Abstract
Purpose :
To identify genes with gene expression (GE) correlating to K12 expression and identify presumptive stem cell-, overexpressed (OEGs) and under-expressed genes (UEGs).
Methods :
NDRI-procured human corneas were cut into thin strips of either, limbus with adjacent periphery (LiPe), or pure Pe. Cell suspensions, generated by the serial Dispase-trypsin process, were cultured for 3 ½ h and the attaching cells were collected. Basal cell purity and limbal and periphery cell content were determined by flow cytometry (IOVS;44:5125). Cells were subjected to single-cell RNA-Seq reading (10x Genomics). The results were gathered in an Excel file. Cells (= columns) with outlying expression levels or expressing melanocyte or blood markers were excluded and the cell-gene matrix was normalized. The K12>0 subset of LiPe cells was ordered from low to high K12 GE and split into twenty 40-cell quantiles. A gene correlation coefficient (CC) index was generated and the (GE weighted) sum for the 50 genes with the highest CC (s50G; CC from .9963 to .9740) was calculated. The Krt12=0 subset ( 603 cells)was then ordered according to the sG50 values and split into Q1 to Q4 quantiles. Under the assumption that the progression of GE values occurring in the K12 ≠ 0 subset hold true for the K12=0 subset, the lowest (Q1) quantile was taken to represent the stem-precursor cell (SCPC) set. Genes with Q1 to Q4 BH-adjusted of 5 % or 1 % FDRs and Q1/Q4 ratios above 2 or below 0.5 were considered SCPC-OEGs or UEGs, respectively.
Results :
Flow cytometry and the % K12=0 cells yielded, 43 % and 37 % basal Li cell content, respectively. The pure Pe sample contained < 0.1 % K12=0 cells. In the K12>0 subset, out of 6586 genes, there were 137 genes with CCs > 0.95 and 364 with CCs > 0.90, positively correlating with the increase in K12 and 40 and 137 negatively correlating at CCs of -0.95 and -0.90, respectively. In the K12=0 subset, our assumption yielded 44 and 25 SCPC OEGs for the 5 % and 1 % FDR levels , respectively, and 337 and 219 UEGs for the 5% and 1 % levels
Conclusions :
K12 correlation analysis, an approach based on the accepted view of the progression of corneal differentiation along the Li to Co axis, provide an alternative and distinct approach for the identification of genes associated with corneal epithelial differentiation
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.