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: Niche factors are important in the maintenance and regulation of stem cells. Limbal stromal cells are potentially a component of limbal stem cell (LSC) niche. We investigated the role of the limbal stromal cells in the ex vivo expansion of limbal stem/progenitor cells. Limbal epithelial cells were cultured as single-cell suspension and cell clusters from dispase II or collagenase A (ColA), or tissue explant. ColA isolated limbal stromal cells along with limbal epithelial cells. In the presence of limbal stromal cells, a higher absolute number of p63α(bright) cells (p<0.05) and a higher proportion of K14 positive epithelial cells were obtained from both ColA and explant tissue cultures. Expansion of the stem/progenitor population from dispase isolation was more efficient in the form of cell clusters than single cell suspension based on the absolute number of p63α(bright) cells. Expansion of the stem cell population is similar in the single cell and cell cluster cultures that are derived from ColA isolation. Our finding suggests that limbal stromal cells and an intact cell-cell contact help to maintain LSCs in an undifferentiated state in vitro during expansion.Experimental Eye Research 09/2013; · 3.03 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: The cornea is covered by a stratified epithelium that is renewed by stem cells located in the peripheral region of the cornea known as the limbus. This stroma of the limbus contains stromal keratocytes that, when expanded in culture, are termed limbal fibroblasts (LFs). It is thought that LFs exhibit similar characteristics to bone marrow mesenchymal stem cells (BM MSCs) and help maintain the epithelial stem cell phenotype in the limbal region. In this study, we aimed at reprogramming SSEA4+ LFs and BM MSCs into corneal epithelial lineage by using a 3-dimensional culture system and embryonic stem cell medium. After enrichment, SSEA4+ cells showed a higher level of stem cell marker expression such as Sox2, Oct4, Nanog, Rex1, ABCG2, and TRA-1-60, and colony forming efficiency than did SSEA4- cells. SSEA4+, as compared to SSEA4- cells, had a greater propensity to form spheres that, in turn, were induced into ectodermal lineage and further differentiated into functional corneal epithelium. Results show that LFs were similar to BM MSCs in marker profiles, and together with the differences noted between SSEA4+ and SSEA4- cells, point to LFsd' being tissue-specific MSCs. However, LFs showed a greater potential for differentiation into corneal epithelium, indicating the potential importance of tissue-specific adult progenitors in their reprogramming capacity into cells of interest. This study opens a new avenue for investigating the molecular mechanism involved in maintaining a limbal stem cell niche and thus a potentially important clinical application to treat corneal epithelial stem cell loss. Stem Cells 2013.Stem Cells 09/2013; · 7.70 Impact Factor