Ex vivo expansion of limbal epithelial stem cells: amniotic membrane serving as a stem cell niche

Identification, maintenance, and expansion of stem cells for subsequent transplantation has become a new strategy for treating many diseases in most medical subspecialties. The stem cells of the corneal epithelium are located in the limbal basal layer and are the ultimate source for constant corneal epithelial renewal. Like those in other tissues, limbal stem cells are supported by a unique stromal microenvironment called the stem cell niche, which consists of certain extracellular matrix components, cell membrane-associated molecules, and cytokine dialogues. Destructive loss of limbal stem cells or dysfunction of their stromal environment renders many corneas with a clinical entity called limbal stem cell deficiency, which is characterized by variable extents of conjunctival ingrowth depending on the severity of limbal damage. A new strategy of treating limbal stem cell deficiency is to transplant a bio-engineered graft by expanding limbal epithelial stem cells ex vivo on amniotic membrane. This review summarizes the published literature data collectively explaining how amniotic membrane is an ideal biological substrate that can help maintain and support the expansion of limbal epithelial stem cells.     

Growth factor changes in ex vivo expansion of human limbal epithelial cells on human amniotic membrane.

PURPOSE: Human limbal epithelial cells cultured on human amniotic membrane have been used for transplantation to treat corneal surface injuries. We determined whether the amniotic basement membrane affects the growth of human limbal epithelial cells through the production of growth factors. METHODS: The epithelial cells grown out from limbal basal epithelium were placed on conventional culture plastic or on the epithelial side of denuded amniotic membrane under serum-free conditions. Culture supernatant was assayed for growth factor release at 24, 48, and 96 hours. RESULTS: The cells grown on both substrata produced similar levels of epidermal growth factor (EGF). Cells grown on amniotic membrane showed enhanced secretion of tissue inhibitor of metalloproteinase type 1 (TIMP1) and reduced production of transforming growth factor beta1 and beta2. Depletion of EGF and TIMPI in cell culture slowed down cell growth and reduced EGF receptor expression, respectively. CONCLUSION: Increased TIMPI influences the proteolytic system in the cell and extracellular matrix interaction, and decreased transforming growth factor beta1 and beta2 may stimulate corneal cell proliferation. We show that the amniotic membrane leads to differential expression of cytokines of limbal epithelial cells cultured on its surface. Such effects may be favorable to the growth and differentiation of the cells when used for ocular surface reconstruction.     

Cryopreservation of human limbal stem cells ex vivo expanded on amniotic membrane

PURPOSE: After cornea transplantation, the donor's limbal zone is currently discarded as medical waste. However, the limbal zone is rich in limbal stem cells and can be used in therapeutic applications of limbus loss. This study aimed to increase the availability of limbal stem cells and develop the optimal conditions of cryopreservation for ex vivo expanded limbal stem cells. METHODS: Pieces of the limbus were cultured on amniotic membrane (AM) to outgrow limbal stem cells as cell sheets for 3 weeks. Different formulas of cryoprotectants were tested to preserve the expanded cell sheets in liquid nitrogen. Before and after cryopreservation, expanded cell sheets were assessed for cellular characteristics by viability, histologic examination, and expression of ABCG2, vimentin, and keratin 3. RESULTS: Expanded cell sheets usually exhibited 3-6 stratified layers after 3-week culture on AM and expressed specific markers of ABCG2 and vimentin for limbal stem cells. The effects of cryopreservation with different cryoprotectants were analyzed by histopathology, stem cell markers, and cell viability. The results showed that the optimal formula of cryoprotectants for expanded limbal cell sheets was 60% Dulbecco modified Eagle medium, 30% fetal bovine serum, and 10% dimethyl sulfoxide. After 8-week cryopreservation in liquid nitrogen, the characteristics of limbal stem cells were maintained, and the average viability of thawed cells was 53.8% +/- 5.8%. CONCLUSIONS: These results showed that limbal stem cells expanded on AM could be cryopreserved and provide a promising source without delay, if banking, for patients with limbal stem cell deficiency in the future.     

Ex vivo expansion of corneal limbal epithelial/stem cells for corneal surface reconstruction

PURPOSE: The management of severe ocular surface disease due to limbal stem cell deficiency has changed dramatically. The concept of limbal stem cells, as the source of corneal epithelium revolutionised the therapeutic approach of ocular surface reconstruction. Deficiency of limbal stem cells results in blinding ocular surface diseases. Grafting viable limbal tissue, from either fellow healthy eye or a donor eye, with the resident stem cell population may replenish limbal stem cells and can restore the corneal surface to normality. Transplanting the limbal tissue can be achieved through a variety of procedures that include cadaveric keratolimbal allograft (KLAL), live or living related conjunctival limbal allograft (Ir-CLAL) and limbal autograft. Advances in tissue engineering techniques have offered a viable alternative to overcome the limitation of limbal tissue available for transplantation. Epithelial stem cells harvested from a small limbal biopsy can be expanded in vitro on a suitable carrier and then transplanted to the diseased cornea to successfully restore the corneal surface. This article is a chronological review of the important steps that brought ex vivo expanded stem cell transplantation in ocular, particularly corneal surface reconstruction. METHODS: The MEDLINE data base was searched for the years 1966-2002, using key words cornea, cell culture, ex-vivo expansion, limbus, stem cell, ocular surface and transplantation. Several articles that were not found by MEDLINE search were taken from references from other articles. Inclusion or exclusion of article was based on the relevance to the subject. CONCLUSIONS: Corneal epithelial reconstruction with ex vivo expanded limbal cells is a potential tool in ocular surface reconstruction, although the technique is currently investigational. Strategies to achieve conjunctival epithelial restoration and tear film replenishment will allow ophthalmic surgeons to truly reconstruct the ocular surface.     

Propagation and phenotypic preservation of rabbit limbal epithelial cells on amniotic membrane

PURPOSE: To describe the phenotypic characteristics of a limbal epithelial cell sheet outgrowth from a limbal explant cultured on amniotic membrane. METHOD: Immunofluorescent staining and confocal microscopy were used to examine the expressions of p63, Ki-67, keratins 3 and 14, connexin 43, and the integrin alpha6/beta4 and alpha3/beta1 subunits in corneal and limbal tissues in a limbal explant and epithelial outgrowth cultured for 2 weeks on amniotic membrane. RESULTS: The expression patterns of p63, Ki-67, keratins, integrins, and connexin 43 in a limbal explant with an epithelial outgrowth cultured for 2 weeks on amniotic membrane resembled those in freshly prepared limbus. Moreover, the distribution of integrin subunits in positive cells of the limbal explant and its epithelial outgrowth was similar to that of the corneal epithelial cells during wound repair. CONCLUSIONS: The epithelial cell sheet grown from a limbal explant on amniotic membrane exhibited a phenotype similar to that of the limbus, suggesting that amniotic membrane is a substrate capable of supporting the propagation and preservation of p63-positive limbal epithelial cells.     

Signaling-transduction pathways required for ex vivo expansion of human limbal explants on intact amniotic membrane

PURPOSE: Ex vivo expansion of limbal epithelial progenitor cells on amniotic membrane (AM) without 3T3 fibroblasts is a new surgical approach to treat limbal stem cell deficiency. Such expansion requires NGF-TrkA-mediated signaling, and this study was conducted to delineate the downstream signaling pathways. METHODS: The human corneolimbal ring was cut into explants and cultured on intact human AM. At day 0 or 10, low-molecular-weight inhibitors were added, whereas the control group received dimethyl sulfoxide (DMSO). The epithelial outgrowth rate was monitored for 17 days, and the epithelial cells were collected for Western blot analysis. RESULTS: In the control, most expansion of human limbal epithelial cells started from the limbus from days 5 to 7 and reached approximately 80% confluence at day 17. Compared with the control, the outgrowth was completely inhibited by 50 microM LY294002 or 50 microM SR13668 and was significantly suppressed by 10 microM U0126, but was not affected by 10 microM of either SB203580 or JNK inhibitor 1. The inhibition of outgrowth by LY294002, SR13668, and U0126 was reversible. Western blot analysis showed that phosphorylation of Akt and FKHRL1was abolished by LY294002 and SR13668, but downregulated by U0126, which also abolished phosphorylation of p44/42 mitogen-activated protein kinase (MAPK). The phosphorylation of p38 and JNK MAPK were downregulated or abolished during ex vivo expansion. CONCLUSIONS: Ex vivo expansion of human limbal epithelial progenitor cells on intact AM is mediated by the survival signaling pathway mediated by PI3K-Akt-FKHRL1 and by the mitogenic MAPK pathway mediated by p44/42 at the expense of p38 and JNK MAPK.     

羊膜为底物角膜缘上皮细胞培养方法的探讨

目的　探讨在羊膜上培养角膜缘上皮细胞 (limbal epithelial cells,LEC)的合适方法。方法　切取大小约 2 mm× 2 mm、厚约 2 0 0 μm含有完整上皮细胞的兔角膜缘组织 ,剪切成 4个 1 mm× 1 mm小的组织块 ,以羊膜为底物分别使用组织块培养法、组织块培养后传代培养法和组织块经消化酶处理后培养法培养兔 L EC,通过倒置显微镜、细胞组织学和扫描电镜观察细胞生长情况。结果　使用上述 3种方法培养的 L EC均可在羊膜上形成密集单层。细胞组织学检查显示在羊膜上培养的 LEC尚可形成多层。扫描电镜观察 LEC立体感强、细胞表面微绒毛丰富。但以组织块经消化酶处理后培养法培养 L EC较为快速。结论在羊膜上上述 3种方法都可用于 LEC的培养 ,但以组织块经消化酶处理后培养法更为实用     

Basement membrane dissolution and reassembly by limbal corneal epithelial cells expanded on amniotic membrane

PURPOSE: To investigate basement membrane (BM) formation during ex vivo expansion of limbal corneal epithelial cells on intact amniotic membrane (iAM) and epithelially denuded (d)AM. METHODS: Human limbal explants were cultured on iAM and dAM. Expression of BM components, including laminin-5, type IV collagen, type VII collagen, perlecan, integrin alpha6, and epithelial cell differentiation markers such as p63, cytokeratin 3 (K3), and cytokeratin 12 (K12), were investigated by immunostaining. Levels of matrix metalloproteinase (MMP)-2 and MMP-9 and tissue inhibitor of matrix metalloproteinase (TIMP)-1 in the conditioned media were determined by ELISA and gelatin zymography. RESULTS: All four BM components were preserved in both iAM and dAM before culturing, but dissolved 1 week afterward when MMP-2 was increased. Epithelial outgrowth correlated with increased expression of MMP-2 and -9 for both cultures. Resynthesis of BM began with laminin-5 followed by other components. This process took place at 1 week on iAM but at 2 weeks on dAM after culturing. At 4 weeks, BM was more maturely deposited as a linear band from the explant toward the leading edge on iAM and temporally correlated with a sharp decline of MMP-9 levels. In contrast, such BM deposition began at the leading edge on dAM only when TIMP-1 levels were increased. Epithelial cell outgrowth on iAM expressed more p63 but less K3 and K12 than did that on dAM. CONCLUSIONS: After dissolution of original amniotic BM, new BM formed by ex vivo expanded human limbal corneal epithelial cells on iAM deposits much faster and is more mature, resulting in regeneration of a limbal epithelial phenotype. In contrast, BM deposition is delayed and remains immature on dAM, resembling wound healing by a corneal epithelial phenotype. Thus, BM resynthesis may be used as another objective readout for assessing the success of ex vivo expansion of limbal epithelial progenitor cells on AM.     

Ultraviolet transmittance of human limbal epithelial cells cultured on human amniotic membranes

PURPOSE: To evaluate ultraviolet (UV) A and B transmittance by human limbal epithelial cells cultured on human amniotic membranes. METHODS: Human limbal epithelial cells were taken from the limbus of donor corneas and were cultured on human amniotic membranes with inactivated 3T3 fibroblasts for 2 to 4 weeks. Then, the cultured cells were examined histologically. Next, cells from different culture periods were irradiated with UV-A (365 nm) or UV-B (302 nm) at energy levels ranging from 50 to 800 microW/cm2, and UV transmittance was measured with a UV light meter. RESULTS: Histological examination revealed a monolayer of corneal epithelial cells on the amniotic membrane after 2 weeks of culture, and a layer of 3-4 cells was formed after 4 weeks. Transmittance of UV-A and UV-B was highest by the amniotic membrane alone, followed in decreasing order by limbal epithelial cells cultured on amniotic membranes for 2 weeks, 3 weeks, and 4 weeks. CONCLUSIONS: These results indicate that UV absorbance increases in proportion to the number of limbal epithelial cell layers in cultures on amniotic membranes. Limbal epithelial cells may need to be cultured until 3-4 layers are formed in order to prevent ocular damage by UV light after transplantation.     

胎儿角膜缘干细胞的培养和生物学特性观察

角膜缘干细胞(limbalstemcells,LSC)及利用角膜缘干细胞移植(limbalstemcellstransplantation,LSCT)重建眼表的研究正在国内外广泛展开。而实行LSCT其前提是必须有LSC供体,尽管当前LSC的来源有自体、同种异体和体外培养的自体及同种异体等几种,但均有其局限性。人胎儿细胞的增殖和分化潜能较成人同类细胞大,此外,胎儿的免疫系统没有发育完善,胎儿组织存在免疫宽容现象[1]。贾卉等[2]研究还发现,胎儿角膜中与免疫反应有关的细胞和细胞间黏附分子较成人少,故胎儿器官、组织或细胞移植较少甚至无免疫排斥反应发生。因此,为探寻一种新的…     

羊膜联合自体角膜缘干细胞移植治疗翼状胬肉

目的探讨羊膜联合自体角膜缘干细胞移植治疗翼状胬肉的效果。方法采用羊膜联合自体角膜缘干细胞移植术治疗翼状胬肉56例(61眼)，随访平均12月。结果56例(61眼)术后均无复发，移植片存活，未见排斥反应发生，治愈率达100％。结论应用羊膜联合自体角膜缘干细胞移植治疗翼状胬肉，不仅取材方便、安全，而且可获得根治性治愈的理想效果，值得临床推广。     

兔骨髓间充质干细胞及人羊膜上皮细胞移植治疗兔角膜缘干细胞缺损的研究

背景角膜缘干细胞缺乏可引起致盲性眼病,但传统的治疗方法疗效欠佳。最近的研究表明,骨髓间充质干细胞（BMSCs）和人羊膜上皮细胞（AECs）有分化成多种细胞的能力,但其对角膜缘于细胞缺乏的疗效仍有待研究和评价。目的观察和对比兔BMSCs和人AECs移植治疗兔角膜缘干细胞缺损的治疗效果。方法将18只新西兰白兔采用随机数字表法分为羊膜基质（AS）移植组、兔BMSCs移植组和人AECs移植组,每组6只。将浸有NaOH溶液的滤纸贴附于角膜表面建立碱烧伤角膜缘干细胞缺损的动物模型。抽取兔髂窝处骨髓并收集人胎盘组织分别分离、制备兔BMSCs和人AECs,通过密度梯度离心加贴壁培养法及胰蛋白酶多次分步消化法获取兔BMSCs和人AECs,采用逆转录聚合酶链反应（RT—PCR）法对培养细胞进行鉴定,再接种于人去上皮AS上,按照动物的分组分别将上述材料缝合至动物模型的角膜表面。术后28d,对各组动物的角膜新生血管（CNV）评分以及角膜混浊度评分进行对比,对角膜组织行组织病理学检查并针对角膜上皮细胞特异性标记物细胞角蛋白3（CK3）行免疫组织化学染色。结果第3代兔BMSCs接种于AS载体上12h后贴附生长,第1代人AECs接种于AS载体上48h后呈单层膜状生长,传代细胞经RT—PCR鉴定符合目标细胞的特征。兔BMSCs移植组术后28d,免疫组织化学染色结果显示,兔BMSCs移植组和人AECs移植组的角膜表面细胞CK3均呈阳性表达,而AS组CK3表达阴性。与AS移植组比较,兔BMSCs移植组和人AECs移植组CNV评分以及角膜混浊度评分明显降低,差异均有统计学意义（BMSCs：Z=-2．983,P=0．003;Z=-2．844,P=0．004;AECs：Z=-2．817,P=0．005;Z=-2．041,P=0．041）。组织病理学检查显示,AS组兔角膜组织有较多的炎性细胞生长,胶原纤维排列紊乱。兔BMSCs组术后角膜表层形成了角膜上皮样的复层结构,无明显杯状细胞,角膜基质内无新生血管生长,炎性细胞减少,胶原纤维排列更加规则,且兔BMSCs移植组角膜透明度明显优于人AECs移植组,差异有统计学意义（Z=-2．091,P=0．037）,而2组间CNV评分的差异无统计学意义（Z=-0．267,P=0．789）。结论移植至兔角膜缘干细胞缺损角膜表面的兔BMSCs和人AECs均能分化为角膜上皮细胞样细胞,可抑制CNV,减轻角膜混浊。在改善角膜透明度方面,兔BMSCs优于人AECs。     

Gap junctional communication in microinjected human limbal and peripheral corneal epithelial cells cultured on intact amniotic membrane

The aim of the study was to determine if human limbal epithelial cells (HLEC) do not form gap junctions (GJ) during ex vivo expansion on preserved and intact human amniotic membrane (AM). Thereby, we attempt to evaluate if characteristic features of the limbal epithelial progenitor cells are preserved on AM. Primary human limbal (HLEC) and peripheral corneal (HPCEC) epithelial cells from limbal and peripheral corneal explants were cultured with SHEM either on intact AM or plastic. After 3-4 weeks, cell cultures were terminated and processed for immunofluorescence. In all cell cultures, formations of GJs were analyzed with a mouse monoclonal antibody to connexin 43 (Cx43) and a rabbit affinity purified antibody against connexin 26 (Cx26). Sections of human limbus and cornea served as positive control. Lucifer yellow (LY) known to be a GJ permeant dye was used to analyse functionality of GJ. Microinjection of LY into single cells was performed with a pressure microinjection device under visual control and with the aid of phase contrast optics. Dye spread of LY into adjacent cells indicating intercellular communication was compared between HLEC and HPCEC cultured either on AM or plastic. In vivo, a punctate pattern of Cx43 was typically found in basal and suprabasal corneal epithelial cells and labeling for Cx26 was observed in all cell layers of the human corneal epithelium, however, subpopulations of limbal basal epithelial cells lacked detectable fluorescence signals for both connexins. In HLEC cultured on AM, a scanty immunolabeling for Cx43 (12.6%) was noted, but HPCEC cultured on AM as well as HLEC cultured on plastic showed a higher labeling index (LI) for Cx43 (42.7 and 52.3%, respectively). A significant lower immunostaining for Cx26 was observed in HLEC cultured on AM (LI: 35.16%) in comparison to HLEC cultured on plastic (68.4%), as well as, HPCEC cultured either on AM or plastic (61% and 79.3%, respectively; p<0.001). Gap junctional communication was evidenced more frequently in HLEC cultured on plastic (51%, p<0.05) in contrast to HLEC cultures on AM, which exhibited a limited dye spread in 29.7% of injected cells. A significant difference in dye coupling was also evidenced between HPCEC on AM (52.9%; p<0.05) and HLEC on AM. Subpopulations of HLEC cultured on AM remain Cx43 and Cx26 negative and without functional GPs indicating that characteristic features of limbal epithelial progenitor cells might be preserved during ex-vivo expansion on AM. These data provide support to the use of the ex-vivo expansion of HLEC as an alternative therapeutic strategy for corneal surface reconstruction in distinct ocular surface diseases.     

Measurement of light transmission of human limbal epithelial cells cultured on human amniotic membranes

PURPOSE: To measure the light transmission properties of human limbal epithelial cell sheets (LECSs) cultured on human amniotic membranes (AMs) and compare them with those of AMs with and without amniotic epithelium. METHODS: Total light transmission of 3 kinds of tissue (LECSs, intact AMs, denuded AMs) was measured in the 250- to 800-nm range by using a spectrophotometer. RESULTS: The percent transmission of each kind of tissue decreased gradually and continually throughout the spectrum as the wavelength shortened and dropped rapidly at 300 nm to less than 20% at 250 nm. All tissues transmitted more than 70% of light in the wavelength region greater than 400 nm and more than 90% in that greater than 600 nm. The percent transmission spectrum of all tissues showed identical curves in the visible light and UV-A regions. However, the percent transmission of LECSs was lower than that of either intact or denuded AMs in the UV-B and UV-C regions. CONCLUSIONS: In the visible and UV-A light region, the percent transmission profiles of amnion-related tissues (LECSs, intact AMs, denuded AMs) are not altered by the presence of either amniotic epithelium or multilayered limbal corneal epithelium. However, the presence of multilayered limbal corneal epithelium, but not amniotic epithelium, on amniotic stroma reduced UV-B and -C transmission significantly. Further study concerning light transmission and other physical properties of LECSs is necessary to fully understand the ocular physiology of eyes grafted with such newly developed bioengineered tissues.