Journal: bioRxiv

Article Title: Epigenomic regulation of human oligodendrocyte myelination properties – relation to age and lineage

doi: 10.1101/2025.09.04.674347

Figure Lengend Snippet: (A) Confocal image of A2B5 − cells obtained from pediatric and adult human patients, stained with OLIG2 antibodies (red), H3K27ac antibodies (green) and DAPI (blue) as a nuclear counterstain. (B) Violin plots of the nuclear intensity of H3K27ac immunoreactivity in OLIG2 + cells in pediatric and adult brains. Data represent the H3K27ac immunoreactivity measured in a total of 450 cells from three biological replicates (****P < 0.0001, unpaired t-test).. (C) Confocal image of A2B5 + cells obtained from pediatric and adult human patients, stained with OLIG2 antibodies (red), H3K27ac antibodies (green) and DAPI (blue) as a nuclear counterstain. (D) Violin plots of the nuclear intensity of H3K27ac immunoreactivity in OLIG2 + cells in pediatric and adult brains. Data represent the H3K27ac immunoreactivity measured in a total of 450 cells from three biological replicates (****P < 0.0001, unpaired t-test). (E) Confocal image of A2B5 − cells obtained from pediatric and adult human patients, stained with OLIG2 antibodies (red), H3K27me3 antibodies (green) and DAPI (blue) as a nuclear counterstain. (F) Violin plots of the nuclear intensity of H3K27me3 immunoreactivity in OLIG2 + cells in pediatric and adult brains. Data represent the H3K27me3 immunoreactivity measured in a total of 600 cells from three biological replicates (****P < 0.0001, unpaired t-test). (G) Confocal image of A2B5 + cells obtained from pediatric and adult human patients, stained with OLIG2 antibodies (red), H3K27me3 antibodies (green) and DAPI (blue) as a nuclear counterstain. (H) Violin plots of the nuclear intensity of H3K27me3 immunoreactivity in OLIG2 + cells in pediatric and adult brains. Data represent the H3K27me3 immunoreactivity measured in a total of 600 cells from three biological replicates (****P < 0.0001, unpaired t-test).

Article Snippet: A2B5 antibody conjugated microbeads (Miltenyi Biotec, Auburn, CA) were then used to select A2B5+ cells.

Techniques: Staining

Journal: bioRxiv

Article Title: Epigenomic regulation of human oligodendrocyte myelination properties – relation to age and lineage

doi: 10.1101/2025.09.04.674347

Figure Lengend Snippet: (A) Confocal image of A2B5− cells obtained from pediatric and adult human patients, stained with OLIG2 antibodies (red), H4K8ac antibodies (green) and DAPI (blue) as a nuclear counterstain. (B) Violin plots of the nuclear intensity of H3K27me3 immunoreactivity in OLIG2 + cells in pediatric and adult brains. Data represent the H4K8ac immunoreactivity measured in a total of 600 cells from three biological replicates (****P < 0.0001, unpaired t-test). (C) Confocal image of A2B5+ cells obtained from pediatric and adult human patients, stained with OLIG2 antibodies (red), H4K8ac antibodies (green) and DAPI (blue) as a nuclear counterstain. (D) Violin plots of the nuclear intensity of H4K8ac immunoreactivity in OLIG2 + cells in pediatric and adult brains. Data represent the H4K8ac immunoreactivity measured in a total of 600 cells from three biological replicates (****P < 0.0001, unpaired t-test).

Article Snippet: A2B5 antibody conjugated microbeads (Miltenyi Biotec, Auburn, CA) were then used to select A2B5+ cells.

Techniques: Staining

Journal: bioRxiv

Article Title: Epigenomic regulation of human oligodendrocyte myelination properties – relation to age and lineage

doi: 10.1101/2025.09.04.674347

Figure Lengend Snippet: (A) Age-related transcriptomic signatures were identified by comparing adult (>18 years) and pediatric (<18 years) samples in two distinct human oligodendrocyte lineage populations: mature oligodendrocytes (A2B5−) and late oligodendrocyte progenitors (A2B5+). Triangles reflect ensheathment potential which is higher in pediatric samples for both late progenitors and mature cells in comparison to their adult counterparts. (B) Genes downregulated in adult samples for each comparison were filtered based on statistical significance (p < 0.05) and log2 fold-change (>1), resulting in a set of differentially expressed genes. Intersection of these sets yielded 1186 overlapping genes; removal of non-coding genes refined this list to 987 protein-coding genes. (C) Gene ontology (GO) analysis was performed using Gprofiler on the 987 genes, and significant GO terms (adjusted p < 0.05) were visualized using a scatter plot generated in Revigo, with representative significant terms highlighted. (D) Significant GO terms were classified into three categories: Myelination related signature (MRS) which includes oligodendrocyte lineage differentiation and myelination related processes), immune-related processes, and stress and cell death. Genes specific to each category were identified for further analysis. (E) Single-sample gene set enrichment analysis (ssGSEA) was conducted on mature oligodendrocyte and late progenitor expression profiles for the identified gene categories (full age-related signature, myelination related, immune, and stress/cell death signatures). Enrichment scores were plotted, and statistical significance between pediatric and adult groups was assessed using t-tests. All signatures showed significantly higher enrichment scores in pediatric samples compared to adults. (F) The ATAC-seq modality of the single-cell multiomic dataset from Velmeshev et al. (2023), including pediatric (ages 1–8 years) and adult (25 and 39 years old) donors, was used to assess accessibility (gene activity) of gene signatures derived from comparing bulk RNA-seq adult human primary cells with pediatric counterparts. For each sample, average gene activity for genes in each group for mature OLs was calculated, and the sum of gene activities per signature per sample was used. A two-sided t-test was used to assess the significance of differences between adults and pediatrics; ** and *** indicate p < 0.005 and p < 0.0005, respectively. (G) We performed a pseudobulk differential analysis on mature OLs (comparing pediatric versus adult) by aggregating normalized gene-activity values (log(CP10k)) to donor-level means, using Individual as the biological replicate. Donors were split into pediatric (age 1 to 8 years old) and adult groups (age > 20), and per-gene differences were tested with Welch’s t-test to accommodate unequal variances and sample sizes. Genes from the overall age and 3 sub-category signatures were extracted and divided into four categories: significantly upregulated (p < 0.05, dark red), upregulated (non-significant, light red), downregulated (non-significant, light blue), and significantly downregulated (p < 0.05, dark blue).

Article Snippet: A2B5 antibody conjugated microbeads (Miltenyi Biotec, Auburn, CA) were then used to select A2B5+ cells.

Techniques: Comparison, Generated, Expressing, Activity Assay, Derivative Assay, RNA Sequencing

Journal: bioRxiv

Article Title: Epigenomic regulation of human oligodendrocyte myelination properties – relation to age and lineage

doi: 10.1101/2025.09.04.674347

Figure Lengend Snippet: (A) Gene ontology using gProfiler on the genes upregulated (log2FC > 1 and p < 0.05) in mature OLs and not in late progenitors in pediatric samples versus adult samples. Top 20 biological processes were selected based on the adjusted p-values and visualized. (B) Gene ontology using gProfiler on the genes upregulated (log2FC > 1 and p < 0.05) in the late progenitors OLs and not in mature OLs in pediatric samples versus adult samples. Top 20 biological processes were selected based on the adjusted p-values and visualized. (C) Age-related transcriptomic signatures were identified by comparing adult (>18 years) and pediatric (<18 years) samples in two distinct human oligodendrocyte lineage populations: mature oligodendrocytes (A2B5−) and late oligodendrocyte progenitors (A2B5+). Genes upregulated in adult samples for each comparison were filtered based on statistical significance (p < 0.05) and log2 fold-change (>1), resulting in a set of differentially expressed genes. Intersection of these sets yielded 520 overlapping genes; removal of non-coding genes refined this list to 284 protein-coding genes. (D) Gene ontology analysis was performed using gProfiler on the 284 genes, and top significant GO terms (adjusted p < 0.05) were visualized. ( E) Gene ontology using gProfiler on the downregulated genes (log2FC > 1 and p < 0.05) in mature OLs and not in late progenitors in pediatric samples versus adult samples. All biological processes were selected based on the adjusted p-values and visualized. (F) Gene ontology using gProfiler on the genes downregulated (log2FC > 1 and p < 0.05) in late progenitors and not in mature cells in pediatric samples versus adult samples. All biological processes were selected based on the adjusted p-values and visualized.

Article Snippet: A2B5 antibody conjugated microbeads (Miltenyi Biotec, Auburn, CA) were then used to select A2B5+ cells.

Techniques: Comparison

Journal: bioRxiv

Article Title: Epigenomic regulation of human oligodendrocyte myelination properties – relation to age and lineage

doi: 10.1101/2025.09.04.674347

Figure Lengend Snippet: (A-C) Single-cell ATAC-seq and single-cell nanoCUT&Tag datasets for human (Kabbe et al., 2024) and single-cell ATAC-seq and snCUT&Tag datasets for mouse (Bartosovic et al., 2021) were used. Accessibility, H3K27ac (activation mark, n=2, samples pooled), and H3K27me3 (repressive mark, n=4, samples pooled) were quantified within ±10 kb TSS regions across all genes (normalized as RPKM values) in OPCs and mature oligodendrocytes from both species. ssGSEA was performed for three gene signatures (age-related, myelination, and immune). Enrichment scores are plotted with fold differences plotted for the conditions. Red represents OPCs and blue indicates mature cells. (D) Transcriptomic comparison between human pediatric (A2B5+) cells and young rat (A2B5+) cells, and human adult (A2B5+) cells and aged rat (A2B5+) cells using normalized read counts. Heatmap visualizations, highlighting markers of OPC and mature oligodendrocytes, indicate higher expression of mature markers in human A2B5+ cells and lower expression of OPC marker genes compared to rat cells. Yellow and blue colors indicate upregulation and downregulation, respectively. Rat transcriptomic data were sourced from Neumann et al. (2019). (E) Schematic illustrating the epigenomic status in OPCs and mature oligodendrocytes in humans versus rodents, emphasizing reduced chromatin accessibility for age-related and myelin-related genes in human cells.

Article Snippet: A2B5 antibody conjugated microbeads (Miltenyi Biotec, Auburn, CA) were then used to select A2B5+ cells.

Techniques: Activation Assay, Comparison, Expressing, Marker