Culture of Oral Mucosal Epithelial Cells for the Purpose of Treating Limbal Stem Cell Deficiency.

The cornea is critical for normal vision as it allows allowing light transmission to the retina. The corneal epithelium is renewed by limbal epithelial cells (LEC), which are located in the periphery of the cornea, the limbus. Damage or disease involving LEC may lead to various clinical presentations of limbal stem cell deficiency (LSCD). Both severe pain and blindness may result. Transplantation of cultured autologous oral mucosal epithelial cell sheet (CAOMECS) represents the first use of a cultured non-limbal autologous cell type to treat this disease. Among non-limbal cell types, CAOMECS and conjunctival epithelial cells are the only laboratory cultured cell sources that have been explored in humans. Thus far, the expression of p63 is the only predictor of clinical outcome following transplantation to correct LSCD. The optimal culture method and substrate for CAOMECS is not established. The present review focuses on cell culture methods, with particular emphasis on substrates. Most culture protocols for CAOMECS used amniotic membrane as a substrate and included the xenogeneic components fetal bovine serum and murine 3T3 fibroblasts. However, it has been demonstrated that tissue-engineered epithelial cell sheet grafts can be successfully fabricated using temperature-responsive culture surfaces and autologous serum. In the studies using different substrates for culture of CAOMECS, the quantitative expression of p63 was generally poorly reported; thus, more research is warranted with quantification of phenotypic data. Further research is required to develop a culture system for CAOMECS that mimics the natural environment of oral/limbal/corneal epithelial cells without the need for undefined foreign materials such as serum and feeder cells.

The effect of hypoxia on the formation of mouse incisor enamel.

OBJECTIVE: The permanently growing mouse incisors exhibit all stages of tooth development along their inciso-apical axis at any time. Any disturbance or injury of the ameloblasts during enamel formation or maturation may result in permanent defects in the finished enamel since the enamel does not undergo repair or remodeling after formation. In order to increase our understanding of how hypoxia affects enamel formation, we induced severe acute hypoxia in adult mice and observed its effects on the enamel in incisors. DESIGN: Incisors from hypoxic mice were obtained 5 and 49 days after the hypoxic insult. Hypoxic and control incisors were dissected out and observed by scanning electron microscopy (SEM). Incisors were subsequently ground longitudinally or transversely, etched, and observed again by SEM. The nature and position of defects were considered in relation to the configuration and dynamics of the incisors. RESULTS: The effect of hypoxia varied considerably, among mice, among incisors, and among ameloblasts. Affected enamel showed hypoplasia with hypomineralization or hypomineralization without hypoplasia. Vascular endothelial growth factor (VEGF) showed considerably stronger labeling in hypoxic compared to control ameloblasts. CONCLUSIONS: The present study demonstrates quantitative and qualitative defects in the enamel reflecting the vulnerability of ameloblasts toward severe acute hypoxia in mouse incisors.

Expression of Clu and Tgfb1 during murine tooth development: effects of in-vivo transfection with anti-miR-214.

Expression of clusterin (Clu) in the murine first molar tooth germ was markedly increased at postnatal developmental stages. The time-course of expression of this gene paralleled those of other genes encoding proteins involved during the secretory phase of odontogenesis, as described previously. Immunohistochemical studies of clusterin in murine molar tooth germs suggested this protein to be located in outer enamel epithelium, regressing enamel organ, secretory ameloblasts, and the dental epithelium connecting the tooth to the oral epithelium at an early eruptive stage. Immunolabelling of transforming growth factor beta-1 (TGF-ß1) revealed it to be located close to clusterin. The levels of expression of Clu and Tgfb1 were markedly decreased following in-vivo transfection with anti-miR-214. In contrast, the expression of several genes associated with regulation of growth and development were increased by this treatment. We suggest that clusterin has functions during secretory odontogenesis and the early eruptive phase. Bioinformatic analysis after treatment with anti-miR-214 suggested that, whilst cellular activities associated with tooth mineralization and eruption were inhibited, activities associated with an alternative developmental activity (i.e. biosynthesis of contractile proteins) appeared to be stimulated. These changes probably occur through regulation mediated by a common cluster of transcription factors and support suggestions that microRNAs (miRNAs) are highly significant as regulators of differentiation during odontogenesis.

Expression of delta-like 1 homologue and insulin-like growth factor 2 through epigenetic regulation of the genes during development of mouse molar.

Delta-like 1 homolog (Dlk1) and insulin-like growth factor 2 (Igf2) are two of six well-studied mouse imprinted gene clusters that are paternally expressed. Their expression is also linked to their maternally expressed non-coding RNAs, encoded by Gene trap locus 2 (Gtl2) and Imprinted maternally expressed transcript (H19), co-located as imprinted gene clusters. Using deoxyoligonucleotide microarrays and real-time RT-PCR analysis we showed Dlk1 and Gtl2 to exhibit a time-course of expression during tooth development that was similar to that of Igf2 and H19. Western blot analysis of proteins encoded by Dlk1 and Igf2 suggested that the levels of these proteins reflected those of the corresponding mRNAs. Immunohistochemical studies of DLK1 in murine molars detected the protein in both epithelial and mesenchymal regions, in developing cusp mesenchyme, and in newly synthesized enamel and dentin tubules. IGF2 protein was detected primarily at prenatal stages, suggesting that it may be active before birth. Analysis of methylation of cytosine-phosphate-guanine (CpG) islands in both Dlk1 and Igf2 suggested the presence of an increasing fraction of hypermethylated bases with increasing time of development. The increased levels of hypermethylation coincided both with the diminished levels of expression of Dlk1 and Igf2 and with decreased levels of DLK1 and IGF2 proteins in the tooth germ, suggesting that their expression is regulated via methylation of CpG islands present in these genes.

Effects of in vivo transfection with anti-miR-214 on gene expression in murine molar tooth germ.

MicroRNAs (miRNAs) are an abundant class of noncoding RNAs that are believed to be important in many biological processes through regulation of gene expression. Little is known of their function in tooth morphogenesis and differentiation. MicroRNA-214 (miR-214), encoded by the polycistronic Dnm30os gene, is highly expressed during development of molar tooth germ and was selected as a target for silencing with anti-miR-214. Mandibular injection of 1-100 pmol of anti-miR-214 close to the developing first molar in newborn mice resulted in significant decrease in expression of miR-214, miR-466h, and miR-574-5p in the tooth germ. Furthermore, levels of miR-199a-3p, miR-199a-5p, miR-690, miR-720, and miR-1224 were significantly increased. Additionally, the expression of 863 genes was significantly increased and the expression of 305 genes was significantly decreased. Among the genes with increased expression was Twist-1 and Ezh2, suggested to regulate expression of miR-214. Microarray results were validated using real-time RT-PCR and Western blotting. Among genes with decreased expression were Amelx, Calb1, Enam, and Prnp; these changes also being reflected in levels of corresponding encoded proteins in the tooth germ. In the anti-miR-214-treated molars the enamel exhibited evidence of hypomineralization with remnants of organic material and reduced surface roughness after acid etching, possibly due to the transiently decreased expression of Amelx and Enam. In contrast, several genes encoding contractile proteins exhibited significantly increased expression. mRNAs involved in amelogenesis (Ambn, Amelx, Enam) were not found among targets of miRNAs that were differentially expressed following treatment with anti-miR-214. It is therefore suggested that effects of miR-214 on amelogenesis are indirect, perhaps mediated by the observed miR-214-dependent changes in levels of expression of numerous transcription factors.

Expression of prion gene and presence of prion protein during development of mouse molar tooth germ.

In order to gain insight into possible cellular functions of the prion protein (PrP) during normal development, the expression of Prnp (encoding the PrP) and the distribution of the PrP were studied in murine tooth germs. Expression of Prnp in the mouse first molar tooth germ was highly dynamic, increasing several-fold during the secretory phase of odontogenesis, exhibiting a time-course of expression similar to that of genes coding for other extracellular proteins [e.g. enamel matrix proteins (Amelx, Ambn, Enam), Aplp1, Clstn1, and Clu]. Western blot analysis suggested that the amounts of PrP and amyloid beta (A4) precursor-like protein 1 (APLP1) in the tooth germ followed time-courses similar to those of the corresponding mRNAs. Immunohistochemical studies of the distribution of PrP in murine molar and incisor tooth germs at embryonic day (E)18.5 suggested that this protein was located in the cervical loop, outer enamel epithelium, pre-ameloblasts, and dental papilla. Different degrees of immunolabelling of pre-ameloblasts on the mesial and distal aspects of a lower molar cusp may be related to different enamel configurations on the two aspects. It is concluded that the dynamic patterns of expression of Prnp, and of distribution of PrP, suggest that PrP may have functions during secretory odontogenesis, perhaps in relation to amelogenesis.

Gene expression and dental enamel structure in developing mouse incisor.

At the mouse incisor tip the initially differentiated ameloblasts produce a thin, prism-free enamel, while further apically, in the immediate adjacent segment, the enamel thickness increases and the four-layered enamel of mouse incisor is formed. Comparative gene-expression profiling was carried out on RNA isolated from these two segments of incisor tooth germs at embryonic day (E)17.5 and at postnatal days (P)0, 1, 2, and 10 using microarrays to measure messenger RNA (mRNA) and microRNA (miRNA) species present in the segments. Validation of expression data was achieved using real-time reverse transcription-polymerase chain reaction (RT-PCR) and western blotting. Bioinformatic data suggested enhanced cellular apoptosis in the incisal tip segment, which, together with diminished expression of the Amelx and Enam genes, may contribute to the production of the thin enamel seen in this tooth segment. For genes exhibiting higher levels of expression in the adjacent segment where complex enamel is being formed, bioinformatic analysis suggested significant associations with cellular functions involving the actin cytoskeleton, cellular development, morphology, and movement. This is suggested to reflect that ameloblasts with Tomes' process are being organized in transverse rows, facilitating the transverse movement that results in prism decussation in the inner enamel of the adjacent segment. Bioinformatic analysis of miRNA expression data lends support to these suggestions.