Investigation Of The Candidate Biomarkers Of Limbal Stem Cells And Their Stemness-related Pathways

Limbal stem cells(LSCs)can be used as an ideal model for stem cell basic research.To date,the specificity of LSCs biomarkers is still controversial.In this study,we firstly aimed to study LSCs biomarkers and their stemness-related pathways in mice based on the immunohistochemical staining,transcriptomics and proteomics analysis.HE staining of the limbal epithelium in 4 weeks old mice was performed.By the HE staining,the palisade of Vogt(POV)was not detectable in limbus,suggesting that mouse LSCs may have their own unique niche structure.Relevant antibodies of corneal epithelial cells(CECs)biomarkers,conjunctival epithelial biomarkers,and LSCs putative biomarkers were used for 4 weeks old mice's limbal epithelium immunohistochemical staining.It was found that the expression of mouse LSCs potential biomarkers has a unique time-space expression pattern,which exhibited agerelated development.Subsequently,specimens with different development stages of the cornea,limbus and conjunctiva of mice from prenatal to postnatal were further collected for HE and immunohistochemical staining in details.The HE staining results suggested that during the prenatal and early postnatal,the development of mouse cornea was dominated by stroma.After P14 eyelid opening,the dominant stromal development of mouse cornea was transformed into the epithelium,indicating that light stimulation might promote the development of CECs.Moreover,no POV structure was observed throughout the developmental process as well.Based on immunohistochemical staining,we found that the expression of LSCs putative biomarkers 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 LSCs interested gene expression products,such as p63 and CK14,still remained weak expression,suggesting that CK15 was suitable to serve as the mouse LSCs biomarkers after P14.We further investigated the LSCs biomarkers based on the transcriptomics and proteomics.The tissue containing limbus(L),central cornea(CC)and conjunctiva from 4 weeks ICR(Institute of Cancer Research)mice were collected for transcriptome Hiseq Xten sequencing.The reliability of the expression of genes from RNA-seq analysis was evaluated by quantitative real-time polymerase chain reaction(qRT-PCR)to be 0.7598(correlation coefficient).A total of 1907 and 395 genes were significantly up-regulated and down-regulated,respectively,in L compared to CC.GO annotation and KEGG pathways analysis were conducted based on these significantly upregulated and down-regulated genes in L.GO terms derived from 1907 up-regulated genes were related to ionic transport,regulation of tissue development,visual perception,and cell adhesion,etc.And the KEGG pathways based on the 1907 upregulated genes analysis showed the extracellular matrix(ECM)-receptor interaction,PI3K-AKT,and MAPK signaling pathways were involved in the maintenance of LSCs stemness.The total proteins were also extracted for proteomic analysis by using Labelfree LC-MS/MS.Reliability of the expression of proteins from proteomic data was evaluated by western blotting.A total of 804 and 76 proteins were significantly upregulated and down-regulated,respectively,in L compared to CC.The immunohistochemical staining was performed for LSCs biomarker staining based on these differentially expressed proteins(DEPs)and found three new LSCs candidate biomarkers(ALDH3B2,SLC5A8,and HSD17B2).By GO annotation and KEGG pathways analysis based on 804 significantly up-regulated proteins,we found the cell adhesion proteins and Focal adhesion pathways might be related to the LSCs stemness phenotype maintaining.In this study,we finally integrated analysis of transcriptomics and proteomics data aimed to reveal the regulatory pathways of LSCs stemness in LSCs niche.Based on the integrated analysis,we obtained the same expression trend DEPs and differentially expressed genes(DEGs),which showed 0.7357(Spearman)correlation.The bioinformatics analysis was then conducted based on these same expression trend DEPs.It was found that the extracellular exosomes played an important role in the communication between CECs and LECs,and the ECM proteins might act as key nodes proteins by activating the Focal adhesion,ECM-receptor interaction,and PI3K-AKT pathways for the maintenance of LSCs stemness-related phenotypes,such as cell cycle,proliferation,differentiation,migration and survival,etc.Above all,this study is the first time to reveal the time spectrum of the expression of LSCs in mice,which could be used for the identification of LSCs at different physiological ages in the lack of a lifelong LSCs-specific biomarker.Firstly,we found three new LSCs candidate biomarkers and several LSCs stemness-related pathways based on the transcriptomics and proteomics analysis.These findings not only provided the important experimental data at transcript and protein levels for LSCs identification,isolation,and stemness maintaining but also laid the foundation for study the heterogeneity of LSCs niche cells.