Chronic inflammatory cells and damaged limbal cells in pterygium

Background

Chronic inflammation in pterygium occurrence has not been explained. Whether damaged limbal basal epithelial cells are associated with pterygium occurrence in black Africans is not clear.

Objective

To explain chronic inflammation in pterygium, and to clarify whether damaged limbal basal epithelial cells were associated with pterygium occurrence in black Africans.

Methods

Chronic inflammatory changes and damaged limbal basal epithelial cells were assessed in 59 samples.

Results

Chronic inflammatory cells were present in 59 pterygia. Inflammatory cell count in 5 (27.8%) of 18 small pterygia was >200 (high) while in 22 (53.7%) of 41 large growths was <200 (low); p = 0.25. The proportion of pterygia with high counts tended to increase with pterygium extent. Twenty (33.9%) of 59 pterygia recurred after surgery. Ten (50%) of 20 samples had high cell counts and 10 (50%), low counts; p = 0.40.P53 expression was detected in 11 (18.6%) of 59 pterygium samples and 5 (71.4%) of 7 controls; p = 0.007. MMP 1 staining was present in 14 (23.7%) of 59 sections and 5 (71.4%) of 7 controls; p = 0.02. MMP2 in 16 (27.1%) cases and 5 (71.4%) controls; p = 0.03. MMP3 was overexpressed in 16 (27.1%) of 59 cases and 5 (71.4%) controls; p = 0.03.

Conclusions

Mild chronic inflammation has a tendency to be more frequent than severe inflammation in pterygia. It is clear that damaged limbal basal epithelial cells are unlikely to be related to pterygium occurrence.     

Chitosan-coated alginate membranes for cultivation of limbal epithelial cells to use in the restoration of damaged corneal surfaces

Some chemicals or thermal burns may result in abnormal reepithelialization by conjunctival epithelial cells and it causes different types of damage on the cornea surface. When reepithelialization does not occur, chronic inflammation and neovascularization develop, often leading to stroma scarring and/or ulceration. The aim of this study is to restore the human corneal surface with autologous corneal epithelial sheets generated by serial cultivation of the limbal epithelial cells over the different compositions of composite membranes. The composite membranes were prepared by coating the alginate membrane with chitosan. In this method, alginate membrane was prepared by precipitation of the sodium alginate solution in calcium chloride solution. Alginate membranes were washed, dried and immersed into the chitosan solutions to prepare composite membranes. The composite membranes were characterized based on their morphology, hydrophilicity, swellability, and chemical structure. In the last part of the study, composite membranes were used as base matrices for limbal epithelial cell cultivation. The cell cultivation on polymeric membranes was investigated as the in vitro studies. In these studies cell attachment, spreading and growth on polymeric membranes were evaluated.     

体外培养人角膜缘上皮细胞抑制激活态角膜基质细胞的生长

背景：角膜受到损伤后,角膜基质细胞激活转变为成纤维细胞,引起角膜基质瘢痕化,导致视力下降甚至丧失。 目的：观察角膜不同部位上皮细胞与角膜基质细胞的相互作用,探索角膜缘上皮细胞群能否抑制激活态角膜基质细胞的生长。 方法：采用酶消化及机械外力相结合的方法获取人角膜中央、角膜旁中央及角膜缘处角膜上皮细胞与浅层角膜基质细胞,进行体外培养。相差显微镜下观察细胞形态及生长变化。待培养角膜上皮细胞与基质细胞发生接触抑制时,记作“0 周”,采用免疫荧光染色技术检测培养细胞中PCNA及p63蛋白的表达。 结果与结论：培养的角膜上皮细胞与成纤维细胞发生接触抑制时,两种细胞间有明显分界线。角膜缘组上皮细胞中PCNA及p63蛋白均有较高的表达;角膜旁中央组PCNA有较高的表达,p63蛋白阴性表达;角膜中央组PCNA表达较低,p63蛋白阴性表达;从鉴定结果中可以得出只有角膜缘组中存在一定比例的角膜缘上皮干细胞。角膜缘组上皮细胞逐渐包围并化解成纤维细胞,在相互作用4周后,成纤维细胞聚集成死细胞团,缺乏角膜缘干细胞的中央组及旁中央组中成纤维细胞生长面积增加,上皮细胞生长受到抑制甚至死亡。说明体外培养的角膜缘上皮细胞群可以抑制激活态角膜基质细胞的生长。     

In vitro phenotypic characterization of human limbal epithelial cells

INTRODUCTION: Human limbal epithelial cells (huLEC) have been used for clinical purposes in ocular surface diseases to promote rapid re-epithelisation and restore corneal epithelium integrity. However, in Mexico this technique has not been fully developed. This study was conducted to characterize the huLEC phenotype expanded in vitro using a cell culture technique. MATERIAL AND METHODS: Cells were obtained from limbal tissue, cultured in KSFM medium and analyzed for the expression of vimentin, K, K19, p63, K12, by flow cytometry and immuno-fluorescence. RESULTS: The phenotype of cultured cells was vimentin+K+K19+ p63+K12-. CONCLUSIONS: Our results suggest that under these culture conditions huLEC maintained their stem cell phenotype. This culture technique could be used for clinical purposes in Mexico.     

Antigen presentation by human uterine epithelial cells to autologous T cells

PROBLEM: Epithelial cells, as sentinels of immune protection in the endometrium, use innate immune mechanisms to protect against infection from pathogenic microbes. Our goal in this study was to assess the ability of human uterine epithelial cells to present antigen to cells of the adaptive immune system. METHOD OF STUDY: Highly purified preparations of uterine epithelial cells from 11 patients were assessed for their ability to present tetanus toxoid (TT) to autologous T cells. Leukocyte contamination in the epithelial cell preparations was numerically and functionally determined. Using confocal microscopy, epithelial cells were tested for the expression of CD40 and CD1d. RESULTS: Purified preparations of endometrial epithelial cells isolated from every patient presented TT recall antigen to autologous T cells. Leukocyte contamination of epithelial cell preparations was insignificant. Uterine epithelial cells express CD40 and CD1d. CONCLUSION: Antigen presentation is an additional aspect of uterine epithelial cell function in maintaining women's health.     

Survival of cultured allogeneic limbal epithelial cells following corneal repair

In this study a technique for determining donor cell fate following corneal grafting was evaluated. Patients treated for limbal deficiency with allogeneic cultured corneal epithelial cells were studied to determine the fate of the grafted cells. The technique was evaluated initially through the use of donor eyes and then applied to the clinical analysis of 7 patients who had received a cultured corneal epithelial allograft. Cells removed from the cornea and any retrieved tissue were analyzed via polymerase chain reaction (PCR) genotyping to determine the origin of the cells populating the patients' healed cornea. A mixture of genotypes was detected in a cornea retrieved from a patient following a fully penetrating keratoplasty who had received a mixture of allogeneic tissue. Donor cells were no longer detected on the corneal surface of all 7 cases beyond 28 weeks postgraft. At these later time points, only patient genotype could be detected. These results demonstrate that PCR genotyping can be used to determine the origin of cells populating the surface of the cornea following the grafting of cultured allogeneic cells and demonstrates that transplanted cultured limbal epithelial cells do not persist on the surface of the host cornea for more than 28 weeks.     

Intrastromal invasion by limbal epithelial cells is mediated by epithelial-mesenchymal transition activated by air exposure

Corneal epithelial stem cells are located in the basal layer of the limbus between the cornea and the conjunctiva. Regulation of these limbal epithelial progenitor cells by the stromal niche dictates corneal surface health. To further characterize this process, limbal explants were cultured at the air-fluid interface, termed air-lifting, to stimulate the niche. As compared to submerged cultures, air-lifting significantly promoted epithelial stratification, migration, proliferation, and intrastromal invasion by limbal epithelial cells. Epithelial intrastromal invasion was noted when the limbal, but not corneal, epithelium was recombined with the limbal stroma containing live, but not dead, cells. Invading limbal basal cells displayed up-regulated nuclear expression of p63 and Ki67, down-regulated E-cadherin and cornea-specific keratin 3, and switched expression of beta-catenin from intercellular junctions to the nucleus and cytoplasm, indicating the activation of the Wnt/beta-catenin pathway. Invaded cells isolated by collagenase from the stroma of air-lifted, but not submerged, explants showed vivid clonal growth on 3T3 fibroblast feeder layers and complete epithelial-mesenchymal transition by expressing nuclear p63 and cytoplasmic S100A4. These findings collectively suggest that epithelial-mesenchymal transition via the Wnt/beta-catenin pathway influences the fate of limbal epithelial cells, likely to be progenitor cells, between regeneration and fibrosis when the stromal niche is activated.     

The porcine limbal epithelial stem cell niche as a new model for the study of transplanted tissue-engineered human limbal epithelial cells

Transparency of the human cornea is dependent upon the integrity of its epithelium and hence a population of limbal epithelial stem cells (LESCs). We have previously shown that LESCs reside in limbal epithelial crypts at the periphery of the human cornea. In this study the anatomy and functionality of the porcine limbus was evaluated for the first time as a novel model of the human limbus. Scanning electron microscopy, confocal microscopy, and histology revealed common structures in the porcine and human limbus in terms of the location and topography of palisades of Vogt and limbal epithelial crypts. Epithelial cells harvested from crypt regions achieved higher colony forming efficiency than cultures established from the noncrypt regions and central cornea. Also, expression of the putative SC markers p63α and integrin β1 brightness was higher in the basal layer of the crypt regions, as shown by immunocytochemistry. De-epithelialized porcine corneas were used as an in vitro organ culture model to study the fate of transplanted human epithelium cultured from the limbus. Multilayered epithelium was observed after ～1 week. Subsequently, wounds were inflicted on the central corneal epithelium. The wounded tissue healed within 5-7 days, and multilayering of the central corneal epithelium was re-established. The transplanted epithelia were repeatedly wounded at least four times and the wounds healed by 1 week. Putative SC marker expression of the transplanted epithelia was confirmed using immunohistochemistry. These results demonstrate that the porcine limbus shares features with the human limbus and as such provides a suitable model for the study of cultured limbal epithelial cell transplantation. These data have significant clinical value as this model can provide information on LESC fate post-transplantation and their ability to respond to injury, which is not possible to study in patients.     

体外扩增的自体脂肪干细胞介导自体脂肪移植的临床研究

目的探讨并评估体外扩增的自体脂肪干细胞介导自体脂肪移植的治疗效果。
　　方法实验组50例患者经过自体脂肪干细胞分离、培养、体外扩增后,介导自体脂肪颗粒注射移植,充填面部塑形、隆胸,随访6~24个月。手术分为两期,第一期：取患者自体脂肪颗粒细胞15~20 ml,进行体外分离、培养、扩增,扩增至第3代,进行细胞免疫表型、分化潜能、遗传稳定性鉴定。第二期：20~25 d 后,抽取一定量的患者自体脂肪颗粒混合第5~6代自体脂肪干细胞移植,用于鼻部、颏部、额部及颞部等面部充填塑形和隆胸。对照组10例患者,单纯自体脂肪移植。
　　结果第3~6代自体脂肪干细胞具有典型的间充质干细胞特征,并保持了遗传稳定性。脂肪移植6~24个月后,回访无明显脂肪吸收现象,塑形效果良好。
　　结论自体脂肪干细胞介导的自体脂肪移植术具有微创、移植脂肪成活率高、成形效果好等优点,是具有发展前途的、值得推荐的再生医学技术。     

Human amniotic epithelial cells as novel feeder layers for promoting ex vivo expansion of limbal epithelial progenitor cells

Human amniotic epithelial cells (HAECs) are a unique embryonic cell source that potentially can be used as feeder layers for expanding different types of stem cells. In vivo, HAECs uniformly expressed pan-cytokeratins (pan-CK) and heterogeneously expressed vimentin (Vim). The two phenotypes expressing either pan-CK(+)/Vim(+) or pan-CK(+)/Vim(-) were maintained in serum-free media with high calcium. In contrast, all HAECs became pan-CK(+)/Vim(+) in serum-containing media, which also promoted HAEC proliferation for at least eight passages, especially supplemented with epidermal growth factor and insulin. Mitomycin C-arrested HAEC feeder layers were more effective in promoting clonal growth of human limbal epithelial progenitors than conventional 3T3 murine feeder layers. Cells in HAEC-supported clones were uniformly smaller, sustained more proliferation, and expressed less CK12 and connexin 43 but higher levels of stem cell-associated markers such as p63, Musashi-1, and ATP-binding cassette subfamily G2 than those of 3T3-supported clones. Subculturing of clonally expanded limbal progenitors from HAEC feeder layers, but not from 3T3 feeder layers, gave rise to uniformly p63-positive epithelial progenitor cells as well as nestin-positive neuronal-like progenitors. Collectively, these results indicated that HAECs can be used as a human feeder layer equivalent for more effective ex vivo expansion of adult epithelial stem cells from the human limbus. Disclosure of potential conflicts of interest is found at the end of this article.     

Characterization of the limbal epithelial stem cell niche: novel imaging techniques permit in vivo observation and targeted biopsy of limbal epithelial stem cells

It is anticipated that stem cell (SC) therapy will enable the regeneration of diseased tissues and organs. Understanding SC niches is an essential step toward realizing this goal. By virtue of its optical transparency and physical separation of SC and transient amplifying cell compartments, the human cornea provides a unique opportunity to visualize and observe a population of adult stem cells, limbal epithelial stem cells (LESCs), in their niche environment. To date, the characteristics of the LESC niche have remained unclear. State-of-the-art imaging techniques were used to construct a three-dimensional (3D) view of the entire human corneal limbus and identify the structural characteristics of the LESC niche. Two distinct candidate LESC niche structures were identified. Cells within these structures express high levels of the putative limbal stem cell markers p63alpha and ABCG2; however, current methods cannot identify for certain which exact cells within this cell population are truly LESCs. These structures could be located and observed in vivo in normal human subjects, but not in patients with clinically diagnosed corneal LESC deficiency. The distribution of these structures around the corneal circumference is not uniform. Biopsies targeted to limbal regions rich in LESC niche structures yielded significantly higher numbers of LESCs in culture. Our findings demonstrate how adult stem cell niches can be identified and observed in vivo in humans and provide new biological insight into the importance of LESC niche structures in maintaining normal LESC function. Finally, the concept of targeted biopsy of adult SC niches improves stem cell yield and may prove to be essential for the successful development of novel adult stem cell therapies. Disclosure of potential conflicts of interest is found at the end of this article.     

酸性成纤维细胞生长因子对顺铂肾损害保护作用的实验研究

目的研究酸性成纤维细胞生长因子(aFGF)对顺铂(DDP)引起的体外培养的肾小管上皮细胞损害的保护作用。方法通过酶消化法获取肾小管上皮细胞,接种于96孔培养板:(1)培养72h后加入一系列浓度的DDP,实验组在DDP作用12h后加入不同浓度aFGF,再培养48h后用WST-8法检测细胞存活率。(2)以DDP建立损伤模型,并在加药后12h加入一定量的aFGF,观察aFGF对损伤的肾小管上皮细胞的保护作用。结果(1)aFGF能使DDP对肾小管上皮细胞的半数抑制浓度(IC50)升高。(2)DDP组与对照组比较,各生化和酶学指标差异均有统计学意义;而aFGF加DDP组与对照组比较,SOD、GSH-Px酶活性差异无统计学意义,MDA、NO升高差异仍有统计学意义。结论aFGF对DDP损伤的肾小管上皮细胞有明显的保护作用。     

人羊膜上皮细胞体外培养重建角膜上皮的实验研究

目的: 探讨人羊膜上皮细胞(human amniotic epithelial cells, HAECs)诱导分化为角膜上皮细胞的可塑性及其重建角膜上皮的可行性。方法: 取足月产人羊膜,通过胶原酶、胰蛋白酶和EDTA消化获得HAECs,将细胞在体外培养扩增、传代,将2~3代HAECs接种于去上皮的兔角膜基质上培养,待HAECs融合形成单层后,置于插入式培养皿中进行气液界面培养14 d。对角膜基质上培养的HAECs用光镜、扫描电镜和透射电镜进行形态学及超微结构观察,以及细胞角蛋白(CK)3/12的免疫组织化学检测。结果: HAECs可在角膜基质上生长,培养1~2 d可形成单层,气液界面培养14 d可形成4~5层细胞的复层上皮,扫描电镜观察细胞表面有丰富的微绒毛,透射电镜下可见上皮细胞之间有桥粒连接,免疫组化检测复层细胞表达CK3/12,所形成的复层结构与正常角膜上皮相似。结论: HAECs有可能作为种子细胞用于组织工程角膜上皮重建。     

自体角膜缘干细胞移植对翼状胬肉患者视觉质量及泪膜功能的影响

目的 探讨翼状胬肉切除术联合自体角膜缘干细胞移植对翼状胬肉患者视觉质量、角膜屈光及泪膜功能的影响。 方法 选取2016年2月~2017年1月在我院治疗的翼状胬肉患者110例,随机分为对照组和观察组,每组55例。对照组给予翼状胬肉切除术治疗,观察组在对照组的基础上给予角膜缘干细胞移植,之后对两组患者视力、角膜屈光度、泪膜功能及术后巩膜并发症进行评估。 结果 与治疗前相比,治疗后两组患者视力、角膜水平曲度及角膜垂直曲度均显著增加,角膜散光度较治疗之前显著下降,且观察组治疗后视力、角膜水平曲度及角膜垂直曲度显著大于对照组,角膜散光度显著低于对照组,差异有统计学意义（P<0.05）;与治疗前相比,治疗后两组患者基础泪液分泌试验（SIT）及泪膜破裂时间（BUT）水平均显著上升,且观察组治疗后显著高于对照组,差异有统计学意义（P<0.05）;术后6个月,观察组眼部刺激症状、巩膜溶解软化及巩膜坏死发生率均显著低于对照组,差异有统计学意义（P<0.05）。 结论 角膜缘干细胞移植联合翼状胬肉切除能有效提高患者视力,增加角膜屈光度,降低患者散光程度,并能有效改善泪膜功能,降低巩膜并发症的发生率。     

Subcutaneous transplantation of autologous oral mucosal epithelial cell sheets fabricated on temperature-responsive culture dishes

The oral mucosa is an attractive cell source for autologous transplantation in human patients who require regenerative therapies of various epithelia. However, the time-course of cellular changes in transplanted oral mucosal epithelia at ectopic sites remains poorly understood. By applying a rat model, we analyzed phenotypic changes in oral mucosal epithelial cell sheets after harvest from temperature-responsive culture dishes and subsequent autologous subcutaneous transplantation. We used monoclonal antibodies to identify epithelial-specific cytokeratins 4, 10, 13, and 14, the stem/progenitor cell marker p63, and proliferating cell nuclear antigen, within the regenerated tissues. Transplanted oral mucosal epithelial cell sheets proliferated during the first week after grafting in conjunction with host inflammation, but then began to degenerate afterward with complete disappearance after 3 weeks. Our findings suggest that host subcutaneous tissues support proliferation and differentiation of the oral mucosal epithelial cell sheets, but are unable to promote maintenance of stem and progenitor cells and therefore cannot produce long-term survivability.     

计算机导航辅助自体干细胞回植治疗早期股骨头坏死

背景：如何利用组织工程技术修复股骨头塌陷的软骨,是塌陷后股骨头坏死治疗的主要研究方向。 目的：观察计算机导航辅助及关节镜监视下髓芯减压自体骨髓间充质干细胞体外培养自体回植治疗早期股骨头缺血性坏死的方法及疗效。 方法：根据世界骨循环研究学会国际骨坏死分期标准,选取早期股骨头缺血性坏死患者104例,均在计算机导航辅助下实施髓芯减压术,同时关节镜监视下进行坏死病灶清除,其中53例在此基础上二期植入体外分离培养的1×109 L-1自体骨髓间充质干细胞2 mL。 结果与结论：经平均30个月的随访及磁共振检查发现,所有患者股骨头坏死体积均明显减小,Harris评分明显提高(P < 0.05);其中给予自体骨髓间充质干细胞回植的患者改善更明显(P < 0.05),其临床成功率和影像学成功率分别达92%和90%,且未见明显的不良反应。说明计算机导航辅助及关节镜监视下髓芯减压自体骨髓间充质干细胞体外培养回植是一种安全、有效的治疗早期股骨头缺血性坏死的方法,效果优于单纯髓芯减压。