经角膜基质细胞诱导的大鼠骨髓间充质干细胞在人羊膜上构建角膜上皮移植片

目的:研究将骨髓间充质干细胞(mesenchymal stem cells,MSC)诱导分化后在羊膜表面培养构建角膜上皮移植片的可行性。方法:取SD大鼠骨髓间充质干细胞和角膜基质细胞,分别培养并传2代后进行共培养,取共培养7d后的MSC覆载于新鲜人羊膜,再培养7d。对MSC及诱导后MSC进行免疫荧光染色和扫描电镜检查;对覆载细胞的羊膜进行HE染色及免疫荧光染色。结果:MSC在体外培养条件下贴壁生长,免疫荧光染色CD29和CD44阳性,CK12阴性。经角膜基质细胞诱导分化后,MSC细胞CK12染色转为阳性。诱导后MSC接种到羊膜表面,迅速贴壁生长,组织学特性无明显改变,CK12染色仍为阳性。结论:经角膜基质细胞诱导的MSC表现出角膜上皮细胞特征,在羊膜上生长后保持不变。     

模拟角膜缘干细胞微环境诱导人多潜能干细胞分化为角膜上皮细胞的研究

目的：探讨模拟角膜缘干细胞(LSCs)微环境诱导人多潜能干细胞(hiPSCs)分化为角膜上皮细胞的可行性。方法：体外建立hiPSCs细胞系，利用transwell体系将hiPSCs与角膜基质细胞共培养模拟角膜缘干细胞微环境，添加小分子骨形态发生蛋白4(BMP4)和特异性转化生长因子β抑制剂(SB431542)，诱导hiPSCs向角膜上皮细胞分化。采用免疫荧光染色、流式细胞方法检测角膜上皮细胞特异标志物 CK3和CK12，角膜上皮细胞前体CK15，角膜缘干细胞标志物ABCG5的表达。结果：hiPSCs体外培养增殖活跃，免疫荧光染色显示干细胞特异性标志物OCT4、SOX2、TRA-1-60、NANOG呈阳性。采用transwell体系将hiPSCs与角膜基质细胞共培养，免疫荧光染色结果显示角膜缘干细胞标志物ABCG5及角膜上皮细胞前体标志物CK15阳性，角膜上皮细胞标志物CK3及CK12阴性； 在共培养的基础上添加小分子BMP4和SB431542，免疫荧光染色及流式细胞检测结果显示角膜上皮细胞特异性标志物CK3阳性表达，且随分化时间延长表达比例增高。结论：模拟角膜缘干细胞微环境同时添加小分子SB431542及BMP4，可成功诱导体外培养的hiPSCs向角膜上皮细胞分化。     

体外诱导人骨髓间充质干细胞向色素上皮样细胞分化的研究

目的 探讨人骨髓间充质干细胞(mesenchymal stem cells,MSC)向色素上皮样细胞诱导、分化的可行性,为视网膜移植提供种子细胞的来源.方法 体外培养人视网膜色素上皮细胞(human retinal pigment epithelium,HRPE)和 MSC,应用免疫细胞化学法检测CD34、CD44 和角蛋白的表达情况,确定2种细胞的来源;通过 Transwell 6孔板非接触共培养的方式,建立 HRPE 和 MSC 共培养体系,显微镜下观察人 MSC 的形态学变化;对于诱导后的 MSC 进行免疫细胞化学鉴定,确定角蛋白的表达情况.结果 MSC呈纤维样克隆样生长,人MSC的CD44(+),CD34(-);HRPE原代培养时细胞内布满色素颗粒,角蛋白表达(+).HRPE和MSC共培养体系中,部分长梭形的MSC逐渐变成多边形,细胞内出现典型的色素颗粒;分化的色素上皮样细胞角蛋白表达阳性.结论 MSC与HRPE在非接触共培养诱导方式下能分化成色素上皮样细胞;分化的细胞具有上皮细胞的特异性标志,诱导后的细胞是否具有HRPE的功能尚有待于进一步研究.     

人角膜基质细胞诱导人骨髓间充质干细胞分化为角膜上皮细胞的初步研究

目的 探讨人骨髓间充质干细胞（MSC）体外分化为角膜上皮细胞的可行性.方法 实验研究.分别取第3代人MSC和自行培养的第3代人角膜基质细胞共同培养1周,实验组在Transwell共培养体系中培养,对照组不放置Transwell小室培养.1周后,观察实验组和对照组中人MSC光镜特征、间接免疫细胞化学染色和电镜结构,对被诱导分化的细胞进行综合鉴别.结果 第3代人MSC和人角膜基质细胞在体外培养条件下均能够较快贴壁生长.两种细胞共同培养1周后,可见部分细胞形态上呈上皮细胞特征,单克隆抗体AE1染色呈阳性,电镜下可见微绒毛、桥粒和张力丝等典型上皮细胞结构特征.结论 体外培养的人MSC在人角膜基质细胞的诱导下可能会分化为人角膜上皮样细胞.     

人骨髓间充质干细胞分化为上皮样细胞的初步研究

目的 应用猪板层角膜做载体将人骨髓间充质干细胞(human bone marrow mesenchymal stem cells,hMSC)跨胚层诱导为上皮样细胞,甚至角膜上皮样细胞,初步探讨hMSC作为构建组织工程角膜种子细胞的可行性.方法 实验研究.用密度梯度离心培养技术结合贴壁培养法分离纯化hMSC并传代,对体外培养的hMSC进行免疫表型鉴定.将传代后的hMSC接种于去上皮的猪角膜基质片前弹力层表面培养诱导分化,免疫荧光检测角膜上皮细胞标志物角蛋白12(CK12)以及角膜缘干细胞标记物ABCG2和CK19的表达.运用体外培养的方法使种植在前弹力层表面的细胞复层化.待细胞融合形成单层后,置入插入式培养皿中进行气液界面培养.培养4周后进行HE染色及免疫组织化学检测,光镜下观察其复层情况.结果 获得的hMSC可以在体外培养扩增,表现出很强的增殖潜能.流式细胞仪示:培养的hMSC CD45阳性率为0.06%,CD34为0.41%,CD44为86.43%,CD29为85.72%,CD105为90.72%.诱导4周后部分细胞表达CK12和CK19,不表达ABCG2.运用气液界面培养法进行体外复层的结果显示可以形成1～2层的上皮样细胞,并很可能为角膜上皮样细胞.结论 在本实验的诱导条件下,hMSC可以分化为上皮样细胞,并很可能为角膜上皮样细胞,hMSC有可能作为组织工程技术角膜上皮重建的种子细胞的选择.     

体外共培养诱导人羊膜间充质干细胞分化为眼表上皮细胞的研究

背景 人羊膜间充质干细胞（hAMSCs）在体外能诱导分化为多种体细胞,但目前有关hAMSCs分化为眼表细胞的研究鲜有报道. 目的 探讨体外共培养诱导hAMSCs向眼表细胞分化的可能性及其分化机制.方法 本研究经广州医科大学第二附属医院伦理委员会批准,在健康产妇的知情同意下从人胎盘中分离hAMSCs并进行培养,采用流式细胞术计数分析CD44、CD45、CD73、CD90在培养细胞中的表达以鉴定细胞,此外通过成骨诱导分化实验和成脂诱导分化实验对培养细胞进行体外分化鉴定.在患者知情同意下获取眼科手术中弃用的人眼球结膜组织,用组织块培养法分离和培养人球结膜成纤维细胞（hBCFs）,将hAMSCs与hBCFs在Transwell培养体系中进行共培养,分为hAMSCs培养组和hAMSCs与hBCFs共培养组,用含质量分数5％胎牛血清（FBS）的DMEM高糖/F12培养基培养7d,采用免疫荧光技术检测hAMSCs中对上皮细胞角蛋白19（CK19）的表达,评价hAMSCs向眼表细胞分化的程度,并检测hAMSCs中α-平滑肌肌动蛋白（α-SMA）的表达,评价其分化过程与间质上皮化转换（MET）过程的关系.结果 传代至3～7代的hAMSCs均为细长形,但随着传代增加,细胞体积增大,细胞中CD44、CD73、CD90表达呈强阳性,但不表达CD45.经成骨或成脂诱导分化后3～4周,细胞中茜素红S（ARS）染色或油红O染色阳性.hAMSCs与hBCFs共培养后1周,hAMSCs形态由细长形变为类上皮细胞型,共培养组的部分细胞中CK19表达阳性,所有细胞α-SMA表达呈微弱阳性,而hAMSCs细胞培养组可见呈绿色荧光的α-SMA阳性细胞,但不表达CK19. 结论 通过体外共培养可诱导hAMSCs向人眼表细胞分化,其分化机制可能与MET过程相关.     

羊膜为载体的人角膜缘上皮细胞移植治疗大鼠角膜碱烧伤的研究

目的研究人角膜缘上皮细胞的生物学特性,探讨体外培养的人角膜缘上皮细胞移植进行眼表重建的临床应用价值及前景。方法体外培养人角膜缘上皮细胞,通过免疫荧光染色法和逆转录聚合酶链反应（RT．PCR）法分析表型,Brdu掺入法检测慢周期（slow—cycling）细胞。建立大鼠角膜碱烧伤模型,随机分为单纯对照组、羊膜移植（AMT）组及羊膜为载体的角膜缘上皮细胞移植（LSAT）组,术后通过裂隙灯显微镜观察、角膜切片HE染色及免疫荧光染色等方法对各组大鼠眼表恢复情况进行评价。结果培养早期大多数角膜缘上皮细胞p63和K19染色阳性,仅少数细胞K3染色阳性;随培养时间的延长,K3阳性细胞增多,部分细胞同时表达p63和K3。RT—PCR示培养的角膜缘上皮细胞中p63和K12均有阳性表达,而角膜上皮细胞仅表达K12。Brdu掺入法检测到慢周期细胞。动物实验表明,LSAT可以显著减轻角膜病变,使角膜恢复完整性和透明性,眼表评分各项指标与AMT组和对照组比较差异均有统计学意义。LSAT组大鼠角膜切片染色示大部分上皮细胞抗人核和K3染色阳性。结论体外培养的人角膜缘上皮细胞中p63不仅表达于角膜缘干细胞,在短暂扩充细胞中也有一定量的表达;慢周期细胞的存在证实了培养的角膜缘上皮细胞中角膜缘干细胞的存在,p63和K19阳性、K3阴性的细胞为角膜缘干细胞;羊膜为载体的体外培养的人角膜缘上皮细胞移植可有效重建眼表,具有较高的临床应用价值与发展前景。     

大鼠骨髓间充质干细胞体外诱导为光感受器样细胞的研究

目的 探讨体外培养的大鼠骨髓问允质干细胞(MSC)诱导为光感受器样细胞的可能性.方法 实验研究.采用贴壁筛选法分离、培养SD大鼠骨髓MSC细胞,经流式细胞仪鉴定后,用含小鼠表皮生长因子(EGF)的培养液诱导MSC细胞8～10 d.用免疫组织化学染色观察诱导后的细胞是否表达视紫红质,并用流式细胞术检测表达视紫红质的细胞比例.结果 传3代的MSC细胞生长曲线呈S型,显示培养细胞具有正常的分裂增殖特性;超过70%的传1代和传2代细胞为CD90单阳性,从传3代绌胞开始CD90单阳性的百分数一直高于93%,即采用贴壁筛选法可获得大量高纯度的大鼠骨髓MSC细胞.经EGF诱导后的细胞有15.32%±0.92%表达视紫红质,而未经诱导的细胞表达视紫红质的比例为1.64%±0.78%.结论 经EGF诱导后,15.32%的大鼠MSC可分化为光感受器样细胞.     

种植人骨髓间充质干细胞的猪角膜板层移植治疗兔角膜损伤的初步研究

目的研究种植人骨髓间充质干细胞（MSCs）的猪角膜基质治疗兔角膜损伤的可能性。方法用全骨髓贴壁法分离纯化人MSCs并传代,流式细胞仪检测免疫表型及诱导成脂、成骨分化鉴定。12只新西兰白兔随机分为2组,实验组取第3代MSCs接种于去上皮的猪角膜基质上,培养4 d后移植到广泛损伤的兔角膜上,对照组单纯移植去上皮猪角膜基质。术后2、4、8周,取各实验眼行组织学检查,观察移植的MSCs及猪角膜基质的存活、转归及移植局部的反应。免疫组织化学、免疫荧光染色检测移植后角膜上皮细胞角蛋白12的表达。结果培养获得的MSCs中CD29阳性者占95.97%,CD44阳性者占96.49%,CD90阳性者占92.79%,CD105阳性者占94.66%,CD34阳性者占0.59%,CD45阳性者占0.36%,符合MCSs的免疫表型,并可以诱导成脂及成骨分化。实验组MSCs接种到去上皮猪角膜基质后贴附、生长迅速,术后植片在植床上存活良好,无排斥反应,角膜较对照组透明,新生血管少,而对照组在移植后发生排斥反应。实验组角膜免疫组织化学及免疫荧光染色均检测出CK12阳性细胞。结论种植MSCs的猪角膜基质移植到损伤兔角膜后可以存活,MSCs可以分化为角膜上皮样细胞,具有构建组织工程角膜的潜能。     

转化生长因子β诱导基因在人角膜组织及细胞中的表达

背景 临床研究表明多数角膜营养不良的发病与转化生长因子β诱导(TGFBI)基因突变有关,但其发病的分子机制尚不完全清楚. 目的 研究TGFBI基因在人角膜组织及体外培养的角膜上皮细胞和基质细胞中的表达,为进一步研究角膜营养不良的发病机制奠定基础. 方法 对人角膜上皮细胞和基质细胞进行培养和传代,应用逆转录聚合酶链反应( RT-PCR)法检测TGFBI mRNA在人角膜组织及细胞中的表达,将供体角膜组织制成石蜡包埋切片,利用免疫组织化学法检测TGFBI蛋白在角膜组织、人角膜上皮细胞和角膜基质细胞中的表达,采用免疫荧光技术检测TGFBI蛋白在细胞爬片的表达. 结果 RT-PCR检测显示人角膜组织和基质细胞中在1274 bp处可见TGFBI mRNA的清晰条带,而角膜上皮细胞中亦有TGFBImRNA表达.免疫组织化学检测显示,TGFBI蛋白在人角膜组织中基质细胞的细胞质中呈阳性表达,但人角膜上皮细胞中未见TGFBI蛋白表达.免疫荧光检测技术显示,人角膜基质细胞胞质中TGFBI蛋白呈红色荧光,而角膜上皮细胞未见TGFBI蛋白表达. 结论 TGFBI主要在人角膜基质层表达,而在上皮层几乎不表达,有助于进一步研究TGFBI在角膜营养不良发病机制中的作用.     

Conjunctival epithelial cells cultured on human amniotic membrane fail to transdifferentiate into corneal epithelial-type cells

PURPOSE: Previous studies on the use of human amniotic membrane (HAM) in rabbit stem cell deficiency models have found the new epithelium growing over the HAM to express cornea-specific keratins (K3 and K12) in 40% of the cases, suggesting that HAM may have induced conjunctival epithelial cells to transdifferentiate into cornea-type epithelial cells. The current study was performed to determine whether HAM could induce transdifferentiation of conjunctival epithelia] cells when cultured in vitro. METHODS: Conjunctival grafts taken from the fornices of New Zealand white rabbits (6-12 weeks old) were placed over HAMs and lifted to an air-media interface using polypropylene double rings. These cultures were maintained in supplemented hormonal epithelial medium with and without 3T3 feeder cells. Rabbit corneal epithelial cells were cultured similarly using strips of keratolimbal grafts placed over HAM. The cultures were terminated at various times between the 8th and 15th day. The cultured epithelial cells were examined histologically and immunohistochemically using monoclonal antibodies AK-2 (to K12 keratin), AM-3 (to goblet cell mucin), and AE-5 (to K3 keratin). RESULTS: Both conjunctival and corneal epithelial cells cultured on HAMs showed multilayered, differentiated epithelial structures. On immunohistochemical examination, both epithelial cells stained positive for AE-5. None of the cultured conjunctival epithelial cells stained positively for AK-2, while the corneal epithelial cells showed positive staining with AK-2. There were no AM-3-positive goblet cells in either epithelial cell culture. There was no difference in the immunohistochemical patterns between cultures with or without 3T3 feeder cells. However, culture without feeder cells seemed to manifest a more degenerative appearance than those with feeders. CONCLUSION: HAM does not induce transdifferentiation of conjunctival epithelial cells into corneal-type epithelial cells under the in vitro culture conditions used in this study.     

RANK, RANKL, OPG, and M-CSF expression in stromal cells during corneal wound healing

PURPOSE: To examine the influx of monocytes into the cornea after epithelial scrape injury and the expression of chemokines that potentially regulate monocyte phenotype in cultured corneal fibroblasts and keratocytes in situ. METHODS: Monocytes were detected by immunocytochemistry for the monocyte-specific antigen CD11b, in unwounded and epithelial scrape-wounded mouse corneas. The receptor activator of NF-kappa B ligand (RANKL), osteoprotegerin (OPG), and monocyte chemotactic and stimulating factor (M-CSF) mRNAs were detected in cultured mouse stromal fibroblasts by RT-PCR and RNase protection assay. RANKL, OPG, and M-CSF proteins were detected in cultured mouse stromal fibroblasts by immunoprecipitation and Western blot analysis. RANKL, RANK, M-CSF, and OPG proteins were detected in unwounded and wounded mouse corneas by immunocytochemistry. Chimeric mice with green fluorescent protein-labeled bone marrow-derived cells underwent corneal scrape injury and were monitored by fluorescence microscopy and immunocytochemistry. RESULTS: A small number of cells expressing the monocyte-specific CD11b antigen were detected in the stromas of unwounded mouse corneas. A larger number of CD11b-positive cells was detected in the stroma at 24 or 48 hours after epithelial scraping injury. Experiments with chimeric mice with fluorescent green protein-labeled, bone marrow-derived cells demonstrated conclusively the origin of these CD11b(+) cells. RANKL, OPG, and M-CSF mRNAs and proteins were detected in cultured mouse stromal fibroblasts. RANKL, M-CSF, and OPG proteins were detected in unwounded corneas, but were expressed at higher levels in stromal cells during the 24- to 48-hour interval after epithelial scrape injury. RANK was detected in stromal cells presumed to be monocytes at 24 and 48 hours after epithelial injury. CONCLUSIONS: Cells expressing the CD11b monocyte-specific antigen appear in the corneal stroma in high numbers by 24 hours after epithelial injury and persist beyond 10 days after wounding. Cultured corneal fibroblasts and keratocytes in situ express RANKL, OPG, and M-CSF cytokines involved in regulating osteoclast differentiation from monocytes in bone. Cells expressing RANK were detected in the stroma at 24 and 48 hours after epithelial injury. The cytokine systems that regulate monocyte transition to osteoclast in bone are upregulated in the cornea in response to epithelial injury and may participate in regulating monocyte phenotype during corneal stromal wound healing.     

Corneal epithelial cells and stromal keratocytes efficently produce CC chemokine-ligand 20 (CCL20) and attract cells expressing its receptor CCR6 in mouse herpetic stromal keratitis

PURPOSE: CC chemokine-ligand 20 (CCL20) is known to be selectively expressed by surface-lining mucosal epithelial cells and skin epidermal keratinocytes and to attract cells such as immature dendritic cells and effector T cells via CCR6. This study evaluated the ability of corneal epithelial cells and stromal keratocytes to produce CCL20 in vitro and in vivo. METHODS: Human corneal epithelial cells (HCE) and corneal keratocytes (HCK) were treated without or with various cytokines and expression of CCL20 mRNA and secreion of its protein were evaluated by RT-PCR and ELISA. Induction of CCL20 mRNA in HCE and HCK was also examined upon in vitro infection with HSV-1. Using a mouse model of herpetic stromal keratitis (HSK), induction of CCL20 expression and accumulation of cells expressing CCR6 were evaluated by RT-PCR and immunohistochemistry. RESULTS: Not only corneal epithelial cells but also stromal keratocytes efficiently expressed CCL20 mRNA and protein upon stimulation with IL-1beta and TNF-alpha. In vitro infection with HSV-1 also induced CCL20 mRNA in both types of cells. In a mouse herpetic stromal keratitis model, prominent accumulation of CCL20 and CCR6 mRNA was revealed in HSV-1-infected corneas. Furthermore, immunohistochemistry demonstrated production of CCL20 by corneal epithelial cells as well as stromal keratocytes and stromal infiltration of DEC205+ dendritic cells, CD4+ T cells and CD8+ T cells. Double staining revealed that CCR6-expressing cells were mostly MHC class II+ dendritic cells. CONCLUSIONS: Not only epithelial cells but also stromal keratocytes are efficient producers of CCL20 in the cornea and recruit CCR6-expressing cells such as dendritic cells into inflamed cornea.     

The Preliminary Experimental Study of Induced Differentiation of Embryonic Stem Cells into Corneal Epithelial Cells

Purpose: To study preliminarily induced differentiation of embryonic stem cells into corneal epithelial cells in vitro.Methods: Murine embryonic stem cells were co-cultured with Rabbit limbal corneal epithelial cells in Transwell system to induce differentiation. Mophological and immunohistochemical examination were implemented.Results: The induced cells from embryonic stem cells have an epithelial appearance. The cells formed a network and were confluent into film gradually after being co-cultured with rabbit limbal corneal epithelial cells for 24 ~ 96 hours. The cells ranged mosaic structure and localized together with clear rim. Most of the cells showed polygonal appearance. Transmission electron microscope showed lots of microvilli on the surface of induced cells and tight junctions between them. These epithelial-like cells expressed the corneal epithelial cell specific marker cytokeratin3/cytokeratin12. Conclusion: The potential mechanism of the differentiation of murine embryonic stem cel     

CD40 expression in normal human cornea and regulation of CD40 in cultured human corneal epithelial and stromal cells

PURPOSE: To determine whether CD40-CD40 ligand (CD40L) interaction plays a role in corneal inflammatory responses, the expression of CD40 and CD40L on normal human cornea was investigated. In addition, using cultured human corneal epithelial (HCE) and human corneal stromal (HCS) cells, the regulation of CD40 expression in human corneal cells investigated, including that induced by proinflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha. METHODS: Frozen optimal cutting temperature (OCT) compound-embedded sections of corneal tissues obtained from 18 normal human corneas were examined by an immunoperoxidase staining technique with anti-CD40 and anti-CD40L monoclonal antibodies (mAbs). Also, cultured HCE and HCS cells, with IFN-gamma (250-1000 U/mL) or TNF-alpha (500-4000 U/mL) treatment for 1 to 4 days and with no treatment, were stained by the immunofluorescence technique with mAbs and analyzed by flow cytometry. RESULTS. The area of positive staining for CD40 showed a topographical difference. The limbal epithelial cells were predominantly positive for CD40. Positive staining was also found to a lesser extent on the cells in the basal layer of peripheral corneal epithelium. Epithelial cells of the central cornea showed no immunoreactivity for CD40. Corneal stromal cells were negative for CD40 in most of the donor tissues (positive: 5 of the 18 corneas). Endothelial cells were distinctly negative for CD40. Cultured HCE cells were also positive but decreased in positive cell number with lengthening culture period. None or less than 5% of the cultured HCS cells were CD40 positive. IFN-gamma enhanced CD40 expression on both cell types. In contrast, TNF-alpha enhanced CD40 on HCE but not on HCS cells. No component cells of normal human cornea or cultured HCE and HCS cells showed immunoreactivity for CD40L. CONCLUSIONS: In the human cornea, CD40 is expressed predominantly on limbal epithelial cells and also on cultured HCE cells with high proliferative potential. In addition, the expression of CD40 is induced on cultured HCE and HCS cells differentially by proinflammatory cytokines, such as IFN-gamma and TNF-alpha.     

Differential regulation of fibronectin synthesis in three different types of corneal cells

The production of fibronectin (FN) and its response to serum or epidermal growth factor (EGF) were investigated in three different types of rabbit corneal cells cultured in vitro. The corneal epithelial cells, stromal fibroblasts (keratocytes) and endothelial cells were separately cultured in different media: basic medium containing minimal serum (0.5%), basic medium with supplementary serum at a final concentration of 10% and basic medium with 100 ng/ml EGF, respectively. FN production by each type of cell was examined either by the immunofluorescent staining method or by the metabolic labeling method followed by immunoprecipitation of FN in the culture medium. Each type of corneal cell produced and secreted FN. FN secretion into the culture medium by keratocytes and by endothelial cells was enhanced by the addition of EGF. However, FN secretion by epithelial cells was lowered by the additional serum or EGF. Furthermore, when the epithelial cells were cultured in the basic medium, DNA synthesis was low but FN secretion was high. These results suggest that the control mechanism of FN production differs between epithelial cells and keratocytes or endothelial cells.     

Induction of epithelial progenitors in vitro from mouse embryonic stem cells and application for reconstruction of damaged cornea in mice

PURPOSE: Severe ocular surface diseases and injuries cause loss of the corneal limbal epithelium, leading to re-epithelialization by bulbar conjunctival cells, resulting in vascularization of the cornea, conjunctival scarring, and loss of visual acuity. In this study, the optimal culture condition for induction of differentiation of epithelial progenitor cells from embryonic stem (ES) cells was determined for use in transplantation to damaged cornea in mice. METHODS: Mouse ES cells were cultured on Petri dishes coated with several extracellular matrix proteins, and the markers for epithelial cells were analyzed with RT-PCR and Western blot analysis. The optimal condition for induction of epithelial progenitor cells was determined, and the progenitors were transplanted onto mouse eyes with corneal epithelia that had been damaged by exposure to n-heptanol. RESULTS: Epithelial progenitors were successfully induced by culturing mouse ES cells on type IV collagen for 8 days. These progenitors expressed keratin (K)12, which is specific to corneal epithelial cells, and cell surface CD44 and E-cadherin, both of which are essential in corneal epithelial wound healing. Complete re-epithelialization of the corneal surface occurred within 24 hours after transplantation. The resultant corneal epithelial cells expressed markers of the grafted cells, and no teratomata were observed during the follow-up period. CONCLUSIONS: Epithelial progenitors were successfully induced in vitro from ES cells and were applicable as grafts for treating corneal epithelial injury. ES cells may become an unlimited donor source of corneal epithelial cells for corneal transplantation and may restore useful vision in patients with a deficiency of limbal epithelial cells. This is an important first trial toward assessing the use of ES cells to reconstruct corneal epithelial cells.