Corneal stromal mesenchymal stem cells: reconstructing a bioactive cornea and repairing the corneal limbus and stromal microenvironment

Corneal stroma-derived mesenchymal stem cells(CS-MSCs) are mainly distributed in the anterior part of the corneal stroma near the corneal limbal stem cells(LSCs). CS-MSCs are stem cells with self-renewal and multidirectional differentiation potential. A large amount of data confirmed that CS-MSCs can be induced to differentiate into functional keratocytes in vitro, which is the motive force for maintaining corneal transparency and producing a normal corneal stroma. CS-MSCs are also an important component of the limbal microenvironment. Furthermore, they are of great significance in the reconstruction of ocular surface tissue and tissue engineering for active biocornea construction. In this paper, the localization and biological characteristics of CS-MSCs, the use of CS-MSCs to reconstruct a tissue-engineered active biocornea, and the repair of the limbal and matrix microenvironment by CS-MSCs are reviewed, and their application prospects are discussed.

Corneal stem cells and tissue engineering: Current advances and future perspectives

Major advances are currently being made in regenerative medicine for cornea. Stem cell-based therapies represent a novel strategy that may substitute conventional corneal transplantation, albeit there aremany challenges ahead given the singularities of each cellular layer of the cornea. This review recapitulates the current data on corneal epithelial stem cells, corneal stromal stem cells and corneal endothelial cell progenitors. Corneal limbal autografts containing epithelial stem cells have been transplanted in humans for more than 20 years with great successful rates, and researchers now focus on ex vivo cultures and other cell lineages to transplant to the ocular surface. A small population of cells in the corneal endothelium was recently reported to have self-renewal capacity, although they do not proliferate in vivo. Two main obstacles have hindered endothelial cell transplantation to date: culture protocols and cell delivery methods to the posterior cornea in vivo. Human corneal stromal stem cells have been identified shortly after the recognition of precursors of endothelial cells. Stromal stem cells may have the potential to provide a direct cell-based therapeutic approach when injected to corneal scars. Furthermore, they exhibit the ability to deposit organized connective tissue in vitro and may be useful in corneal stroma engineering in the future. Recent advances and future perspectives in the field are discussed.

An updated narrative review of treatment for limbal epithelial stem cell deficiency

Background and Objective:Nearly 30 years have passed since limbal stem cell deficiency(LSCD)was first identified by pioneers and given clinical attention.LSCD remains a difficult disease to treat.It can potentially lead to blinding.At present,understanding of limbal stem cells(LSCs)has deepened and various treatment options for LSCD have been devised.The objective of this review is to summarize basic knowledge of LSCD and current treatment strategies.Methods:PubMed search was performed to find studies published in English on LSCs and LSCD including original reports and reviews.Literatures published from 1989 to 2022 were reviewed.Key Content and Findings:LSCs are enigmatic stem cells for which no specific marker has been discovered yet.Although LSCD is not difficult to diagnose,it is still challenging to treat.An important advancement in the treatment of LSCD is the provision of guidelines for selecting systematic surgical treatment according to the patient’s condition.It is also encouraging that stem cell technologies are being actively investigated for their potential usefulness in the treatment of LSCD.Conclusions:Although various treatment options for LSCD have been developed,it should be kept in mind that the best chance of treatment for LSCD is in the early stage of the disease.Every effort should be made to preserve as many LSCs as possible in the early treatment of LSCD.

The Limbal Niche and Its Role in Maintaining Corneal Regeneration

In recent years, stem cells have been a focal point in research designed to evaluate the efficacy of ophthalmologic therapies, specifically those for corneal conditions. The corneal epithelium is one of the few regions of the body that maintains itself using a residual stem cell population within the adjacent limbus. Stem cell movement has additionally captivated the minds of researchers due to its potential application in different body regions. The cornea is a viable model for varying methods to track stem cell migratory patterns, such as lineage tracing and live imaging from the limbus. These developments have the potential to pave the way for future therapies designed to ensure the continuous regeneration of the corneal epithelium following injury via the limbal stem cell niche. This literature review aims to analyze the various methods of imaging used to understand the limbal stem cell niche and possible future directions that might be useful to consider for the better treatment and prevention of disorders of the cornea and corneal epithelium. .

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.

Human amniotic membrane transplantation: Different modalities of its use in ophthalmology

The amniotic membrane(AM) is the inner layer of the fetal membranes and consist of 3 different layers: the epithelium, basement membrane and stroma which further consists of three contiguous but distinct layers: the inner compact layer, middle fibroblast layer and the outermost spongy layer. The AM has been shown to have anti-inflammatory, anti-fibrotic, anti-angiogenic as well as anti-microbial properties. Also because of its transparent structure, lack of immunogenicity and the ability to provide an excellent substrate for growth, migration and adhesion of epithelial corneal and conjunctival cells, it is being used increasingly for ocular surface reconstruction in a variety of ocular pathologies including corneal disorders associated with limbal stem cell deficiency, surgeries for conjunctival reconstruction, as a carrier for ex vivo expansion of limbal epithelial cells, glaucoma surgeries and sceral melts and perforations. However indiscriminate use of human AM needs to be discouraged as complications though infrequent can occur. These include risk of transmission of bacterial, viral or fungal infections to the recipient if the donors are not adequately screened for communicable diseases, if the membrane is not processed under sterile condi-tions or if storage is improper. Optimal outcomes can be achieved only with meticulous case selection. This review explores the ever expanding ophthalmological indications for the use of human AM.

Transplantation of corneal stem cells cultured on amniotic membrane for corneal burn: experimental and clinical study

OBJECTIVE: To investigate the proliferation and differentiation of cultured corneal stem cells and determine the effect of corneal stem cells cultured on amniotic membranes on the limbal area for treating corneal burns. METHODS: The proliferation and differentiation of corneal stem cells in vitro had been examined using colony-forming efficiency and immunohistochemistry. The stem cells had been cultured on amniotic membranes and transplanted to the limbal area for treating corneal burns. RESULTS: Corneal stem cells had a high proliferation capacity in primary and first passage, cytokeratin 3 was not expressed in primary culture but partly in first passage. The stem cells could proliferate to form cell layer on an amniotic membrane. When transplanted, stem cells could survive on limbus. After transplantation, ocular inflammation resolved, the cornea re-epithelialized, the stromal opacity reduced, the superficial neovascularity was lessened and the conjunctival fornix re-established. CONCLUSIONS: Ocular surface conditions could be improved by allograft of corneal stem cells cultured on amniotic membranes.

Transplantation of human limbal cells cultivated on amniotic membrane for reconstruction of rat corneal epithelium after alkaline burn

BACKGROUND: The transplantation of limbal epithelial cells cultivated on amniotic membrane is a newly developed treatment for limbal stem cell deficiency. The purpose of our study was to investigate the biological characteristics of limbal epithelial cells and evaluate the effect of transplantation of cultivated human limbal epithelial cells on ocular surface reconstruction in limbal stem cell deficiency rat model. METHODS: Human limbal cells were isolated and cultivated in vitro. Cytokeratins 3, 12, and 19 (K3, K12 and K19) and p63 were detected by immunofluorescent staining or RT-PCR. BrdU labelling test was used to identify the slow cycling cells in the cultures. Limbal stem cell deficiency was established in rat cornea by alkali burn. Two weeks after injury, the rats received transplants of human limbal stem cells cultivated on amniotic membrane carrier. The therapeutic effect was evaluated by slit lamp observation, Hemotoxin and Eosin (HE) staining and immunofluorescent staining. RESULTS: On day 7 in primary culture, p63 and K19 were strongly expressed by most cells but only a few cells expressed K3. On days 14 and 21, p63 and K19 were still expressed by a majority of cells, but the expressive intensity of p63 decreased in a number of cells, while the proportion of K3 positive cells increased slightly and some cells coexpressed p63 and K3. RT-PCR showed that gene expression of both p63 and K12 were positive in cultivated limbal cells, but in mature superficial epithelial cells, only K12 was detected. BrdU labelling test showed that most cells were labelled with BrdU after 7 days' labelling and BrdU label retaining cells were observed after chasing for 21 days with BrdU free medium. For in vivo test, slit lamp observation, HE staining and immunofluorescent staining showed that the rats receiving transplant of human limbal stem cells cultivated on amniotic membrane grew reconstructed corneas with intact epithelium, improved transparency and slight or no neovascularization. A majority of epithelial cells of the reconstructed cornea were positive to antihuman nuclear antibody and cells expressing K3 were found mainly in superfacial epithelium. CONCLUSIONS: Limbal stem cells can be cultivated in vitro: the cells are characterized by high proliferation and slow cycling and identified as p63/K19 positive and K3/K12 negative. During culture, some stem cells can proliferate and differentiate into mature cornea epithelial cells. Amniotic membrane is a suitable carrier for limbal stem cells. Transplantation of human limbal stem cells cultivated on amniotic membrane can functionally reconstruct rat cornea with limbal stem cell deficiency.

人脐带间充质干细胞移植治疗角膜碱烧伤的实验研究

目的:研究人脐带间充质干细胞(hUCMSCs)移植治疗兔角膜碱烧伤的疗效,分析多形核中性白细胞(PMNs)的浸润和血管内皮生长因子(VEGF)的表达变化。方法:将75只健康日本白兔随机分成A、B、C组,每组25只,均建立右眼角膜碱烧伤模型。A组兔右眼角膜碱烧伤后立即行角膜病灶区羊膜负载hUCMSCs覆盖术;B组兔右眼角膜碱烧伤后立即行角膜病灶区羊膜覆盖术;C组兔右眼角膜碱烧伤后未进行处理。角膜碱烧伤后3、7、14、21、28d,裂隙灯下观察实验兔角膜恢复情况并照相,对角膜新生血管(CNV)生长情况进行评分,并分离角膜组织制作病理切片,通过苏木素-伊红(HE)染色观察PMNs浸润情况,采用免疫组化染色法测定VEGF的表达。结果:角膜碱烧伤后14d时,A组CNV较B组生长明显缓慢,A组病灶区周围CNV生长评分明显低于B组(P<0.05)。碱烧伤后3d时,角膜PMNs数量增加,浸润角膜基质层,7d时稍有下降,14d时达到峰值,后渐进性下降,碱烧伤后早期浸润在病灶区角膜基质内,后期浸润范围与溃疡面积相同,碱烧伤后各时间点A组和B组角膜PMNs密度均明显低于C组(P<0.05),且14、21d时,A组均明显低于B组(P<0.05)。碱烧伤后各组角膜VEGF表达水平均在7~14d时达到峰值,28d时明显降低,碱烧伤后各时间点A组和B组VEGF表达水平均明显低于C组(P<0.05),且7、14、21d时,A组均明显低于B组(P<0.05)。结论:羊膜负载hUCMSCs移植治疗兔角膜碱烧伤可减少CNV形成,抑制碱烧伤后角膜血管化,角膜病理损伤及血管化与PMNs和VEGF密切相关。

自体角膜缘干细胞移植与常规翼状胬肉切除术疗效比较

目的通过翼状胬肉单纯切除术及翼状胬肉切除加自体角膜缘干细胞移植术2种手术方式治疗翼状胬肉,比较其治愈及复发情况及角膜上皮修复时间,评价其疗效。方法将86例(88眼)翼状胬肉患者随机分为翼状胬肉单纯切除术组(A组)43例44眼及翼状胬肉切除加自体角膜缘干细胞移植术组(B组)43例44眼,组间比较采用χ2检验比较其疗效。结果A组10例10眼复发,B组仅有1例1眼复发,2组比较差异有统计学意义(P<0.05);A组角膜上皮修复时间平均为(5.15±0.92)d,B组角膜上皮修复时间平均为(3.36±0.82)d,2组比较差异有统计学意义(P<0.05)。结论翼状胬肉切除加自体角膜缘干细胞移植术可以降低复发率,缩短角膜上皮修复时间,是治疗翼状胬肉较为安全有效的方法。

翼状胬肉角膜缘干细胞移植与羊膜移植的比较

目的:对比观察翼状胬肉切除加自体角膜缘干细胞移植术(PECT)或加羊膜移植术这2种方法治疗翼状胬肉的效果。方法:翼状胬肉91例分为胬肉切除加自体角膜缘干细胞移植术24例,胬肉切除加羊膜移植术67例。术后随访3a,比较2组的恢复情况及复发倾向。结果:胬肉切除角膜缘加干细胞移植术后仅5例术眼有异物感或不适,无任何复发倾向。而羊膜移植术后有18例术眼有异物感或不适,15例眼睑轻度水肿及结膜充血水肿,且2例有复发倾向,无1例睑球粘连。结论:翼状胬肉切除加自体角膜缘干细胞移植术或加羊膜移植术均为治疗翼状胬肉的可靠方法,但以前者更佳。

自体角膜缘干细胞移植与结膜瓣转位治疗翼状胬肉的对比研究

目的探讨自体角膜缘干细胞移植与结膜瓣转位治疗翼状胬肉的疗效。方法将126例(214眼)翼状胬肉患者随机分为2组:角膜缘干细胞移植组58例92眼,结膜瓣转位组68例122眼,分别在手术显微镜下行胬肉切除联合自体角膜缘干细胞移植术和胬肉切除联合结膜瓣转位术,术后随访1a,观察并比较2组复发率和并发症情况。结果术后1a,自体角膜缘干细胞移植组4眼复发,复发率为4.35%,结膜瓣转位组23眼复发,复发率为18.85%,2组术后复发率比较差异有显著统计学意义(P=0.002<0.01)。角膜缘干细胞组无一眼发生睑球粘连,而结膜瓣转位组有9眼出现睑球粘连。结论自体角膜缘干细胞移植是治疗翼状胬肉的一种有效方法。

不同术式治疗原发性翼状胬肉的临床效果分析

目的:比较分析羊膜移植术、自体角膜缘干细胞移植术及术中应用0.2g/L丝裂霉素C治疗原发性翼状胬肉的临床效果。方法:回顾性分析我院232例曾行原发性翼状胬肉手术的患者,其中82例行羊膜移植术治疗,90例行自体角膜缘干细胞移植术治疗,60例在术中应用0.2g/L丝裂霉素C治疗,比较三种术式术后复发率及并发症。结果:自体角膜缘干细胞移植术和术中应用0.2g/L丝裂霉素C治疗原发性翼状胬肉的复发率低于羊膜移植术(P<0.05);而自体角膜缘干细胞移植术和术中应用0.2g/L丝裂霉素C之间复发率无差异(P>0.05)。角膜上皮缺损,流泪和畏光在0.2g/L丝裂霉素组发生比例较其他两组高(P<0.05),在自体角膜缘干细胞移植组和羊膜移植组两组间差异无统计学意义(P>0.05)。结膜充血及结膜下出血在三组间无统计学差异(P>0.05)。结论:与其他两种术式相比,自体角膜缘干细胞移植术治疗原发性翼状胬肉,有着复发率低、并发症少的优点。

深低温保存角膜缘干细胞活性实验研究

目的 评价深低温冷冻保存兔眼角膜缘干细胞活性。方法 12只 (12眼 )新西兰白兔 ,制备 2mm周边角膜和 2mm球结膜的环形浅层角膜缘植片及角膜干细胞缺乏眼表疾病模型。角膜缘植片通过程序降温仪程序降温后 - 196℃深低温保存 ,30天和 6 0天后行角膜缘自体移植。组化及免疫组化证实深低温保存的角膜缘干细胞活性。结果 角膜干细胞缺乏导致角膜上皮愈合延迟 ,上皮结膜化 ,PAS染色阳性 ,AE5染色弱阳性 ;基质水肿 ,角膜血管化。深低温保存角膜缘干细胞自体移植能促进角膜上皮愈合 ,为角膜表型 ,PAS染色阴性 ,AE5染色阳性。基质水肿逐渐吸收 ,新生血管消退、变细。结论 深低温保存法能保存角膜缘干细胞活性 ;能随时按需为临床提供活性角膜缘材料 ;为深低温保存角膜缘干细胞移植治疗临床眼表疾病提供了实验依据。

HLA匹配角膜缘干细胞联合羊膜移植修复翼状胬肉

背景:HLA匹配的角膜缘干细胞联合羊膜移植治疗翼状胬肉是眼科医生治疗的新理念,该方法治疗后并发症少、康复快、复发率低,尤其是能减少移植排斥反应等已被广泛接受。目的:探讨HLA匹配的角膜缘干细胞联合羊膜移植治疗翼状胬肉疗效。方法:选取2013年3月1日至2014年4月30日行HLA匹配的角膜缘干细胞联合羊膜移植治疗的翼状胬肉患者47例(47眼)作为试验组;回顾性分析2010年3月1日至2012年12月1日采用单纯翼状胬肉切除方法治疗的翼状胬肉患者40例(47眼)作为对照组,对两组患者治愈和复发情况进行比较。结果与结论:试验组47例,全部顺利完成治疗,无并发症,其中1例切口感染,特殊对症治疗后均已好转出院,全部患者均无移植排斥反应。试验组治愈45眼,复发2眼,复发率为4.3%。对照组47眼中治愈32眼,复发15眼,复发率31.9%。结果表明HLA匹配的角膜缘干细胞联合羊膜移植治疗翼状胬肉并发症少、复发率低,是治疗翼状胬肉一种行之有效的方法。

骨髓间充质干细胞移植重建碱烧伤眼表的实验研究

目的探索人骨髓间充质干细胞(MSCs)的诱导分化条件,观察负载人MSCs的羊膜移植重建碱烧伤大鼠眼表的疗效。方法传三代人MSCs分组培养,不同条件刺激后种于羊膜表面。碱烧伤后1个月大鼠随机分A、B、C和D组:A组(A1、A2、A3、A4)移植不同条件诱导的人MSCs,B组移植人角膜缘干细胞(LSCs),C组移植羊膜,D组未治疗。术后观察眼表并评分,2周和1个月时行印迹细胞学(IC)检查,1个月时切片行苏木精-伊红染色,免疫组织化学和免疫荧光检测溴脱氧尿嘧啶(BrdU)、ABCG2、K3/K12、Cx43和血管内皮生长因子(VEGF)。结果术后A、B组较C、D组角膜新生血管和炎性细胞减少、透明度增加(P<0.05)。IC检查示A、B组未见杯状细胞,C、D组可见杯状细胞。A、B组均形成3层以上上皮细胞,C组3层左右,D组为1～2层。免疫组织化学和免疫荧光检查示:A、B组角膜表面可检测到BrdU、ABCG2阳性细胞;A组Cx43均为阴性,A4组中有2个标本K3/K12为弱阳性,其余A组标本均为阴性,B组K3/K12、Cx43均为阳性;A、B组VEGF均较低,C组较强,D组最强。结论人MSCs与人LSCs相同,可成功重建碱烧伤大鼠眼表。其眼表重建功能可能与抗炎和抗血管化作用有关。

自体角膜缘干细胞移植术治疗复发性翼状胬肉疗效观察

目的：观察自体角膜缘干细胞移植术治疗复发性翼状胬肉疗效。方法：把66例81眼复发性翼状胬肉患者随机分为两组,治疗组：34例44眼行翼状胬肉切除联合自体角膜缘干细胞移植术;对照组：32例37眼行翼状胬肉切除联合带蒂结膜瓣转移术。胬肉复发的定义：血管纤维组织重新侵入角膜。结果：术后随访12-24mo。治疗组治愈率98%,复发率2%;对照组治愈率84%,复发率16%。两组复发率有统计学意义（χ^2=4.62,P〈0.05）,两组均无明显并发症。结论：自体角膜缘干细胞移植术治疗复发性翼状胬肉疗效高,复发率低于带蒂结膜瓣转移术。

角膜缘部干细胞对角膜上皮创伤愈合的影响

利用角膜缘上皮移植、结膜移植和非手术的方法分别对角膜上皮创伤伴随角膜缘损伤的兔眼进行实验研究,结果表明角膜缘上皮移植组上皮愈合时间短(平均8.5天),且无杯状细胞,无或极少量新生血管;结膜移植组,上皮愈合时间延长(平均11.5天),含有杯状细胞和新生血管;非手术组形成角膜血管翳性混浊。而不伴随角膜缘损伤仅有角膜中央上皮创伤的兔眼愈合时间最短(平均3.5天),愈合后无新生血管及杯状细胞。结果证实角膜缘部干细胞的存在并对角膜上皮再生有直接作用。

自体角膜缘部上皮移植治疗翼状胬肉

采用自体角膜缘部上皮移植治疗原发性胬肉５５眼，复发性胬肉６眼。经３～６个月随访，原发性胬肉全部治愈无复发，复发性胬肉有一例复发。研究表明本手术法与结膜移植法相比有复发率低、眼部不充血、角膜上皮恢复时间短、角膜无新生血管等优点，并对自体角膜缘部上皮移植治疗翼状胬肉的机理进行了探讨。

翼状胬肉模型的建立

目的人为地造成局部角膜缘干细胞缺损以及慢性结膜炎症的眼表模型,探讨翼状胬肉的形成原因。方法40只新西兰大白兔随机分为A、B、C、D 4组,设右眼为实验眼,左眼为对照眼。采用手术切除1个象限角巩膜缘组织的方法产生角膜缘干细胞缺损模型,眼部以稀盐酸(A、B组)或滑石粉(C、D组)定期点眼产生慢性角结膜炎模型。实验眼在同一眼上观察同时产生角膜缘干细胞缺损和慢性角结膜炎模型后的形态学变化,对照眼分别采用两种模型之一的相应形态学改变。于2、4、8、12周每只眼记录角膜上皮缺损、球结膜充血和结膜新生物情况,评分,并随机每组取材2只作病理。结果切除角膜缘干细胞的兔眼,可以在短期内在切除角膜缘干细胞的方向上出现类胬肉增殖组织样改变。类胬肉增殖组织的形成、程度和是否有局部的炎症反应以及角膜上皮完整性的破坏呈正相关性。结论局限性角膜缘干细胞的缺损和长期慢性炎症刺激的同时作用可能是翼状胬肉形成的主要原因。