Figure 2. Fusing hThOs and hCOs produces assembloids that form reciprocal synapses.

(A) Reporter line validation for hCOs. Top: Schematic of SLC17A7 (VGLUT1) exon 12 in the TP-190a-VGLUT1-tdTomato reporter line, which was used to generate all hCOs. Bottom: VoxHunt deconvolution analysis of bulk RNA-seq data from D70 hCOs using E13 mouse brain data from the Allen Brain Atlas as a reference. Organoids were visually categorized as positive or negative for tdTomato fluorescence prior to sequencing. The tdTomato RNA level for each sample is indicated in TPM (transcripts per million). Each stacked bar indicates one bulk RNA-seq sample derived from 2–3 pooled organoids.

(B) The snRNA-seq analysis of hCOs. Left: UMAP plot with cluster annotations. ExN: excitatory neuron, DL: deep layer, UL: upper layer, Un.: unknown. Right: Dot plot showing subplate marker expression by cluster. Avg Exp: normalized average expression, % Cells: percentage of cells expressing a marker within a cluster.

(C) VoxHunt analysis mapping hCOs (all clusters) onto the E15 mouse brain.

(D) Pseudotime analysis of the neural cell trajectory (Cycling Progenitors to UL ExNs, DL ExNs, and Subplate/DL ExNs) from hCOs.

(E) UMAP plots of glutamatergic markers SLC17A6 (VGLUT2) and SLC17A7 (VGLUT1). Color indicates normalized transcript level.

(F) Traces showing the voltage and AP responses in a cell recorded from an hCO.

(G) Fluorescence and bright field image of a TC assembloid at 5 days postfusion (dpf).

(H) Representative fluorescence images for 2-dimensional fusion assay. Thalamic neurons (magenta, right chamber) and cortical neurons (green, left chamber) extend processes from their respective chambers, across the barrier region (dashed yellow lines), and into the opposite chamber starting at D9. Elaborate processes extending from the opposite sides can be seen in both halves by D61.

(I) Fluorescence image of an hCO co-transduced with hSyn-GFP and hSyn-V5-Mito-APEX2 lentiviruses.

(J) Schematic and TEM image of an APEX2⁺ mitochondrion (circled in magenta) in a TC synapse. Pre: presynaptic compartment, post: postsynaptic compartment.

(K) Schematic and TEM image of an APEX2⁺ mitochondrion (circled in green) in a CT synapse. APEX2⁻ mitochondria are indicated by asterisks (*).

Scale bars (F): 10 mV, 2.5 s.

Data in (B-E) were produced by snRNA-seq analysis of 12,008 nuclei from D90 hThOs.

See Figures S1 and S5 for additional data validating the TP-190a-VGLUT1-tdTomato reporter line and hCOs, respectively. See Figure S4 for additional data related to electrophysiological properties and synapses in hCOs. See Figure S6 for NeuronChat analysis.