Single-cell RNA sequencing in cornea research:Insights into limbal stem cells and their niche regulation

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.

Comparative analysis of different feeder layers with 3T3 fibroblasts for culturing rabbits limbal stem cells

AIM： To explore the possibility of human umbilical cord mesenchymal stem cells（h UCMSCs）, human umbilical vein endothelial cells（h UVECs）, human dental pulp stem cells（h DPSCs） and human periodontal ligament stem cells（h PDLSCs） serving as feeder cells in co-culture systems for the cultivation of limbal stem cells.METHODS： Different feeder layers were cultured in Dulbecco＇s modified Eagle＇s medium（DMEM）/F12 and were treated with mitomycin C. Rabbits limbal stem cells（LSCs） were co-cultured on h UCMSCs, h UVECs, h DPSCs, h PDLSCs and NIH-3T3, and then comparative analysis were made between each group to see their respective colony-forming efficiency（CFE） assay and immunofluorescence（IPO13,CK3/12）.RESULTS： The efficiency of the four type cells in supporting the LSCs morphology and its cellular differentiation was similar to that of NIH-3T3 fibroblasts as demonstrated by the immunostaining properties analysis, with each group exhibiting a similar strong expression pattern of IPO13, but lacking CK3 and CK12 expression in terms of immunostaining. But h UCMSCs, h DPSCs and h PDLSCs feeder layers were superior in promoting colony formation potential of cells when compared to h UVECs and feedercell-free culture.CONCLUSION： hUCMSCs, hDPSCs and hPDLSCs can be a suitable alternative to conventional mouse NIH-3T3 feeder cells, so that risk of zoonotic infection can be diminished.

Investigation of immunogenicity of cryopreserved limbal stem cells

AIM: To investigate changes in immunogenicity of cryopreserved limbal stem cells. METHODS: Cryopreserved limbal stem cells, fresh primary limbal stem cells and blank controls were inoculated subcutaneously in C57BL-6 mice and the percentage of CD25 cells in limbal explants was determined by flow cytometry at day 21 post inoculation. Morphological studies were performed by light and electron microscopy of limbal explant sections. RESULTS: The number of regional and systemic lymphocytes derived from cryopreserved limbal stem cells was lower than that from fresh primary limbal stem cells. CONCLUSION: Lymphocytes derived from cryopreserved limbal stem cells showed changes in immunogenicity, but the significance is unknown. The cryopreservation and thawing methods await further study.

Therapeutic effect of secretome from TNF-αstimulated mesenchymal stem cells in an experimental model of corneal limbal stem cell deficiency

AIM:To explore the secretome efficacy in tumor necrosis factor(TNF)-αstimulated mouse mesenchymal stem cells(MSCs)in a murine model of corneal limbal alkali injury.METHODS:Corneal limbal stem cell deficiency(LSCD)was created in the eyes of male C57 mice.Concentrated conditioned medium from TNF-αstimulated MSCs(MSC-CMT)was applied topically for 4 wk,with basal medium and conditioned medium from MSCs as controls.Corneal opacification,corneal inflammatory response,and corneal neovascularization(NV)were evaluated.Corneal epithelial cell apoptosis,corneal conjunctivation,and inflammatory cell infiltration were assessed with TUNEL staining,CK3 and Muc-5 AC immunostaining,and CD11 b immunofluorescence staining,respectively.The effect of TSG-6 was further evaluated by knockdown with short hairpin RNA(sh RNA).RESULTS:Compared to the controls,topical administration of MSC-CMT significantly ameliorated the clinical symptoms of alkali-induced LSCD,with restrained corneal NV,reduced corneal epithelial cell apoptosis,and inhibition of corneal conjunctivation.In addition,MSC-CMT treatment significantly reduced CD11 b+inflammatory cell infiltration,and inhibited the expression of pro-inflammatory cytokines(IL-1β,TNF-αand IL-6).Furthermore,the promotion of corneal epithelial reconstruction by MSC-CMT was largely abolished by TSG-6 knockdown.CONCLUSION:Our study provides evidence that MSCCMT enhances the alleviation of corneal alkali injuries,partially through TSG-6-mediated anti-inflammatory protective mechanisms.MSC-CMT may serve as a potential strategy for treating corneal disorders.

A narrative review of limbal stem cell deficiency&severe ocular surface disease

Background and Objective:Limbal stem cell deficiency(LSCD)describes the clinical condition when there is dysfunction of the corneal epithelial stem/progenitor cells and the inability to sustain the normal homeostasis of the corneal epithelium.The limbal stem cells are located in a specialized area of the eye called the palisades of Vogt(POV).There have been significant advances in the diagnosis and management of LSCD over the past decade and this review focuses on the pathophysiology of LSCD,its clinical manifestations,diagnosis,and causes.Methods:Papers regarding LSCD were searched using PubMed to identify the current state of diagnosis and causes of LSCD published through to June 2022.Key Content and Findings:LSCD is clinically demonstrated by a whorl-epitheliopathy,loss of the POV,and conjunctivalization of the cornea.The diagnosis of this condition is based on clinical examination and aided by the use of impression cytology,in vivo confocal microscopy,and anterior segment optical coherence tomography(asOCT).There are many causes of LSCD,but those which are most common include chemical injuries,aniridia,contact lens wear,and Stevens-Johnson syndrome(SJS).Conclusions:While this condition is most commonly encountered by corneal specialists,it is important that other ophthalmologists recognize the possibility of LSCD as it may arise in other co-morbid eye conditions.

Cell-based therapies for limbal stem cell deficiency: a literature review

Background and Objective:Limbal stem cell deficiency(LSCD)is characterized by the insufficiency of limbal stem cells to maintain the corneal epithelium.Severe cases of LSCD may be treated with limbal transplantation from healthy autologous or allogeneic limbal tissue.Multiple cell-based therapies have been studied as alternative treatments to improve success rates and minimize immunosuppressive regimens after allogeneic transplants.In this review,we describe the success rates,and complications of different cell-based therapies for LSCD.We also discuss each therapy’s relative strengths and weaknesses,their history in animal and human studies,and their effectiveness compared to traditional transplants.Methods:PubMed was searched for publications using the terms LSCD,cell-based therapy,cultivated limbal epithelial transplantation(CLET),cultivated oral mucosal epithelial transplantation(COMET),and mesenchymal stem cells from 1989 to August 2022.Inclusion criteria were English language articles.Exclusion criteria were non-English language articles.Key Content and Findings:current cell-based therapies for LSCD are CLET and non-limbal epithelial cells.Non-limbal epithelial cell methods include COMET,conjunctival epithelial autografts,and mesenchymal stem/stromal cells(MSCs).Moreover,several alternative potential sources of non-limbal cells have described,including induced pluripotent stem cells(iPSCs),human embryonic stem cells(hESCs),human dental pulp stem cells,hair follicle bulge-derived epithelial stem cells,amniotic membrane epithelial cells,and human umbilical cord lining epithelial cells.Conclusions:Cell-based therapies are a promising treatment modality for LSCD.While CLET is currently the only approved cell-based therapy and is only approved in the European Union,more novel methods have also been shown to be effective in human or animal studies thus far.Non-limbal epithelial cells such as COMET are also an alternative treatment to allogeneic transplants especially as a surface stabilizing procedure.iPSCs are currently being studied in early phase trials and have the potential to revolutionize the way LSCD is treated.Lastly,cell-based therapies for restoring the limbal niche such as mesenchymal stem cells have also shown promising results in the first human proof-of-concept study.Several potential sources of non-limbal cells are under investigation.

Focus on limbal stem cell deficiency and limbal cell transplantation

Limbal stem cell deficiency(LSCD)causes severe vision impairment and can lead to blindness,representing one of the most challenging ocular surface disorders.Stem cell deficiency can be congenital or,more often,acquired.The categorization of ocular surface transplantation techniques is crucial to achieving treatment homogeneity and quality of care,according to the anatomic source of the tissue being transplanted,genetic source,autologous or allogenic transplantation(to reflect histocompatibility in the latter group),and cell culture and tissue engineering techniques.The aim of this minireview is to provide a summary of the management of LSCD,from clinical characteristics and therapeutic outcomes to the development of novel therapeutic approaches.The manuscript also briefly summarizes recent findings in the current literature and outlines the future challenges to overcome in the management of the major types of ocular surface failure.

Limbal epithelial stem cells in corneal surface reconstruction

Cornea serves as the partial front barrier and major light reflection organ of the eye.The integrity of corneal surface is essential for ocular function.Injuries or congenital diseases could significantly destruct the homeostasis of the ocular surface,especially the microenvironment of limbal epithelial stem cells(LESCs),and will eventually cause dysfunction of corneal regeneration and diminish of LESCs.The loss of LESCs by different reasons are named limbal stem cell deficiency(LSCD),which is one of the leading cause of vision loss worldwide.To restore the corneal surface,LESC transplantation in the form of tissue or cell cultures is currently a viable and promising method to treat LSCD.In this review,we aim to introduce the characters and niche of LESCs,and discuss different aspects of its application in cornea surface reconstruction.

Ocular stem cells:a narrative review of current clinical trials

·Stem cells are undifferentiated cells showcasing a remarkable capacity of self-replenishing and differentiating into mature cells.Their ability to proliferate connotes that a designated stem cell source is capable of generating an unrestricted number of mature cells.The ever-increasing comprehension of position,activity,and function of ocular stem cells has led to rapid progress and incessant improvement of possible procedures and therapies.A narrative review was conducted to summarize the current evidence on clinical trials and respective literature,regarding current evolution in the field of ocular regenerative medicine.We tried to ascertain the safety of experimental and clinical procedures,their effectiveness,and the ethical repercussion of their use.

Transplantation of tissue-engineered human corneal epithelium in limbal stem cell deficiency rabbit models

AIM: To evaluate the biological functions of tissue-engineered human corneal epithelium (TE-HCEP) by corneal transplantation in limbal stem cell deficiency (LSCD) rabbit models. METHODS: TE-HCEPs were reconstructed with DiI-labeled untransfected HCEP cells and denuded amniotic membrane (dAM) in air-liquid interface culture, and their morphology and structure were characterized by hematoxylin-eosin (HE) staining of paraffin-sections, immunohistochemistry and electron microscopy. LSCD models were established by mechanical and alcohol treatment of the left eyes of New Zealand white rabbits, and their eyes were transplanted with TE-HCEPs with dAM surface outside by lamellar keratoplasty (LKP). Corneal transparency, neovascularization, thickness, and epithelial integrality of both traumatic and post transplantation eyes were checked once a week by slit-lamp corneal microscopy, a corneal pachymeter, and periodic acid-Schiff (PAS) staining. At day 120 post surgery, the rabbits in each group were sacrificed and their corneas were examined by DiI label observation, HE staining, immunohistochemistry and electron microscopy. RESULTS: After cultured for 5 days on dAM, HCEP cells, maintaining keratin 3 expression, reconstructed a 6-7 layer TE-HCEP with normal morphology and structure. The traumatic rabbit corneas, entirely opaque, conjunctivalized and with invaded blood vessels, were used as LSCD models for TE-HCEP transplantation. After transplantation, obvious edema was not found in TE-HCEP-transplanted corneas which became more and more transparent, the invaded blood vessels reduced gradually throughout the monitoring period. The corneas decreased to normal thickness on day 25, while those of dAM eyes were over 575 mu m in thickness during the monitoring period. A 45 layer of epithelium consisting of TE-HCEP originated cells attached tightly to the anterior surface of stroma was reconstructed 120 days after TE-HCEP transplantation, which was similar to the normal control eye in morphology and structure. In contrast, intense corneal edema, turbid, invaded blood vessels were found in dAM eyes, and no multilayer epithelium was found but only a few scattered conjunctiva-like cells appeared. CONCLUSION: The TE-HCEP, with similar morphology and structure to those of innate HCEP, could reconstruct a multilayer corneal epithelium with normal functions in restoring corneal transparency and thickness of LSCD rabbits after transplantation. It may be a promising HCEP equivalent for clinical therapy of corneal epithelial disorders.

Quantification of corneal neovascularization after ex vivo limbal epithelial stem cell therapy

AIM: To assess cultured limbal epithelial stem cell transplantation in patients with limbal stem cell deficiency by analyzing and quantifying corneal neovascularization.METHODS: This retrospective, interventional case series included eight eyes with total limbal stem cell deficiency. Ex vivo limbal epithelial stem cells were cultured on human amniotic membrane using an animalfree culture method. The clinical parameters of limbal stem cell deficiency, impression cytology, and quantification of corneal neovascularization were evaluated before and after cultured limbal stem cell transplantation. The area of corneal neovascularization,vessel caliber(VC), and invasive area(IA) were analyzed before and after stem cell transplantation by image analysis software. Best-corrected visual acuity(BCVA),epithelial transparency, and impression cytology were also measured.RESULTS: One year after surgery, successful cases showed a reduction(improvement) of all three parameters of corneal neovascularization [neovascular area(NA), VC, IA], while failed cases did not. NA decreased a mean of 32.31%(P =0.035), invasion area29.37%(P =0.018) and VC 14.29%(P =0.072). BCVA improved in all eyes(mean follow-up, 76 ±21mo).Epithelial transparency improved significantly from 2.00 ±0.93 to 0.88±1.25(P =0.014). Impression cytology showed that three cases failed after limbal epithelial stem cell therapy before 1y of follow-up.CONCLUSION: This method of analyzing andmonitoring surface vessels is useful for evaluating the epithelial status during follow-up, as successful cases showed a bigger reduction in corneal neovascularization parameters than failed cases. Using this method,successful cases could be differentiated from failed cases.

Induced pluripotent stem cells as a potential therapeutic source for corneal epithelial stem cells

Corneal blindness caused by limbal stem cell deficiency(LSCD) is one of the most common debilitating eye disorders. Thus far, the most effective treatment for LSCD is corneal transplantation, which is often hindered by the shortage of donors. Pluripotent stem cell technology including embryonic stem cells(ESCs) and induced pluripotent stem cells(iPSCs) have opened new avenues for treating this disease. iPSCs-derived corneal epithelial cells provide an autologous and unlimited source of cells for the treatment of LSCD. On the other hand, iPSCs of LSCD patients can be used for iPSCs-corneal disease model and new drug discovery. However, prior to clinical trial, the efficacy and safety of these cells in patients with LSCD should be proved. Here we focused on the current status of iPSCs-derived corneal epithelial cells used for cell therapy as well as for corneal disease modeling. The challenges and potential of iPSCs-derived corneal epithelial cells as a choice for clinical treatment in corneal disease were also discussed.

Chronic ocular GVHD:limbal and conjunctival stem cell allografts from the same hematopoietic stem cell donor

Dear Editor,I am Dr.Elias F Jarade from the Beirut Eye and ENT Specialist Hospital, Beirut, Lebanon. I write to describe a novel surgical technique in the management of chronic ocular graft-versus-host disease（GVHD）.

Midterm outcomes of penetrating keratoplasty following allogeneic cultivated limbal epithelial transplantation in patients with bilateral limbal stem cell deficiency

AIM:To evaluate the midterm outcomes of penetrating keratoplasty(PK)following allogeneic cultivated limbal epithelial transplantation(CLET)for bilateral total limbal stem cell deficiency(LSCD).METHODS:Ten patients(10 eyes)with bilateral LSCD were enrolled in this prospective noncomparative case series study.Each participant underwent PK approximately 6 mo after a CLET.Topical tacrolimus,topical and systemic steroids,and oral ciclosporin were administered postoperatively.Best-corrected visual acuity(BCVA),intraocular pressure(IOP),ocular surface grading scores(OSS),corneal graft epithelial rehabilitation,persistent epithelial defect(PED),immunological rejection,and graft survival rate were assessed.RESULTS:The time interval between PK and allogeneic CLET was 6.90±1.29(6-10)mo.BCVA improved from 2.46±0.32 log MAR preoperatively to 0.77±0.55 log MAR post-PK(P<0.001).Kaplan-Meier analysis of mean graft survival revealed graft survival rates of 100%at 12 and 24 mo and 80.0%at 36 mo.PEDs appeared in 5 eyes at different periods post-PK,and graft rejection occurred in 4 eyes.The total OSS decreased from 12.4±4.4 before allogeneic CLET to 1.4±1.51 after PK.CONCLUSION:A sequential therapy design of PK following allogeneic CLET can maintain a stable ocular surface with improved BCVA despite the relatively high graft rejection rate.

Medical and surgical approach to ocular surface reconstruction

Ocular surface disease(OSD)can have a severe impact on patients as it can lead to visual impairment and persistent discomfort.Ocular surface reconstruction(OSR)is an approach to the management of ocular diseases that cause structural damage to the ocular surface.OSR encompasses both medical and surgical treatment options.In this review,we discuss the medical and surgical strategies used in OSR.Medical management often aims to treat tear insufficiency,inflammation,and keratinization.Surgical treatments may be employed for a variety of reasons,including failure of medical management.This may include improving the oculo-palpebral structures in order to improve lid positioning and tear film.Additional therapies focus on improving tear production,such as through salivary gland transplantation.In situations where the ocular surface is so severely damaged that there is loss of limbal stem cells,limbal stem cell transplant(LSCT)may be indicated.Other surgeries such as amniotic membrane transplant(AMT)and conjunctival flaps(CFs)can help promote corneal healing.Finally,in severe situations where the cornea is beyond salvage,corneal transplantation,such as a penetrating keratoplasty(PKP),can be considered.OSR often requires a combination of medical and surgical approaches targeted to each specific patient’s presentation in order to achieve optimal outcomes.

Cryopreserved limbal lamellar keratoplasty for peripheral corneal and limbal reconstruction

This study aimed to evaluate the outcomes and described the recovery process of cryopreserved limbal lamellar keratoplasty（CLLK） for peripheral corneal and limbal diseases. Thirteen eyes of 12 patients with a mean age of 41±23.9 y were included. The average follow-up was 12.1±5.6 mo. Stable ocular surface was achieved in all eyes at last follow-up. Epithelialization originated from both recipient and graft in 9 eyes. We conclude that CLLK compensates for the shortage of donor corneas and cryopreserved limbal grafts provide epithelialization sources in ocular surface reconstruction.

Axenfeld-Reiger syndrome: A search for the missing links

BACKGROUND Axenfeld-Rieger syndrome(ARS)is a rare cause of congenital glaucoma and may result in loss of vision.ARS is mostly autosomal dominant in nature characterized by developmental abnormalities in the angle of anterior chamber and iris of the eye,also associated with structural abnormalities in the body.AIM To study and observe the demographics and clinical findings in a very rare ocular disease known as ARS.METHODS Case records of symptomatic patients attending Ophthalmology outpatient department and diagnosed to have ocular hypertension or glaucoma in 3 years from March 2017 to March 2020 were evaluated to search for cases diagnosed with ARS.Records of all patients diagnosed with ARS were then analysed for demographic and clinical characterization as well as management and success of therapy.RESULTS Eight out of ten patients with positive clinical signs were symptomatic and had glaucoma.One of these patients had limbal stem cell deficiency and another had vernal keratoconjunctivitis.CONCLUSION Clinical characterization of ARS is important for making a definitive diagnosis and determining prognosis.

多孔复合水凝胶负载角膜缘干细胞重建眼表

干细胞疗法是一种用于重建角膜缘干细胞缺乏症(LSCD)患者眼表的新技术.然而,目前报道的干细胞疗法治疗LSCD仅局限于二维(2D)培养体系,不能模拟干细胞在体内生存的原生微环境.本研究基于多孔明胶甲基丙烯酰(GelMA)/丝素蛋白甲基丙烯酸缩水甘油酯(SilMA),制备了一种可注射的光交联水凝胶用于负载角膜缘干细胞(LSCs)以重建眼表.多孔GelMA/SilMA水凝胶具有优异的透明度和良好的粘附性.更重要的是,该水凝胶保持了兔LSCs的活力,支持了兔LSCs的粘附,并促进了它们在体外3D培养环境中的迁移.蛋白质组学分析证实,多孔GelMA/SilMA水凝胶3D细胞培养体系能够保持LSCs的自我更新和分化能力.此外,组织学分析表明,负载LSCs的多孔GelMA/SilMA水凝胶能够有效的促进LSCD再上皮化.多孔水凝胶能够简化连续细胞培养的相关操作,以构建具有细胞均匀分布的眼表.这些发现为干细胞疗法和再生医学用于眼表重建提供了有价值的见解.