Abstract
Purpose :
Thus far, our studies using Confetti and the K5-H2B-GFP transgenic mice suggest that there are two stem/progenitor cell populations that independently give rise to the ductal and meibum synthesizing meibocyte populations within the meibomian gland. The purpose of this study was to use single cell RNA sequencing analysis of mouse meibomian glands to further identify/characterize these different cell lineages.
Methods :
The tarsal plates from the upper and lower eyelids of 6, 6-month-old, male C57Bl/6 mice divided into two groups were collected and single cells isolated. Cells were then submitted to the UCI Genomics High Throughput Facility and raw sequencing data demultiplexed and processed using Cellranger (10× Genomics version 3.1.0). Preliminary analysis and visualization of the data were performed using R version 4.0.5 and Seurat version 4.0.5. For all datasets, cells with <200 and >6000 genes, and >5% mitochondrial genes detected were removed. We performed integrated analysis of the 2 datasets using SCTransform normalization separately for each dataset, selected 2000 informative features, and performed integration using the FindIntegrationAnchors function. Cells were visualized using UMAP (Uniform Manifold Approximation and Projection). Pseudotemporal ordering of meibocytes and determination of pseudotime dependent transcription factors was performed using Monocle version 2.18.0.
Results :
A total of 7259 cells from two different samples were analyzed and 19560 genes detected across all cells. UMAP analysis identified 5 distinct cell clusters, including 1) a Krt 6a positive ductal epithelial cluster, 2) a Krt6a and AWAT2 negative and PPARg positive undifferentiated meibocyte cluster, 3) a AWAT2 positive differentiated meibocyte cluster, 4) a Krt6a and Ki67 positive ductal epithelial progenitor cluster and 5) a Krt6a negative and Ki67 positive meibocyte progenitor cell cluster. Trajectory analysis also identified transcription factors unique to these two different meibomian gland cell lineages.
Conclusions :
Our findings support the hypothesis that there are two separate progenitor cell populations that give rise separately to ductal epithelial cells and meibum synthesizing meibocytes. Understanding the regulatory mechanisms that control these separate pathways is needed to establish appropriate cell culture models to evaluate mechanisms that regulate meibum synthesis.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.