Single-cell transcriptomics identifies limbal stem cell population and cell types mapping its differentiation trajectory in limbal basal epithelium of human cornea.

PURPOSE: This study aimed to uncover novel cell types in heterogenous basal limbus of human cornea for identifying LSC at single cell resolution. METHODS: Single cells of human limbal basal epithelium were isolated from young donor corneas. Single-cell RNA-Sequencing was performed using 10x Genomics platform, followed by clustering cell types through the graph-based visualization method UMAP and unbiased computational informatic analysis. Tissue RNA in situ hybridization with RNAscope, immunofluorescent staining and multiple functional assays were performed using human corneas and limbal epithelial culture models. RESULTS: Single-cell transcriptomics of 16,360 limbal basal cells revealed 12 cell clusters belonging to three lineages. A smallest cluster (0.4% of total cells) was identified as LSCs based on their quiescent and undifferentiated states with enriched marker genes for putative epithelial stem cells. TSPAN7 and SOX17 are discovered and validated as new LSC markers based on their exclusive expression pattern and spatial localization in limbal basal epithelium by RNAscope and immunostaining, and functional role in cell growth and tissue regeneration models with RNA interference in cultures. Interestingly, five cell types/states mapping a developmental trajectory of LSC from quiescence to proliferation and differentiation are uncovered by Monocle3 and CytoTRACE pseudotime analysis. The transcription factor networks linking novel signaling pathways are revealed to maintain LSC stemness. CONCLUSIONS: This human corneal scRNA-Seq identifies the LSC population and uncovers novel cell types mapping the differentiation trajectory in heterogenous limbal basal epithelium. The findings provide insight into LSC concept and lay the foundation for understanding the corneal homeostasis and diseases.

A Novel Ratiometric Fluorescent Probe for Mercury (II) ions and Application in Bio-imaging.

Since the accumulation of mercury (II) ions in the environment and ecosystem causes serious problems to environment and disease, the recognition of Hg2+ ions and its bio-imaging is of high importance. In sight of the advantages of fluorescence probes, a new probe (PMH) was facilely synthesized by incorporating phenylimidazole fluorophore and 3-methyl-2- benzothiazolinone hydrazone hydrochloride monohydrate. The PMH probe exhibited a ratiometric response for Hg2+ ions with fluorescence intensity increasing at 520 nm and decreasing at 445 nm simultaneously. The PMH probe interacted with Hg2+ ions in seconds with high optical stability and showed good selectivity over other metal ions. In addition, the probe has excellent biocompatibility and imaging performance in cells and zebrafish.

A novel rhodamine B-based "off-on'' fluorescent sensor for selective recognition of copper (II) ions.

A novel rhodamine based sensor RBH was developed and synthesized by rhodamine B with 3-methyl-2-benzothiazolinone hydrazone, which exhibited high selectivity and sensitivity for sensing Cu2+ ions in the presence of other important relevant metal ions in aqueous acetonitrile. As an "off-on" fluorescent sensor, RBH exhibited a UV-vis absorption increase at 570 nm and a 132-fold significant fluorescence enhancement at 580 nm with a distinct color change from colorless to purple upon the addition of Cu2+ ions. The fluorescence intensity of RBH is linear with the Cu2+ ions concentration with low limit of detection. Furthermore, the sensor RBH has been successfully utilized for fluorescence imaging of Cu2+ ions in living cells. The sensor could potentially recognize Cu2+ ions in biological system.

Conformationally Induced Off-On Cell Membrane Chemosensor Targeting Receptor Protein-Tyrosine Kinases for in Vivo and in Vitro Fluorescence Imaging of Cancers.

Molecules capable of monitoring receptor protein-tyrosine kinase expression could potentially serve as useful tools for cancer diagnosis due to the overexpression of tyrosine kinases during tumor growth and metastasis. In this work, a conformationally induced "off-on" tyrosine kinase cell membrane fluorescent sensor (SP1) was designed and evaluated for the detection and imaging of receptor protein-tyrosine kinases in vivo and in vitro. SP1 consists of sunitinib and pyrene linked via hexamethylenediamine and displays quenched fluorescence as a dimer. The fluorescence of SP1 is restored in the presence of receptor protein-tyrosine kinases upon strong interaction with SP1 at the target terminal. The unique signal response mechanism enables SP1 use for fluorescence microscopy imaging of receptor protein-tyrosine kinases in the cell membranes of living cells, allowing for the rapid differentiation of cancer cells from normal cells. SP1 can be used to visualize the chick embryo chorioallantoic membrane and mouse model tumors, suggesting its possible application for early cancer diagnosis.