[Show abstract][Hide abstract] ABSTRACT: TCF4, a key transcription factor of Wnt signaling system, has been recently found to be essential for maintaining stem cells. However, its signaling pathway is not well elucidated. This study was to explore the functional roles and signaling pathway of TCF4 in maintaining adult stem cell properties using human corneal epithelial stem cells as a model. With immunofluorescent staining and real-time polymerase chain reaction, we observed that TCF4 was exclusively expressed in the basal layer of human limbal epithelium where corneal epithelial stem cells reside. TCF4 was found to be well colocalized with ABCG2 and p63, two recognized epithelial stem/progenitor cell markers. Using in vitro culture models of primary human corneal epithelial cells, we revealed that TCF4 mRNA and protein were upregulated by cells in exponential growth stage, and RNA interference by small interfering RNA-TCF4 (10-50 nM) transfection blocked TCF4 signaling and suppressed cell proliferation as measured by WST-1 assay. TCF4 silence was found to be accompanied by downregulated proliferation-associated factors p63 and survivin, as well as upregulated cyclin-dependent kinase inhibitor 1C (p57). By creating a wound healing model in vitro, we identified upregulation and activation of β-catenin/TCF4 with their protein translocation from cytoplasm to nuclei, as evaluated by reverse transcription-quantitative real-time polymerase chain reaction, immunostaining, and Western blotting. Upregulated p63/survivin and downregulated p57 were further identified to be TCF4 downstream molecules that promote cell migration and proliferation in wound healing process. These findings demonstrate that transcription factor TCF4 plays an important role in determining or maintaining the phenotype and functional properties of human corneal epithelial stem cells.
[Show abstract][Hide abstract] ABSTRACT: There is a great interest in using epithelium generated in vitro for tissue bioengineering. Mouse 3T3 fibroblasts have been used as a feeder layer to cultivate human epithelia including corneal epithelial cells for more than 3 decades. To avoid the use of xeno-components, we evaluated human fibroblasts as an alternative feeder supporting human corneal epithelial regeneration. Five human fibroblast cell lines were used for evaluation with mouse 3T3 fibroblasts as a control. Human epithelial cells isolated from fresh corneal limbal tissue were seeded on these feeders. Colony forming efficiency (CFE) and cell growth capacity were evaluated on days 5-14. The phenotype of the regenerated epithelia was evaluated by morphology and immunostaining with epithelial markers. cDNA microarray was used to analyze the gene expression profile of the supportive human fibroblasts. Among 5 strains of human fibroblasts evaluated, two newborn foreskin fibroblast cell lines, Hs68 and CCD1112Sk, were identified to strongly support human corneal epithelial growth. Tested for 10 passages, these fibroblasts continually showed a comparative efficiency to the 3T3 feeder layer for CFE and growth capacity of human corneal epithelial cells. Limbal epithelial cells seeded at 1 × 10(4) in a 35-mm dish (9.6 cm(2)) grew to confluence (about 1.87-2.41 × 10(6) cells) in 12-14 days, representing 187-241 fold expansion with over 7-8 doublings on these human feeders. The regenerated epithelia expressed K3, K12, connexin 43, p63, EGFR and integrin β1, resembling the phenotype of human corneal epithelium. DNA microarray revealed 3 up-regulated and 10 down-regulated genes, which may be involved in the functions of human fibroblast feeders. These findings demonstrate that commercial human fibroblast cell lines support human corneal epithelial regeneration, and have potential use in tissue bioengineering for corneal reconstruction.
PLoS ONE 01/2012; 7(6):e38825. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Allergic diseases affect a large population. Pollen, an ubiquitous allergen, is the trigger of seasonal rhinitis, conjunctivitis, and asthma, as well as an exacerbating factor of atopic dermatitis. However, the underlying mechanism by which pollen induces thymic stromal lymphopoietin (TSLP)-triggered allergic inflammation through epithelial innate immunity is largely unknown.
We sought to explore whether short ragweed (SRW) pollen induces TSLP/OX40 ligand (OX40L)/OX40 signaling through Toll-like receptor (TLR) 4-dependent pathways in patients with allergic disease.
Three models were used for this study, a well-characterized murine model of allergic conjunctivitis induced by SRW pollen, a topical challenge model on the murine ocular surface, and a culture model of primary human corneal epithelium exposed to aqueous extract of defatted SRW pollen (SRWe).
The topical challenges with SRW pollen generated typical allergic conjunctivitis in BALB/c mice. Clinical signs, stimulated TSLP/OX40L/OX40 signaling, and T(H)2 cytokine levels in the ocular mucosa and draining cervical lymph nodes were significantly reduced or eliminated in TLR4-deficient (Tlr4-d) or myeloid differentiation primary response gene 88 (MyD88) knockout (MyD88(-/-)) mice compared with those seen in their wild-type littermates. SRWe stimulated TSLP production by ocular epithelia in wild-type but not Tlr4-d or MyD88(-/-) mice. SRWe-stimulated TSLP was blocked by TLR4 antibody and nuclear factor κB inhibitor in murine and human corneal epithelia.
For the first time, we have shown that SRW pollen, acting as a functional TLR4 agonist, initiates TLR4-dependent TSLP/OX40L/OX40 signaling, which triggers T(H)2-dominant allergic inflammation. These findings shed light on the understanding of mucosal epithelial innate immunity and create new therapeutic targets to cure allergic diseases.
The Journal of allergy and clinical immunology 08/2011; 128(6):1318-1325.e2. · 12.05 Impact Factor