诱导胚胎干细胞向角膜上皮细胞分化的实验研究

探索胚胎干细胞在表层角膜缘基质诱导下向角膜上皮细胞分化的可能性. 体外培养带GFP标记的 ES-D3细胞, 并利用视黄酸进行预诱导, 然后将预诱导后的细胞接种在表层角膜缘基质上, 细胞融合形成单层后, 随机分为3组进行研究: 第1组传代后直接进行检测; 第2组在 气-液界面上培养10天, 然后植入裸鼠皮下以进行体内诱导; 第3组作为对照组, 不给予GFP-ES-D3细胞特殊诱导条件, 细胞自由分化. 诱导分化的细胞植入裸鼠皮下体2周后没有畸胎瘤形成. 诱导分化的细胞呈现上皮样外观, 体内诱导组和体外诱导组免疫组织化学染色均检测到CK3, P63和PCNA表达阳性, 电子显微镜检查可见两组细胞表面都有微绒毛和细胞间紧密连接形成. 实验对照组部分细胞脱落和死亡, 大部分表现神经样细胞的树突样外观, 小部分未死亡的贴壁细胞呈多态性, 这些结果表明胚胎干细胞在特定条件下经表层角膜基质诱导能够分化为角膜上皮细胞. 胚胎干细胞诱导分化有可能为眼表重建和组织工程化角膜的构建提供上皮种子细胞.     

诱导胚胎干细胞向角膜上皮细胞分化的实验研究

探索胚胎干细胞在表层角膜缘基质诱导下向角膜上皮细胞分化的可能性.体外培养带GFP标记的ES-D3细胞,并利用视黄酸进行预诱导,然后将预诱导后的细胞接种在表层角膜缘基质上,细胞融合形成单层后,随机分为3组进行研究:第1组传代后直接进行检测;第2组在气-液界面上培养10天,然后植入裸鼠皮下以进行体内诱导;第3组作为对照组,不给予GFP-ES-D3细胞特殊诱导条件,细胞自由分化.诱导分化的细胞植入裸鼠皮下体2周后没有畸胎瘤形成.诱导分化的细胞呈现上皮样外观,体内诱导组和体外诱导组免疫组织化学染色均检测到CK3,P63和PCNA表达阳性,电子显微镜检查可见两组细胞表面都有微绒毛和细胞间紧密连接形成.实验对照组部分细胞脱落和死亡,大部分表现神经样细胞的树突样外观,小部分未死亡的贴壁细胞呈多态性,这些结果表明胚胎干细胞在特定条件下经表层角膜基质诱导能够分化为角膜上皮细胞.胚胎干细胞诱导分化有可能为眼表重建和组织工程化角膜的构建提供上皮种子细胞.     

山羊角膜缘干细胞荧光蛋白(Venus)细胞株的建立及角膜上皮植片构建

角膜缘干细胞是角膜上皮更新与修复的来源,角膜上皮受损严重常会导致角膜盲。尽管近几年通过角膜缘干细胞移植术(LSCT)治愈角膜上皮受损的临床应用已被推广,但是对于角膜缘干细胞移植受损机体后的修复机理并不明确。为了实现角膜缘干细胞移植后的活体追踪,使用G418筛选标记有Venus荧光蛋白的角膜缘干细胞株(GLSC-V),并以其为种子细胞接种于去上皮羊膜上,体外培养21d构建成荧光角膜上皮植片。荧光倒置显微镜下观察GLSC-V的细胞质和细胞核均有绿色荧光表达,在体外培养荧光至少持续3个月。免疫荧光检测GLSC-V细胞P63、Integrinβ1均呈阳性表达,对GLSC-V细胞及未转染的GLSCs进行半定量RT-PCR检测显示,两组细胞皆未表达终末分化角膜上皮细胞基因k3、k12,GLSC-V中p63及pcna较未转染组细胞略上调,venus强表达。经HE染色观察构建的人工角膜组织由5～6层上皮细胞组成,组织中上表皮细胞个数少、体积大且呈扁平状;基底部细胞密集、体积小且成立方状。经免疫荧光检测仅组织基底部最基层细胞表达P63,上表皮细胞不表达。该人工角膜与正常角膜上皮组织结构特性相似,可用于移植,为研究角膜缘干细胞修复严重受损角膜上皮机理奠定基础。     

氟尿嘧啶诱发人支气管损伤修复过程及支气管干细胞的定位

目的观察离体人支气管损伤修复过程,进行支气管干细胞的定位。方法取肺癌手术切除的人支气管的正常部分进行组织培养,应用氟尿嘧啶(5-FU)诱发支气管上皮损伤,动态观察修复过程,用免疫组化SP法检测PCNA,β1-整合素及CK-19的表达,同时进行Hoechst33342荧光染色。结果1．5-FU作用12h后人支气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,PCNA染色阴性,证明为G0期细胞。其中部分细胞Hoechst33342染色阴性。2．将5-FU去除3～6h后,细胞形态变为扁平,PCNA染色见核染色阳性的细胞与阴性细胞(Go期细胞)间隔分布;12h后细胞变为立方,细胞数目逐渐增多,到48～72h恢复假复层柱状上皮。3．β1-整合素及CK-19在已分化细胞中呈阳性反应。结论在5-FU的打击下,进入增殖期细胞死亡、脱落,仅余Go期细胞,其中含有支气管干细胞。正是这些干细胞增殖分化使支气管上皮修复。     

大鼠体内气管损伤修复过程及气管干细胞的定位研究

目的观察大鼠体内气管损伤修复过程,进行气管干细胞的定位.方法应用氟尿嘧啶(5-FU)诱发在体气管上皮损伤,动态观察修复过程;对损伤后气管上皮细胞行Hoechst33342荧光染色,并用RT-PCR法检测ABC转运蛋白ABCG2/bcrp1基因.结果1.5-FU作用30min后大鼠气管上皮细胞绝大部分脱落,可见少量间隔分布的类似裸核的细胞呈钉状位于基底膜上,免疫组化检测增殖细胞核抗原阴性,证明为G0期细胞.其中部分细胞Hoechst33342染色阴性,为侧群(side population,SP)细胞.2.将5-FU 去除3-6h后,上皮细胞形态变为扁平,9-12h 后细胞变为立方,细胞数目逐渐增多,24h上皮细胞数更多,连接成片,可见纤毛,48h 接近恢复假复层纤毛柱状上皮.3.RT-PCR检测ABCG2/Bcrp1阳性反应产物长度为272bp.结论5-FU打击后,残余的G0期气管上皮细胞中含有干细胞.     

宫颈异倍体细胞Ki67和P16蛋白的表达

目的通过测定宫颈上皮同一异倍体细胞内Ki67和P16蛋白的表达,了解宫颈病变的严重程度。方法将宫颈细胞经DABI,Ki67和P16蛋白的免疫荧光处理,通过不同的激发光测定着色的细胞数。结果在8例宫颈病变中（2例宫颈炎,1例CIN1,2例CIN2,2例CIN3及1例宫颈癌）,除2例宫颈炎病例外,其余6例均可见〉5c的异倍体细胞。在6例中共发现有55个〉5c异倍体细胞,其中有38个Ki67阳性细胞（69％）和12个P16阳性细胞（21．8％）。10个Ki67和P16共阳性的细胞（18．2％）。结论高级别宫颈鳞状上皮内瘤变中异倍体细胞,可出现Ki67和P16蛋白阳性表达。     

过表达TPX2对人宫颈癌Hela细胞增殖和周期的影响及机制

该文的目的是研究过表达Xklp2靶蛋白(targeting protein for Xklp2,TPX2)对人宫颈癌Hela细胞体外增殖和细胞周期的影响及相关机制。构建TPX2过表达慢病毒载体(LV11-TPX2)及阴性对照(LV11-NC),选取稳定感染过表达慢病毒载体(LV11-TPX2)的Hela细胞作为实验组,将稳定感染(LV11-NC)的Hela细胞作为阴性对照组。未感染病毒的人宫颈癌Hela细胞作为空白对照组(CON)。CCK-8法及克隆形成实验检测各组Hela细胞体外增殖能力,FCM法检测各组Hela细胞的细胞周期分布变化。Western blot检测TPX2通路中Aurora A、eg5、P53蛋白质及增殖和细胞周期相关蛋白Ki67、CyclinB2、PCNA的水平。结果显示:与空白对照组及阴性对照组比较,LV11-TPX2感染组Hela细胞增殖能力明显增强(P0.05);LV11-TPX2组形成的克隆数目明显多于阴性对照组及空白对照组(P0.05)。LV11-TPX2组S期及G_2/M期细胞所占比例明显增加(P0.05)。LV11-TPX2感染组Hela细胞中Aurora A、eg5、Ki67、CyclinB2、PCNA蛋白质水平明显上调(P0.05),P53蛋白质明显下调(P0.05)。以上结果表明,TPX2基因过表达能促进宫颈癌细胞的增殖,S期及G_2/M期细胞所占比例明显增加,可能与其下调P53蛋白质水平及上调Aurora A、eg5、Ki67、CyclinB2、PCNA蛋白水平有关。     

宫颈HPV16持续感染阶段宫颈P16和Ki67的表达及其与宫颈癌变的关系

目的:探讨宫颈人乳头状瘤病毒(HPV)16持续感染阶段宫颈P16和Ki67的表达及其与宫颈癌变的相关性。方法:采用P16/Ki67免疫组化双染法检测102例HPV16持续感染者、136例非持续感染者宫颈组织P16、Ki67蛋白的表达,并根据免疫组化结果分组为双染阳性组、双染阴性组。所有患者随访观察2年,比较两组患者的结局及宫颈癌前病变的发生率。结果:P16、Ki67及P16/Ki67双染的阳性率分别为40.3%、44.5%及34.0%,HPV16持续感染患者P16、Ki67及P16/Ki67双染的阳性率均显著高于非持续感染患者(P0.05)。HPV16持续感染患者的P16、Ki67蛋白表达呈显著正相关(P0.05)。HPV16持续感染患者中,双染阳性组的病情持续和进展比例明显高于双染阴性组,也明显高于HPV16非持续感染(双染阴性组、双染阳性组)患者(P0.05)。HPV16持续感染患者中,双染阳性组进展为HSIL及以上病变发生率为32.5%(13/40),显著高于双染阴性组6.5%(P0.05)。结论:P16,Ki67双染阳性在HPV16持续感染阶段与宫颈上皮内病变疾病进展成正相关,对HPV16持续感染进展为宫颈高度病变有预警价值,可作为HPV16阳性早期治疗的敏感指标。     

猴表皮干细胞横向分化为角膜上皮细胞的研究

表皮干细胞可以作为角膜上皮细胞的替代物, 在自体眼表修复及组织工程生物角膜的构建中将产生不可估量的作用. 将体外分离培养的2 ~ 4代猴表皮干细胞和人角膜缘基质组织及角膜上皮细胞进行共培养(Transwell法), 于共培养前后使用流式细胞仪、RT-PCR和免疫组织 化学技术对其分化情况进行检测和鉴定. 并于第2, 4, 6, 8和10天进行免疫组织化学染色观察表皮干细胞转分化的比率. 实验采用条件培养基诱导法作为对照. 表皮干细胞在共培养前表达表皮干细胞标志, K15和整合素β1阳性, K3/K12阴性; 共培养后转为表达K3/K12, 从基因水平和蛋白质水平表现出角膜上皮细胞的特征, 但是条件培养基诱导法阳性率较低. 可见, 表皮干细胞具有可塑性, 在角膜缘基质组织及角膜上皮细胞的调控下, 可以横向分化为类角膜上皮细胞, 有可能重建角膜上皮, 进行自体生物角膜的构建.     

培养基血清浓度对人表皮干细胞增殖分化的影响

目的:比较不同血清浓度培养体系对表皮干细胞增殖分化的影响.方法:采用两步酶消化法和IV型胶原差速贴壁相结合的方法获得人原代表皮干细胞,分别以0%、5%、10%、15%和20%血清浓度的培养基在96孔板中进行培养.观察表皮干细胞形态,克隆形成及增殖的情况,应用四甲基偶氯唑蓝(MTT)比色法检测各组细胞存活和生长情况,分析量效和时效关系;持续传代培养细胞,每次传代的同时取适量细胞,用免疫细胞化学的方法进行表皮干细胞和表皮细胞相应标志物(K19、K14和K10)的测定.结果:表皮干细胞在各种血清浓度的培养基内均能形成克隆,增殖良好.用四甲基偶氮唑蓝(MTT)比色法测定,所得相同时间点各组OD值在统计学上没有差异(P>0.05),表皮干细胞生长速度各组间无差异.第1代表皮干细胞K19均有表达,而K14和K10表达均为阴性;其后高血清浓度(15%、20%)培养基中细胞较低血清浓度(0%、5%)先出现K14、K10蛋白的表达;培养至第10代是各组细胞均出现K10高表达,而K19、K14表达阴性.结论:在低血清浓度(0%、5%)的培养基中表皮干细胞生长良好,且能够相对较好保持表皮干细胞的特性.     

Stratified epithelial sheets engineered from a single adult murine corneal/limbal progenitor cell

The limbal region of the adult cornea contains stem cells which are ultimately responsible for regeneration of the corneal epithelium during wound repair. However, primarily-isolated murine corneal/limbal epithelial cells rapidly senesce on plastic in a serum-free low [Ca(2+)] medium, suggesting only transit amplifying cells are promoted. We developed a novel expansion method by seeding at a low cell density (<500 cells/cm(2)) and prolonging each culture time beyond the lifespan of transit amplifying cells (4 weeks). Expanded cells were uniformly small, negative to K12 keratin, but positive for p63 nuclear staining, and could be subcultured beyond 100 passages. After limiting dilution, one clone (TKE2) was selected that exhibited single cell clonal expansion with a doubling time of 34.2 hrs, and had normal karyotyping, but no anchorage-independent growth. A single cell could be continually expanded to a confluent monolayer on denuded amniotic membrane and became stratified by exposing to the air-medium interface. The resultant stratified epithelium expressed K14 keratin, involucrin, connexin 43 and p63, but not K12 keratin or Pax 6. However, expression of K12 could be up-regulated by increasing extracellular calcium concentration and addition of foetal bovine serum (FBS) at P12, but less so at P85. Therefore, this murine lim-bal/corneal epithelium-derived progenitor cell line still retained the plasticity for adopting corneal lineage differentiation, could be useful for investigating limbal niche cues that may promote corneal epithelial fate decision.     

Rat limbal epithelial side population cells exhibit a distinct expression of stem cell markers that are lacking in side population cells from the central cornea

The side population (SP) phenotype is shared by stem cells in various tissues and species. Here we demonstrate SP cells with Hoechst dye efflux were surprisingly collected from the epithelia of both the rat limbus and central cornea, unlike in human and rabbit eyes. Our results show that rat limbal SP cells have a significantly higher expression of the stem cell markers ABCG2, nestin, and notch 1, compared to central corneal SP cells. Immunohistochemistry also revealed that ABCG2 and the epithelial stem/progenitor cell marker p63 were expressed only in basal limbal epithelial cells. These results demonstrate that ABCG2 expression is closely linked to the stem cell phenotype of SP cells.     

Investigation of K14/K5 as a stem cell marker in the limbal region of the bovine cornea

Background

Identification of stem cells from a corneal epithelial cell population by specific molecular markers has been investigated previously. Expressions of P63, ABCG2 and K14/K5 have all been linked to mammalian corneal epithelial stem cells. Here we report on the limitations of K14/K5 as a limbal stem cell marker.

Methodology/Principal Findings

K14/K5 expression was measured by immunohistochemistry, Western blotting and Real time PCR and compared between bovine epithelial cells in the limbus and central cornea. A functional study was also included to investigate changes in K5/14 expression within cultured limbal epithelial cells undergoing forced differentiation. K14 expression (or its partner K5) was detected in quiescent epithelial cells from both the limbal area and central cornea. K14 was localized predominantly to basal epithelial cells in the limbus and suprabasal epithelial cells in the central cornea. Western blotting revealed K14 expression in both limbus and central cornea (higher levels in the limbus). Similarly, quantitative real time PCR found K5, partner to K14, to be expressed in both the central cornea and limbus. Following forced differentiation in culture the limbal epithelial cells revealed an increase in K5/14 gene/protein expression levels in concert with a predictable rise in a known differentiation marker.

Conclusions/Significance

K14 and its partner K5 are limited not only to the limbus but also to the central bovine cornea epithelial cells suggesting K14/K5 is not limbal specific in situ. Furthermore K14/K5 expression levels were not lowered (in fact they increased) within a limbal epithelial cell culture undergoing forced differentiation suggesting K14/K5 is an unreliable maker for undifferentiated cells ex vivo.     

Long-Term Cultures of Human Cornea Limbal Explants Form 3D Structures Ex Vivo – Implications for Tissue Engineering and Clinical Applications

Long-term cultures of cornea limbal epithelial stem cells (LESCs) were developed and characterized for future tissue engineering and clinical applications. The limbal tissue explants were cultivated and expanded for more than 3 months in medium containing serum as the only growth supplement and without use of scaffolds. Viable 3D cell outgrowth from the explants was observed within 4 weeks of cultivation. The outgrowing cells were examined by immunofluorescent staining for putative markers of stemness (ABCG2, CK15, CK19 and Vimentin), proliferation (p63α, Ki-67), limbal basal epithelial cells (CK8/18) and differentiated cornea epithelial cells (CK3 and CK12). Morphological and immunostaining analyses revealed that long-term culturing can form stratified 3D tissue layers with a clear extracellular matrix deposition and organization (collagen I, IV and V). The LESCs showed robust expression of p63α, ABCG2, and their surface marker fingerprint (CD117/c-kit, CXCR4, CD146/MCAM, CD166/ALCAM) changed over time compared to short-term LESC cultures. Overall, we provide a model for generating stem cell-rich, long-standing 3D cultures from LESCs which can be used for further research purposes and clinical transplantation.     

Hypoxia is a key regulator of limbal epithelial stem cell growth and differentiation

The aim of this study was to determine whether the growth and differentiation of limbal epithelial stem cell cultures could be controlled through manipulation of the oxygen tension. Limbal epithelial cells were isolated from corneoscleral disks, and cultured using either feeder cells in a growth medium supplemented with serum (3T3 system) or without feeder cells in a dedicated serum-free medium (EpiLife). During the culture, the cells were maintained either at ambient oxygen tension (20%) or at different levels of hypoxia (15, 10, 5, and 2% oxygen). The effect of oxygen on cell growth, progression through cell cycle, colony forming efficiency (CFE), and expression of stem cell (ABCG2 and p63α) and differentiation (CK3) markers was determined throughout the culture period of up to 18 days. Low oxygen levels favored a stem cell phenotype with a lower proliferative rate, high CFE, and a relatively higher expression of ABCG2 and p63α, while higher levels of oxygen led not only to decreased CFE but also to increased proportion of differentiated cells positive for CK3. Hypoxic cultures may thus potentially improve stem cell grafts for cultured limbal epithelial transplantation (CLET).     

Preservation of immunorecognition by transferring cells from 10% neutral buffered formalin to 70% ethanol

Abstract

Prolonged fixation of cells and tissues in 10% neutral buffered formalin (NBF) may decrease immunorecognition in some antigen-antibody pairs. Short fixation in 10% NBF followed by transfer to 70% ethanol has been used to overcome these effects, but the effects of this transfer on immunorecognition have not been explored adequately. We used two cell lines, DU145 (prostate cancer) and SKOV3 (ovarian cancer), grew them on coverslips and fixed them with 10% NBF at room temperature for 5 min and 12, 15, 18, 36, 108 and 180 h. Aliquots of the same cells were fixed in 10% NBF for 12 h, then transferred to 70% ethanol for 3, 6, 24, 96 and 168 h. Immunostaining with PCNA, Ki67-MIB-1, cytokeratins AE1/AE3 and EGFr was done concomitantly. In both cell lines, immunorecognition decreased between 18 and 36 h of fixation in 10% NBF for PCNA, Ki67-MIB-1 and cytokeratins AE1/AE3. By 108 to 180 h of 10% NBF exposure, there was complete loss of immunorecognition of PCNA and extensive loss of Ki67-MIB-1 and cytokeratins AE1/AE3. The effects on EGFr immunorecognition were less. Transfer to 70% ethanol after fixation for 12 h in 10% NBF preserved immunorecognition of the antibodies.     

Y-27632, a ROCK Inhibitor,Promoted Limbal Epithelial Cell Proliferation and Corneal Wound Healing

Transplantation of ex vivo cultured limbal epithelial cells is proven effective in restoring limbal stem cell deficiency. The present study aimed to investigate the promoting effect of Y-27632 on limbal epithelial cell proliferation. Limbal explants isolated from human donor eyes were expanded three weeks on culture dishes and outgrowth of epithelial cells was subsequently subcultured for in vitro experiments. In the presence of Y-27632, the ex vivo limbal outgrowth was accelerated, particularly the cells with epithelial cell-like morphology. Y-27632 dose-dependently promoted the proliferation of in vitro cultured human limbal epithelial cells as examined by phase contrast microscopy and luminescent cell-viability assay 30 hours after the treatment. The colony forming efficacy determined 7 days after the treatment was enhanced by Y-27632 also in a dose-dependent manner. The number of p63- or Ki67-positive cells was dose-dependently increased in Y-27632-treated cultures as detected by immunofluorescent staining and western blotanalysis. Cell cycle analysis by flow cytometric method revealed an increase in S-phase proliferating cells. The epithelial woundclosure rate was shown to be faster in experimental group received topical treatment withY-27632 than the sham control using a rat corneal wounding model. These resultsdemonstrate that Y-27632 can promote both the ex vivo and in vitro proliferation oflimbal epithelial cell proliferation. The in vivo enhanced epithelial wound healingfurther implies that the Y-27632 may act as a new strategy for treating limbal stem cell deficiency.     

Hyperproliferation of normally quiescent keratinocytes in non-lesional psoriatic skin due to high calcium concentration (an organotypic culture model)

Calcium plays an important role in the regulation of different functions of keratinocytes. In the present work we studied the effect of different extracellular calcium concentrations (0.01 mM-2.0 mM) on the proliferation and differentiation of human keratinocytes in normal human and non-lesional psoriatic skin. Using explant culture model, the proliferative and differentiated subsets of keratinocytes were detected by specific antibodies related to cell proliferation [beta-1 integrin (CD29), proliferating cell antigen (Ki67), proliferating cell nuclear antigen (PCNA)] and differentiation [differentiated cell cytokeratins (K1/K10) and differentiating cell antigen (lectin Ulex europaius agglutinin, UEA-1)]. After 4 days of culturing at high Ca2+ (2.0 mM) we observed marked hyperproliferation among the normally quiescent keratinocytes of non-lesional psoriatic skin. In normal uncultured and cultured skin and in uncultured and two-day-cultured non-lesional psoriatic skin both at normal (1.2 mM) and at high (2.0 mM) Ca2+ concentration only one layer of basal CD29+/Ki67+/K1/K10-/UEA-1- cell was observed. In sections from non-lesional psoriatic skin cultured for 4 days in the presence of high Ca2+ (2.0 mM) this cell population has expanded from at least three layers above the basement membrane. This expanded cell population of the 4-day high Ca2+ cultured non-lesional skin showed clear PCNA positive staining on frozen sections with the strongest positivity among the most basal localized cells. These data suggest that (i) extracellular Ca2+ concentration can influence the proliferation of basal ("stem") keratinocytes, (ii) the proliferative response to high Ca2+ concentration of psoriatic non-lesional basal keratinocytes differs from that of normal basal keratinocytes, (iv) changes in the extracellular Ca2+ milieu might play a role in the induction of the hyperproliferative psoriatic lesion.     

Serum-Free and Xenobiotic-Free Preservation of Cultured Human Limbal Epithelial Cells

Aim/Purpose of the Study

To develop a one-week storage method, without serum and xenobiotics, that would maintain cell viability, morphology, and phenotype of cultured human limbal epithelial sheets.

Materials and Methods

Human limbal explants were cultured on intact human amniotic membranes for two weeks. The sheets were stored in a hermetically sealed container at 23°C in either a serum-free medium with selected animal serum-derived compounds (Quantum 286) or a xenobiotic-free medium (Minimal Essential Medium) for 4 and 7 days. Stored and non-stored cultures were analyzed for cell viability, amniotic membrane and epithelial sheet thickness, and a panel of immunohistochemical markers for immature cells (ΔNp63α, p63, Bmi-1, C/EBP∂, ABCG2 and K19), differentiated cells (K3 and Cx43), proliferation (PCNA), and apoptosis (Caspase-3).

Results

The cell viability of the cultures was 98 ± 1% and remained high after storage. Mean central thickness of non-stored limbal epithelial sheets was 23 ± 3 μm, and no substantial loss of cells was observed after storage. The non-stored epithelial sheets expressed a predominantly immature phenotype with ΔNp63α positivity of more than 3% in 9 of 13 cultures. After storage, the expression of ABCG2 and C/EBP∂ was reduced for the 7 day Quantum 286-storage group; (P = 0.04), and Bmi-1 was reduced after 4 day Quantum 286-storage; (P = 0.02). No other markers varied significantly. The expression of differentiation markers was unrelated to the thickness of the epithelia and amniotic membrane, apart from ABCG2, which correlated negatively with thickness of limbal epithelia (R = -0.69, P = 0.01) and ΔNp63α, which correlated negatively with amniotic membrane thickness (R = -0.59, P = 0.03).

Conclusion

Limbal epithelial cells cultured from explants on amniotic membrane can be stored at 23°C in both serum-free and xenobiotic-free media, with sustained cell viability, ultrastructure, and ΔNp63α-positivity after both 4 and 7 days.     

Cultivation and Characterization of Cornea Limbal Epithelial Stem Cells on Lens Capsule in Animal Material-Free Medium

A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.