## 3 Results
### Organoid Responsiveness to Pasireotide Correlates with SSTR2 and SSTR5 Expression
ScRNA-Seq Reveals the Existence of Unique Proliferative Cell Populations in iPSC\({}^{\text{CDH23}}\) Cultures When Compared to iPSCs\({}^{\text{ctrl}}\)  
Using Seurat to identify cell clusters, as well as Uniform Manifold Approximation and Projection 9 UMAP, clustering analysis identified 16 distinct cell populations/clusters consisting of known marker genes. Clusters 1, 5, and 7 of the iPSC\(s^{\text{CDH23}}\) were distinct from the iPSC\({}^{\text{ctrl}}\) cultures (Figure 7a,b). Pituitary stem cells were characterized in iPSC\({}^{\text{ctrl}}\) and iPSC\({}^{\text{CDH23}}\) cultures (Figure 7b). Clusters 1 and 5 expressed markers consistent with the corticotroph subtype cell lineage (Figure 5c). Markers of dysregulated cell cycles and increased proliferation were identified in cell cluster 7 (Figure 7c). Expression of the E2 factor (E2F) family of transcription factors, which are downstream effectors of the retinoblastoma (RB) protein pathway and play a crucial role in cell division control, were identified in distinct cell cluster 7, which was identified within the iPSC\({}^{\text{CDH23}}\) cultures (Figure 7c). Stem cell markers were also upregulated in cell cluster 7, and identified within the iPSC\({}^{\text{CDH23}}\) cultures (Figure 7c). Using Cytobank software to analyze organoids collected 30 days post-differentiation, cells were gated on live CK20 positive singlets, and 9000 events per sample were analyzed by the viSNE algorithm. ViSNE plots are shown in two dimensions with axes identified by tSNE- 1 and tSNE-2, and each dot representing a single cell positioned in the multidimensional space (Figure 7d). Individual flow cytometry standard files were concatenated into single flow cytometry standard files, from which 12 spatially distinct populations were identified (Figure 7e). Overlaying cell populations identified by traditional gating strategies onto viSNE plots identified unique cell populations within the iPSC\({}^{\text{CDH23}}\) cultures (Figure 7e). There were distinct cell populations between the iPSCand iPSC\({}^{CDH23}\) organoids, in addition to expression of hormone and cell lineage markers such as ACTH, TPit, PRL, and PIT1 (Figure 7e). The cell populations that exhibited high expression of Ki67 within the iPSC\({}^{ctrl}\) organoid cultures included SOX2+ and PIT1+ populations (Figure 7f). The highly proliferating cell populations within the iPSC\({}^{CDH23}\) organoid cultures included those that expressed CD90+/VIM+/CXCR4+ (mesenchymal stem cells), CXCR4+/SOX2+ (stem cells), TPit+ (corticotroph cell lineage), CD133+/CD31+ (endothelial progenitor cells), and CK20+/VIM+/CXCR4+ (hybrid epithelial-mesenchymal stem cells) (Figure 7f). Overall, the iPSC\({}^{CDH23}\) organoids were significantly more proliferative compared to the iPSC\({}^{ctrl}\) cultures (Figure 7f). Immunofluorescence staining of iPSC\({}^{CDH23}\) organoids revealed increased mRNA expression of TPit and POMC, which correlated with increased ACTH protein compared to iPSCs\({}^{ctrl}\) (Supplemental Figure S6). As shown in Supplemental Figure S6b,c, iPSC\({}^{CDH23}\) cultures also exhibited a significant increase in the expression of GH and LH (Supplemental Figure S6b,c). Collectively, Figure 7 demonstrates that the development of pituitary organoids generated from iPSCs of CD patients may reveal the existence of cell populations which, potentially, contribute to the support of adenoma growth and progression, as well as an expansion of stem and progenitor cells that may be the targets for tumor recurrence. Figure 7: Single cell analysis of iPSC\({}^{ctrl}\) and iPSC\({}^{CDH23}\) cultures 15 and 30 days post-directed differentiation. (**a**) UMAP plots showing identified cell clusters 0–16 in iPSC\({}^{ctrl}\) and iPSC\({}^{CDH23}\) cultures 15 days post-directed differentiation. (**b**) Violin plots of representative identified markers of the corticotroph cell lineage, where 2 subpopulations were observed among iPSC\({}^{ctrl}\) and iPSC\({}^{CDH23}\) cultures. Arrows highlight clusters 1, 5, and 7.