Olfactory ensheathing cells from adult female rats are hybrid glia that promote neural repair

Abstract

Olfactory ensheathing cells (OECs) are unique glial cells found in both central and peripheral nervous systems where they support continuous axonal outgrowth of olfactory sensory neurons to their targets. Previously we reported that following severe spinal cord injury, OECs transplanted near the injury site modify the inhibitory glial scar and facilitate axon regeneration past the scar border and into the lesion. To better understand the mechanisms underlying the reparative properties of OECs, we used single-cell RNA-sequencing of OECs from adult rats to study their gene expression programs. Our analyses revealed five diverse OEC subtypes, each expressing novel marker genes and pathways indicative of progenitor, axonal regeneration, secreted molecules, or microglia-like functions. We found substantial overlap of OEC genes with those of Schwann cells, but also with microglia, astrocytes, and oligodendrocytes. We confirmed established markers on cultured OECs, and localized select top genes of OEC subtypes in olfactory bulb tissue. We also show that OECs secrete Reelin and Connective tissue growth factor, extracellular matrix molecules which are important for neural repair and axonal outgrowth. Our results support that OECs are a unique hybrid glia, some with progenitor characteristics, and that their gene expression patterns indicate functions related to wound healing, injury repair and axonal regeneration.

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Summary

This manuscript explores the transcriptomic identities of olfactory ensheathing cells (OECs), glial cells that support life-long axonal growth in olfactory neurons, as they relate to spinal cord injury repair. The authors show that transplantation of cultured, immunopurified rodent OECs at a spinal cord injury site can promote injury-bridging axonal regrowth. They then characterize these OECs using single-cell RNA sequencing, identifying five subtypes and proposing functional roles that include regeneration, wound healing, and cell-cell communication. They identify one progenitor OEC subpopulation and also report several other functionally relevant findings, notably, that OEC marker genes contain mixtures of other glial cell type markers (such as for Schwann cells and astrocytes), and that these cultured OECs produce and secrete Reelin, a regrowth-promoting protein that has been disputed as a gene product of OECs.

This manuscript offers an extensive, cell-level characterization of OECs, supporting their potential therapeutic value for spinal cord injury and suggesting potential underlying repair mechanisms. The authors use various approaches to validate their findings, providing interesting images that show the overlap between sprouting axons and transplanted OECs, and showing that OEC marker genes identified using single-cell RNA sequencing are present in vivo, in both olfactory bulb tissue and spinal cord after OEC transplantation.

Despite the breadth of information presented, however, further quantification of results and explanation of experimental approaches would be needed to support some of the authors' claims. Additionally, a more thorough discussion is needed to contextualize their findings relative to previous work.

(1) Important quantification is lacking for the data presented. For example, multiple figures include immunohistochemistry or immunocytochemistry data (Figures 1, 5, 6), but they are presented without accompanying measures like fractions of cells labeled or comparisons against controls. As a result, for axons projecting via OEC bridges in Figure 1, it is unclear how common these bridges are in the presence or absence of OECs. For Figure 6., it is unclear whether cells having an alternative OEC morphology coincide with progenitor OEC subtype marker genes to a statistically significant degree. Similar quantification is missing in other types of data such as Western blot images (Fig. 9) and OEC marker gene data (for which p-values are not reported; Table S2).

The addition of quantitative measures and, where appropriate, statistical comparisons with p-values or other significance measures, would be important for supporting the authors' claims and more rigorously conveying the results.

(2) Some aspects of the experimental design that are relevant to the interpretation of the results are not explained. For example, OECs appear to be collected from only female rats, but the potential implications of this factor are not discussed.

Additionally, it is unclear from the manuscript to what degree immunopurified cells are OECs as opposed to other cell types. The antibody used to retain OECs, nerve growth factor receptor p75 (Ngfr-p75), can also be expressed by non-OEC olfactory bulb cell types including astrocytes [1-3]. The possible inclusion of Ngfr-p75-positive but non-OEC cell types in the OEC culture is not sufficiently addressed. Such non-OEC cell types are also not distinguished in the analysis of single-cell RNA sequencing data (only microglia, fibroblasts, and OECs are identified; Figure 2). Thus, it is currently unclear whether results related to the OEC subtype may have been impacted by these experimental factors.

(3) The introduction, while well written, does not discuss studies showing no significant effect of OEC implantation after spinal cord injury. The discussion also fails to sufficiently acknowledge this variability in the efficacy of OEC implantation. This omission amplifies bias in the text, suggesting that OECs have significant effects that are not fully reflected in the literature. The introduction would need to be expanded to properly address the nuance suggested by the literature regarding the benefits of OECs after spinal cord injury. Additionally, in the discussion, relating the current study to previous work would help clarify how varying observations may relate to experimental or biological factors.

(a) Cragnolini, A.B. et al., Glia, (2009), doi: 10.1002/glia.20857.
(b) Vickland H. et al., Brain Res., (1991), doi: 10.1016/0006-8993(91)91659-O.
(c) Ung K. et al., Nat Commun., (2021), doi: 10.1038/s41467-021-25444-3.

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Olfactory ensheathing cells from adult female rats are hybrid glia that promote neural repair

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