人角膜缘干细胞体外培养的增殖与分化研究

目的了解角膜缘干细胞体外培养的增殖分化规律。方法组织块法培养细胞,测定细胞克隆形成率（ＣＦＥ）,免疫荧光染色检测干细胞表达角质蛋白Ｋ３的状况。结果原代培养２１天左右细胞生长达到饱和,传第１代７～１０天形成单层,ＣＦＥ为９．５２％±４．９７％;传第２代７天,ＣＦＥ为４．２５％±２．１０％（Ｐ＜０．０１）。正常角膜缘基底细胞不表达角质蛋白Ｋ３;原代培养的干细胞亦不表达Ｋ３,传第１代细胞有部分表达。结论人角膜干细胞位于角膜缘基底部,培养的角膜缘干细胞早期具有较高的增殖力并保持干细胞的分化特性。     

体外不同区域角膜上皮细胞对5—FU耐受性的研究

为体外培养角膜上皮移植选取细胞来源，本文应用溴代脱氧尿嘧啶（Ｂｒｄｕ，胸腺嘧啶的类似物）渗入ＤＮＡ合成，抗—Ｂｒｄｕ单克隆荧光抗体标记细胞法，在流式细胞仪（ＦＡＣＳｔａｒｐｌｕｓ）上检测离体情况下兔角膜不同区域上皮细胞对５－ＦＵ耐受情况，以确定长周期细胞的部位。结果显示：经５－ＦＵ处理后的角膜周边及角膜中央部上皮细胞增殖明显受到抑制，而角膜缘细胞对５－ＦＵ毒性则有相对耐受性。由此得出：角膜缘上皮细胞群中有长周期的角膜干细胞存在。因而，行体外培养角膜上皮时，应选取角膜缘部的细胞。     

角膜缘干细胞培养上清液对血管内皮细胞增殖的影响

目的：观察角膜缘干细胞培养液对血管内皮细胞增殖的影响。

方法：分别培养角膜缘干细胞及球结膜上皮细胞,并通过免疫组化检测AE-5蛋白鉴别角膜缘干细胞。搜集两组培养细胞的上清液加入培养的人脐静脉血管内皮细胞中,并设立对照组。培养24h后通过MTT法检测细胞增殖情况并进行统计学分析。

结果：角膜缘干细胞AE-5染色呈阴性,而球结膜上皮细胞为阳性。加入角膜缘干细球结膜上皮细胞和对照组培养液的血管内皮细胞MTT值分别为2.097±0.079,1.981±0.034和1.990±0.044。三组间有显著性差异(F=9.169,P=0.000)。加入角膜缘干细胞培养上清液的血管内皮细胞增殖活性明显高于其他两组(P=0.005和P=0.007)。

结论：角膜缘干细胞培养液能够促进血管内皮细胞的增殖,这可能是角膜缘干细胞的特征。本研究从功能学角度为角膜缘干细胞理论提供更多的依据。     

人角膜上皮干细胞的低钙培养及碱性成纤维细胞生长因子对其增殖的影响

目的 探讨如何获取较纯的人角膜上皮干细胞,以及碱性成纤维细胞生长因子（fibroblast growth factor,bFGF)对人角膜缘上皮干细胞增殖的影响,为干细胞的移植奠定基础。方法 采用低钙培养法对人角膜缘体外培养,将培养的细胞行AE5免疫荧光染色,观察培养细胞的分化状态,并将其分别置于不同浓度的bFGF中,通过数码照相技术和计算机图像分析系统,检测角膜干细胞的增殖。结果 体外培养的细胞以角膜上皮干细胞为主;不同浓度的bFGF(1-100ng/ml)可促进体外培养的人角膜缘上皮干细胞的增殖（P＜0．001）,而各实验组间比较,差异无显著性（P＞0．05）。结论 将无钙培养液与低钙培养基组合,可获得较纯的、未分化的角膜干细胞;bFGF对人角膜上皮干细胞的增殖具有重要的促进作用。计算机图像分析系统适用定量检测呈膜状生长的上皮细胞增殖,是一种新颖、实用、准确的检测手段。     

上皮性钙黏附蛋白在人角膜上皮中的表达研究

目的 研究上皮性钙黏附蛋白（E-cadherin）在人角膜上皮中的分布。方法低钙培养基培养人角膜缘干细胞．并取人角膜中央上皮及角膜边缘上皮组织，分别使用免疫组织化学及免疫细胞化学方法检测E-cadherin在人角膜中央上皮、角膜边缘上皮及低钙培养的人角膜缘干细胞中的分布情况。结果E-cadherin在角膜边缘上皮全层细胞即处于分化阶段的短暂扩充细胞中大量表达，在抑制干细胞分化、促进增殖的低钙培养的人角膜缘干细胞中表达减少，在终末分化细胞即角膜中央上皮细胞中没有表达。结论E-cadherin在处于分化阶段的角膜上皮细胞中有大量的表达。     

LY294002联合奥曲肽对bFGF诱导的兔晶状体上皮细胞增殖的影响

目的　探讨ＬＹ２９４００２联合奥曲肽对碱性成纤维细胞生长因子（ｂａｓｉｃｆｉｂｒｏｂｌａｓｔｇｒｏｗｔｈｆａｃｔｏｒ,ｂＦＧＦ）诱导的兔晶状体上皮细胞（ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌｌｓ,ＬＥＣｓ）增殖的影响。方法　兔眼ＬＥＣｓ经原代和传代培养后,共分为４组:空白对照组、增殖对照组、实验药物组、增殖药物组。空白对照组使用仅含无血清ＤＭＥＭ培养;增殖对照组使用加ｂＦＧＦ（１０ｍｇ·Ｌ－１）的无血清ＤＭＥＭ;实验药物组培养时在不加ｂＦＧＦ增殖的情况下分别单独应用１０－５ｍｏｌ·Ｌ－１ＬＹ２９４００２、单独应用１０－９ｍｏｌ·Ｌ－１奥曲肽和联合应用１０－５ｍｏｌ·Ｌ－１ＬＹ２９４００２与１０－９ｍｏｌ·Ｌ－１奥曲肽来培养;增殖药物组在加ｂＦＧＦ增殖的情况下分别单独应用１０－５ｍｏｌ·Ｌ－１ＬＹ２９４００２、单独应用１０－９ｍｏｌ·Ｌ－１奥曲肽和联合应用１０－５ｍｏｌ·Ｌ－１ＬＹ２９４００２和１０－９ｍｏｌ·Ｌ－１奥曲肽进行细胞培养,每组重复５次。各组分别培养４８ｈ。ＭＴＴ比色法测定吸光度（Ａ值）分析各组细胞的生长抑制率,流式细胞仪检测各周期细胞的百分率。结果　增殖对照组与空白对照组相比,吸光度Ａ值升高（Ｐ＜０．０５）;实验药物组中各组分别与空白对照组相比,吸光度Ａ值均有不同程度下降（均为Ｐ＜０．０５）;增殖药物组中各组分别与增殖对照组相比,吸光度Ａ值明显下降（均为Ｐ＜０．０５）;生长抑制率与吸光度Ａ值趋势相同。经流式细胞仪分析,实验药物组中联合用药组与两个单独用药组相比Ｇ０／Ｇ１期细胞比例增高,Ｓ期细胞比例降低（均为Ｐ＜０．０５）;增殖药物组中,联合用药组与两个单独用药组相比也出现Ｇ０／Ｇ１期细胞比例增高,Ｓ期细胞比例降低（均为Ｐ＜０．０５）。结论　ＬＹ２９４００２联合奥曲肽较单独用药对ＬＥＣｓ的增殖抑制作用更强。这为临床筛选药物防治后发性白内障提供依据。     

角膜缘组织定位培养和冷冻后培养的实验研究

目的验证角膜缘干细胞的组织学定位,探讨低温冷冻保存对其增殖活性的影响。方法取新鲜角膜缘上皮组织和相应部位浅层巩膜组织各10块进行体外细胞培养,对比观察细胞生长情况。取冷冻保存的角膜缘上皮组织14例,观察体外培养后细胞生长情况。通过免疫组化方法检测冷冻保存的角膜缘上皮细胞的增殖活性和培养后单层细胞K3角蛋白的表达。结果10例新鲜角膜缘上皮组织培养后,7例有上皮细胞生长,1周形成细胞单层;10例浅层巩膜组织培养后未见细胞生长。14例冷冻角膜缘上皮组织培养后,4例有上皮细胞生长,9d形成细胞单层。5例冷冻角巩膜环组织冰冻切片中,3例可见角膜缘上皮基底细胞PCNA表达阳性。培养细胞对K3角蛋白特异性的AE-5单克隆抗体免疫反应阳性。结论角膜缘干细胞定位于角膜缘上皮基底部,低温冷冻保存的角膜缘干细胞组织可以保持增殖活性,体外培养后生长分化成为角膜上皮。     

表皮生长因子对兔角膜上皮细胞生长作用的实验研究

研究了表皮生长因子（ＥＧＦ）对角膜上皮细胞的生长影响。用氚标的胸腺嘧啶核甘（３ＨＴｄＲ）掺入法证明ＥＧＦ单独对角膜上皮细胞无生长促进作用，而当和角膜基质块同时培养时，ＥＧＦ能明显促进角膜上皮细胞生长，不同浓度ＥＧＦ的每分钟放射性计数（ＣＰＭ）差异均具有显著性意义（Ｐ＜０．０５或Ｐ＜０．００１），并且随ＥＧＦ浓度增加对角膜上皮细胞促生长作用增强。提示ＥＧＦ能促进角膜上皮细胞的生长作用，但需要角膜基质的协同作用。     

角膜上皮干细胞定位特征的免疫组织化学研究

利用单克隆抗体ＡＥ５与分化型角膜上皮细胞中角蛋白Ｋ３特异性结合,研究缺乏分化标志特征的角膜上皮干细胞定位特点,应用免疫组织化学方法显示Ｋ３阳性表达的区域分布于除角膜缘上皮基底部以外所有角膜上皮细胞中,角膜上皮干细胞存在于角膜缘基底部ＡＥ５抗体反应阴性细胞中,即角膜干细胞位于角膜缘上皮层基底部。     

肿瘤坏死因子α和表皮生长因子对晶状体上皮细胞增殖的作用

目的研究肿瘤坏死因子α（ＴＮＦα）和表皮生长因子（ＥＧＦ）对人、牛晶状体上皮细胞增殖的影响。方法采用ＭＴＴ比色法检测ＴＮＦα和ＥＧＦ对晶状体上皮细胞（ｌｅｎｓｅｐｉｔｈｅｌｉａｌｃｅｌ，ＬＥＣ）增殖的作用。结果０．１Ｕ／ｍｌ的ＴＮＦα即可明显促进人、牛晶状体上皮细胞的增殖；ＥＧＦ为１、１０ｎｇ／ｍｌ时可明显促进牛晶状体上皮细胞增殖。结论细胞因子ＴＮＦα、ＥＧＦ通过促进ＬＥＣ的增殖参与后囊混浊的形成     

Hypoxia enhances the expansion of human limbal epithelial progenitor cells in vitro

PURPOSE: To demonstrate the effects of hypoxia on proliferation and differentiation of human limbal epithelial cells in vitro. METHODS: Primary human limbal epithelial cells were harvested from the rim of donor corneas. Colony-forming efficiency (CFE) and cell proliferation were observed in standard (20% O(2)) or hypoxic (2% O(2)) culture conditions. Cell cycle, forward scatter (FSC) and side scatter (SCC) of cells were analyzed by flow cytometry. Proliferating cells were also observed by pulse labeling (2 hours) with BrdU and Ki67 staining. Apoptosis was detected by TUNEL assay. Isolated colonies were examined by immunohistochemistry against K15, p63, involucrin, and K3. Involucrin expression was also analyzed by Western blot analysis. RESULTS: Both CFE and proliferation of limbal epithelial cells was significantly enhanced in hypoxia. Flow cytometry revealed a higher fraction of hypoxic cells in the G(0)/G(1)-phase and fewer cells in the S-phase, compared with normoxia. However, there was no difference in the uptake of BrdU during a 2-hour pulse, suggesting that hypoxic colonies contained rapidly cycling cells. Apoptotic cells were sparse in both groups, and hypoxic cells showed lower FSC compared with normoxic cells. Although there was no difference in the staining pattern of K15, p63, and Ki67, cells cultivated in normoxia expressed higher levels of the differentiation markers involucrin and K3. Significantly higher involucrin expression was also observed by Western blot. CONCLUSIONS: Hypoxic culture (2%) enhances proliferation while inhibiting differentiation of limbal epithelial cells in vitro.     

Efficient cryopreservative conditions for cultivated limbal and conjunctival epithelial cells

PURPOSE: To examine the effects of cryopreservation on the viability of cultivated corneal limbal and conjunctival epithelial cells and to evaluate the optimal conditions for cryopreservation. METHODS: The cultivated human limbal epithelial cells (HLECs) were stored in media including 20%, 50%, and 90% fetal bovine serum (FBS) and 10% dimethyl sulfoxide (DMSO) at -196 degrees C for 1 week. The cultivated rabbit conjunctival epithelial cells were stored in 10%, 20%, and 50% FBS with 10% glycerol or DMSO as a cryoprotectant at -196 degrees C for 1 week. After thawing, cell viability was assessed using the trypan blue vital staining and 3-[4,5-dimethylthiazol-2-yl]-2,5-dephenyl tetrazolium bromide (MTT) assay. Immunofluorescent staining was performed with cytokeratin 3/12 antibody. Colony-forming efficiency (CFE) was evaluated 2 weeks after culture. RESULTS: HLECs cryopreserved with 50% FBS showed the highest cell viability, whereas those with 20% FBS revealed the lowest survival rate (87.1% +/- 0.8% and 79.8% +/- 4.01%, respectively; P = 0.030). CFE of HLECs was 2.13 +/- 1.35%, 2.31 +/- 2.23%, and 1.94 +/- 0.72% in cells with 20%, 50%, and 90% FBS, respectively (P > 0.05). For conjunctival epithelial cells, the cell viability was the highest with 50% FBS and 10% glycerol (95.0% +/- 4.27%), and the lowest survival rate was observed in the condition of 10% FBS and 10% DMSO (80.0% +/- 5.49%). CFE of cryopreserved conjunctival epithelial cells was 14.1% +/- 1.9% in cells with 20% FBS and glycerol and 13.5% +/- 2.0% in those with 20% FBS and DMSO (P > 0.05). HLECs expressed CK3/12 after cryopreservation in all conditions examined. CONCLUSIONS: The best results were yielded by 50% FBS for cell viability in HLECs. Glycerol seems to be superior to DMSO in cell viability of the rabbit conjunctival epithelium after cryopreservation.     

角膜缘niche细胞与基质细胞维持角膜缘干细胞功能的对比研究

目的　比较角膜缘niche细胞（limbal niche cells，LNCs）与角膜缘基质细胞（limbal stromal cells，LSCs）在维持角膜缘干细胞功能上的不同特性。方法　将LNCs和LSCs分别从6个角膜缘组织分离，并在相同的条件下培养、传代。LNCs与LSCs经丝裂霉素C（mitomycin C,MMC）处理后分为LNCs组与LSCs组作为饲养细胞分别与角膜缘干细胞共培养，比较两组角膜缘干细胞克隆形成率（colony-forming efficiency,CFE）、上皮细胞复层化以及细胞标志物和部分基因的表达。结果　LNCs组角膜缘干细胞CFE（6.57±1.54）%高于LSCs组（1.43±0.47）%。 LNCs组细胞复层上皮数（4~5层）多于LSCs组（2~3层）。角膜缘干细胞克隆与免疫荧光染色及mRNA半定量分析结果显示，LNCs组比LSCs组表达了更多干细胞标志物ΔNp63，能更有效地维持角膜缘干细胞的细胞特性。逆转录PCR分析结果显示，LNCs组与LSCs组都分泌了一些维持角膜缘干细胞生长的生长因子，但LNCs组比LSCs组高表达上皮型钙黏蛋白（E-cadherin），低表达营养神经素3（NT3），能更好地支持角膜上皮增殖。结论　LNCs比LSCs能更好地支持角膜缘干细胞的生长及维持其干细胞特性。     

Isolation of putative corneal epithelial stem cells from cultured limbal tissue

PURPOSE: To investigate methods of isolating putative corneal epithelial stem cells from cultured limbal tissue. METHODS: Three extraction techniques were compared to identify an efficient method of obtaining a large number of viable corneal epithelial stem cells from the limbus. Limbal tissues were extracted by incubation at 37 degrees C or 4 degrees C for 1 or 16 hours, respectively, with 1.2 U/ml dispase/trypsin or by treatment with 0.05% trypsin and 0.01% ethyldiaminetetraacetic acid (EDTA) at 37 degrees C in single procedure. Collected cells were cultured on NIH/3T3-seeded plates, and colony forming efficiency (CFE) was evaluated. Fluorescence activated cell sorting (FACS) was performed with a Coulter EPICS 753 after incubation with Hoechst 33342 and propidium iodide (PI). Hoechst negative cells were obtained using gates exhibiting low Hoechst blue with a 424/44 nm BP filter. Gated cells of each fraction were re-cultured to assess the capability of colony formation. RESULTS: The mean numbers of viable cells obtained from treatment with dispase and trypsin was 3 x 10(4) cell/ml and 8.06 x 10(5) cell/ml at 37 degrees C and 4 degrees C incubations; the number increased to 1.21 x 10(6) cell/ml with a trypsin/EDTA treatment (p < 0.05). CFE was 9.67 +/- 2.13% and 6.63 +/- 2.35% in rabbit and human cells, respectively. Likewise, the Hoechst negative fraction was 3.61 +/- 0.42% and 5.21 +/- 4.91% in rabbit and human cells, respectively. The sorted Hoechst negative cells were cultured through four passages, forming small round colonies. In rabbit cells, the CFEs of Hoechst negative and positive fractions after FACS, were 12.67 +/- 2.24% and 1.17 +/- 6.13%, respectively (p < 0.05). CONCLUSIONS: Putative corneal epithelial stem cells were efficiently isolated from limbal tissue using a trypsin/EDTA extraction and FACS. This technique may be very useful in tissue engineered stem cell therapy.     

A serum-free clonal growth assay for limbal, peripheral, and central corneal epithelium.

The stem cells and transient amplifying cells of the corneal epithelium are thought to be localized in the limbal and corneal basal epithelium, respectively. To study the differential regulation of proliferation of these progenitor cells, a defined, serum-free, clonal growth assay was developed for central (CC) and peripheral (PC) corneal and limbal (L) epithelial cells. After incubation in Dispase II (1.2 U/ml; 1 hr for PC and CC and 3 hr for L) and subsequent brief trypsin-ethylenediaminetetraacetic acid digestion, 18 or 180 single cells/cm2 were seeded in MCDB 151 medium supplemented with insulin, transferrin, selenium, hydrocortisone, epidermal growth factor (EGF), phosphoethanolamine, ethanolamine, and calcium. During the first week of culture, the cells gradually developed an increasing number of colonies, and the mean colony-forming efficiency on day 6 for L was 4.2 +/- 2.4%, significantly lower than 11.4 +/- 5.9% for PC and 12.8 +/- 7.6% for CC (P less than 0.003). Colony morphology was identical in L, PC, and CC with small, elongated cells more cohesive in the center but more migratory in the periphery. There were no differences in immunofluorescent staining with monoclonal antibody AE-5, indicating the corneal derivation of all colonies. Cultures could be passaged on day 14 and grown for more than 3 weeks with increasing desquamation. Addition of a mixture of trace metals to yield MCDB 153 did not enhance growth; increased selenium concentrations were inhibitory. Elimination of EGF from the supplement abolished most of the clonal growth. The lower rate of L proliferation might be explained by the absence of serum and stromal mitogens. This culture system seems preferentially to support transient amplifying cells and allows investigation of the differentiation of isolated corneal stem cells to transient amplifying cells or the proliferation and differentiation of transient amplifying cells by various factors without the interaction of undefined serum components or paracrine influences from other cells.     

The side population cells in the rabbit limbus sensitively increased in response to the central cornea wounding

PURPOSE: Side population (SP) cells are known to reside in the limbus as putative corneal epithelial stem cells. This study was performed to demonstrate the presence and the characteristics of SP cells in the rabbit limbal epithelium and explore their sensitivity in response to the central cornea wounding. METHODS: To sort out the SP cells, freshly isolated rabbit limbal and central corneal epithelial cells were subjected to Hoechst 33342 dye efflux assay. For characterization of the sorted SP cells, RT-PCR analysis, semi-dry three-dimensional (3-D) cell culture, and transplantation in nude mice were performed. To explore wound sensitivity of the limbal SP cells, the rabbit central cornea was wounded by direct contact of a 6-mm paper disk soaked with 1 N NaOH, and changes in the population size of the SP cells and the colony-forming efficiency (CFE) was monitored on days 1, 2, and 5 after wounding. RESULTS: The SP cells were present in the rabbit limbal epithelium with an incidence of 0.73% +/- 0.14% (n = 8) and were smaller in cell size than the major population (MP) cells, quiescent in the cell cycle, and in the undifferentiated state. The SP cells were able to regenerate the cornea-like structure with basal enrichment of p63-positive cells by in vitro 3-D culture and in vivo transplantation, all of which were best achieved by the whole population (WP) of cells comprising SP and MP cells. After central cornea wounding, this rare population of the limbal SP cells increased in size fivefold on day 1 and then decreased on day 2. The transient increase in the SP cells was subsequently followed by the propagation of an increase in CFE in the limbal MP cells on day 2 and then in the corneal MP cells on day 5. In the hematopoietic colony-forming assay, the limbal SP cells gave approximately eightfold higher CFU than the limbal MP cells. CONCLUSIONS: The SP cells identified in the rabbit limbus are an undifferentiated and noncycling rare epithelial cell population, which sensitively respond to the central cornea wounding by their transient increase in the population size.     

去除型人皮肤成纤维饲细胞系的建立及其重建角膜表层的研究

目的　探讨去除型人皮肤成纤维饲细胞系的建立及其重建角膜表层的相关研究。方法　将增强型绿色荧光蛋白（EGFP）、端粒酶逆转录酶（hTERT）和单纯疱疹病毒胸苷激酶（HSV-TK）3种基因导入人皮肤成纤维细胞，建立荧光标记的永生化的可去除型TERT+TK-D人源饲细胞系。将创建的细胞系经丝裂霉素C处理后作为饲养细胞与人角膜缘干细胞共培养，并与3T3饲细胞的培养结果作比较。结果　TERT+TK-D细胞系在体外经过6个月的连续传代后仍然表达绿色荧光蛋白，保持旺盛的分裂能力，对更昔洛韦敏感。TERT+TK-D组的克隆形成率（colony forming efficiency,CFE）为（11.77±0.21)%，与3T3组CFE［（12.8±1.61)］%比较，差异无统计学意义（P=0.332）；经过共培养，两组都形成了4~5层复层上皮细胞片。角膜缘干细胞克隆和上皮细胞片的免疫荧光染色及Real-time PCR定量分析结果显示，TERT+TK-D组角蛋白K3表达低于3T3组；PCR结果证实TERT+TK-D饲细胞在更昔洛韦作用下凋亡、裂解，没有混入培养获得的角膜上皮细胞片中。结论　转基因荧光标记的永生化的去除型人皮肤成纤维饲细胞有望替代3T3细胞用于角膜再生医疗。     

Phenotypic characterization of human corneal epithelial cells expanded ex vivo from limbal explant and single cell cultures

Cultivated human corneal epithelial cells have been successfully used for corneal reconstruction. Explant and single cell systems are currently used for human corneal epithelial cultivation. This study was conducted to characterize the phenotypes of human corneal epithelial cells expanded ex vivo by these two culture systems with regard to their growth potential, morphology and antigen expression patterns. Human corneal epithelial cells were expanded by limbal explant culture or limbal single cell suspension culture on a mitomycin C treated 3T3 fibroblast feeder layer. The phenotypes of primary cultured cells were evaluated by morphology and immunohistochemical staining with antibodies for proposed keratinocyte stem cell markers (p63, EGFR, K19 and integrin beta1) and differentiation markers (K3, involucrin and gap junction protein connexin 43). BrdU labeling was performed to identify the label-retaining cells. Human corneal epithelial cells were grown from limbal tissues preserved as long as 16 days by both culture systems. The growth rate depended on the tissue freshness, the time from death to preservation and the time from death to culture, but not on the donor age. Cell growth was observed in 96.2% (n = 43) of single cell suspension cultures and in 90.8% (n = 213) of explant cultures. The cell expansion was confluent in 10-14 days in single cell suspension cultures and 14-21 days in explant cultures. The cell morphology in single cell suspension culture was smaller, more compact and uniform than that in explant culture. Immunostaining showed a greater number of the small cells expressing p63, EGFR, K19 and integrin beta1, while more larger cells stained positively for K3, involucrin and connexin 43 in both culture systems. BrdU-label retaining cells were identified in 2.3+/-0.7% of explant cultures and 3.73+/-1.5% of single cell cultures chased for 21 days. In conclusion, the limbal rims are a great treasure for ex vivo expansion of human corneal epithelial cells. The phenotypes of corneal epithelial cells, ranging from basal cells to superficial differentiated cells, are well maintained in both culture systems. Slow-cycling BrdU-label retaining cells, that are characteristic of stem cells, were identified in the cultures.     

The phenotype of limbal epithelial stem cells

PURPOSE: The purpose of this study was to identify phenotypic markers of human limbal stem cells in fetal and adult corneas. METHODS: RNA from microscopically dissected superficial limbal and central fetal (18 weeks) corneas was amplified and used to generate P(32)-labeled, reverse-transcribed antisense RNA that was linearly amplified and hybridized to a focused stem cell cDNA microarray. Differential gene expression of fetal limbus was compared with the expression of central cornea. Microarray differential expression experiments were performed on P63-expressing primary cultured limbal epithelial cells (passage 1; Pa1) and primary cells passaged 5 times (Pa5). Semiquantitative RT-PCR assay and immunohistochemistry were performed on fetal and adult corneas and cultured primary limbal epithelial cells, to confirm the results of the microarray experiments. Slow-cycling (pulsed bromodeoxyuridine label-retaining) limbal epithelium in corneal organ culture was studied for the expression of four selected upregulated limbal genes. RESULTS: Of the 266 genes tested, 33 were differentially overexpressed (more than twofold) in the fetal limbus (compared with central cornea) and primary cultured limbal epithelium compared with primary cells after 5 passages. Cytokeratin 15 (CK15) and cytokeratin 14 (CK14) are expressed in limbal basal epithelium and P-cadherin (CDH3) and Wnt-4 expression was restricted to basal and immediate parabasal limbal epithelium of both the adult and fetal corneas). Bromodeoxyuridine label retaining epithelium in corneal organ culture (slow-cycling cells) expressed the four selected limbal upregulated genes. CONCLUSIONS: For the first time, a focused stem cell pathway microarray analysis has been performed on fetal cornea and cultured limbal explant epithelium. CK15, CK14, CDH3, and Wnt-4 are expressed in the basal limbal epithelial cells.     

Novel enzymatic isolation of an entire viable human limbal epithelial sheet

OBJECTIVE. To develop a reproducible method of isolating an intact viable human limbal epithelial sheet. METHODS. Human pigmented limbus was incubated at 4 degrees C for 18 hours in supplemental hormonal epithelial medium (SHEM) containing 50 mg/mL dispase II and 100 mM sorbitol. A loose limbal epithelial sheet was separated by a spatula. The remaining stroma was digested and subcultured. The viability of isolated cells was assessed. Isolated epithelial sheets and remaining stroma were subjected to immunostaining. Sheets 1.5 mm in length were cultured in SHEM on plastic until confluence, and cell extracts were subjected to Western blot analysis. RESULTS. Intact limbal epithelial sheets were consistently isolated. Pigmented palisades of Vogt revealed large superficial squamous cells and small basal cuboidal cells. No epithelial cells grew from the remaining stroma. Mean viability was 80.7% +/- 9.1%. The basal epithelium was negative to keratin 3 and connexin 43, but was scatter positive for p63. The epithelial sheet showed negative staining for laminin 5 and collagen VII, but interrupted linear basal staining for collagen IV. The remaining stroma showed negative staining for laminin 5, positive linear staining for collagen IV in the basement membrane, and diffuse staining for collagen VII in the superior stroma subjacent to the basement membrane. Western blot analysis revealed that cells originating from the limbal sheets expressed keratin 3 and p63. CONCLUSIONS. An intact limbal epithelial sheet can be consistently and reproducibly isolated and contains stem cell characteristics in the basal epithelium by degrading laminin 5 and part of collagen IV, and disassembling collagen VII.