Figure 1.

Generation and characterization of brainstem organoids (HBSOs)

(A) Schematic showing the derivation of HBSOs from hPSCs. Representative phase-contrast images showing the HBSOs derived from the control hPSCs at different stages of differentiation.

(B) qRT-PCR analyses show the relative expressions of rostro-caudal (HOXA2, HOXB1, HOXB2, GBX2, and OTX2) and dorsoventral (PAX7, OLIG3, NGN, and PHOX2B) markers in the day-20 control HBSOs in comparison to the day-60 HCOs. Data are represented as mean ± SEM of three independent pools of ≥4 organoids. Unpaired t test; NS, non-significant.

(C) Immunostaining of PHOX2B and markers of the forebrain (TBR1), midbrain (FOXA2, OTX2), and hindbrain (ISL1) in the day-60 control HBSOs.

(D) Immunostaining of PHOX2B, TH, VGLUT2, and 5-HT in the day-60 control HBSOs. The inset on the top-right corner is an enlargement of a square region in the image. RTN (PHOX2B⁺ VGLUT2⁺ TH⁻), A5-like (PHOX2B⁺; TH⁺; VGLUT2^–), and C1-like (PHOX2B⁺; TH⁺; VGLUT2⁺) neurons are marked by a filled arrowhead, open arrowhead, and an arrow, respectively.

(E) Schematic showing MEA analysis of day-60 control HBSOs.

(F) Firing rate of HBSO neurons in response to different CO₂ levels and RTN blocker (KCNQ channel activator, retigabine). The mean spike firing rate of each MEA recording was normalized with the first recording undertaken in 5% CO₂ condition to calculate the fold change ± SEM from ≥9 independent experiments per group. One-way ANOVA.

(G) UMAP projection of all 23,088 individual cells from day-60 HBSOs, colored by cell types.

(H) Violin plots showing the key markers of six main cell types. OPC, oligodendrocyte progenitor cell.

(I) Violin plots showing the key markers of six neuronal subtypes.

(J) Expression of hindbrain markers (HOXA2, HOXB2, and NKX2-2) in the HBSO cells.

(K) Relative expression of regional markers TOX3 (r5) and PRDX6 (r6) in HCOs and HBSOs.

(L) Pathway scores of hindbrain development in HCOs and HBSOs. p value (non-parametric Wilcoxon rank-sum test) from comparing HBSOs and HCOs is shown. ^∗∗∗∗p value < 0.0001. See also Figure S1 and Data S1.