Transcriptome analysis identifies the differentially expressed genes related to the stemness of limbal stem cells in mice.

Limbal stem cells (LSCs) reside in the basal layer of limbal epithelial cells (LECs). They are crucial for maintenance of corneal epithelium homeostasis and corneal wound healing. Their stemness is determined by their gene expression pattern. Despite of several positive identifiers have been reported, the unique biomarker for LSCs still remain elusive. Differentially expressed genes (DEGs) between stem cells and differentiated cells affect the fate of stem cells via specific signaling pathway. In order to understand the DEGs in the LSCs, RNA-seq was firstly conducted using a mouse model. A total of 1907 up-regulated DEGs and 395 down-regulated DEGs were identified in the limbus (L) compared to central cornea (CC) and conjunctiva (Cj). Reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) and immunofluorescence staining. The expression pattern of putative biomarkers was considered to be age-related. In up-regulated DEGs GO analysis, 570 gene ontology (GO) terms were significantly enriched. Five groups of genes related with biological processes from these significantly enriched GO terms comprised ionic transport, regulation of tissue development, muscle contraction, visual perception, and cell adhesion, which were clustered as a weighted similar network. Whereas, in down-regulated DEGs GO analysis, 61 GO terms were significantly enriched and only one group of ATP biosynthesis and metabolic process were clustered. Furthermore, we identified 55 signaling pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) database based on up-regulated genes and 14 KEGG pathways based on down-regulated genes. In this study, we provide a landscape of the expression of putative LSCs biomarkers and stemness-related signaling pathways in a mouse model. Our findings could aid in the identification of LSC niche factors that may be related to the stemness of the LSCs.

Dynamic spatiotemporal expression pattern of limbal stem cell putative biomarkers during mouse development.

Limbal stem cells (LSCs), a subpopulation of limbal epithelial basal cells, are crucial to the homeostasis and wound healing of corneal epithelium. The identification and isolation of LSCs remains a challenge due to lack of specific LSCs biomarkers. In this study, Haematoxylin-eosin (HE), 4', 6-diamidino-2-phenylindole (DAPI), and immunohistochemistry (IHC) stains were performed on the pre- and post-natal limbus tissues of mice which has the advantage of more controllable in term of sampling age relative to human origin. By morphological analysis, we supported that there is an absence of the Palisades of Vogt (POV) in the mouse. The development of prenatal and neonatal cornea was dominated by its stroma, whereas after eyelids opened at P14, the corneal epithelial cells (CECs) quickly go stratification in response to the liquid-air interface. Based on IHC staining, we found that the expression of LSCs putative biomarkers in limbal epithelial basal cells appeared in chronological order as follows: Vim = p63 > CK14 > CK15 (where = represents same time; > represents earlier), and in corneal epithelial basal cells were weakened in chronological order as follows: Vim > p63 > CK15 > CK14, which might also represent the stemness degree. Furthermore, the dynamic spatial expression of the examined LSCs putative biomarkers during mouse development also implied a temporal restriction. The expression of Vim in epithelial cells of mouse ocular surface occurred during E12-E19 only. The expression of CK15 was completely undetectable in CECs after P14, whereas the others putative molecular markers of LSCs, such as p63 and CK14, still remained weak expression, suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14. In this study, our data demonstrated the dynamic spatiotemporal expression pattern of LSCs putative biomarkers in mouse was age-related and revealed the time spectrum of the expression of LSCs in mouse, which adds in our knowledge by understanding the dynamic expression pattern of biomarkers of stem cells relate to maintenance of their stemness.

An Insight into the Difficulties in the Discovery of Specific Biomarkers of Limbal Stem Cells.

Keeping the integrity and transparency of the cornea is the most important issue to ensure normal vision. There are more than 10 million patients going blind due to the cornea diseases worldwide. One of the effective ways to cure corneal diseases is corneal transplantation. Currently, donations are the main source of corneas for transplantation, but immune rejection and a shortage of donor corneas are still serious problems. Graft rejection could cause transplanted cornea opacity to fail. Therefore, bioengineer-based corneas become a new source for corneal transplantation. Limbal stem cells (LSCs) are located at the basal layer in the epithelial palisades of Vogt, which serve a homeostatic function for the cornea epithelium and repair the damaged cornea. LSC-based transplantation is one of the hot topics currently. Clinical data showed that the ratio of LSCs to total candidate cells for a transplantation has a significant impact on the effectiveness of the transplantation. It indicates that it is very important to accurately identify the LSCs. To date, several putative biomarkers of LSCs have been widely reported, whereas their specificity is controversial. As reported, the identification of LSCs is based on the characteristics of stem cells, such as a nuclear-to-cytoplasm ratio (N/C) ≥ 0.7, label-retaining, and side population (SP) phenotype. Here, we review recently published data to provide an insight into the circumstances in the study of LSC biomarkers. The particularities of limbus anatomy and histochemistry, the limits of the current technology level for LSC isolation, the heterogeneity of LSCs and the influence of enzyme digestion are discussed. Practical approaches are proposed in order to overcome the difficulties in basic and applied research for LSC-specific biomarkers.

Practical Application of Aptamer-Based Biosensors in Detection of Low Molecular Weight Pollutants in Water Sources.

Water pollution has become one of the leading causes of human health problems. Low molecular weight pollutants, even at trace concentrations in water sources, have aroused global attention due to their toxicity after long-time exposure. There is an increased demand for appropriate methods to detect these pollutants in aquatic systems. Aptamers, single-stranded DNA or RNA, have high affinity and specificity to each of their target molecule, similar to antigen-antibody interaction. Aptamers can be selected using a method called Systematic Evolution of Ligands by EXponential enrichment (SELEX). Recent years we have witnessed great progress in developing aptamer selection and aptamer-based sensors for low molecular weight pollutants in water sources, such as tap water, seawater, lake water, river water, as well as wastewater and its effluents. This review provides an overview of aptamer-based methods as a novel approach for detecting low molecular weight pollutants in water sources.