Fig. 5: The enTsOrg grafts show developmental patterns characterized by thoracic spinal segment and D–V features.

a, Spot plots of the distributions of the different partitions of the A–P axis (including cervical, thoracic and lumbar regions) in organoid transplant areas (7 weeks after transplantation) by transcriptome analysis referring to human embryonic spinal cord dataset (GSE136719), with a pie diagram visualizing the proportions, n = 2. b, Schematic diagram of spinal segment (upper row). UMAP plots showing the expression of A–P axis related Hox genes in the enTsOrg and sOrg (bottom row). c, Heat map of Hox gene expression determined by ST-seq. d, qPCR analysis of thoracic-specific Hox genes in spinal cord tissues at 7 weeks; expression levels were normalized to the sOrg group using the 2^−ΔΔCt method. Two-sided Welch’s t-test, n = 3 or 4 biological replicates. e, Immunofluorescence timeline of thoracic-specific marker (HOXC9), cholinergic neuron marker (ChAT) and MN marker (ISL1) in the transplanted organoids with the qualification analysis of the HOXC9⁺/ChAT⁺ or HOXC9⁺/ISL1⁺ cells proportion in total cells. Two-way ANOVA followed by uncorrected Fisher’s LSD (3 wpi, 5 wpi and 14 wpi, n = 3 biological replicates; 7 wpi, n = 5 biological replicates). f, Schematic diagram of D–V patterning of the neural tube and spinal cord. g, UMAP plots of progenitor and neural domains in D–V patterning of the neural tube and spinal cord, with a pie diagram visualizing the proportion of these cell types by ST-seq at 7 weeks. h, Bubble plots of D–V patterning-specific gene marker expression of sOrg and enTsOrg. The bubble size indicates the mean scaled gene expression level. i, Violin plot of the expression levels of D–V patterning-specific genes in different D–V regions of enTsOrg determined by ST-seq. j–l, Immunofluorescence images of ventral-specific markers (NKX2.2 (j) and FoxA2 (k)) and a dorsal-specific marker (Pax2 (l)) in the enTsOrg and sOrg groups at 7 weeks. Heat maps showing the distribution and expression levels of related markers. The magnified areas are shown in the white boxes; white dotted lines separate the dorsal and ventral sides. Scale bars, 100 μm in e, and 200 μm in j, k and l. Data represent the mean ± s.e.m. in d and e. RP, roof plate; dI2, dorsal interneuron 2; dI4, dorsal interneuron 4; FP, floor plate; HOXC9, homeobox C9; Pax2, paired box 2.

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