Ex vivo preservation and expansion of human limbal epithelial stem cells on amniotic membrane cultures

BACKGROUND/AIM: Amniotic membrane (AM) transplantation effectively expands the remaining limbal epithelial stem cells in patients with partial limbal stem cell deficiency. The authors investigated whether this action could be produced ex vivo. METHODS: The outgrowth rate on AM was compared among explants derived from human limbus, peripheral cornea, and central cornea. For outgrowth of human limbal epithelial cells (HLEC), cell cycle kinetics were measured by BrdU labelling for 1 or 7 days, of which the latter was also chased in primary cultures, secondary 3T3 fibroblast cultures, and in athymic Balb/c mice following a brief treatment with a phorbol ester. Epithelial morphology was studied by histology and transmission electron microscopy, and phenotype was defined by immunostaining with monoclonal antibodies to keratins and mucins. RESULTS: Outgrowth rate was 0/22 (0%) and 2/24 (8.3%) for central and peripheral corneal explants, respectively, but was 77/80 (96.2%) for limbal explants (p <0.0001). 24 hour BrdU labelling showed a uniformly low (that is, less than 5%) labelling index in 65% of the limbal explants, but a mixed pattern with areas showing a high (that is, more than 40%) labelling index in 35% of limbal explants, and in all (100%) peripheral corneal explants. Continuous BrdU labelling for 7 days detected a high labelling index in 61.5% of the limbal explants with the remainder still retaining a low labelling index. A number of label retaining cells were noted after 7 day labelling followed by 14 days of chase in primary culture or by 21 days of chase after transplantation to 3T3 fibroblast feeder layers. After exposure to phorbol 12-myristate 13-acetate for 24 hours and 7 day labelling, HLEC transplanted in athymic mice still showed a number of label retaining basal cells after 9 days of chase. HLEC cultured on AM were strongly positive for K14 keratin and MUC4 and slightly positive in suprabasal cells for K3 keratin but negative for K12 keratin, AMEM2, and MUC5AC. After subcutaneous implantation in athymic mice, the resultant epithelium was markedly stratified and the basal epithelial cells were strongly positive for K14 keratin, while the suprabasal epithelial cells were strongly positive for K3 keratin and MUC4, and the entire epithelium was negative for K12 keratin and MUC5A/C. CONCLUSIONS: These data support the notion that AM cultures preferentially preserve and expand limbal epithelial stem cells that retain their in vivo properties of slow cycling, label retaining, and undifferentiation. This finding supports the feasibility of ex vivo expansion of limbal epithelial stem cells for treating patients with total limbal stem cell deficiency using a small amount of donor limbal tissue.     

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.     

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.     

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.     

In vitro antiangiogenic activity in ex vivo expanded human limbocorneal epithelial cells cultivated on human amniotic membrane

PURPOSE: To compare the in vitro antiangiogenic activities of ex vivo expanded human limbocorneal epithelial (HLE) cells cultivated on preserved human amniotic membrane (AM) and to identify factors responsible for the activities. METHODS: The antiangiogenic effects were compared of culture media conditioned by AM, HLE cells, or HLE cells cultivated on intact AM (HLE/IAM), on denuded AM (HLE/DAM), or on DAM cocultured with 3T3 fibroblasts (HLE/DAM/3T3). A monolayer culture of human umbilical vein endothelial cells (ECs) was used in a proliferation and migration assay. ECs suspended in type I collagen gel were used to assess capillary tube formation. Quantitative analyses of tissue inhibitor of metalloproteinase (TIMP)-1, thrombospondin (TSP)-1, pigment epithelium-derived factor (PEDF), and endostatin (proteolytic fragment of collagen XVIII) were performed by ELISA. Immunoconfocal microscopy was performed to localize the site of endostatin expression in HLE cells and AM. RESULTS: HLE cell- but not AM-conditioned medium (CM) inhibited the proliferation and migration of ECs, and coculture of HLE cells, but not of AM, with ECs inhibited capillary tube formation. Although some data from HLE cells alone are not significantly different from the control, increased inhibitory activity was expressed by HLE/IAM and HLE/DAM and was most significantly expressed by HLE/DAM/3T3. Quantitation of TIMP-1, TSP-1, PEDF, and endostatin revealed that only the level of endostatin showed an increased expression by HLE cells cultivated on AM. Neutralizing antibody to endostatin substantially abrogated the inhibitory effect on EC proliferation and migration, but was less effective on EC differentiation. Endostatin signal was more prominent in the basement membrane zone of HLE cells cultivated on denuded AM than in those cultivated on intact AM. CONCLUSIONS: The antiangiogenic effect of HLE cells was enhanced when they were cultivated on AM and cocultured with 3T3 fibroblasts, and endostatin-related antiangiogenic factor may play a major role. This highlights the significance of cell-matrix and cell-cell interaction in the regulation of antiangiogenic factor secretion by HLE cells.     

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     

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.     

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.     

Factors affecting outcome following transplantation of ex vivo expanded limbal epithelium on amniotic membrane for total limbal deficiency in rabbits

PURPOSE: To determine factors affecting the outcome of corneal surface reconstruction in rabbits with total limbal stem cell deficiency (LSCD), by using autologous limbal epithelial stem cells (LSC) ex vivo, expanded on rabbit amniotic membrane (AM). METHODS: Left eyes of 52 rabbits were rendered totally limbal stem cell deficient by n-heptanol debridement of the entire corneal epithelium followed by surgical removal of 360 degrees of limbal rim. After cytologic verification of LSCD, the fibrovascular pannus of each cornea was removed. Group I (n = 10) received a rabbit AM transplant, whereas groups II, III, and IV (n = 42) underwent transplantation of LSCs cultured on rabbit AM (LSC-AM graft) derived from a small limbal biopsy specimen from the right eye. Clinical outcome was graded as a success if a smooth, avascular corneal surface was restored, a partial success if more than two quadrants of corneal surface were smooth, or a failure if the corneal surface was revascularized and irregular. RESULTS: A long-term follow-up of more than 1 year was achieved. Compared with the 100% failure rate in group I, inclusion of expanded LSCs resulted in variable success rates in groups II, III, and IV (all P < 0.001). Kaplan-Meier survival analysis showed that different suturing techniques, subconjunctival injection of long-acting steroid, and tarsorrhaphy used in groups II (n = 17) and III (n = 13) did not significantly alter the outcome (P = 0.89). However, the use of a larger graft and human AM as a temporary patch with the explant retained for 1 week in group IV (n = 12) significantly improved the success rate to 83% (P = 0.002). Among eyes showing clinical failure, there was a significant correlation between the logarithm of the first day when an epithelial defect was noted and the time of graft failure (r(2) = 0.60, P < 0.001). Furthermore, the presence of severe lid deformity was borderline significant when correlated with failure cases in all four groups (P = 0.069). CONCLUSIONS: Ex vivo expansion of LSCs can be achieved by using rabbit AM culture. Such expanded LSCs can successfully reconstruct corneal surfaces affected by total LSCD. This animal model is useful to investigate culturing variables affecting epithelial stemness so that surgical reconstruction of corneas with total LSCD can be successfully performed. Furthermore, this model can be used to test the feasibility of gene therapies targeting LSCD in the future.     

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.     

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.     

The fate of limbal epithelial progenitor cells during explant culture on intact amniotic membrane

PURPOSE: The clinical success of treating corneas with total limbal stem cell deficiency using limbal biopsy explants cultured on intact amniotic membrane (iAM) relies on ex vivo expansion of limbal epithelial progenitor cells. However, the ultimate fate of limbal epithelial progenitor cells in the explant remains unclear. METHODS: Human limbal explants were cultured on iAM for 2 weeks and then removed and transferred to a new iAM until passage 3. The outgrowth surface area of each passage was measured and compared. For each passage, clonogenicity on 3T3 fibroblasts feeder layers was compared among progenitor cells removed from the outgrowth, the explant surface, and the remaining stroma. Cryosections of the explant and the outgrowth were detected with p63, vimentin, pancytokeratin, and the basement membrane components type VII and IV collagen and laminin 5 antibodies. RESULTS: The outgrowth surface area significantly decreased from passage (P)1 to P3. The total number of epithelial cells that were isolated from the explant surface also decreased from before culture (P0) to P1, became stable from P1 to P2, but was uncountable at P3. Clonogenicity significantly declined from P1 to P3 for the epithelium derived from the explant surface and the outgrowth epithelium; the extent was less in the former than in the latter at P2 and P3. In addition, groups of epithelial cells invaded the limbal stroma of the explants from P1 to P3; p63(+)/pancytokeratin(-) and p63(+)/vimentin(+) cells also presented in the limbal stroma. Increasing fibroblast, but not epithelial, colonies were observed from cells isolated from the remaining limbal stroma when seeded on 3T3 fibroblast feeder layers from P1 to P3. CONCLUSIONS: During ex vivo expansion on iAM, some limbal epithelial progenitor cells indeed migrate onto iAM from the explant surface, whereas some also invade the limbal stroma, very likely undergoing epithelial-mesenchymal transition. This new information should be taken into account in formulating new strategies to improve the expansion protocol.     

Expression of Delta Np63 in response to phorbol ester in human limbal epithelial cells expanded on intact human amniotic membrane

PURPOSE: To evaluate the effect of phorbol 12-myristate 13-acetate (PMA) on the expression of Delta Np63 in human limbal epithelial cells (HLECs) during ex vivo expansion on amniotic membrane (AM). METHODS: Primary HLECs were cultured either on AM or plastic surfaces and were treated with 1 micro g/mL PMA for 24 hours. Expression of Delta Np63 and the differentiation-associated gap junctional protein connexin 43 (Cx43) were studied by laser scanning microscopy. RESULTS: The labeling index (LI) of Delta Np63 was higher in HLECs cultured on AM than in HLECs grown on plastic (81.4% +/- 12.2% and 66.6% +/- 16.5%, respectively; P < 0.001). After PMA treatment, Delta Np63 expression in HLECs on plastic dramatically decreased to 20.4% +/- 11.4%. However, HLECs cultured on AM showed only a moderate decrease in Delta Np63 expression (56.4% +/- 10.9%, P < 0.001) after PMA treatment. It was also observed that 72.8% +/- 17.5% of the Delta Np63-positive cells in untreated HLECs cultured on plastic coexpressed Cx43, in contrast to only 21.9% +/- 3.7% of the Delta Np63-positive cells in HLECs cultured on AM (P < 0.001). The latter indicates that growth over AM preserves limbal phenotype, whereas growth over plastic surface induces or allows transition toward corneal peripheral phenotype. CONCLUSIONS: Delta Np63 protein is typically detected in human corneal epithelial cells with high proliferative capacity including, limbal epithelial stem cells (SCs) and probably also transient amplifying cells (TACs). AM supports Delta Np63 protein expression in HLECs and maintains a higher resistance against phorbol ester-induced differentiation, indicating that characteristic signs of limbal epithelial progenitor cells may be preserved during ex vivo expansion on AM.