Fig. 3: Stage-dependent role of CD45⁺ immune cells in promoting vascular morphogenesis.

a, Schematic diagrams showing the strategy to isolate CD45⁺;CD11b⁺ immune cells from the CTX and GE of prenatal human brain from GW15–23. These CD45⁺;CD11b⁺ cells are subjected to bulk RNA-seq and scRNA-seq, followed by bioinformatics analyses. The transcriptomic data are validated using immunohistochemistry (IHC), immunofluorescence microscopy (IF) and RNAscope-based in situ hybridization. Finally, CD45⁺;CD11b⁺ cells are further characterized using high-dimensional flow cytometry and 3D Matrigel HUVEC assays. b, A volcano plot showing the genes enriched in CD45⁺ cells from GW20–23 (right) and those enriched in cells from GW14–19 (left). Adjusted P values and fold changes were calculated using DESeq2. By default in DESeq2, the P values attained by the Wald test are corrected for multiple testing using the Benjamini–Hochberg method. The genes shown were filtered to be below the adjusted P value of 0.05 and above a fold change of 1.2 between GW14–19 and GW20–23 comparisons (highlighted by the dashed lines). c, GSEA reveals GO terms enriched in CD45⁺ cells from GW14–19 and GW20–23. The data in b and c are from 21 independent biological samples. NES, normalized enrichment score; FDR, false discovery rate. d, Images taken from InCucyte S3 Live Imaging Device of HUVEC in Matrigel-based branching morphogenesis at 3, 6, 12, 24 and 48 h after plating. The conditions include 20,000 HUVEC alone and 20,000 HUVEC cocultured with 20,000 GW14–19 or GW20–23 CD45⁺ cells from prenatal human brain. e, Quantification of average and total endothelial branch lengths formed by HUVEC. Statistics use a two-tailed, unpaired Student’s t-test, and the data represent the mean ± standard error of the mean. The P values represent comparisons between HUVEC coincubated with CD45⁺ cells versus HUVEC only. Not significant comparisons are not shown. n indicates the number of independent biological samples used for quantification. For each biological sample, at least three technical replicates are used.