角膜缘干细胞的临床和基础研究
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摘要
第一部分应用活体共聚焦显微镜进行角膜缘干细胞缺失的临床分级研究
【目的】观察角膜缘干细胞缺失中央角膜上皮和角膜缘上皮在活体共聚焦显微镜下的改变，从细胞水平建立一套角膜缘干细胞缺失的临床分级系统。
【方法】根据裂隙灯显微镜下荧光素染色的不同阳性表现，将临床诊断为角膜缘干细胞缺失的18例24眼初步分成4个临床阶段组，各组均利用海德堡视网膜断层扫描仪3代Rostock角膜模块(HRT3 RCM)进行活体眼表组织成像。图像信息集中阅读分析。
【结果】5个形态学指标被发现有助于分析中央角膜区和角膜缘区的上皮层改变：角膜上皮细胞层数的改变；角膜上皮细胞大小和活动性改变；外侵细胞：如炎症细胞、树突状细胞；角膜上皮层内和基底膜下神经丛的改变；角膜上皮层内血管的出现。每个临床阶段组都有其对应的共聚焦显微镜下细胞水平的改变，且组与组之间中央角膜区和角膜缘区的上皮层有明显的形态学和结构学差异。角膜缘干细胞缺失临床分级系统首次从活体眼表单个细胞水平创立。
【结论】角膜缘干细胞缺失中央角膜上皮和角膜缘上皮在细胞水平的形态学和结构学差异与其临床体征有较高一致性，活体共聚焦显微镜可作为诊断极早期角膜缘干细胞缺失和辅助其分级的有用工具。
第二部分人胚胎干细胞标记物在角膜缘上皮和角膜上皮的表达研究
【目的】研究5个典型人胚胎干细胞标记物碱性磷酸酶(ALP)、Nanog、Oct-4、阶段特异性胚胎抗原(SSEA)-1和SSEA-4在角膜缘上皮和角膜上皮的表达。
【方法】可移植级别人角巩膜组织进行冷冻切片，免疫组织化学方法从蛋白水平检测ALP、Nanog、Oct-4、SSEA-1和SSEA-4在角膜缘上皮和角膜上皮的表达，RT-PCR法从mRNA水平进一步确认SSEA-4、Nanog和Oct-4在角膜和角膜缘的表达。
【结果】免疫组织化学法研究显示SSEA-4广泛表达于角膜缘上皮和角膜上皮的各层，ALP散在表达于少量角膜缘前基质层细胞中。SSEA-4的表达经RT-PCR得到确认。免疫组织化学法和RT-PCR均显示SSEA-1和Oct-4不表达与角膜缘上皮和角膜上皮。RT-PCR在mRNA水平检测到Nanog在角膜缘和角膜的表达，但免疫组织化学法在蛋白水平未检测到Nanog在角膜缘和角膜的表达。
【结论】SSEA-4广泛表达于成年角膜缘上皮和角膜上皮的各层中，提示SSEA-4不能作为一个理想的角膜缘干细胞标记物。Nanog是否表达于角膜和角膜缘仍需进一步确证。
第三部分应用可诱导转基因大鼠模型进行角膜缘干细胞研究的初步探索
【目的】探讨一种可直接药物诱导转基因大鼠模型用于角膜缘干细胞研究的可行性。
【方法】可Doxycycline直接诱导型GFP转基因大鼠(ROSA26-rtTA-Collal-tetOP-H2BGFP)模型被引入。GFP基因的表达通过日常喂给水中添加Doxycycline直接激活，Doxycycline持续添加21天后停止。大鼠于1月和4月大被处以安乐死，这里的1月和4月指从开始喂给Doxycycline的日期到大鼠安乐死之间的间隔时间，安乐死大鼠在立体生物显微镜下行立体荧光照相，然后分别剜出左右眼球，左眼球进行冰冻切片，免疫组织化学染色研究；右眼球进行角巩膜铺片，荧光显微镜照相和激光共聚焦显微镜照相分析。
【结果】GFP绿色可见荧光广泛表达于Doxycycline诱导的转基因1月大鼠的全身多个组织，眼表表达显著，且可见明显的高表达角膜缘条带。经Doxycycline诱导的转基因4月大鼠眼表GFP表达减退，但可见角膜缘表达强度仍高于周边角膜和中央角膜，漩涡状高表达条带连接角膜缘和角膜中央，经激光共聚焦显微镜分析，这些高表达条带内的细胞形状特殊，走行于角膜缘和角膜区翼状上皮细胞和基底上皮细胞之间。
【结论】经Doxycycline诱导的4月转基因大鼠角膜缘区的GFP高阳性表达提示眼球表面分化缓慢的细胞群位于角膜缘，连接角膜缘和角膜中央的漩涡状GFP高表达条带中提示分化缓慢的细胞从角膜缘漩涡状移行入中央角膜，补给脱落的角膜上皮细胞。可Doxycycline直接诱导型转基因大鼠是研究角膜缘干细胞的有效模型。
PartⅠStaging System for Limbal Stem Cells DeficiencyUsing in Vivo Confocal Microscopy
Objective To describe the corneal and limbal epithelial changes in patients with imbalstem cells deficiency (LSCD) using in vivo laser scanning confocal microscopy (LSCM),and to classify the stages of LSCD at the cellular level.
Method This is a prospective, noncomparative clinical study. Patients were selectedaccording to their clinical presentations. Twenty four eyes of 18 subjects with LSCD wereclassified into 4 stages groups initially according to their cornea status. In vivo imagings ofthe corneal and limbal epithelium in all the 24 eyes were obtained with HRT 3 Rostockcornea module LSCM. The LSCM images were reviewed.
Results Five useful parameters were found to evaluate all the LSCM images: theepithelium layers; epithelial cells size and activity; invading cells; epithelial andsubepithelial nerve, intraepithelial vessels. Both the central cornea and limbus showed 4different stages changes which correlated with the initial clinical 4 stages group. Thestaging system for LSCD at the cellular level was established.
Conclusion The morphological and structural changes of corneal and limbal epithelium at the cellular level correlated with clinical presentations at different stages in LSCD.Confocal microscopy might be a useful tool to detect very early stage of LSCD and couldaid in the staging of LSCD.
PartⅡExpression of Human Embryonic Stem Cells Markers in theLimbal and Corneal Epithelial Cells
Objective To investigate the expression of five human embryonic stem cells (hESCs)markers, Alkaline phosphatase (ALP), Stage-specific embryonic antigen (SSEA)-1,SSEA-4, Nanog and Oct-4, in the limbal and corneal epithelial cells.
Method Frozen sections were obtained from human corneoscleral tissues.Immunohistochemistry study for five hESCs markers, ALP, SSEA-1, SSEA-4, Nanog andOct-4, was performed to evaluate their expression at the protein level in the human limbusand cornea. The mRNA expression of SSEA-4, Nanog and Oct-4 was further confirmed byreverse transcription-PCR (RT-PCR).
Results Immunohistochemistry study showed that SSEA-4 was present in all layers ofthe limbal and corneal epithelial cells and ALP was detected in a small subgroup of stromalcells in the anterior limbus. The expression of SSEA-4 was confirmed by RT-PCR. SSEA-1and Oct-4 were not detected at the protein nor the mRNA level in the limbus and cornea.Nanog mRNA transcript was detected in the cornea and limbus, but Nanog protein was notdetected using immunostaining.
Conclusion The expression of SSEA-4 in mature corneal epithelial cells indicates thatit is not a marker for corneal epithelial progenitor/stem cells. Whether Nanog is expressedon the ocular surface needs to be further confirmed at the protein level.
PartⅢUsing an Inducible Transgenic Mice Model toStudy Limbal Stem Cells
Objective To establish an inducible GFP transgenic mice model to study the limbalstem cells.
Method An inducible GFP transgenic mice (ROSA26-rtTA-Col1a1-tetOP-H2BGFP)model was selected and the expression of GFP was turned on for 21 days starting atdifferent periods and followed by 1 month and 4 months, which was counted from the datethe GFP was turned on. After the mice were euthanized, the left eyes were frozen with OCTand immunohistochemistry study for GFP expression pattern was performed on the frozensections. The sclerocornea tissues from the right eyes were isolated, mounted and observedunder confocal and fluorescence microscopy. Images were processed.
Results Expression of GFP was present on the whole ocular surface in both 1 monthand 4 months mice and stronger GFP expression band was seen in the limbus. In 4 monthsmice, the expression of GFP was weaker than 1 month and parts of higher GFP expressionlimbal cells were organized in a radial stripe fashion towards the center of the corneaforming a vortex pattern. Three-dimensional reconstructions of the vortex pattern confocalimages revealed that the stripe cells were seen in the basal and suprabasal corneal epitheliallayers and different from the other cells in the peripheral and central cornea.
Conclusion The epithelial cells in the limbus retained the higher GFP expression in 4months after the GFP was turned on indicating that they are the slowest cycling cells. Theradial vortex pattern suggests that these epithelial cells might migrate from the limbustowards the center of the cornea. This mouse model is a useful tool to study the in vivohomeostasis of corneal epithelial cells.
【目的】观察角膜缘干细胞缺失中央角膜上皮和角膜缘上皮在活体共聚焦显微镜下的改变，从细胞水平建立一套角膜缘干细胞缺失的临床分级系统。
【方法】根据裂隙灯显微镜下荧光素染色的不同阳性表现，将临床诊断为角膜缘干细胞缺失的18例24眼初步分成4个临床阶段组，各组均利用海德堡视网膜断层扫描仪3代Rostock角膜模块(HRT3 RCM)进行活体眼表组织成像。图像信息集中阅读分析。
【结果】5个形态学指标被发现有助于分析中央角膜区和角膜缘区的上皮层改变：角膜上皮细胞层数的改变；角膜上皮细胞大小和活动性改变；外侵细胞：如炎症细胞、树突状细胞；角膜上皮层内和基底膜下神经丛的改变；角膜上皮层内血管的出现。每个临床阶段组都有其对应的共聚焦显微镜下细胞水平的改变，且组与组之间中央角膜区和角膜缘区的上皮层有明显的形态学和结构学差异。角膜缘干细胞缺失临床分级系统首次从活体眼表单个细胞水平创立。
【结论】角膜缘干细胞缺失中央角膜上皮和角膜缘上皮在细胞水平的形态学和结构学差异与其临床体征有较高一致性，活体共聚焦显微镜可作为诊断极早期角膜缘干细胞缺失和辅助其分级的有用工具。
第二部分人胚胎干细胞标记物在角膜缘上皮和角膜上皮的表达研究
【目的】研究5个典型人胚胎干细胞标记物碱性磷酸酶(ALP)、Nanog、Oct-4、阶段特异性胚胎抗原(SSEA)-1和SSEA-4在角膜缘上皮和角膜上皮的表达。
【方法】可移植级别人角巩膜组织进行冷冻切片，免疫组织化学方法从蛋白水平检测ALP、Nanog、Oct-4、SSEA-1和SSEA-4在角膜缘上皮和角膜上皮的表达，RT-PCR法从mRNA水平进一步确认SSEA-4、Nanog和Oct-4在角膜和角膜缘的表达。
【结果】免疫组织化学法研究显示SSEA-4广泛表达于角膜缘上皮和角膜上皮的各层，ALP散在表达于少量角膜缘前基质层细胞中。SSEA-4的表达经RT-PCR得到确认。免疫组织化学法和RT-PCR均显示SSEA-1和Oct-4不表达与角膜缘上皮和角膜上皮。RT-PCR在mRNA水平检测到Nanog在角膜缘和角膜的表达，但免疫组织化学法在蛋白水平未检测到Nanog在角膜缘和角膜的表达。
【结论】SSEA-4广泛表达于成年角膜缘上皮和角膜上皮的各层中，提示SSEA-4不能作为一个理想的角膜缘干细胞标记物。Nanog是否表达于角膜和角膜缘仍需进一步确证。
第三部分应用可诱导转基因大鼠模型进行角膜缘干细胞研究的初步探索
【目的】探讨一种可直接药物诱导转基因大鼠模型用于角膜缘干细胞研究的可行性。
【方法】可Doxycycline直接诱导型GFP转基因大鼠(ROSA26-rtTA-Collal-tetOP-H2BGFP)模型被引入。GFP基因的表达通过日常喂给水中添加Doxycycline直接激活，Doxycycline持续添加21天后停止。大鼠于1月和4月大被处以安乐死，这里的1月和4月指从开始喂给Doxycycline的日期到大鼠安乐死之间的间隔时间，安乐死大鼠在立体生物显微镜下行立体荧光照相，然后分别剜出左右眼球，左眼球进行冰冻切片，免疫组织化学染色研究；右眼球进行角巩膜铺片，荧光显微镜照相和激光共聚焦显微镜照相分析。
【结果】GFP绿色可见荧光广泛表达于Doxycycline诱导的转基因1月大鼠的全身多个组织，眼表表达显著，且可见明显的高表达角膜缘条带。经Doxycycline诱导的转基因4月大鼠眼表GFP表达减退，但可见角膜缘表达强度仍高于周边角膜和中央角膜，漩涡状高表达条带连接角膜缘和角膜中央，经激光共聚焦显微镜分析，这些高表达条带内的细胞形状特殊，走行于角膜缘和角膜区翼状上皮细胞和基底上皮细胞之间。
【结论】经Doxycycline诱导的4月转基因大鼠角膜缘区的GFP高阳性表达提示眼球表面分化缓慢的细胞群位于角膜缘，连接角膜缘和角膜中央的漩涡状GFP高表达条带中提示分化缓慢的细胞从角膜缘漩涡状移行入中央角膜，补给脱落的角膜上皮细胞。可Doxycycline直接诱导型转基因大鼠是研究角膜缘干细胞的有效模型。
PartⅠStaging System for Limbal Stem Cells DeficiencyUsing in Vivo Confocal Microscopy
Objective To describe the corneal and limbal epithelial changes in patients with imbalstem cells deficiency (LSCD) using in vivo laser scanning confocal microscopy (LSCM),and to classify the stages of LSCD at the cellular level.
Method This is a prospective, noncomparative clinical study. Patients were selectedaccording to their clinical presentations. Twenty four eyes of 18 subjects with LSCD wereclassified into 4 stages groups initially according to their cornea status. In vivo imagings ofthe corneal and limbal epithelium in all the 24 eyes were obtained with HRT 3 Rostockcornea module LSCM. The LSCM images were reviewed.
Results Five useful parameters were found to evaluate all the LSCM images: theepithelium layers; epithelial cells size and activity; invading cells; epithelial andsubepithelial nerve, intraepithelial vessels. Both the central cornea and limbus showed 4different stages changes which correlated with the initial clinical 4 stages group. Thestaging system for LSCD at the cellular level was established.
Conclusion The morphological and structural changes of corneal and limbal epithelium at the cellular level correlated with clinical presentations at different stages in LSCD.Confocal microscopy might be a useful tool to detect very early stage of LSCD and couldaid in the staging of LSCD.
PartⅡExpression of Human Embryonic Stem Cells Markers in theLimbal and Corneal Epithelial Cells
Objective To investigate the expression of five human embryonic stem cells (hESCs)markers, Alkaline phosphatase (ALP), Stage-specific embryonic antigen (SSEA)-1,SSEA-4, Nanog and Oct-4, in the limbal and corneal epithelial cells.
Method Frozen sections were obtained from human corneoscleral tissues.Immunohistochemistry study for five hESCs markers, ALP, SSEA-1, SSEA-4, Nanog andOct-4, was performed to evaluate their expression at the protein level in the human limbusand cornea. The mRNA expression of SSEA-4, Nanog and Oct-4 was further confirmed byreverse transcription-PCR (RT-PCR).
Results Immunohistochemistry study showed that SSEA-4 was present in all layers ofthe limbal and corneal epithelial cells and ALP was detected in a small subgroup of stromalcells in the anterior limbus. The expression of SSEA-4 was confirmed by RT-PCR. SSEA-1and Oct-4 were not detected at the protein nor the mRNA level in the limbus and cornea.Nanog mRNA transcript was detected in the cornea and limbus, but Nanog protein was notdetected using immunostaining.
Conclusion The expression of SSEA-4 in mature corneal epithelial cells indicates thatit is not a marker for corneal epithelial progenitor/stem cells. Whether Nanog is expressedon the ocular surface needs to be further confirmed at the protein level.
PartⅢUsing an Inducible Transgenic Mice Model toStudy Limbal Stem Cells
Objective To establish an inducible GFP transgenic mice model to study the limbalstem cells.
Method An inducible GFP transgenic mice (ROSA26-rtTA-Col1a1-tetOP-H2BGFP)model was selected and the expression of GFP was turned on for 21 days starting atdifferent periods and followed by 1 month and 4 months, which was counted from the datethe GFP was turned on. After the mice were euthanized, the left eyes were frozen with OCTand immunohistochemistry study for GFP expression pattern was performed on the frozensections. The sclerocornea tissues from the right eyes were isolated, mounted and observedunder confocal and fluorescence microscopy. Images were processed.
Results Expression of GFP was present on the whole ocular surface in both 1 monthand 4 months mice and stronger GFP expression band was seen in the limbus. In 4 monthsmice, the expression of GFP was weaker than 1 month and parts of higher GFP expressionlimbal cells were organized in a radial stripe fashion towards the center of the corneaforming a vortex pattern. Three-dimensional reconstructions of the vortex pattern confocalimages revealed that the stripe cells were seen in the basal and suprabasal corneal epitheliallayers and different from the other cells in the peripheral and central cornea.
Conclusion The epithelial cells in the limbus retained the higher GFP expression in 4months after the GFP was turned on indicating that they are the slowest cycling cells. Theradial vortex pattern suggests that these epithelial cells might migrate from the limbustowards the center of the cornea. This mouse model is a useful tool to study the in vivohomeostasis of corneal epithelial cells.
引文
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5. Li, W., Y. Hayashida, et al. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res 2007; 17 (1): 26-36.
6. Vascotto, S. G., M. Griffith. Localization of candidate stem and progenitor cell markers within the human cornea, limbus, and bulbar conjunctiva in vivo and in cell culture. Anat Rec A Discov Mol Cell Evol Biol 2006; 288 (8): 921-31.
7. Chee, K. Y., A. Kicic, et al. Limbal stem cells: the search for a marker. Clin Experiment Ophthalmol 2006; 34 (1): 64-73.
8. Millan, J. L, W. H. Fishman. Biology of human alkaline phosphatases with special reference to cancer. Crit Rev Clin Lab Sci 1995; 32 (1): 1-39.
9. Cavaleri, F., H. R. Scholer. Nanog: A New Recruit to the Embryonic Stem Cell Orchestra. Cell 2003; 113 (5): 551-552.
10. Chambers, I., D. Colby, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003; 113 (5): 643-55.
11. Mitsui, K., Y. Tokuzawa, et al. The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells. Cell 2003; 113 (5): 631-642.
12. Niwa, H., J. Miyazaki, et al. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24 (4): 372-6.
13. Pesce, M., H. R. Scholer. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 2001; 19 (4): 271-8.
14. Solter, D., B. B. Knowles. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A 1978; 75 (11): 5565-9.
15. Kannagi, R., N. A. Cochran, et al. Stage-specific embryonic antigens (SSEA-3 and-4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. Embo J 1983; 2 (12): 2355-61.7.
16. Polisetty, N., A. Fatima, et al. Mesenchymal cells from limbal stroma of human eye. Mol Vis 2008; 14: 431-42.
17. de Paiva, C. S., Z. Chen, et al. ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells 2005; 23 (1): 63-73.
18. Pellegrini, G., E. Dellambra, et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A 2001; 98 (6): 3156-61.
19. Moore, K. A., I. R. Lemischka. Stem cells and their niches. Science 2006; 311 (5769): 1880-5.
1. Potten, C. S., R. Schofield, et al. A comparison of cell replacement in bone marrow, testis and three regions of surface epithelium. Biochim Biophys Acta 1979; 560 (2):281-99.
2. Tumbar, T., G. Guasch, et al. Defining the epithelial stem cell niche in skin. Science 2004; 303 (5656): 359-63.
3. Cotsarelis, G., T.-T. Sun, et al. Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990; 61 (7): 1329-1337.
4. Arai, F., A. Hirao, et al. Tie2/angiopoietin-l signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004; 118 (2): 149-61.
5. Zhang, J., C. Niu, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003; 425 (6960): 836-41.
6. Potten, C. S., W. J. Hume, et al. The segregation of DNA in epithelial stem cells. Cell 1978; 15 (3): 899-906.
7. Urbanek, K., D. Cesselli, et al. Stem cell niches in the adult mouse heart. Proc Natl Acad Sci U S A 2006; 103 (24): 9226-31.
8. Welm, B. E., S. B. Tepera, et al. Sca-l(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 2002; 245 (1): 42-56.
9. Cotsarelis, G., S. Z. Cheng, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.Cell 1989; 57 (2): 201-9.
10. Foudi, A., K. Hochedlinger, et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 2009; 27 (1): 84-90.
11. Kanda, T., K. F. Sullivan, et al. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 1998; 8 (7): 377-85.
12. Meek, K. M., N. J. Fullwood. Corneal and scleral collagens-a microscopist's perspective. Micron 2001; 32 (3): 261-72.
13. Nagasaki, T., J. Zhao. Centripetal movement of corneal epithelial cells in the normal adult mouse. Invest Ophthalmol Vis Sci 2003; 44 (2): 558-66.
1. Berliner ML. Biomicroscopy of the Eye. Hafner Publishing Company, New York,1966.
2. Vogt A. Textbook and Atlas of Slit Lamp Microscopy of the Living Eye. J. P. Wayenborgh, Bonn, 1981.
3. Martonyi CL, Bahn CF, Meyer RF. Clinical Slit Lamp Biomicroscopy and Photo Slit Lamp Biomicrography. Time One Ink, Ltd, Ann Arbor, 1985.
4. Minsky M. Memoir on inventing the confocal scanning microscope. Scanning 1988; 10:128-138.
5. Cavanagh HD, Petroll WM, Alizadeh H, et al. Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease. Ophthalmol 1993; 100:1444-1454.
6. Semwogerere D, Weeks ER. Confocal microscopy. In: Wnek GE, Bowlin GL, editors. Encyclopedia of biomaterials and biomedical engineering. London: Taylor & Francis;2005. pp. 1-10.
7. Webb RH: Confocal optical microscopy. Rep Prog Phys 1996; 59: All-All.
8. Masters, B. R., M. Bohnke. Confocal microscopy of the human cornea in vivo. Int Ophthalmol 2001; 23 (4-6): 199-206.
9. Masters, B. R., M. Bohnke. Three-dimensional confocal microscopy of the living human eye. Annu Rev Biomed Eng 2002; 4: 69-91.
10. Wilson T: Confocal Microscopy. Academic Press, London, 1990.
11. Corle TR, Kino GS. Confocal Scanning Optical Microscopes and Related Imaging Systems. Academic Press, San Diego, 1996.
12. Janknecht, P., J. Funk. Optic nerve head analyser and Heidelberg retina tomograph: accuracy and reproducibility of topographic measurements in a model eye and in volunteers. Br J Ophthalmol 1994; 78 (10): 760-8.
13. Weinberger, D., H. Stiebel, et al. Three-dimensional measurements of idiopathic macular holes using a scanning laser tomograph. Ophthalmology 1995; 102 (10):1445-9.
14. Eckard, A., J. Stave, et al. In vivo investigations of the corneal epithelium with the confocal Rostock Laser Scanning Microscope (RLSM). Cornea 2006; 25 (2): 127-31.
15. Guthoff R.F., Baudouin C, Stave J. Atlas of Confocal Laser Scanning In-vivo Microscopy in Ophthalmology. Springer, 2006.
16. Patel, D. V., C. N. McGhee. Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review. Clin Experiment Ophthalmol 2007; 35 (1): 71-88.
17. Matsuda H. Electron microscopic study on the corneal nerve with special reference to its endings. Jpn J Ophthalmol 1968; 12:163-73.
18. Ueda S, del Cerro M, LoCascio JA, et al. Peptidergic and catecholaminergic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study. Acta Ophthalmol 1989; 192 (Suppl.): 80-90.
19. Muller LJ, Vrensen GF, Pels L et al. Architecture of human corneal nerves. Invest Ophthalmol Vis Sci 1997; 38: 985-94.
20. Oliveira-Soto L, Efron N. Morphology of corneal nerves using confocal microscopy. Cornea 2001; 20: 374-84.
21. Patel DV, McGhee CNJ. Mapping of the normal human corneal sub-basal nerve plexus by in vivo laser scanning confocal microscopy. Invest Ophthalmol Vis Sci 2005; 46: 4485-8.
22. Grupcheva CN, Wong T, Riley AF, et al. Assessing the sub-basal nerve plexus of the living healthy human cornea by in vivo confocal microscopy. Clin Experiment Ophthalmol 2002; 30:187-90.
23. Erie JC, McLaren J, Hodge DO, et al. The effect of age on the corneal subbasal nerve plexus. Cornea 2005; 24: 705-9.
24. Mustonen RK, McDonald MB, et al. Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy. Cornea 1998; 17: 485-492.
25. Kaufman SC, Musch DC, Belin MW, et al. Confocal microscopy: a report by the American Academy of Ophthalmology. Ophthalmology 2004; 111: 396-406.
26. Mustonen RK, McDonald MB, et al. Normal human corneal cell populations evaluated by in vivo scanning slit confocal microscopy. Cornea 1998; 17: 485-492.
27. Vanathi M, Tandon R, Sharma N, et al. In-vivo slit scanning confocal microscopy of normal corneas in Indian eyes. Indian J Ophthalmol 2003; 51: 225-30.
28. Patel S, McLaren J, Hodge D, et al. Normal human keratocyte density and corneal thickness measurement by using confocal microscopy in vivo. Invest Ophthalmol Vis Sci 2001; 42: 333-9.
29. Bourne WM, Nelson LR, Hodge DO. Central corneal endothelial cell changes over a ten-year period. Invest Ophthalmol Vis Sci 1997; 38: 779-782.
30. Berlau J, Becker HH, Stave J, et al. Depth and age-dependent distribution of keratocytes in healthy human corneas: a study using scanning-slit confocal microscopy in vivo. J Cataract Refract Surg 2002; 28: 611-16.
31. Guthoff RF, Zhivov A, Stachs O. In vivo confocal microscopy, an inner vision of the cornea-a major review. Clinical and Experimental Ophthalmology 2009; 37:100-117.
32. Winchester K, Mathers WD, Sutphin JE, et al. Diagnosis of Acanthamoeba keratitis in vivo with confocal microscopy. Cornea 1995; 14:10-17.
33. Pfister DR, Cameron JD, Krachmer JH, et al. Confocal microscopy findings of Acanthamoeba keratitis. Am J Ophthalmol 1996; 121:119-28.
34. Nakano E, Oliveira M, Portellinha W, et al. Confocal microscopy in early diagnosis of Acanthamoeba keratitis. J Refract Surg 2004; 20 (Suppl.): S737-40.
35. Kobayashi A, Ishibashi Y, Oikawa Y, et al. In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis. Cornea 2008; 27 (4): 439-45.
36. Mathers WD, Sutphin JE, Folberg R, et al. Outbreak of keratitis presumed to be caused by Acanthamoeba. Am J Ophthalmol 1996; 121:129-42.
37. Florakis GJ, Moazami G, Schubert H, et al. Scanning slit confocal microscopy of fungal keratitis. Arch Ophthalmol 1997; 115:1461-1463.
38. Avunduk AM, Beuerman RW, Varnell ED, et al. Confocal microscopy of Aspergillus fumigatus keratitis. Br J Ophthalmol 2003; 87: 409-410.
39. Brasnu E, Bourcier T, Dupas B, et al. In vivo confocal microscopy in fungal keratitis. Br J Ophthalmol 2007; 91:588-91.
40. Bozkurt B, Irkec M. In vivo laser confocal microscopic findings in patients with epithelial basement membrane dystrophy. Eur J Ophthalmol 2009; 19 (3): 348-54.
41. Toshida H, Uesugi Y, Ebihara N, et al. In vivo observations of a case of chlorpromazine deposits in the cornea using an HRT Ⅱ Rostock corneal module.Cornea 2007; 26(9): 1141-3.
42. Traversi C, Martone G, Malandrini A, et al. In vivo confocal microscopy in recurrent granular dystrophy in corneal graft after penetrating keratoplasty. Clin Experiment Ophthalmol 2006; 34 (8): 808-10.
43. Labbe A, Nicola RD, Dupas B, et al. Epithelial basement membrane dystrophy:evaluation with the HRT Ⅱ Rostock Cornea Module. Ophthalmology 2006; 113 (8):1301-8.
44. Rosenberg ME, Tervo TM, Petroll WM, et al. In vivo confocal microscopy of patients with corneal recurrent erosion syndrome or epithelial basement membrane dystrophy.Ophthalmology 2000; 107: 565-73.
45. Patel DV, Grupcheva CN, McGhee CNJ. Meesmann's corneal dystrophy imaged by in vivo confocal microscopy. Cornea 2005; 24: 669-73.
46. Werner LP, Werner L, Dighiero P, et al. Confocal microscopy in Bowman and stromal corneal dystrophies. Ophthalmology 1999; 106:1697-704.
47. Chiou AG, Beueraian RW, Kaufman SC, et al. Confocal microscopy in lattice comeal dystrophy. Graefes Arch Clin Exp Ophthalmol 1999; 237: 697-701.
48. Mustonen RK, McDonald MB, Srivannaboon S, et al. In vivo confocal microscopy of Fuchs' endothelial dystrophy. Cornea 1998; 17: 493-503.
49. Chiou AG, Kaufman SC, Beuerman RW, et al. Confocal microscopy in cornea guttata and Fuchs' endothelial dystrophy. Br J Ophthalmol 1999; 83:185-9.
50. Grupcheva CN, Craig JP, Sherwin T, et al. Differential diagnosis of comeal oedema assisted by in vivo confocal microscopy. Clin Experiment Ophthalmol 2001; 29:133-7.
51. Patel DV, Grupcheva CN, McGhee CNJ. In vivo confocal microscopy in posterior polymorphous dystrophy. Cornea 2005; 24: 550-4.
52. Cheng LL, Young AL, Wong AKK, et al. Confocal microscopy of posterior polymorphous endothelial dystrophy. Cornea 2005; 24: 599-602.
53. Rajan M, Watters W, Patmore A, et al. In vitro human corneal model to investigate stromal epithelial interactions following refractive surgery J Catract Refract Surg 2005; 31:1789-1801.
54. Kaufman SC, Kaufman HE. How has confocal microscopy helped us in refractive surgery? Current Opinion in Ophthalmology 2006; 17: 380-388.
55. Chew SJ, Beuerman RW, Kaufman HE, et al. In vivo confocal microscopy of corneal wound healing after excimer laser photorefractive keratectomy. CLAO J 1995; 21:273-280.
56. Erie JC, Nau CB, McLare JW, et al. Long-term keratocyte deficits in the corneal stroma after LASIK. Ophthalmol 2004; 111: 1356-1361.
57. Tsiklis NS, Kymionis GD, Pallikaris A1, et al. Endothelial cell density after photorefractive keratectomy for moderate myopia using a 213 nm solid-state laser system. J Cataract Refract Surg. 2007; 33 (11): 1866-70.
58. Sonigo B, Iordanidou V, Chong-Sit D, et al. In vivo corneal confocal microscopy comparison of intralase femtosecond laser and mechanical microkeratome for laser in situ keratomileusis. Invest Ophthalmol Vis Sci 2006; 47 (7): 2803-11.
59. Mazzotta C, Traversi C, Baiocchi S, et al. Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy m vivo: early and late modifications. Am J Ophthalmol 2008; 146 (4): 527-533. Epub 2008 Jul 30.
60. Caporossi A, Baiocchi S, Mazzotta C, et al. Parasurgical therapy for keratoconus by riboflavin-ultraviolet type A rays induced cross-linking of corneal collagen:preliminary refractive results in an Italian study. J Cataract Refract Surg 2006; 32 (5):837-45.
61. Niederer R, Perumal D, Sherwin T, et al. Corneal innervation and cellular changes after corneal transplantation: an in vivo confocal microscopy study. Invest Ophthalmol Vis Sci 2007; 48: 621-626.
62. Resch MD, Imre L, Tapaszto B, et al. Confocal microscopic evidence of increased Langerhans cell activity after corneal metal foreign body removal. Eur J Ophthalmol.2008; 18 (5): 703-7.
63. Chiou AG, Kaufman SC, Kaz K, et al. Characterization of epithelial downgrowth by confocal microscopy. J Cataract Refract Surg 1999; 25:1172-1174.
64. Patel SV, McLaren JW, Hodge DO, et al. Confocal microscopy in vivo in corneas of long-term contact lens wearers. Invest Ophthalmol Vis Sci 2002; 43: 995-1003.
65. Oliveira-Soto L, Efron N. Morphology of corneal nerves in soft contact lens wear. A comparative study using confocal microscopy. Ophthalmic Physiol Opt 2003; 23:163-74.
66. Jalbert Ⅰ, Stapleton F. Effect of lens wear on corneal stroma:preliminary findings.Aust NZ J Ophthalmol 1999; 27: 211-13.
67. Toshida H, Murakami A. An atypical case of microcysts associated with silicone hydrogel contact lens: findings on in vivo confocal laser microscopy. Eye Contact Lens 2009; 35 (3): 156-8.
68. Iordanidou V, Sultan G, Boileau C, et al. In vivo corneal confocal microscopy in marfan syndrome. Cornea 2007; 26 (7): 787-92.
69. Rosenberg ME, Tervo TM, Immonen IJ, et al. Corneal structure and sensitivity in type 1 diabetes mellitus. Invest Ophthalmol Vis Sci 2000; 41: 2915-21.
70. Zhao C, Lu S, Truffert A, et al. Corneal nerves alterations in various types of systemic polyneuropathy, identified by in vivo confocal microscopy. Klin Monatsbl Augenheilkd 2008; 225 (5): 413-7.
2. Sun, T. T., R. M. Lavker. Corneal epithelial stem cells: past, present, and future. J Investig Dermatol Symp Proc 2004; 9 (3): 202-7.
3. Li, W., Y. Hayashida, et al. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res 2007; 17 (1): 26-36.
4. Chee, K. Y., A. Kicic, et al. Limbal stem cells: the search for a marker. Clin Experiment Ophthalmol 2006; 34 (1): 64-73.
5. Stave, J., G. Zinser, et al. Modified Heidelberg Retinal Tomograph HRT. Initial results of in vivo presentation of corneal structures. Ophthalmologe 2002; 99 (4): 276-80.
6. Eckard, A., J. Stave, et al. In vivo investigations of the corneal epithelium with the confocal Rostock Laser Scanning Microscope (RLSM). Cornea 2006; 25 (2): 127-31.
7. Kobayashi, A., Y. Ishibashi, et al. In vivo and ex vivo laser confocal microscopy findings in patients with early-stage acanthamoeba keratitis. Cornea 2008; 27 (4):439-45.
8. Chen, Z., D. Shijing, et al. Clinical and experimental findings in Acanthamoeba keratitis with Heidelberg Retina Tomograph Ⅲ-RCM. Ophthalmic Physiol Opt 2008;28 (2): 163-7.
9. Brasnu, E., T. Bourcier, et al. In vivo confocal microscopy in fungal keratitis. Br J Ophthalmol 2007; 91 (5): 588-91.
10. Mazzotta, C, C. Traversi, et al. Corneal healing after riboflavin ultraviolet-A collagen cross-linking determined by confocal laser scanning microscopy in vivo: early and late modifications. Am J Ophthalmol 2008; 146 (4): 527-533.
11. Sonigo, B., V. Iordanidou, et al. In vivo corneal confocal microscopy comparison of intralase femtosecond laser and mechanical microkeratome for laser in situ keratomileusis. Invest Ophthalmol Vis Sci 2006; 47 (7): 2803-11.
12. Bozkurt, B., M. Irkec. In vivo laser confocal microscopic findings in patients with epithelial basement membrane dystrophy. Eur J Ophthalmol 2009; 19 (3): 348-54.
13. Traversi, C, G. Martone, et al. In vivo confocal microscopy in recurrent granular dystrophy in corneal graft after penetrating keratoplasty. Clin Experiment Ophthalmol 2006; 34 (8): 808-10.
14. Labbe, A., R. D. Nicola, et al. Epithelial basement membrane dystrophy: evaluation with the HRTII Rostock Cornea Module. Ophthalmology 2006; 113 (8): 1301-8.
15. Toshida, H., A. Murakami. An atypical case of microcysts associated with silicone hydrogel contact lens: findings on in vivo confocal laser microscopy. Eye Contact Lens 2009; 35 (3): 156-8.
16. Tsiklis, N. S., G. D. Kymionis, et al. Endothelial cell density after photorefractive keratectomy for moderate myopia using a 213 nm solid-state laser system. J Cataract Refract Surg 2007; 33 (11): 1866-70.
17. Zhao, C, S. Lu, et al. Corneal nerves alterations in various types of systemic polyneuropathy, identified by in vivo confocal microscopy. Klin Monatsbl Augenheilkd 2008; 225 (5): 413-7.
18. Iordanidou, V., G. Sultan, et al. In vivo corneal confocal microscopy in marfan syndrome. Cornea 2007; 26 (7): 787-92.
19. Jurowska-Liput, J., P. Krzyzanowska, et al. Evaluation on the cellular level the filtering bleb following trabeculectomy, using the HRT Ⅱ Cornea Module. Klin Oczna 2008;110 (10-12): 343-6.
20. Donisi, P. M., P. Rama, et al. Analysis of limbal stem cell deficiency by corneal impression cytology. Cornea 2003; 22 (6): 533-8.
1. Schermer, A., S. Galvin, et al. Differentiation-related expression of a major 64K corneal keratin in vivo and in culture suggests limbal location of corneal epithelial stem cells. J Cell Biol 1986; 103 (1): 49-62.
2. Cotsarelis, G., S. Z. Cheng, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.Cell 1989; 57 (2): 201-9.
3. Lavker, R. M., S. C. Tseng, et al. Corneal epithelial stem cells at the limbus: looking at some old problems from a new angle. Exp Eye Res 2004; 78 (3): 433-46.
4. Sun, T. T., R. M. Lavker. Corneal epithelial stem cells: past, present, and future. J Investig Dermatol Symp Proc 2004; 9 (3): 202-7.
5. Li, W., Y. Hayashida, et al. Niche regulation of corneal epithelial stem cells at the limbus. Cell Res 2007; 17 (1): 26-36.
6. Vascotto, S. G., M. Griffith. Localization of candidate stem and progenitor cell markers within the human cornea, limbus, and bulbar conjunctiva in vivo and in cell culture. Anat Rec A Discov Mol Cell Evol Biol 2006; 288 (8): 921-31.
7. Chee, K. Y., A. Kicic, et al. Limbal stem cells: the search for a marker. Clin Experiment Ophthalmol 2006; 34 (1): 64-73.
8. Millan, J. L, W. H. Fishman. Biology of human alkaline phosphatases with special reference to cancer. Crit Rev Clin Lab Sci 1995; 32 (1): 1-39.
9. Cavaleri, F., H. R. Scholer. Nanog: A New Recruit to the Embryonic Stem Cell Orchestra. Cell 2003; 113 (5): 551-552.
10. Chambers, I., D. Colby, et al. Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells. Cell 2003; 113 (5): 643-55.
11. Mitsui, K., Y. Tokuzawa, et al. The Homeoprotein Nanog Is Required for Maintenance of Pluripotency in Mouse Epiblast and ES Cells. Cell 2003; 113 (5): 631-642.
12. Niwa, H., J. Miyazaki, et al. Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet 2000; 24 (4): 372-6.
13. Pesce, M., H. R. Scholer. Oct-4: gatekeeper in the beginnings of mammalian development. Stem Cells 2001; 19 (4): 271-8.
14. Solter, D., B. B. Knowles. Monoclonal antibody defining a stage-specific mouse embryonic antigen (SSEA-1). Proc Natl Acad Sci U S A 1978; 75 (11): 5565-9.
15. Kannagi, R., N. A. Cochran, et al. Stage-specific embryonic antigens (SSEA-3 and-4) are epitopes of a unique globo-series ganglioside isolated from human teratocarcinoma cells. Embo J 1983; 2 (12): 2355-61.7.
16. Polisetty, N., A. Fatima, et al. Mesenchymal cells from limbal stroma of human eye. Mol Vis 2008; 14: 431-42.
17. de Paiva, C. S., Z. Chen, et al. ABCG2 transporter identifies a population of clonogenic human limbal epithelial cells. Stem Cells 2005; 23 (1): 63-73.
18. Pellegrini, G., E. Dellambra, et al. p63 identifies keratinocyte stem cells. Proc Natl Acad Sci U S A 2001; 98 (6): 3156-61.
19. Moore, K. A., I. R. Lemischka. Stem cells and their niches. Science 2006; 311 (5769): 1880-5.
1. Potten, C. S., R. Schofield, et al. A comparison of cell replacement in bone marrow, testis and three regions of surface epithelium. Biochim Biophys Acta 1979; 560 (2):281-99.
2. Tumbar, T., G. Guasch, et al. Defining the epithelial stem cell niche in skin. Science 2004; 303 (5656): 359-63.
3. Cotsarelis, G., T.-T. Sun, et al. Label-retaining cells reside in the bulge area of pilosebaceous unit: Implications for follicular stem cells, hair cycle, and skin carcinogenesis. Cell 1990; 61 (7): 1329-1337.
4. Arai, F., A. Hirao, et al. Tie2/angiopoietin-l signaling regulates hematopoietic stem cell quiescence in the bone marrow niche. Cell 2004; 118 (2): 149-61.
5. Zhang, J., C. Niu, et al. Identification of the haematopoietic stem cell niche and control of the niche size. Nature 2003; 425 (6960): 836-41.
6. Potten, C. S., W. J. Hume, et al. The segregation of DNA in epithelial stem cells. Cell 1978; 15 (3): 899-906.
7. Urbanek, K., D. Cesselli, et al. Stem cell niches in the adult mouse heart. Proc Natl Acad Sci U S A 2006; 103 (24): 9226-31.
8. Welm, B. E., S. B. Tepera, et al. Sca-l(pos) cells in the mouse mammary gland represent an enriched progenitor cell population. Dev Biol 2002; 245 (1): 42-56.
9. Cotsarelis, G., S. Z. Cheng, et al. Existence of slow-cycling limbal epithelial basal cells that can be preferentially stimulated to proliferate: implications on epithelial stem cells.Cell 1989; 57 (2): 201-9.
10. Foudi, A., K. Hochedlinger, et al. Analysis of histone 2B-GFP retention reveals slowly cycling hematopoietic stem cells. Nat Biotechnol 2009; 27 (1): 84-90.
11. Kanda, T., K. F. Sullivan, et al. Histone-GFP fusion protein enables sensitive analysis of chromosome dynamics in living mammalian cells. Curr Biol 1998; 8 (7): 377-85.
12. Meek, K. M., N. J. Fullwood. Corneal and scleral collagens-a microscopist's perspective. Micron 2001; 32 (3): 261-72.
13. Nagasaki, T., J. Zhao. Centripetal movement of corneal epithelial cells in the normal adult mouse. Invest Ophthalmol Vis Sci 2003; 44 (2): 558-66.
1. Berliner ML. Biomicroscopy of the Eye. Hafner Publishing Company, New York,1966.
2. Vogt A. Textbook and Atlas of Slit Lamp Microscopy of the Living Eye. J. P. Wayenborgh, Bonn, 1981.
3. Martonyi CL, Bahn CF, Meyer RF. Clinical Slit Lamp Biomicroscopy and Photo Slit Lamp Biomicrography. Time One Ink, Ltd, Ann Arbor, 1985.
4. Minsky M. Memoir on inventing the confocal scanning microscope. Scanning 1988; 10:128-138.
5. Cavanagh HD, Petroll WM, Alizadeh H, et al. Clinical and diagnostic use of in vivo confocal microscopy in patients with corneal disease. Ophthalmol 1993; 100:1444-1454.
6. Semwogerere D, Weeks ER. Confocal microscopy. In: Wnek GE, Bowlin GL, editors. Encyclopedia of biomaterials and biomedical engineering. London: Taylor & Francis;2005. pp. 1-10.
7. Webb RH: Confocal optical microscopy. Rep Prog Phys 1996; 59: All-All.
8. Masters, B. R., M. Bohnke. Confocal microscopy of the human cornea in vivo. Int Ophthalmol 2001; 23 (4-6): 199-206.
9. Masters, B. R., M. Bohnke. Three-dimensional confocal microscopy of the living human eye. Annu Rev Biomed Eng 2002; 4: 69-91.
10. Wilson T: Confocal Microscopy. Academic Press, London, 1990.
11. Corle TR, Kino GS. Confocal Scanning Optical Microscopes and Related Imaging Systems. Academic Press, San Diego, 1996.
12. Janknecht, P., J. Funk. Optic nerve head analyser and Heidelberg retina tomograph: accuracy and reproducibility of topographic measurements in a model eye and in volunteers. Br J Ophthalmol 1994; 78 (10): 760-8.
13. Weinberger, D., H. Stiebel, et al. Three-dimensional measurements of idiopathic macular holes using a scanning laser tomograph. Ophthalmology 1995; 102 (10):1445-9.
14. Eckard, A., J. Stave, et al. In vivo investigations of the corneal epithelium with the confocal Rostock Laser Scanning Microscope (RLSM). Cornea 2006; 25 (2): 127-31.
15. Guthoff R.F., Baudouin C, Stave J. Atlas of Confocal Laser Scanning In-vivo Microscopy in Ophthalmology. Springer, 2006.
16. Patel, D. V., C. N. McGhee. Contemporary in vivo confocal microscopy of the living human cornea using white light and laser scanning techniques: a major review. Clin Experiment Ophthalmol 2007; 35 (1): 71-88.
17. Matsuda H. Electron microscopic study on the corneal nerve with special reference to its endings. Jpn J Ophthalmol 1968; 12:163-73.
18. Ueda S, del Cerro M, LoCascio JA, et al. Peptidergic and catecholaminergic fibers in the human corneal epithelium. An immunohistochemical and electron microscopic study. Acta Ophthalmol 1989; 192 (Suppl.): 80-90.
19. Muller LJ, Vrensen GF, Pels L et al. Architecture of human corneal nerves. Invest Ophthalmol Vis Sci 1997; 38: 985-94.
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