Fig. 5: Late-stage stimulation promoted the structural integration between organoids and the host via OBCIs.

A This schematic illustrates the timeline for late-stage stimulation (60 dpt, left), and detailed parameter (right); (B) This schematic establishes the graft-host or host-graft synaptic structure; (C) Coronal sections of mice brains are shown at 120 dpt in the different groups. Scale bar: 500 μm; (D)(a-j) High magnification views of the transplanted organoid on a coronal section show a high density of GFP⁺ projections. (a) contralateral cortex; (b-c) ipsilateral cortex, adjacent graft; (d) ipsilateral corpus callosum; (e-f) contralateral cortex; (g) contralateral corpus callosum and hippocampus; (h) ipsilateral hippocampus; (i) contralateral ventral posterolateral thalamus nucleus (VPL); (j) ipsilateral VPL. Scale bar: 50 μm in (a), (c-e) and (g-h),10 μm in (b), 20 μm in (f, i, j); (E) The GFP⁺ projections are quantified in ipsilateral brain regions at 120 dpt in BO-ET and BO-ET-ES, n = 3 grafts; (F, H) Staining for MAP2/ VGLUT1 and MAP2/GAD65/67 in grafts at 120 dpt. Scale bar: 20 μm; (G, I) VGLUT1 and GAD65/67 density is quantified at 120 dpt in three groups, n = 3 grafts, *P < 0.05, **P < 0.01, ***P < 0.001; (J) Dendritic branches from BO, BO-ET, and BO-ET-ES grafts at 120 dpt are shown. The red triangle marks the dendritic spines. Scale bar: 5 μm; (K) Dendritic spine density is quantified at 120 dpt, n = 3 grafts, *P < 0.05; (L) Staining for hSYN (red), PSD95 (green), and GFP (white) at 120 dpt. The red arrow indicates the colocalization of hSYN and PSD95. Scale bar: 5 μm; (M) Quantification of the number of hSYN/PSD95 colocalized puncta at 120 dpt, n = 3 grafts, ***P < 0.001. Data are shown as mean ± SD, n = 5 images/3 mice in (G, I, K, M). Statistical significance was tested by One-way ANOVA with Tukey’s multiple comparison for G, I, K, M. The schematic illustrates in Fig. 5B (BioRender.com/d87u058) and 5D (BioRender.com/t24s318) were created with BioRender.com.