"This may have important clinical implications when bioengineering tissues for clinical use, as it provides a safer and xenobiotic-free culture system. 43 Cultivation of limbal cells was also studied by Shahdadfar et al (2012) on amniotic membrane. Although in his study he did not use CBS but he compared FBS with human serum as the sole supplement and found them equally efficient for ex vivo growth of limbal epithelial cells. "
[Show abstract][Hide abstract] ABSTRACT: A simple, reproducible, animal-material free method for cultivating and characterizing cornea limbal epithelial stem cells (LESCs) on human lens capsule (LC) was developed for future clinical transplantation. The limbal tissue explants (2×2×0.25 mm) were harvested from 77 cadavers and expanded ex vivo on either cell culture plates or LC in medium containing human serum as the only growth supplement. Cell outgrowth at the edge of the explants was observed within 24 hours of cultivation and achieved viable outgrowth (>97% viability as measured by MTT assay and flow cytometry) within two weeks. The outgrowing cells were examined by genome-wide microarray including markers of stemness (p63α, ABCG2, CK19, Vimentin and Integrin α9), proliferation (Ki-67), limbal epithelial cells (CK 8/18 and 14) and differentiated cornea epithelial cells (CK 3 and 12). Immunostaining revealed the non-hematopoietic, -endothelial and -mesenchymal stem cell phenotype of the LESCs and the localization of specific markers in situ. Cell adhesion molecules, integrins and lectin-based surface carbohydrate profiling showed a specific pattern on these cells, while colony-formation assay confirmed their clonal potency. The LESCs expressed a specific surface marker fingerprint (CD117/c-kit, CXCR4, CD144/VE-Cadherin, CD146/MCAM, CD166/ALCAM, and surface carbohydrates: WGA, ConA, RCA, PNA and AIL) which can be used for better localization of the limbal stem cell niche. In summary, we report a novel method combining the use of a medium with human serum as the only growth supplement with LC for cultivating, characterizing and expanding cornea LESCs from cadavers or alternatively from autologous donors for possible treatment of LESC deficiency.
PLoS ONE 09/2012; 7(10):e47187. DOI:10.1371/journal.pone.0047187 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: This study was conducted to perform global transcriptomic profiling and biomarker discovery on undisturbed human corneal epithelial subpopulations using a novel approach combining laser capture microdissection (LCM) and RNA sequencing. LCM facilitated the harvest of four cellular compartments along the corneal epithelial differentiation pathway: Stroma, basal limbal crypt (BLC), superficial limbal crypt (SLC), and cornea. Compartments were analyzed using massive parallel sequencing and bioinformatics. Specificity of LCM was confirmed by imaging, expression of epithelial markers, and gene ontology analysis of biological processes. Interestingly, many significant upregulated genes in SLC mapped to processes involved in regulation of vasculature like sFLT1. In contrast, BLC had many upregulated genes mapping to neurogenic and developmental processes. The primitive nature of BLC was confirmed by KEGG pathway analysis. We present full gene lists of unique expressed genes in both BLC and cornea representing candidate biomarkers. Possible novel regulators of limbal epithelial stem cells (LESCs) include Lrig1 and SOX9. The presented molecular insight in LESC biology is expected to be beneficial for both research and clinical translation.
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