Design of sgRNA and ssODN to edit TBX19K146R mutation

A, Illustration of the TBX19 gene (HGNC: 11596; NCBI: NG_008244.1; Ensembl: ENSG00000143178; Human GRCh38) and TBX19 protein.

B, Wild-type (WT) sequence containing target site, cut site, target and PAM sequences.

C, ssODN design to edit in a missense K146R mutant of TBX19 using CRISPR/Cas9.

D, Sequence analysis of a TBX19K146R/K146R hiPSC clone 63 obtained by Sanger sequencing after screening by CAPS. This clone was subsequently used in this work to differentiate into pituitary organoids (see below).

E, Summary of our strategy procedure:

Step 1: Production of the knock-in hiPSC lines by CRISPR/Cas9 genome editing.

Step 2: Differentiation into the pituitary organoids from mutant hiPSC lines in parallel with WT line using 3D culture, then comparison of the development of organoids between the two groups.

CAPS: cleaved amplified polymorphic sequences; hiPSC: human induced pluripotent stem cells; KI: knock-in; PAM: protospacer-adjacent motif; sgRNA: single guide RNA; ssODN: single-stranded oligo DNA nucleotides; WT: wildtype; 3D: three dimensions.

Time course of organoid growth and gene expression in WT and TBX19K146R/K146R organoids

A, Culture protocol and schematic to generate pituitary organoids in three-dimensional (3D) culture from hiPSC. Organoids were collected at days (d) 0, 6, 18, 27, 48, 75, and 105 during differentiation to analyze.

B, Bright-field microscopy views of WT organoid examples at different time points throughout differentiation. Scale bars are indicated in each image.

C-G, Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (in black line) and TBX19K146R/K146R organoids (in green line). Data show means ± standard error of the mean (SEM); n=1 on day (d)0, n=3 samples per group (8 organoids per sample) at the time points d6 and d18, n=4-8 organoids per group at each subsequent time point (d27, d48, d75 and d105). Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p < 0.05 (*), p< 0.01 (**).

C, Relative quantification expression of HESX1, one of the earliest pituitary placode markers. The expression of HESX1 was not significantly different in TBX19K146R/K146R organoids vs. WT.

D, Relative quantification expression of PITX1, an anterior pituitary progenitor marker in the ectodermal primordium. PITX1 was significantly downregulated in TBX19K146R/K146R organoids by d48 (** p= 0.0022) and d75 (* p= 0.0159).

E, Relative quantification expression of LHX3, a pituitary progenitor transcription factor. LHX3 was significantly lower in TBX19K146R/K146R organoids as compared to WT on d27 (** p= 0.004), d48 (** p= 0.0022) and d75 (** p=0.0079).

F, Relative quantification expression of TBX19, a corticotroph lineage marker. There was no significant difference in TBX19 expression between the two groups (p= 0.195, d75).

G, Relative quantification expression of POMC, a differentiated corticotroph marker. POMC was significantly downregulated in TBX19K146R/K146R organoids by d48 (* p= 0.026) and thereafter (* p= 0.0159 on d75; * p= 0.0317 on d105).

Impairment of corticotroph development in TBX19K146R/K146R organoids as compared with control

A, C, Immunostaining of LHX3 and CDH1 (E-cadherin) expression in epithelial cells, typical of Rathke’s pouch ectoderm in early pituitary primordia, was reduced in TBX19K146R/K146R organoids vs wild-type on day 48 (n=10 organoids for each group). Scale bars : 10 μm.

B, D, Immunostaining showed that ACTH and TBX19 expression was reduced in TBX19K146R/K146R organoids vs wild-type on day 105 (n=10 organoids for each group). Scale bars: 10 μm.

3D reconstruction of whole Wild type (WT) and TBX19K146R/K146R organoids on day 105

A, A representative image of whole-mount immunostaining against ACTH in a cleared WT organoid on d105 using light-sheet microscopy. Scale bar: 200 μm.

B, A representative image of whole-mount immunostaining against ACTH in a cleared TBX19K146R/K146R organoid on d105 as above, showing impaired corticotroph differentiation. Scale bar: 200 μm.

C, The number of corticotroph cells per mm3 was significantly decreased in TBX19K146R/K146R organoids (* p= 0.0159). Means ± SEM for n=4 WT, n=5 TBX19K146R/K146R organoids. Mann-Whitney test (unpaired, two-tailed, nonparametric).

Design of sgRNA and ssODN to introduce a NFKB2D865G mutation

A, Illustration of NFKB2 gene (HGNC: 7795; Ensembl: ENSG00000077150; Human GRCh38) and NFKB2 protein.

B, WT sequence containing target site, cut site, target and PAM sequence.

C, ssODN design to introduce the missense mutation D865G into NFKB2 using CRISPR/Cas9.

D, Sequence analysis of a representative NFKB2D865G/D865G hiPSC clone 7 obtained by Sanger sequencing after screening by CAPS. This clone was subsequently used in this work to differentiate into pituitary organoids (see below).

CAPS: cleaved amplified polymorphic sequences; hiPSC: human induced pluripotent stem cells; KI: knock-in; PAM: protospacer-adjacent motif; sgRNA: single guide RNA; ssODN: single-stranded oligo DNA nucleotides; WT: wildtype.

Time course of organoid growth and gene expression in WT and NFKB2D865G/D865G organoids

A-E, Relative quantification (RQ) mRNA expression analysis for key markers of pituitary organoids during differentiation: WT (in black line) and NFKB2D865G/D865G organoids (in green line). Data show means ± standard error of the mean (SEM); n=1 on day (d)0, n=3 samples per group (8 organoids per sample) at the time points d6 and d18, n=4-6 organoids per group at each following time point (d27, d48, d75 and d105). Mann-Whitney t-test (unpaired, two-tailed, nonparametric). p < 0.05 (*), p< 0.01 (**).

A, Relative quantification expression of HESX1. The expression of HESX1 was not significantly different in NFKB2D865G/D865G organoids vs. WT.

B, PITX1 was significantly downregulated in NFKB2D865G/D865G organoids by d27 (** p= 0.0022) and d48 (* p= 0.0022).

C, LHX3 was significantly lower in NFKB2D865G/D865G organoids as compared to WT on d27 (** p= 0.0087), and d48 (** p= 0.0087).

D, TBX19 was significantly increased in NFKB2D865G/D865G organoids by d48 (* p= 0.026) and d105 (p= 0.0095).

E, POMC was significantly downregulated in NFKB2D865G/D865G organoids by d105 (* p= 0.019).

F, There was no significant difference in volume between WT and NFKB2D865G/D865G organoids (p= 0.6126). Data show means ± SEM; n=7 in the WT group, n=8 in the mutant group. Mann-Whitney test (unpaired, two-tailed).

Impairment of pituitary development in NFKB2D865G/D865G organoids

A, B, Immunostaining of LHX3 and NFKB2 expression in the early pituitary-type epithelium was reduced in NFKB2D865G/D865G organoids vs WT on day 48 (n=10 organoids for each group). Scale bars: 10 μm

C, D, Immunostaining showed that by day 105, there was complete absence of corticotroph cells co-expressing ACTH and TBX19 in NFKB2D865G/D865G organoids versus WT organoids on day 105 (n=10 organoids for each group). Note the population of cells expressing TBX19 without ACTH in the mutant example. Scale bars: 10 μm

3D reconstruction of whole NFKB2D865G/D865G or WT organoids on day 105

A, A representative image of whole-mount immunostaining against ACTH in a cleared WT organoid on d105 using light-sheet microscopy. Scale bar: 200 μm.

B, A representative image of whole-mount immunostaining against ACTH in a cleared NFKB2D865G/D865G organoid on d105 as above, showing impaired corticotroph differentiation. Scale bar: 100 μm.

C, The number of corticotroph cells per mm3 was significantly decreased in NFKB2D865G/D865G organoids (* p= 0.0007). Means ± SEM for n=6 WT organoids, n=8 NFKB2D865G/D865G organoids. Mann-Whitney test (unpaired, two-tailed, nonparametric).

Whole transcriptome profiles of WT vs NFKB2D865G/D865G organoids on day 48

A, Heat map showing global differential gene expression between WT and NFKB2D865G/D865G organoids (n=5 for each group).

B, Volcano plot of genes showing differential gene expression between WT and NFKB2D865G/D865G organoids. Red dots in the left panel indicate 143 genes involved in pituitary-hypothalamic development. Grey dots indicate all other genes detected in RNA-seq.

C, Volcano plot of genes showing differential gene expression between WT vs NFKB2D865G/D865G organoids. Green dots indicate genes coding for 21 key transcription factors (TF) whose expression is significantly changed (fold change < 0.5 or > 2, padj < 0.05). Red dots indicate fold change in expression between 0.5 and 2. Grey dots indicate all other genes detected in RNA-seq.

Pit-Hpt: pituitary-hypothalamic; KI: knock-in; TF: transcription factors

RNA-seq expression data for a list of 143 genes known to have a functional influence on pituitary-hypothalamic development. Differentially expressed genes (padj < 0.05) in NFKB2D865G/D865G organoids are highlighted in green.