BACKGROUND: Dysfunction or deficiency of corneal epithelium results in vision impairment or blindness in severe cases. The rapid and effective regeneration of corneal epithelial cells relies on the limbal stem cells (LSCs). However, the molecular and functional responses of LSCs and their niche cells to injury remain elusive.
METHODS: Single-cell RNA sequencing was performed on corneal tissues from normal mice and corneal epithelium defect models. Bioinformatics analysis was performed to confirm the distinct characteristics and cell fates of LSCs. Knockdown of Creb5 and OSM treatment experiment were performed to determine their roles of in corneal epithelial wound healing.
RESULTS: Our data defined the molecular signatures of LSCs and reconstructed the pseudotime trajectory of corneal epithelial cells. Gene network analyses characterized transcriptional landmarks that potentially regulate LSC dynamics, and identified a transcription factor Creb5, that was expressed in LSCs and significantly upregulated after injury. Loss-of-function experiments revealed that silencing Creb5 delayed the corneal epithelial healing and LSC mobilization. Through cell-cell communication analysis, we identified 609 candidate regeneration-associated ligand-receptor interaction pairs between LSCs and distinct niche cells, and discovered a unique subset of Arg1+ macrophages infiltrated after injury, which were present as the source of Oncostatin M (OSM), an IL-6 family cytokine, that were demonstrated to effectively accelerate the corneal epithelial wound healing.
CONCLUSIONS: This research provides a valuable single-cell resource and reference for the discovery of mechanisms and potential clinical interventions aimed at ocular surface reconstruction.

Human embryonic stem cell (hESC)- and human induced pluripotent stem cell (hiPSC)-derived retinal pigment epithelium (RPE) therapies are promising alternatives for the treatment of retinal degenerative diseases caused by RPE degeneration. The generation of autologous RPE cells from human adult donors, which has the advantage of avoiding immune rejection and teratoma formation, is an alternative cell resource to gain mechanistic insight into and test potential therapies for RPE degenerative diseases. Here, we found that limbal stem cells (LSCs) from hESCs and adult primary human limbus have the potential to produce RPE cells and corneal stromal stem cells (CSSCs). We showed that hESC-LSC-derived RPE cells (LSC-RPE) expressed RPE markers, had a phagocytic function, and synthesized tropical factors. Furthermore, during differentiation from LSCs to RPE cells, cells became pigmented, accompanied by a decrease in the level of LSC marker KRT15 and an increase in the level of RPE marker MITF. The Wnt signaling pathway plays a role in LSC-RPE fate transition, promotes MITF expression in the nucleus, and encourages RPE fate transition. In addition, we also showed that primary LSCs (pLSCs) from adult human limbus similar to hESC-LSC could generate RPE cells, which was supported by the co-expression of LSC and RPE cell markers (KRT15/OTX2, KRT15/MITF), suggesting the transition from pLSC to RPE cells, and typical polygonal morphology, melanization, RPE cell marker genes expression (TYR, RPE65), tight junction formation by ZO-1 expression, and the most crucial phagocytotic function. On the other hand, both hESC-LSCs and pLSCs also differentiated into CSSCs (LSC-CSSCs) that expressed stem cell markers (PAX6, NESTIN), presented MSC features, including surface marker expression and trilineage differentiation capability, like those in human CSSCs. Furthermore, the capability of pLSC-CSSC to differentiate into cells expressing keratocyte marker genes (ALDH3A1, PTGDS, PDK4) indicated the potential to induce keratocytes. These results suggest that the adult pLSC is an alternative cell resource, and its application provides a novel potential therapeutic avenue for preventing RPE dysfunction-related retinal degenerative diseases and corneal scarring.

Limbal stem/progenitor cells (LSPCs) play a crucial role in maintaining corneal health by regulating epithelial homeostasis. Although PM2.5 is associated with the occurrence of several corneal diseases, its effects on LSPCs are not clearly understood.
In this study, we explored the correlation between PM2.5 exposure and human limbal epithelial thickness measured by Fourier-domain Optical Coherence Tomography in the ophthalmologic clinic. Long- and short-term PM2.5 exposed-rat models were established to investigate the changes in LSPCs and the associated mechanisms.
We found that people living in regions with higher PM2.5 concentrations had thinner limbal epithelium, indicating the loss of LSPCs. In rat models, long-term PM2.5 exposure impairs LSPCs renewal and differentiation, manifesting as corneal epithelial defects and thinner epithelium in the cornea and limbus. However, LSPCs were activated in short-term PM2.5-exposed rat models. RNA sequencing implied that the circadian rhythm in LSPCs was perturbed during PM2.5 exposure. The mRNA level of circadian genes including Per1, Per2, Per3, and Rev-erbα was upregulated in both short- and long-term models, suggesting circadian rhythm was involved in the activation and dysregulation of LSPCs at different stages. PM2.5 also disturbed the limbal microenvironment as evidenced by changes in corneal subbasal nerve fiber density, vascular density and permeability, and immune cell infiltration, which further resulted in the circadian mismatches and dysfunction of LSPCs.
This study systematically demonstrates that PM2.5 impairs LSPCs and their microenvironment. Moreover, we show that circadian misalignment of LSPCs may be a new mechanism by which PM2.5 induces corneal diseases. Therapeutic options that target circadian rhythm may be viable options for improving LSPC functions and alleviating various PM2.5-associated corneal diseases.

BACKGROUND: Limbal stem cells (LSCs) are essential for maintaining corneal transparency and ocular surface integrity. Many external factors or genetic diseases can lead to corneal limbal stem cell deficiency (LSCD), resulting in the loss of barrier and corneal epithelial cell renewal functions. Stem cell transplantation is one of the primary treatments for LSCD, including limbal transplantation and cultivated limbal epithelial transplantation. In addition, a variety of non-limbal stem cell lines have been experimented with for LSCD treatment. Biological scaffolds are also used to support in vitro stem cell culture and transplantation. Here, we review the mechanisms of corneal maintenance by LSCs, the clinical stage and surgical treatment of LSCD, the source of stem cells, and the biological scaffolds required for in vitro culture.
METHODS: This study is a narrative retrospective study aimed at collecting available information on various aspects of surgical treatments for LSCD. Relevant literature was searched in a range of online databases, including Web of Science, Scopus, and PubMed from 2005 to March, 2023.
RESULTS: A total of 397 relevant articles were found, and 49 articles with strong relevance to the studies in this paper were obtained and analyzed. Moreover, 11 of these articles were on the concept of LSCD and the mechanism of LESCs maintaining the corneal epithelium, 3 articles on the staging and grading of LSCD, 17 articles on cell transplantation methods and donor cell sources, and 18 articles on scaffolds for delivering stem cells. We also summarized the advantages and disadvantages of different cell transplantation methods and the benefits and limitations of scaffolds based on the above literature.
CONCLUSIONS: The treatment of LSCD is determined by the clinical stage and whether it involves monocular or binocular eyes. Appropriate surgical techniques should be taken for LSCD patients in order to reconstruct the ocular surface, relieve symptoms, and restore visual function. Meanwhile, biological scaffolds assist in the ex vivo culture and implantation of stem cells.

Limbal stem cell deficiency (LSCD) is an ocular surface disease resulting from a reduction and/or dysfunction of limbal stem cells. The symptoms of LSCD are non-specific and can be difficult to distinguish from other ocular surface diseases through slit-lamp examination. Impression cytology is currently considered the gold standard for LSCD diagnosis; however,it is a qualitative method with low sensitivity. Nonetheless,emerging imaging techniques offer quantitative diagnosis and staging of LSCD. This review article examines four imaging methods and their associated parameters for diagnosing LSCD: optical coherence tomography,which measures corneal epithelial thickness; optical coherence tomography angiography,which detects corneal neovascularization; in vivo confocal microscopy,which measures corneal epithelial thickness,subbasal nerve density,and corneal basal cell density; and future applications of full-field/spectral-domain optical coherence tomography.
角膜缘干细胞缺乏（LSCD）是由于角膜缘干细胞数量或功能异常导致的角膜上皮稳态失衡，其症状缺乏特异性，且裂隙灯检查难以与其他眼表疾病鉴别。印迹细胞学实验室检查只能在一定范围内进行定性诊断。近年来，出现了新型影像学检查，为LSCD的诊断和分期带来了新的曙光。本文归纳总结了多种检查方法，包括眼前节光学相干层析成像术观察角膜上皮厚度和角膜缘Vogt栅栏状结构、眼前节相干光层析血管成像术检测角膜新生血管、角膜活体共聚焦显微镜测量角膜上皮厚度、基底细胞密度和基底膜下神经，以及全场/频域光学相干层析成像术在LSCD诊断方面的应用。旨在为LSCD的临床诊疗及相关研究奠定基础。.

OBJECTIVE: This study aimed to investigate corneal limbus changes in patients with type 2 diabetes mellitus (DM) using in vivo confocal microscopy (IVCM) and explore the correlation between their ocular manifestations and systemic status.
METHODS: Fifty-five patients with type 2 DM and 20 age-matched controls were included. The following IVCM parameters were compared between the 2 groups: palisades of Vogt (POV), corneal epithelial thickness (CET), basal cell density (BCD), subbasal nerve plexus, and dendritic cell density. All subjects underwent blood and urine sampling for laboratory analysis, including fasting blood glucose, glycated hemoglobin, total cholesterol (TC), triglycerides (TG), low-density lipoprotein (LDL) cholesterol, high-density lipoprotein (HDL) cholesterol, C-reactive protein, urinary albumin-to-creatinine ratio, urine albumin, and urine creatinine. The correlations between IVCM parameters and blood biomarkers were detected. Receiver operating characteristic curve was used for selecting the cutoff value of risk factors for corneal stem cell injury in patients with DM.
RESULTS: Compared with controls, patients with DM displayed a significant reduction of POV (superior region, P = 0.033; inferior region, P = 0.003; nasal region, P < 0.001; temporal region, P < 0.001), central CET (44.8 ± 3.6 μm vs. 51.9 ± 3.6 μm, P < 0.001), central corneal BCD (7415.5 ± 563.2 cells/mm 2 vs. 9177.9 ± 977.8 cells/mm 2 , P < 0.001), and peripheral corneal BCD (6181.3 ± 416.5 cells/mm 2 vs. 8576.3 ± 933.2 cells/mm 2 , P < 0.001). Dendritic cell density (41.0 ± 33.7 cells/mm 2 vs. 24.6 ± 7.8 cells/mm 2 , P = 0.001) was significantly higher in the DM group. The following weak correlations were shown between IVCM parameters and blood biomarkers: central corneal BCD was negatively correlated with DM duration (r = -0.3, P = 0.024), TC (r = -0.36, P = 0.007), and LDL (r = -0.39, P = 0.004). The presence of POV in the superior region was negatively correlated with TC (r = -0.34, P = 0.011) and LDL (r = -0.31, P = 0.022). Cutoff values of 1.215 mmol/L for HDL, 1.59 mmol/L for TG, or 4.75 mmol/L for TC were established to distinguish patients with a high risk from a low risk for stem cell damage.
CONCLUSIONS: Patients with type 2 DM displayed a lower positive rate of typical POV and a decrease in BCD, CET, and subbasal nerve density. The most relevant indicators for stem cell phenotypes were DM duration, TC, and LDL. Lipid status in diabetic patients could be a predictor of risk for developing corneal limbal stem cell deficiency. Further studies with larger sample sizes or basic research are needed to verify the results.

The corneal epithelium is composed of stratified squamous epithelial cells on the outer surface of the eye, which acts as a protective barrier and is critical for clear and stable vision. Its continuous renewal or wound healing depends on the proliferation and differentiation of limbal stem cells (LSCs), a cell population that resides at the limbus in a highly regulated niche. Dysfunction of LSCs or their niche can cause limbal stem cell deficiency, a disease that is manifested by failed epithelial wound healing or even blindness. Nevertheless, compared to stem cells in other tissues, little is known about the LSCs and their niche. With the advent of single-cell RNA sequencing, our understanding of LSC characteristics and their microenvironment has grown considerably. In this review, we summarized the current findings from single-cell studies in the field of cornea research and focused on important advancements driven by this technology, including the heterogeneity of the LSC population, novel LSC markers and regulation of the LSC niche, which will provide a reference for clinical issues such as corneal epithelial wound healing, ocular surface reconstruction and interventions for related diseases.

Objective: To analyze the clinical features and treatment outcomes of eyes with contact lens-induced limbal stem cell deficiency (CL-iLSCD). Methods: This cross-sectional study involved patients diagnosed with CL-iLSCD at the Eye, Ear, Nose & Throat Hospital of Fudan University between October 2018 and September 2022. A total of 17 patients (25 eyes) with a mean age of (36.4±6.9) years were enrolled. Among them, 14 were females (82.4%). Corneal and limbal abnormalities, especially the range of epitheliopathy, were observed under a slit lamp biomicroscope with fluorescein staining. Anterior segment optical coherence tomography and in vivo laser scanning confocal microscopy were performed to obtain the central corneal epithelial thickness, density of basal epithelial cells and corneal nerve fiber length. The clinical features of CL-iLSCD, along with their treatment outcomes and related risk factors, were analyzed. Results: All patients wore soft contact lenses, with an average daily wearing time of (10.5±2.5) hours and a median wearing duration of 10 (4 to 30) years. Ocular symptoms, including decreased vision, ocular discomfort or pain, redness, and photophobia, were present in 22 eyes (88.0%). The most characteristic clinical sign of CL-iLSCD was comb-or whorl-pattern late fluorescein staining under cobalt blue light, which was most commonly seen at the superior limbus (25/25, 100.0%). Additionally, reductions in central corneal epithelial thickness, basal cell density, and corneal nerve fiber length were observed. A comprehensive score was assigned to each eye based on clinical findings and in vivo imaging biomarkers. LSCD was mild, moderate, and severe in 5, 11, and 8 eyes, respectively. A history of misdiagnosis was found in 20 eyes (80.0%). After discontinuing the use of contact lenses and receiving medical treatment, significant improvement was observed in all eyes, with 13 eyes fully recovered. Conclusions: The symptoms and clinical signs of CL-iLSCD can be subtle at the early stage. Discontinuing contact lens wear and medication are effective to treat CL-iLSCD.
目的： 探讨角膜接触镜相关角膜缘干细胞功能障碍（CL-iLSCD）的临床特征及治疗。 方法： 回顾性病例系列研究。纳入于2018年10月至2022年9月在复旦大学附属眼耳鼻喉科医院角膜病门诊确诊为CL-iLSCD并接受药物治疗和随访的17例患者（25只眼）的资料，其中男性3例，女性14例，年龄为（36.4±6.9）岁。采用裂隙灯显微镜结合荧光素染色观察角膜和角膜缘尤其是上皮病变范围，眼前节相干光层析成像术测量中央角膜上皮厚度，活体激光共聚焦显微镜测量角膜上皮基底细胞密度和角膜神经纤维长度，记录患者的临床表现、诊疗经过、转归和相关因素等。 结果： 所纳入的患者有4~30年软性角膜接触镜戴镜史，中位数为10年；每日戴镜时间为（10.5±2.5）h。88.0%（22/25）的患眼有视力下降、眼部不适或疼痛、眼红和畏光等症状。常见体征为角膜上皮梳齿状和（或）漩涡状迟发性荧光素染色，上方角膜缘（11~1点钟方位）受累最多（25/25，100.0%），并伴随中央角膜上皮厚度、上皮基底细胞密度和神经纤维长度降低等活体影像学表现异常。结合影像学参数综合等级评估为轻度、中度和重度角膜缘干细胞功能障碍的各为5、11和8只眼。80.0%（20/25）的患眼曾发生误诊。停戴角膜接触镜并给予以人工泪液和糖皮质激素为基础的药物治疗后，所有患眼的症状和体征改善明显，其中13只眼治愈，12只眼好转。 结论： CL-iLSCD的症状缺乏特异性，且早期体征较为隐匿。停戴角膜接触镜和药物治疗是目前该病有效的治疗方案。.

The cornea serves as an important barrier structure to the eyeball and is vulnerable to injuries, which may lead to scarring and blindness if not treated promptly. To explore an effective treatment that could achieve multi-dimensional repair of the injured cornea, the study herein innovatively combined modified mRNA (modRNA) technologies with adipose-derived mesenchymal stem cells (ADSCs) therapy, and applied IGF-1 modRNA (modIGF1)-engineered ADSCs (ADSCmodIGF1) to alkali-burned corneas in mice. The therapeutic results showed that ADSCmodIGF1 treatment could achieve the most extensive recovery of corneal morphology and function when compared not only with simple ADSCs but also IGF-1 protein eyedrops, which was reflected by the healing of corneal epithelium and limbus, the inhibition of corneal stromal fibrosis, angiogenesis and lymphangiogenesis, and also the repair of corneal nerves. In vitro experiments further proved that ADSCmodIGF1 could more significantly promote the activity of trigeminal ganglion cells and maintain the stemness of limbal stem cells than simple ADSCs, which were also essential for reconstructing corneal homeostasis. Through a combinatorial treatment regimen of cell-based therapy with mRNA technology, this study highlighted comprehensive repair in the damaged cornea and showed the outstanding application prospect in the treatment of corneal injury.

This 15-year-old male patient has been diagnosed with osteogenesis imperfecta through genetic testing after birth and has poor vision. His full corneas in both eyes are unevenly thinned and bulging in a spherical shape, with the right eye being more severe. He underwent a limbal stem cell-sparing lamellar keratoplasty in the right eye, resulting in improved vision with a corrected visual acuity of 0.5, a decrease in corneal curvature, and a significant increase in corneal thickness. The surgery had a satisfactory outcome. The condition of the left eye is progressing and will require further surgical treatment.
患者男性，15岁，生后基因检测确诊为成骨不全，视力不佳。该患者双眼全角膜不均匀薄变，呈球形扩张，右眼更重。行右眼保留角膜缘的板层角膜移植术，术后右眼视力提升，矫正视力为0.5，角膜曲率下降，角膜厚度显著增加，手术效果满意；左眼病情进展将进一步手术治疗。.