Fig. 3: Early-stage stimulation promoted the differentiation of organoid grafts via OBCIs.

A This schematic illustrates the timeline for organoid implantation (0 dpt), electrode insertion, 1 M stimulation, and recording signals. The right figure shows detailed parameters of electrical stimulation; (B) Coronal sections of mice brains demonstrate organoid growth in the BO, BO-ET, and BO-ET-ES group at 60 dpt. BO, organoid implantation; BO-ET, organoid and electrode implantation without regulation; BO-ET-ES, BO-ET with electrical stimulation. Scale bar: 500 μm; (C) The organoid volumes are quantified in different groups, n = 6 grafts, **P < 0.01; (D) The immunofluorescence staining for Ki67 (green) and human nuclei (HN, red) in three groups at 60 dpt. Scale bar: 50 μm; (E) Quantification of Ki67⁺ cells show an increase in BO-ET-ES, n = 6 grafts, **P < 0.01; (F) Immunofluorescence for HN and CD31 in different groups at 60 dpt. Scale bar: 100 μm (left) and 30 μm (right); (G)The vascular structures (CD31) are quantified, n = 6 grafts, **P < 0.01; (H, J) Neural progenitors and mature neurons are identified with PAX6 (H) and NeuN (J) at 120 dpt. Scale bar: 50 μm in H, and 100 μm in J. (I, K) The expression of PAX6⁺ and NeuN⁺ cells are quantified, n = 3 grafts, *P < 0.05, **P < 0.01; (L) Immunofluorescence staining for CTIP2 (L) and SATB2 (N) at 120 dpt. Scale bar: 50 μm in L, and 100 μm in N; (M, O) Quantification of the CTIP2⁺HN⁺ cells and SATB2⁺/GFP⁺ cells are shown, n = 3 grafts, **P < 0.01; (P) hSYN (red) and PSD95 (green) is displayed in the three groups at 180 dpt. Scale bar: 10 μm (left), and 3 μm (right); (Q) Quantification of the number of hSYN/PSD95 colocalized puncta at 180 dpt in the three groups, n = 3 grafts, *P < 0.05, **P < 0.01. Data are shown as mean ± SD, n = 8 images/6 mice in (C, E, G), n = 5 images/3 mice in (I, M, O, Q). n = 6 images/3 mice in (K). Statistical significance was tested by One-way ANOVA with Tukey’s multiple comparison for C, E, G, I, K, M, O, Q.