Time course of organoid growth and gene expression in WT and TBX19 KI organoids
A, Culture protocol and outline to generate pituitary organoids in three-dimensional (3D) culture from hiPSC. Organoids were collected at days (d) 0, 6, 18, 27, 48, 75, 105 during differentiation to analyze.
B, Bright-field microscopy views of WT organoid examples at different time points throughout differentiation. Scale bars indicated in each image.
C, Number of replicates per time point and per genotype in experiments depicted in following graphs. Asterisks indicate that 7-8 organoids were grouped for each sample. For other points, each sample consists of a single organoid.
D-H, Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (in black line) and TBX19 KI organoids (in red line). Relative quantification of each target gene was obtained by the 2−ΔΔCt method from qRT-PCR results (see Methods). Data show means ± standard error of the mean (SEM, Mann-Whitney t-test [unpaired, two-tailed, nonparametric]). p < 0.05 (*), p< 0.01 (**).
D, Relative quantification of HESX1 expression, the earliest pituitary placode marker assessed. The expression of HESX1 is significantly downregulated in TBX19 KI organoids vs. WT at d18 and d27.
E, Relative quantification expression of PITX1, a pituitary progenitor marker. PITX1 was significantly downregulated in TBX19 KI organoids by d48 and d75.
F, Relative quantification expression of LHX3, a pituitary progenitor marker. LHX3 was significantly lower in TBX19 KI organoids as compared to WT from d48 onwards.
G, Relative quantification expression of TBX19, a critical transcriptional determinant for corticotroph differentiation. TBX19 expression is higher in TBX19 KI organoids at d105. H, Relative quantification expression of POMC, a corticotroph marker. POMC was significantly downregulated in TBX19 KI organoids from d48 onwards.