately leading to retinal pigment epithelium (RPE) and photoreceptor cell death [150]. There are vari- ous other forms of retinal dystrophies; however, a more detailed description is beyond the scope of this chapter.

To date, there are limited treatment options for most retinal dystrophies, which usually progress to vision loss; thus, SC-based therapies might enable replacement or regeneration of the damaged cells (RPE or photoreceptor cells) in the near future.

The first clinical trial in humans was done by Schwarz et al. in 2012 [99] and 2015 [100]. In a study by Li et al., human ESC-derived retinal pigment epithelial (hESC- RPE) cell suspension was injected subretinally in seven early-stage Stargardt disease patients [101]. Over 60 months after transplantation, no systemic or local adverse reactions were observed, except for transiently high intraocular pressure in two cases, with improved or stable visual acuity [101]. Thus, the authors concluded that hESC- RPE transplantation in early-stage Stargardt disease was safe and well-tolerated [101]. Similarly, in another study, no serious adverse events over a 3-year period were found, suggesting long-term safety and feasibility of subretinal hESC-RPE transplantation, with the majority of patients remaining stable visual acuity [102]. However, in a study reported in 2023, which was conducted in 12 eyes with advanced Stargardt disease, no significant improvement in best-corrected visual acuity was observed during 1 year follow-up period after hESC-RPE transplantation [103]. A prospective clinical case series investigated the safety and efficacy of suprachoroidal adipose tissue-derived MSCs implantation in four patients with Stargardt disease with promising 6-month follow-up results [104]. Weiss and Levy reported recent results from the Stem cell ophthalmology treatment study (SCOTS) in which autologous bone marrow-derived MSCs in the treatment of Stargardt disease were used by various local ocular or systemic intravenous injections, concluding that the treatment might be beneficial after 1 year follow-up period [105]. Another study evaluated the therapeutic potential and safety of intravitreal use of a bone marrow mononuclear fraction containing CD34+ cells in patients with Stargardt-type macular dystrophy and showed discrete improvements in visual acuity and microperimetry in the treated eye compared to the untreated one [106].

In advanced RP, one study examined the safety of subretinal adipose-derived MSC implantation in 11 patients [107]. While systemic complications were absent, choroi- dal neovascular membrane and epiretinal membrane formation were observed [107]. Although no significant changes were noted in visual acuity or electroretinography recordings, one patient showed visual acuity and visual field improvement, and three reported enhanced brightness perception [107]. The findings highlight the short- term safety of this treatment but emphasize the need for caution in their application, suggesting further studies to optimize delivery techniques and evaluate long-term effects [107]. In a phase I clinical trial with 14 participants having advanced RP, intravitreal bone marrow-derived MSC injection was evaluated [108]. Mild, transient adverse events were noted during 12 months of follow-up [108]. While at the begin- ning, visual acuity improved significantly, it returned to baseline levels at 12 months; however, other tests did not indicate any disease progression. In one patient, diffuse vitreous hemorrhage required surgical intervention, which then resulted in restored vision [108]. Another phase 3 clinical study included 82 RP patients and reported promising short-term results (improvements in visual acuity, visual field, and electroretinography recordings with no serious complications) using suprachoroidal injection of umbilical cord-derived MSCs [109].

A recent three-level meta-analysis study identified seven cell-based therapy studies for Stargardt disease (including 40 eyes) and two studies for RP (including 44 eyes) until 2023, and their analysis highlighted that the application of cell therapy could enhance visual acuity in treated patients [148].

• Age-related macular degeneration

Age-related macular degeneration (AMD) is a prevalent chronic retinal degenera- tive disorder characterized by pathological alterations in the macula and its adjacent vasculature (abnormalities in the photoreceptor/RPE/Bruch’s membrane/choroidal complex), predominantly affecting the elderly population [151]. It leads to central vision loss, often resulting in geographic atrophy and/or neovascularization [151].

AMD can be classified into two types: dry and wet [151]. While dry AMD is more common, wet AMD typically leads to more severe vision loss and usually develops over a period of weeks to months [151].

Since Peyman et al. first described transplantation of RPE as an intact sheet in 1991 [152], tremendous progress has been made in the field of regenerative medicine, with various SC-based treatment options being clinically tested for AMD [148]. The first clinical trials in humans were done by Schwarz et al. in patients with AMD and Stargardt disease [99]. Successful integration of hESC-derived RPE cells into the host RPE layer was observed without abnormal growth or immune rejection [99]. Vision improved in both patients, highlighting the safety and therapeutic potential of hESC- derived RPE cells for retinal degenerative diseases [99, 100]. Since then, in a recent meta-analysis study, already 140 eyes in 12 clinical studies underwent various treat- ment strategies including suprachoroidal [104], intravitreal, subretinal, subfoveal

[110] cell suspension injections or subretinal implantation of a bioengineered sheet of cells on human-vitronectin-coated polyester membrane or on a perylene membrane [111, 148]. Da Cruz et al. demonstrated safety and feasibility of subretinal transplan- tation of a manufactured hESC-RPE monolayer on a synthetic basement membrane (human-vitronectin-coated polyester membrane) for the treatment of wet AMD in two patients [112]. In most clinical studies, RPE cells were differentiated from ESCs

[110, 113–115] or included adipose-derived [104], umbilical cord-derived [116] or bone marrow-derived MSCs [105, 106]. Another promising SC source is iPSC, as they have the capacity to generate unlimited and readily available autologous RPE cells.

These pluripotent SCs can proliferate indefinitely in laboratory settings while retain- ing their ability to differentiate into various cell types [117, 118]. Takagi et al. reported a case of iPSC-derived RPE cell transplantation in a patient with wet AMD [117].

Although the treatment did not improve vision, it remained stable for 4 years after transplantation without significant side effects [117].

Advancements are also observed in research focusing on EV use in retinal dis- eases. In China, a clinical trial is registered and currently ongoing (NCT03437759) to evaluate the safety and efficacy of MSC-derived exosomes in promoting the closure of macular holes (full-thickness defect in the macula). The trial involves 44 participants diagnosed with early phase 1 macular holes, who will receive MSC-derived exosomes via local intravitreal injection; however, the results have not been published yet.

4.3   Optic nerve

Retinal ganglion cells (RGCs) are central nervous system neurons located in the innermost layers of the retina, responsible for transmitting visual signals from the retina to the brain via their axons [119]. RGC axonal injury leads to neuronal death and loss of function with limited capacity to regenerate [119]. Successful optic nerve regeneration requires a source of healthy RGCs to replace damaged cells [119]. Research into nervous system regeneration is particularly intriguing and is still primarily conducted using animal models [119].

The first step in developing SC-based strategies for optic nerve regeneration requires a reliable and high-volume source of healthy RGC production [153].

Currently, there are two main methods for generating RGCs from pluripotent SC (hESC or iPSCs): organoid differentiation, which involves the development of self-organizing three-dimensional miniature organs in vitro from pluripotent SCs, and planar differentiation, where RGCs are trans-differentiated from SCs [153].

Unfortunately, simple intravitreal injection of cultured RGCs is not enough for their integration into proper retinal layers [153]. However, research by Wu et al. dem- onstrated that human iPSCs promote the survival of transplanted retinal ganglion cells (RGCs) and enhance neurite extension. Their ex vivo studies showed that RGCs cotransplanted with iPSCs onto adult rat retina explants had significantly better survival and longer neurite growth compared to RGC-only transplants [154].

• Glaucoma

Glaucoma, a common optic neuropathy, is the leading cause of irreversible blindness [119]. Current glaucoma treatments focus on lowering intraocular pressure [119]. In the context of new glaucoma treatment strategy development, cell therapy has three primary goals: providing trophic and neuroprotective support, replacing lost cells and restoring function [119]. MSCs for glaucoma therapy have been widely investigated in animal models. Intravitreal injections of MSCs in a rodent model of glaucoma showed alignment of injected MSCs along the internal limiting membrane that survived for several weeks, promoting RGC survival [36]. A study compared human MSC treatment with their MSC-derived EVs alone in a rat model of optic nerve injury and concluded that cell treatment provided better RGCs neuroprotec- tion than EVs alone, suggesting that cellular-based therapies may offer superior

neurotrophic support [37]. In a study by Pan et al. [124], exosomes derived from umbilical cord MSCs were investigated in a rat model of optic nerve compression and revealed that while exosomes enhanced the survival of RGCs, they did not facilitate axonal regeneration. Results of another study showed that bone marrow MSC- derived EVs promoted significant neuroprotection of RGCs in a rat model of glau- coma and prevented thinning of the retinal nerve fiber layer [125]. Regular injections of EVs were effective, and RNA sequencing identified specific miRNA upregulated in these EVs, suggesting a potential mechanism for their neuroprotective function [125].

In another study, mouse iPSC and mouse ESC-derived RGCs were transplanted into both healthy and glaucomatous mouse retinas, with a remarkable survival and integration success rate exceeding 65% [38]. These transplanted RGCs exhibited prolonged survival for up to 12 months post-transplantation [38]. Notably, the transplanted cells demonstrated the ability to polarize within the host retina and form axonal processes [38].

To date, only one clinical study investigated the effect of a single intravitreal injection of bone marrow-derived MSCs in two patients with advanced-stage open- angle glaucoma (NCT02330978) [120]. Despite treatment, no improvements were observed in visual acuity or visual field [120]. ERG tests conducted at baseline and subsequent weeks showed stable responses in one patient, while the other developed retinal detachment with proliferative vitreoretinopathy; thus, further research is warranted [120].

5. Future directions and conclusions

Advances in regenerative medicine, including the development of new SC-based therapies, have revolutionized the treatment possibilities of some ocular disorders, as discussed in this chapter; however, several issues remain to be overcome before these methods can be used routinely in day-to-day clinical practice.

The utilization of pluripotent ESCs has been ethically and politically controver- sial since 1998 because it involves the use of human preimplantation embryos [10]. This hurdle can be overcome with the use of human iPSCs; however, their produc- tion is very costly and still prevents a more widespread use. Another concern with pluripotent SC transplantation is their growth control after transplantation and, thus, their possible role in malignant transformation [10]. It is, therefore, crucial to perform strict tumorigenicity testing before clinical application. Although a healthy eye is known as an immune-privileged site, it can be lost due to ocular disease states. Thus, transplantation of allogeneic SCs might need proper immunosuppression to be successful.

Another important and still unanswered challenge in treating retinal and optic nerve diseases is how to establish functional connections and proper integration of transplanted cells into the diseased tissue to enable the formation of a coherent visual image [155]. Although EV could overcome some of these issues and enable cell-free therapy, a better understanding of the precise therapeutic mechanisms is needed, as well as strict toxicological and microbiological studies, optimization of their thera- peutic cargo, etc. [156].

As SC-based therapies are classified as advanced therapy medicinal products, the critical quality of the final product needs to be strictly defined with standardization and optimization of work protocols before clinical use. Thus, future directions might include research that would enable more widespread availability of the treatment

by providing GMP-compliant, accessible, reliable, validated, pretested, and robust SC-based therapies for treating various advanced ocular diseases.

To sum up, cell therapy for retinal dystrophy develops rapidly; however, we should remain cautious with the completion of all necessary safety studies before treating patients. We should warn patients against practices that offer quick solutions on the Internet and are charging high prices for unapproved treatments for these potentially blinding diseases. In the coming years, results from well-conducted clinical trials will determine the best strategies of treatment for each of these diseases as well as the modes of immune suppression when required.

Acknowledgements

Funding was provided by the Slovenian Research Agency (project number J3-50107).

Conflict of interest

The authors declare no conflict of interest.

Author details

Zala Lužnik Marzidovšek¹*, Janina Simončič², Petra Schollmayer¹, Elvira Maličev³, Primož Rožman³ and Marko Hawlina^1,4

• Eye Hospital, University Medical Centre Ljubljana, Ljubljana, Slovenia
• Medical Faculty, University of Ljubljana, Ljubljana, Slovenia
• Blood Transfusion Centre of Slovenia, Ljubljana, Slovenia
• Department of Ophthalmology, Medical Faculty, University of Ljubljana, Ljubljana, Slovenia

*Address all correspondence to: zala.luznik@kclj.si

© 2024 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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