Limbal epithelial stem cells (LESCs) are the sole source for the long-term maintenance of corneal epithelial homeostasis and the repair of major injuries. Conversely, limbal stem cell deficiency (LSCD) is a pathological condition characterized by depletion or dysfunction of LESCs, leading to clinical manifestations, such as corneal epithelial defects, opacity, and neovascularization. The ideal therapeutic strategy involves replacement of the necessary stem cells. Since its first report in 1997 1, cultivated limbal epithelial transplantation (CLET) has become a crucial modality for treating severe LSCD and is considered a milestone technique in the field of regenerative medicine. However, CLET is patient- and donor-dependent because autologous transplantation necessitates a healthy contralateral eye, and allogeneic transplantation requires immunosuppression and is constrained by donor shortages. Consequently, the clinical burden markedly exceeds the current availability of stem cells. Recently, substantial strides have been made in stem cell therapies for corneal diseases, with stem cells from diverse sources being investigated and successively entering clinical trials. Looking ahead, the next generation of transplantation strategies will focus on optimizing culture systems, exploiting novel seed cells, such as induced pluripotent stem cells (iPSCs), and exploring multi-dimensional therapeutic pathways. While the transplantation procedure itself is readily achievable, the permanent restoration of the corneal epithelium currently hinges on a single predictive metric: the proportion of stem cells within the graft. In future cultivation strategies, mere expansion of the number of stem cells will be insufficient. Instead, precise enrichment of bona fide stem cells is imperative to dramatically improve transplantation success rates and long-term therapeutic efficacy. A landmark study 2 previously identified ABCB5 as a specific protein marker for LESCs. Using magnetic bead sorting to enrich for ABCB5-positive cells, transplantation achieved complete and long-term restoration of corneal structure and transparency. Compared with conventional CLET, the enrichment of ABCB5-positive cells offers superior long-term durability by ensuring a higher concentration of bona fide stem cells. This molecularly-targeted approach potentially reduces the risk of late-stage graft failure often associated with depletion of the stem cell pool in traditional non-enriched grafts. The standardized workflow for the isolation and enrichment of LESCs via specific molecular markers is illustrated schematically in Figure 1. Currently, a multicenter clinical trial (NCT03549299) investigating allogeneic ABCB5-positive LESCs for the treatment of LSCD is underway. The lack of standardized production protocols and quality control remains a critical barrier hindering the widespread translation of stem cell therapies from bench to bedside. Recently, a clinical trial 3 in the United States established a standardized and safe solution that uses a xenobiotic-free, serum-free, and antibiotic-free culture system. iPSCs are derived from reprogrammed adult somatic cells and can be harvested autologously, thereby significantly reducing the risk of immune rejection associated with allogeneic transplantation. Furthermore, iPSCs can be generated from diverse sample sources, which theoretically allows for unlimited production to meet transplantation demands. Notably, the world's first clinical trial using human iPSC-derived corneal epithelial cell sheets for the treatment of LSCD 4 has been completed in Japan. The study effectively validated the safety and efficacy of this therapy in humans, offering a revolutionary new therapeutic strategy to address the challenges of donor shortages and the treatment of bilateral LSCD. From a broader clinical perspective, a 2025 global landscape review in Cell Stem Cell reported that as of late 2024, there were 115 interventional trials using human pluripotent stem cell (hPSC)-derived products worldwide, with ocular indications being one of the primary targets 5. However, it is crucial to note that iPSCs possess intrinsic tumorigenicity and are subject to genomic instability as well as potential mutations introduced during the reprogramming process. Consequently, rigorous safety screening is mandatory prior to clinical application. To circumvent the limitations of conventional allogeneic grafting, researchers have investigated a diverse array of stem cell-based strategies. Notably, an ongoing clinical trial (NCT06700655) is evaluating the co-transplantation of LESCs and corneal stromal stem cells to achieve the simultaneous restoration of both the corneal epithelium and stromal transparency. Although various other stem cell sources, such as mesenchymal stem cells, human embryonic stem cells, and cultured oral mucosal epithelial cells, are alternative options, their clinical translation is hindered by the challenges of large-scale standardized expansion under Good Manufacturing Practices. In these environments, maintaining consistent cellular potency across different production lots is difficult and further complicated by diverging regulatory pathways. While autologous CLET is often managed as a tissue transplant procedure, allogeneic products, such as iPSC-derived cells, are strictly categorized as Advanced Therapy Medicinal Products, necessitating rigorous standardized potency testing and stringent donor eligibility criteria to ensure safety and lot-to-lot consistency. Although iPSCs remain a promising “corneal substitute,” achieving routine clinical use necessitates a rigorous safety framework to address potential long-term biological risks. This includes high-resolution pre-transplantation quality control, such as whole-genome sequencing to detect oncogenic mutations, as well as extended post-transplantation monitoring for late-onset tumorigenesis and immunological rejection. These integrated safety and manufacturing protocols are essential to ensure reliable application of next-generation biological products in regenerative ophthalmology. To assist clinicians and researchers in navigating the therapeutic options, a comparative analysis of these technologies, including their manufacturing complexity and key clinical endpoints, is summarized in Table 1. The therapeutic landscape of LSCD is currently undergoing a marked paradigm shift, transitioning from traditional donor-dependent tissue grafting toward technology-driven personalized regeneration (Figure 2). As discussed, this next-generation approach is a synergistic process that integrates precise cell selection, exploitation of novel seed cells, and engineering of functionalized microenvironments. The substitution of live cells poses inherent risks of immune rejection and tumorigenicity. In contrast, exosomes secreted by stem cells possess comparable bioactivity and contribute to the regeneration of the corneal microenvironment. An ongoing clinical trial (NCT06543667) is evaluating the use of LESC-derived exosome eye drops for the treatment of dry eye. This cell-free approach represents a potential future strategy for the management of mild-to-moderate LSCD. To achieve successful ocular surface restoration, selecting the appropriate stem cell type must be synergistically integrated with optimized delivery methods to ensure precise targeting and optimal graft survival. Although amniotic membrane transplantation has been used traditionally as a natural carrier, its clinical application is hampered by batch-to-batch variability and the risk of disease transmission. In response to these limitations, the field is shifting toward technology-driven solutions that better mimic the native microenvironment. Advanced nanomaterials, such as functionalized hydrogels and nanofibrous scaffolds, offer robust support by replicating the extracellular matrix. This synergistic interaction allows these scaffolds to mimic the native limbal niche. This approach creates a biomimetic microenvironment that provides essential cues to sustain cell viability and preserve the stemness of transplanted cells. The evolution of delivery systems further encompasses complex structural engineering. While 3D bioprinted constructs are sophisticated vehicles for LESCs to facilitate their structural and functional integration into host tissues 6, compartmentalized 3D bioprinting has recently enabled the precise spatial arrangement of distinct hPSC-LESC subpopulations 7. This allows for the reconstruction of the complex architectural hierarchy of the limbal niche, providing a high-fidelity platform for both regenerative therapy and corneal disease modeling. In parallel with these scaffold-based approaches, an autologous cultivated limbal epithelial cell sheet known as Nepic can be directly transplanted onto the patient's corneal surface without a carrier 8. The safety and efficacy of Nepic have been validated in clinical trials, leading to its approval as a regenerative medical product in Japan. While these bioengineered materials hold broad application prospects for optimizing the limbal niche, their potential toxicity and metabolic impact on the recipient microenvironment must not be overlooked. Beyond graft optimization, the clinical success of limbal stem cell transplantation (LSCT) is critically contingent upon the recipient ocular surface environment. This immunological dimension is particularly pronounced in inflammatory contexts, such as Stevens–Johnson syndrome, in which chronic inflammation leads to the profound breakdown of ocular immune privilege. In such diseased states, the limbal niche is replaced by conjunctivalization and extensive neovascularization, which facilitates infiltration of host immune cells and significantly reduces the survival of allogeneic grafts. Furthermore, the persistent pro-inflammatory cytokine milieu, characterized by elevated levels of factors such as interleukin-1 and tumor necrosis factor-alpha, directly suppresses LESC proliferation and disrupts the delicate balance of stem cell maintenance. This immune dysregulation is a primary barrier to therapeutic success, necessitating strategies that go beyond simple cell replacement to include robust modulation of the host's local immune status 5. Future therapeutic strategies should transcend passive cellular replacement to actively mitigate persistent inflammation and pathological neovascularization within the host tissue. The development of multifunctional scaffolds capable of simultaneous stem cell delivery and the controlled release of anti-inflammatory or anti-angiogenic agents represents an essential advancement. By modulating the recipient microenvironment, these synergistic therapeutic platforms can reconstitute a permissive niche, which enhances long-term viability and functional integration of the transplanted graft. Ultimately, the transition toward next-generation LSCT requires a holistic approach in which the choice of seed cells is no longer viewed in isolation. Instead, the efficacy of the therapy is defined by the dynamic interplay between cellular potency, biophysical cues provided by the scaffold, and active modulation of the recipient ocular surface environment. Addressing these fundamental scientific challenges is essential for the next generation of LSCT. Specifically, identifying predictive molecular markers, such as ABCB5, will allow clinicians to gauge long-term graft survival more accurately prior to transplantation 2. Furthermore, it is paramount to unravel the mechanisms of graft failure by transitioning from a focus on simple cell loss toward investigating the complex balance between permanent functional integration and transient paracrine support 5, 7. Finally, characterizing the immunological status of the recipient's ocular surface remains a decisive factor because modulating the host microenvironment to mitigate chronic inflammation and neovascularization is just as critical as the potency of the seed cells 5. To provide a holistic perspective on these advancements, we propose a technology-driven roadmap that integrates cell source selection, advanced manufacturing, and targeted microenvironment modulation (Figure 3). Novel stem cell culture and transplantation protocols have reached a turning point in the treatment of LSCD. However, several clinical questions remain unresolved, particularly regarding the long-term functional integration of transplanted cells versus transient paracrine effects. Furthermore, defining the optimal therapeutic window for intervention in acute versus chronic LSCD is essential for maximizing sustainable visual recovery. Because many clinical trials remain in their preliminary stages, safety must be prioritized as the paramount outcome measure, accompanied by rigorous regulatory measures and long-term postoperative monitoring to ensure the sustainability of visual improvement. Liqin Huang: writing – original draft, investigation, conceptualization, methodology, validation, visualization. Liangbo Chen: conceptualization, methodology, investigation, validation, visualization, writing – review and editing. The authors have nothing to report. The authors have nothing to report. The authors have nothing to report. The authors have nothing to report. The authors declare no conflicts of interest. Data sharing is not applicable to this article as no datasets were generated or analyzed.
Huang et al. (Fri,) studied this question.