2.1.1. Source from cultivated limbal epithelium

Treatment for LSCD primarily relies on limbal autografting techniques, including conjunctival limbal autografting (CLAU), cultivated limbal epithelium transplantation (CLET), and simple limbal epithelial transplantation (SLET).⁵

CLAU, first described by Kenyon and Tseng in 1989, is associated with various complications such as infection, scarring, and chronic inflammation due to the large graft area.⁶ To improve treatment outcomes and mitigate risks, several improvements, including CLET and SLET, have been developed utilizing either autologous or allogenic tissue. Reports have shown that CLET requires a smaller dose of donor’s limbal epithelium, reducing the risk of complications, and favorable results have been reported.7, 8, 9, 10 Some researchers reported that autologous CLET has high long-term survival rates and greater safety compared to allogenic CLET.¹¹ In some cases of partial LSCD where stem cell transplantation may not be necessary, Sharma et al. have suggested the use of amniotic membrane transplantation (AMT) alone as a therapeutic modality, which has comparable curative effect to cultivated LSCs transplantation.¹² Sangwan et al. introduced a novel surgical technique called SLET, involving the extraction of a small biopsy of healthy eye limbal tissue. This tissue is then divided into 8–10 pieces and placed on fresh human amniotic membrane adhered to the diseased eye’s scraped corneal bed. SLET simplifies the procedure as cell expansion occurs in vivo, reducing treatment time and costs.¹³ An ongoing clinical trial (ISRCTN12217540) is comparing the effectiveness of CLET and SLET in treating LSCD in ocular burn patients. Another innovative approach involves the transplantation of LSCs cultured on the concave surface of a siloxane-hydrogel extended-wear contact lens, which has shown promising results. The contact lens approach offers the advantages of being autologous, xeno-free, cost-effective, and easily accessible.¹⁴ Additionally, Miguel et al. have developed a tissue-engineered scaffold using a fibrin and agarose mixture, where corneal epithelial cells, corneal fibroblasts, and corneal endothelial cells are co-cultured.¹⁵ These co-cultured fibrin-agarose scaffolds exhibit optimal mechanical and optical behavior similar to native human corneas, and have demonstrated high biocompatibility in animal models.¹⁶

2.1.2. Source from cultivated conjunctival epithelium

Besides the corneal epithelium, the conjunctival epithelium can be used as an alternative source of cells for ocular surface reconstruction. Conjunctival epithelium and corneal epithelial cells are the most similar stratified epithelial tissues that exist in the body.¹⁷ Patients with a range of ocular surface problems have had successful conjunctival regeneration through the transplantation of autologous conjunctival epithelial cells that are cultivated outside the body.^18,19 With positive results noted, this method has also been applied to corneal surface reconstruction in patients with LSCD.²⁰ In summary, the conjunctival epithelium, with its close resemblance to corneal epithelial cells, offers a promising cell source for ocular surface reconstruction.

2.1.3. Source from other stem cell resources

According to current knowledge, the pool of stem cells capable of regenerating corneal tissue for the treatment of LSCD encompasses OME, DPSC, HFSC, iPSC, and ESC. OME, a non-limbal cell type utilized for therapeutic purposes, has gained broad application in the treatment of LSCD, with researchers worldwide reporting encouraging outcomes.21, 22, 23, 24, 25, 26, 27 Instances of Stevens-Johnson syndrome, having an immunological basis and potentially affecting the oral mucosa, are included.²⁸ DPSCs share important characteristics with LSCs in the eye and can be administered via a contact lens.²⁹ The current study provides evidence that HFSCs can differentiate into a phenotype phenotypically similar to corneal epithelial cells for reconstructing the ocular surface in LSCD mice.³⁰ Owing to uncontrolled proliferation, non-specific differentiation, transplantation rejection, tumorigenesis, and ethical concerns, clinical trials applying ESCs as grafts have several limitations. Previous studies have demonstrated different strategies for corneal epithelial cell differentiation from human iPSCs.^31,32 Nevertheless, there is a lack of reported clinical studies on the use of iPSCs for treating LSCD.

5.1.1. Regenerative treatment for age-related macular degeneration (AMD)

AMD is a degenerative disease that leads to blindness and is primarily caused by the loss or dysfunction of RPE. The absence of RPE results in the degeneration of a majority of the photoreceptors located above, resulting in a significant and progressive loss of vision. Results from preclinical and clinical studies suggest that RPE replacement techniques may be able to impede the disease’s progression and restore vision. While attempts have been made to transplant primary RPE in humans, the outcomes have been inconsistent in terms of graft survival and improvement of visual acuity.⁸¹ Utilizing stem cell derived cells offers significant advantages, such as an almost limitless cell supply and the ability to control their differentiation to guarantee optimal safety and potency prior to transplantation.

To find out if transplanting hESC-derived RPE into the subretinal space is safe and tolerable, numerous prospective clinical investigations were carried out.82, 83, 84, 85 These studies have reported improvements in BCVA, indicating promising early results for the use of transplanted hESC-RPE cells in alleviating AMD. In a phase I clinical study, patients with dry AMD showed an improvement in vision from 21 ETDRS letters to 28 following subretinal transplantation of hESC-derived RPE. Additionally, within 4 months, the hESC-derived RPE cells showed no evidence of hyperproliferation, tumorigenicity or apparent rejection.⁸⁶ In another study, a highly mismatched donor RPE monolayer, implanted subretinally into a highly degenerate retina, demonstrated long-term survival and function for 2 years. The study also provided evidence of limited immunogenicity for the allogeneic hESC-RPE cells.⁸⁷ It is thought that a very modest number of CD8 cytotoxic T cells in the implant location let these highly mismatch RPE cells survive and avoid being destroyed. In addition to the subretinal injection of hRPE, Kashani et al. have invented a new clinical-grade retinal implant composed of hESC-derived RPE that is cultivated on an ultrathin, synthetic parylene substrate designed to mimic Bruch’s membrane.⁸⁸ Results at 1 year indicate that the implant is safe and well tolerated in individuals with advanced dry AMD.⁸⁹ Regarding cell delivery methods, the injection of RPE cell suspension has a drawback because it only distributes a small number of cells over a damaged basal lamina, with unpredictable results.^90,91 An alternative technique to the injection of RPE cell suspension is the transplantation of RPE-choroid sheets. Although there is a significant chance of postoperative complications with this approach, it provides a consistent cell sheet of polarized RPE on its basal lamina. Christiane and colleagues assessed the outcome of the two RPE transplantation methods.⁹² Their findings demonstrated comparable anatomical and functional outcomes for both techniques.

5.1.2. Regenerative treatment for Stargardt’s disease (STGD1)

Stargardt’s disease (STGD1) is one of the most common hereditary forms of juvenile macular degeneration. Initially, patients’ central vision is impaired. As the lipofuscin in apical RPE cells accumulate, the patients reported diminished vision, which results in secondary choroidal neovascularization and legal blindness.⁹³

In order to evaluate the long-term safety and effectiveness of subretinal injection of hESC-RPE cell suspension in the early stage of STGD1, many clinical trials were carried out,94, 95, 96 in which none of the patients experienced adverse reactions systemically or locally. However, changes of long-term visual function were mixed. Another study conducted a detailed analysis of retinal anatomy and function using microperimetry and spectral-domain OCT.⁹⁷ While no evidence of uncontrolled growth or inflammatory responses was found in any of the 12 participants, microperimetry indicated no benefits at 12 months for any of them. There was a possibility of injury since one person taking the maximum dose experienced localized retinal thinning and reduced sensitivity in the hyperpigmented region. Therefore, it is essential to consider the careful implementation of protective treatments following a longer duration of observation due to the slow rate of progressive degeneration in RDDs. In addition to hESCs, MSCs are also utilized for the treatment of STGD1. A phase II study involved the suprachoroidal implantation of ADMSCs in four patients. There were no signs of systemic or ocular complications, and all STGD1 patients showed improvements in visual field, and visual acuity during the six-month follow-up.⁹⁸ However, the study’s patient population size was insufficient to permit a conclusive statistical analysis.

5.2.1. Regenerative treatment for retinitis pigmentosa (RP)

RP encompasses a range of inherited diseases characterized by problems adjusting to darkness and night blindness in adolescence, loss of the mid-peripheral visual field in early adulthood, and eventual loss of central vision as a result of severe rod and cone photoreceptor degeneration.¹⁰³ RP is a prevalent hereditary retinal disease, affecting approximately 1 in 4000 individuals.¹⁰³

The first clinical trial to investigate the long-term safety of transplanting human fetal neuroretinal cells into RP patients was conducted by Taraprasad and colleagues in 1999.¹⁰⁴ The study reported no complications in the host eyes. While initial follow-up showed improved light sensitivity, this improvement diminished between 3 and 13 months. In a similar trial, Liu et al. observed significant improvement in visual acuity for five out of eight patients between 2 and 6 months post-RPCs injection, but this improvement was not maintained at the 12-month mark.¹⁰⁵ Another clinical trial demonstrated visual acuity score improvements for 50% of RP patients treated with neural RPC layers and RPE transplantation,¹⁰⁶ providing evidence of the safety and benefits of retinal implants. Using the questionnaire, Rubens et al. examined the quality of life of RP patients who underwent intravitreal injection of autologous bone marrow MSCs (ABMSCs). At the three-month mark, they observed a statistically significant increase in the patients’ quality of life, but this improvement did not last at the 12-month mark.¹⁰⁷ Administration of ADMSCs did not result in significant benefits and was associated with some ocular complications such as choroidal neovascular membrane and epiretinal membrane.¹⁰⁸ Additionally, a preliminary study investigating intravitreal administration of autologous bone marrow CD34⁺ cells demonstrated short-term safety, but its efficacy remains inconclusive.¹⁰⁹ Aekkachai et al. reported improvements in BCVA after intravitreal injection of bone marrow-derived mesenchymal stem cells (BM-MSCs) in participants with advanced RP, although the effects returned to baseline at 12 months.¹¹⁰ Another study reported infusion of human umbilical cord mesenchymal stem cells (UCMSCs) through the dorsal hand vein resulted in maintained or improved visual acuity for 81.3% of RP patients for 12 months.¹¹¹ A prospective phase-III clinical study evaluating the efficacy of subtenon space transplantation of Wharton’s jelly-derived mesenchymal stem cells (WJ-MSCs) enrolled 34 eyes from 32 RP patients. The study showed improvements in mean outer retinal thickness, horizontal ellipsoid zone width, and BCVA after 6 months, which remained stable at 1-year follow-up.¹¹² Ongoing research is investigating the use of NPC-derived astrocytes (NCT04284293) and NPC-derived RPE (NCT03073733 and NCT02320812) for the treatment of RP.¹⁰⁰

Despite promising results, one limitation of these studies is the challenge of obtaining sufficient human fetal retina donor tissue to treat large clinical populations.

5.2.2. Regenerative treatment for diabetic retinopathy (DR)

DR, a common complication of diabetes, is the main factor contributing to avoidable blindness in people in their working years.¹¹³ DR is classified as either proliferative or non-proliferative based on its stage of development.¹¹⁴ During the early non-proliferative stage, hyperglycemia damages the blood-retinal barrier, resulting in blood leakage. These changes initiate ischemic alterations in the surrounding retina. With the progression of hypoperfusion in the retinal capillary bed, proliferative diabetic retinopathy occurs. This condition is regarded as an ineffective response to ischemia, characterized by the development of new vessels at the papilla, on the retina, and the iris.¹¹⁴ Eventually, the proliferation of new vessels induced by localized retinal ischemia can result in tractional retinal detachment and subsequent vision loss.

In a pilot clinical trial, researchers evaluated the efficacy and safety of intravenous ABMSCs.¹¹⁵ A total of 17 patients with either non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR) contributed 34 analyzed eyes. Throughout the 6-month follow-up period, there were no instances of severe complications. Additionally, the NPDR group exhibited reductions in macular thickness and a notable improvement in baseline BCVA, whereas the PDR group did not demonstrate such changes. ABMSCs may provide a secure and efficient therapeutic alternative for DR, with the optimal therapeutic window appearing to be during the NPDR.

5.3.1. Regenerative treatment for glaucoma

Glaucoma, a neurodegenerative disease that causes a gradual loss of RGC. Although lowering IOP is the standard therapy for glaucoma, some individuals may not obtain adequate outcomes with maximum dose.

One proposed method of glaucoma stem cell treatment is the secretion of neuro-trophic factors by stem cells, which can promote the survival of RGC in animal models.116, 117, 118 Furthermore, stem cells are being studied as a potential source of replacement for the injured RGC.¹¹⁹ Although several studies suggested safety and potential utility of MSC transplantation,^117,120 further clinical trials are still needed to demonstrate visual function improvement of this advanced therapy. There is a distinct obstacle existing compared to RPE and photoreceptor cell replacement: in order for the transplants or replacements to be successful, the transplanted cell must not only integrate into the ganglion cell layer, but also differentiate into RGC-like cells and generate axons from the cell to the optic nerve. Although it does not apply to iPSC, it has been shown that RGC-like cells derived from ESC and MSC can migrate and integrate into the retina.