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Rare cell populations can be challenging to characterize using microfluidic single-cell RNA sequencing (scRNA-seq) platforms. Typically, the population of interest must be enriched and pooled from multiple biological specimens for efficient collection. However, these practices preclude the resolution of sample origin together with phenotypic data and are problematic in experiments in which biological or technical variation is expected to be high (e.g., disease models, genetic perturbation screens, or human samples). One solution is sample multiplexing whereby each sample is tagged with a unique sequence barcode that is resolved bioinformatically. We have established a scRNA-seq sample multiplexing pipeline for mouse retinal ganglion cells using cholesterol-modified-oligos and utilized the enhanced precision to investigate cell type distribution and transcriptomic variance across retinal samples. As single cell transcriptomics are becoming more widely used to research development and disease, sample multiplexing represents a useful method to enhance the precision of scRNA-seq analysis.
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Introduction: Glaucoma, a disease of retinal ganglion cell (RGC) injury and potentially devastating vision loss, is associated with both ocular hypertension (OHT) and reduced ocular blood flow. However, the relationship between OHT and retinal capillary architecture is not well understood. In this project, we studied microvasculature damage in mice exposed to mild levels of induced OHT. Methods: Mild OHT was induced with the microbead model for 2 weeks. At this time point, some retinas were immunostained with CD31 (endothelium), Collagen IV (basement membrane), and RBPMS (RGCs) for z-stack confocal microscopy. We processed these confocal images to distinguish the three retinal capillary plexi (superficial, intermediate, and deep). We manually counted RGC density, analyzed vascular complexity, and identified topographical and spatial vascular features of the retinal capillaries using a combination of novel manual and automated workflows. Other retinas were dissociated and immunopanned to isolate RGCs and amacrine cells (ACs) for hypoxia gene array analysis. Results: RGC counts were normal but there was decreased overall retinal capillary complexity. This reduced complexity could be explained by abnormalities in the intermediate retinal capillary plexus (IRCP) that spared the other plexi. Capillary junction density, vessel length, and vascular area were all significantly reduced, and the number of acellular capillaries was dramatically increased. ACs, which share a neurovascular unit (NVU) with the IRCP, displayed a marked increase in the relative expression of many hypoxia-related genes compared to RGCs from the same preparations. Discussion: We have discovered a rapidly occurring, IRCP-specific, OHT-induced vascular phenotype that precedes RGC loss. AC/IRCP NVU dysfunction may be a mechanistic link for early vascular remodeling in glaucoma.
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Transmissible cancers, in which cancer cells themselves act as an infectious agent, have been identified in Tasmanian devils, dogs, and four bivalves. We investigated a disseminated neoplasia affecting geographically distant populations of two species of mussels (Mytilus chilensis in South America and M. edulis in Europe). Sequencing alleles from four loci (two nuclear and two mitochondrial) provided evidence of transmissible cancer in both species. Phylogenetic analysis of cancer-associated alleles and analysis of diagnostic SNPs showed that cancers in both species likely arose in a third species of mussel (M. trossulus), but these cancer cells are independent from the previously identified transmissible cancer in M. trossulus from Canada. Unexpectedly, cancers from M. chilensis and M. edulis are nearly identical, showing that the same cancer lineage affects both. Thus, a single transmissible cancer lineage has crossed into two new host species and has been transferred across the Atlantic and Pacific Oceans and between the Northern and Southern hemispheres.