Fig. 2 ∣. PCCOs incorporate PLCs into cortical brain organoids.

a, PCCO generation protocol. b, Light sheet fluorescence microscopy image of GFP⁺ PLCs invading cortical organoids. Bar, 800 μm; arrowheads, PLCs. c, GFP⁺ PLC penetration into the CTIP2⁺ cortical plate-like zone. Nuclei were stained with DAPI. Bars, 400 μm; arrowheads, PLCs. d, Merged UMAP shows PCCO cellular compositions; eight cell clusters are annotated according to standard marker gene expression. Split UMAP shows the contribution of cortical brain organoids and PCCOs. RGC, radial glia cell. e. Cell fraction plot for proportionate cell clusters in cortical brain organoids and PCCOs. IPC, intermediate progenitor cell. f, Feature plots for cortical brain organoids and PCCOs. BCL11B, deep-layer neurons; EOMES, intermediate progenitors; GFAP, astrocytes; ATCA2, CSPG4 (NG2) and PDGFRB, PLCs. g, Star-shaped astrocytic morphology seen in PCCOs but not cortical brain organoids. GFP, PLCs. Scale bar, 400 μm; enlargement bar, 100 μm. The double arrowhead indicates the astrocytic end feet connection. The single arrowhead indicates star-shaped astrocytes. h, Laminin-β1 expression and colocalization adjacent to GFP⁺ PLCs, evident only in PCCOs. Bar, 400 μm; enlargement bar, 100 μm. The single arrowheads indicate laminin (in gray). i, Quantification of h. Cells with adjacent laminin-β1 were counted in ImageJ. DAPI⁺ nuclei were used as reference; n = 12 includes 3 PCCOs derived from 2 healthy individuals in unrelated families and H1 cells and 4 image regions from each section. A two-tailed t-test with Šidák multiple comparisons test correction was used to determine significance ****P < 0.0001, P = 0000147. j, Immunostaining for anti-laminin-β1 and anti-GFAP colocalized with GFP⁺ PLCs in PCCOs. Right: zoom-in of small region. The bottom row shows two-color staining. Scale bar, 40 μm. The single arrowheads indicate laminin-β1 (gray). The double arrowheads indicate the astrocytic end feet connection. DAPI (blue) was used to stain nuclei. PLCs were derived from NCSCs generated with H9 cells. k, Model of pericyte basement membrane-astrocyte structure in PCCOs. PLCs express laminin and promote astrocytic maturation and end feet attachment to PLCs. b, n = 8 includes 4 biological replicates (PCCOs generated from H1, iPSC-Line1, iPSC-Line2 and iPSC-Line3) and 2 technical replicates (two PCCOs for each of the biological replicates). c,g–i, n = 12 includes 4 biological replicates (PCCOs generated from H1, iPSC-Line1, iPSC-Line2 and iPSC-Line3) and 3 technical replicates (3 PCCOs for each of the biological replicates). See replications in Extended Data Figs. 9 and 10.