Transcriptomic comparison of young and aged choroid at the bulk and single-cell levels (single cells)

Purpose: The goals of this study were to identify cell specific expression patterns from infant and aged choroid, with a particular emphasis on how endothelial gene expression changes with age. Methods: Independent libraries were prepared for infant and adult choroidal cells from four human donors. Macular and peripheral samples from single-cell donors 18-21 were acquired, analyzed, and uploaded in a previous experiment (GSE135922). In single-cell donors 22-25, new reads were acquired from a 12-mm macula-centered punch. Choroidal punches gently dissociated in collagenase II for 1 hours before cryopreservation. Thawed cells were enriched endothelial cells by incubating with anti-CD31-microbeads. Both CD31-positive and CD31-negative libraries from single-cell donors 22-25 were sequenced on an Illumina NovaSeq system. Sequenced reads were mapped to the human genome build hg19 with CellRanger(v3.0.1) and filtered to remove cells likely to be doublets or cells with a high proportion of mitochondrial reads. Libraries from single-cell donors 22-25 were aggregated with donors 18-21 using canonical correlation analysis. Feature selection was performed with the variance stabilizing transformation before integration was performed with Seurat (v3.0.1). Results: A total of 37,070 cells were recovered after filtering with 415,740,738 corresponding reads. A total of 17,229 cells were recovered from infant libraries while 19,841 cells were recovered from the adult libraries. Clusters were assigned to known choroidal cell types based on expression of distinguishing marker genes. Differential expression analysis identified genes enriched in each population of cells originating from infant versus adult libraries. Particular emphasis was given to genes enriched in infant choriocapillaris endothelial cells. Conclusions: This study provides a transcriptomic comparison of infant and aged human donor choroid at the bulk and single-cell levels. The aged choroid, in particular the aged vasculature, is enriched in pro-inflammatory genes. Overall design: mRNA profiles for thousands of cells from infant and adult choroidal isolates were generated from four donor eyes (single-cell donors 22-25) using 10X Genomics Chromium single-cell system followed by sequencing on an Illumina NovaSeq.