Contributions
Abstract: P774
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Neurobiology
Introduction: Functional studies of human oligodendrocytes (OLs) rely on cells that have been maintained or generated in-vitro. Tissue culture is also required to derive OLs from induced pluripotent stem cells. Single cell RNA sequencing (scRNA-seq) allows for transcriptomic analysis of cell populations without the need for initial fractionation.
Objectives: We aimed to apply a single cell RNA sequencing (scRNA-seq) based analysis to profile the transcriptome of human OLs immediately ex-vivo (EV) and to compare the profile with human OLs that had been maintained in dissociated culture (IV).
Methods: Dissociated OLs were derived from white matter from normal appearing adult human brain surgical specimens using a combination of trypsin digestion and Percoll gradient separation. Single cells were then either sequenced the same day (EV) or placed in a culture medium comprised of DMEM/F12+N1+B27+PDGF+FGF for 6 days and then sequenced (IV). Single-cell RNA-sequencing was performed using a droplet sequencing approach (10X Chromium). Principal component analysis (PCA) and t-distributed stochastic neighbour embedding (t-SNE) were used for dimensionality reduction, allowing for a visual representation of distinct cell populations, which were then validated using characteristic lineage marker genes. We performed differential expression analysis comparing the EV and IV OL populations, correcting for multiple tests. Differences in gene expression were considered significant if they reached a q-value < 0.01 and | log fold change | > 2.
Results: The EV sample was comprised mainly (> 90%) of OLs and microglia in approximately equal proportion, with a rare cluster of PDGFR-alpha expressing cells and AQP4 expressing cells. Of all significantly differentially expressed genes, ~80% were upregulated in the IV compared to the EV conditions. Gene enrichment analysis showed that the significantly differentially expressed genes are involved in processes including cell metabolism, morphology, and survival. However, genes characteristic of mature OLs (MBP, MOBP, CNP, PLP1) were all downregulated in the IV samples.
Conclusion: Our scRNA-seq analysis provides a distinct molecular signature for EV OLs and shows that the differentially expressed genes between EV and IV OLs are involved in key biological pathways. The EV transcriptomic profile will allow for future comparison with cells isolated from multiple sclerosis tissue samples or from stem cell sources.
Disclosure: Kelly Perlman: nothing to disclose
Charles P. Couturier: nothing to disclose
Qiao-Ling Cui: nothing to disclose
Luke Healy: nothing to disclose
Caroline Esmonde-White: nothing to disclose
Jiannis Ragoussis: nothing to disclose
Kevin Petrecca: nothing to disclose
Jack Antel: nothing to disclose
Abstract: P774
Type: Poster Sessions
Abstract Category: Pathology and pathogenesis of MS - Neurobiology
Introduction: Functional studies of human oligodendrocytes (OLs) rely on cells that have been maintained or generated in-vitro. Tissue culture is also required to derive OLs from induced pluripotent stem cells. Single cell RNA sequencing (scRNA-seq) allows for transcriptomic analysis of cell populations without the need for initial fractionation.
Objectives: We aimed to apply a single cell RNA sequencing (scRNA-seq) based analysis to profile the transcriptome of human OLs immediately ex-vivo (EV) and to compare the profile with human OLs that had been maintained in dissociated culture (IV).
Methods: Dissociated OLs were derived from white matter from normal appearing adult human brain surgical specimens using a combination of trypsin digestion and Percoll gradient separation. Single cells were then either sequenced the same day (EV) or placed in a culture medium comprised of DMEM/F12+N1+B27+PDGF+FGF for 6 days and then sequenced (IV). Single-cell RNA-sequencing was performed using a droplet sequencing approach (10X Chromium). Principal component analysis (PCA) and t-distributed stochastic neighbour embedding (t-SNE) were used for dimensionality reduction, allowing for a visual representation of distinct cell populations, which were then validated using characteristic lineage marker genes. We performed differential expression analysis comparing the EV and IV OL populations, correcting for multiple tests. Differences in gene expression were considered significant if they reached a q-value < 0.01 and | log fold change | > 2.
Results: The EV sample was comprised mainly (> 90%) of OLs and microglia in approximately equal proportion, with a rare cluster of PDGFR-alpha expressing cells and AQP4 expressing cells. Of all significantly differentially expressed genes, ~80% were upregulated in the IV compared to the EV conditions. Gene enrichment analysis showed that the significantly differentially expressed genes are involved in processes including cell metabolism, morphology, and survival. However, genes characteristic of mature OLs (MBP, MOBP, CNP, PLP1) were all downregulated in the IV samples.
Conclusion: Our scRNA-seq analysis provides a distinct molecular signature for EV OLs and shows that the differentially expressed genes between EV and IV OLs are involved in key biological pathways. The EV transcriptomic profile will allow for future comparison with cells isolated from multiple sclerosis tissue samples or from stem cell sources.
Disclosure: Kelly Perlman: nothing to disclose
Charles P. Couturier: nothing to disclose
Qiao-Ling Cui: nothing to disclose
Luke Healy: nothing to disclose
Caroline Esmonde-White: nothing to disclose
Jiannis Ragoussis: nothing to disclose
Kevin Petrecca: nothing to disclose
Jack Antel: nothing to disclose