Comparative analysis of human-derived feeder layers with 3T3 fibroblasts for the ex vivo expansion of human limbal and oral epithelium.
ABSTRACT Corneal transplantation with cultivated limbal or oral epithelium is a feasible treatment option for limbal stem cell deficiency (LSCD). Currently utilized co-culture of stem cells with murine 3T3 feeder layer renders the epithelial constructs as xenografts. To overcome the potential risks involved with xenotransplantation, we investigated the use of human-derived feeder layers for the ex vivo expansion of epithelial (stem) cells. Human limbal and oral epithelium was co-cultured with mouse 3T3 fibroblasts, human dermal fibroblasts (DF), human mesenchymal stem cells (MSC), and with no feeder cells (NF). Cell morphology was monitored with phase-contrast microscopy, and stem cell characteristics were assessed by immunohistochemistry, real-time PCR for p63 and ABCG2, (stem cell markers), and by colony-forming efficiency (CFE) assay. Immunohistochemical analysis detected positive staining for CK3 (cornea specific marker) and Iβ1 and p63 (putative stem cell markers) in all culture conditions. The level of Iβ1 and p63 was significantly higher in both limbal and oral cells cultured on the 3T3 feeder, as compared to the MSC or NF group (p<0.01). This level was comparable to the cells cultured on DF. Expression of p63 and ABCG2 in limbal and oral epithelial cells in the 3T3 and DF groups was significantly higher than that in the MSC or NF group (p<0.01). No statistical difference was detected between 3T3 and DF groups. The CFE of both limbal and oral cells co-cultured on 3T3 fibroblasts was comparable to cells grown on DF, and was significantly higher than that of cells co-cultured with MSC or NF (p<0.01). Epithelial cells grown on a DF feeder layer maintained a stem cell-like phenotype, comparable to cells grown on a 3T3 feeder layer. In conclusion, DF provides a promising substitute for 3T3 feeder cells during cultivation of xenobiotic-free corneal equivalents.
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ABSTRACT: Corneal epithelium is maintained throughout life by well-orchestrated proliferation of limbal epithelial stem cells (LESCs), followed by migration and maturation centripetally towards the ocular surface. Disturbance of LESCs can potentially lead to a blinding condition, which can be reversed by reconstitution of a functional LESC pool. The current clinical procedures are effective to some degree, however, deeper knowledge of the molecular interplay within the limbal niche is necessary to achieve a fully satisfactory patient outcome. The present study was thus undertaken to carry out a comprehensive transcriptome analysis of four distinct human limbal compartments, including basal limbal crypts (BLCs), superficial limbal crypts (SLCs), cornea, and the supporting stroma, with the aid of laser capture microdissection and deep RNA sequencing. The tissue harvest pipeline was rigorously optimized so that the exposure to cold ischemia would be less than five minutes. The global gene ontology analysis confirmed existence of primitive cells in BLCs, migratory and activated cells in SLCs, and differentiated cells in cornea. Interestingly, many significantly upregulated genes in SLCs mapped to processes involved in regulation of vasculature, such as sFLT1. In contrast, BLCs exhibited many genes mapping to neurogenic processes and processes related to cell development. The primitive nature of BLCs was, furthermore, confirmed by the KEGG pathway analysis, and some potential regulators of LESCs were revealed, such as Lrig1 and SOX9. The analysis also yielded comprehensive lists of uniquely expressed genes in both BLCs and cornea, which may be useful to identify possible biomarkers. In conclusion, the current investigation provides new insight into the relationship between distinct cell populations within the limbal niche, identifies candidates to be verified for novel biological functions, and yields a wealth of information for prospective data mining.PLoS ONE 11/2013; 8(5):e64244. · 3.53 Impact Factor
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ABSTRACT: Purpose: To preserve limbal stem cell (LSC) function in vitro with xenobiotic-free culture conditions. Methods: Limbal epithelial cells were isolated from 139 donors using 15 variations of 3 dissociation solutions. All culture conditions were compared to the baseline condition of murine 3T3-J3 feeders with xenobiotic (Xeno) Keratinocyte Growth Medium at 20% O2. Five Xeno and Xeno-free media with increasing concentrations of calcium and Epidermal Growth Factor (EGF) were evaluated at 5%, 14% and 20% O2. Human MRC-5, dermal (fetal, neonatal or adult) and limbal stromal fibroblasts were compared. Statistical analysis was performed on number of maximum serial weekly passages, percentage of aborted colonies, Colony Forming Efficiency (CFE), p63α(bright) cells and RT-PCR ratio of p63α/K12. Immunocytochemistry and RT-PCR for p63α, ABCG2, Bmi1, C/EBPδ, K12 and MUC1 were performed to evaluate phenotype. Results: Dispase/TrypLE was the isolation method that consistently showed the best yield, viability and CFE. On 3T3-J2 feeders, Xeno-free medium with calcium 0.1mM and EGF 10ng/ml at 20% O2 supported more passages with equivalent percentage of aborted colonies, p63α(bright) cells and p63α/K12 RT-RCR ratio compared to baseline Xeno-media. With this Xeno-free medium, MRC-5 feeders, showed the best performance, followed by fetal, neonatal, adult HDF, and limbal fibroblasts. MRC-5 feeders supported serial passages with sustained high expression of progenitor cell markers at levels as robust as the baseline condition without significant difference between 20% and 5% O2. Conclusions: LSC function can be maintained in vitro under appropriate Xeno-free conditions.Investigative ophthalmology & visual science 09/2013; · 3.43 Impact Factor
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ABSTRACT: Recent breakthroughs in regenerative medicine have generated enthusiasm and many efforts to explore new therapeutic potentials of both somatic and pluripotent stem cells. About 30 years passed since a discovery of a method of producing a great number of human epidermal keratinocytes by cultivation from a small skin biopsy, many possibilities are now envisaged for therapeutic application of different cultured cell types. The importance of stem cell content was proven for many tissues or organs in different pathologies. Ocular burns cause depletion of limbal stem cells, which lead to corneal opacification and visual loss. Most of available treatments are palliative and focused on the relief of the devastating clinical picture. This review is focused on recent developments cell based therapy of limbal stem cell deficiency. All findings can provide support for improvement and standardization of the cure for this disabling disease. Stem Cells 2013.Stem Cells 08/2013; · 7.70 Impact Factor