Extended Data Fig. 4: Astrocyte and oligodendrocyte cluster analysis and spatial transcriptomics in MS lesions.

a, Differential spatial expression patterns of astroglial GFAP in subcortical versus cortical demyelination by immunohistochemistry (left); t-SNE plots visualizing astrocyte-specific genes corresponding to all (RFX4), protoplasmic (SLC1A2, GPC5) and fibrous or reactive astrocytes (GFAP, CD44). Quantification of RFX4⁺ in situ hybridization signals per nuclei in GM and WM of control samples validates RFX4 as a canonical astrocyte marker (control, n = 5); quantification of GPC5⁺ and CD44⁺ in situ hybridization signals per RFX4⁺ astrocytes validates GPC5 as protoplasmic GM and CD44 as fibrous WM marker. Two-tailed Mann–Whitney U-tests were performed. Data are mean ± s.e.m. b, Upregulation of astroglial CRYAB, MT3 (black arrowheads) and endothelin type B receptor transcript EDNRB (white arrowhead) in reactive astrocytes in subcortical lesions. c, t-SNE plots showing OL-specific expression of myelin-encoding genes MBP, CNP and transcription factor ST18; note co-expression of ST18 with PLP1 in control WM by in situ hybridization. d, Visualization of enriched GO terms in myelinating OLs based on DGE analysis. Binomial test with FDR correction was used to calculate FDR-corrected P values using 151 genes differentially expressed in OLs. e, Co-expression spatial transcriptomic studies confirming upregulation of heat-shock protein 90 transcript HSP90AA1 in both progenitor (PDGFRA-expressing) and myelinating (PLP1-expressing) OLs at lesion rims (PPWM, black arrowheads). The black asterisk indicates a blood vessel. For t-SNE and violin plots, data shown from 9 control and 12 MS samples. For astrocyte violin plots, 1,571 control and 3,810 MS nuclei are shown. Box plots inside violin plots represent median and standard deviation of gene expression. For in situ hybridization and immunohistochemistry experiments, representative images from from three control and four MS individual tissue sections are shown.