Single cell RNA-sequencing reveals five subtypes of olfactory bulb-derived olfactory ensheathing cells that secrete Reelin

Olfactory ensheathing cells (OECs) are the only glial cells that support the olfactory sensory neurons which undergo adult neurogenesis and continually project their axons to glomeruli in the olfactory bulbs. We used single cell RNA sequencing to study the gene expression programs of OECs and to determine the diversity of purified OECs previously shown to promote spinal cord injury repair. Our analyses revealed five subtypes of OECs, each expressing unique marker genes and pathways indicative of progenitor, axonal regeneration, migration, or microglia-like functions. As expected, we found substantial overlap of OEC genes with those of Schwann cells, but also with astrocytes, oligodendrocytes and microglia. We experimentally confirmed the classic marker genes of the OEC subtypes and provide evidence that Reelin and Connective Tissue Growth Factors are secreted by multiple OEC subtypes. Our results support that adult OECs are a hybrid glia including some with progenitor characteristics and that they likely carry out diverse functions related to injury repair and axonal regeneration. Olfactory bulbs were collected from 8-10 weeks old GFP-labeled Sprague-Dawley rats. Primary cultures were generated from both olfactory bulbs from each rodent. Immunopurification was carried out using anti-GFRp75. Flasks with purified OECs and leftover cells were rinsed with HBSS and dissociated with trypsin-EDTA as above. Cells were washed in D/F-FBS-P/S medium and further resuspended with silconized pipettes. Media and cells were washed and centrifuged at 365 g for 10 min, and resuspended. After the final wash, cells for sequencing were resuspended in PBS+BSA in volumes of 1200 cells/µL. 4 flasks of cells (2 purified OECs and 2 ‘leftovers’) were generated on each of 2 different culture dates for scRNA-seq, yielding 8 samples in total (4 purified OECs and 4 ‘leftovers’)