Modeling corticotroph deficiency with pituitary organoids supports the functional role of NFKB2 in human pituitary differentiation
Abstract
Background
Deficient Anterior pituitary with common Variable Immune Deficiency (DAVID) syndrome, combining adrenocorticotropic hormone deficiency (ACTHD) and primary hypogammaglobulinemia, is caused by NFKB2 heterozygous mutations. Nuclear factor kappa B (NFKB) signaling is a key regulator of the immune system; however, the underlying mechanism of its association with endocrine symptoms remains unknown. The role of NFKB2 in the development of the human pituitary was called into question by Nfkb2 -deficient Lym1 mice, which have normal pituitary functions.
Purpose
The aim of this study was to create a human disease model to define the role of NFKB2 in human pituitary development.
Methods
We established pituitary organoids in three-dimensional (3D) culture after directed differentiation from CRISPR/Cas9-edited human induced pluripotent stem cells (hiPSC). First, we conducted a proof-of-concept study, introducing a homozygous TBX19 K146R/K146R missense pathogenic variant in hiPSC, an allele found in patients with congenital isolated ACTHD. We then used the same method to produce NFKB2 D865G/D865G mutant organoids, harboring the pathogenic missense variant previously identified in DAVID patients. This mutation causes a failure of NFKB2 p100 phosphorylation that blocks processing to form active NFKB2 p52. We further characterized pituitary organoid development with bulk RNA sequencing and validated findings with quantitative RT-PCR and by immunofluorescence in section and whole organoids.
Results
Analysis of wild-type (WT) organoids demonstrated that this in vitro model recapitulates corticotroph cell differentiation. TBX19 K146R/K146R organoids conserved early expression of HESX1 , but had significantly decreased PITX1 , TBX19 , LHX3, and POMC transcription . NFKB2 D865G/D865G organoids also had dramatically reduced corticotrophs. Furthermore, NFKB2 D865G/D865G significantly perturbs the expression of 67 genes known to contribute to pituitary development, among which 39 transcription factors. Differential expression was found for several growth factor genes or genes associated with the epithelial-to-mesenchymal transition and terminal endocrine differentiation.
Conclusion
We used a combination of CRISPR/Cas9 editing and refinement of a 3D organoid culture protocol to model human ACTHD due to TBX19 or NFKB2 mutations. The NFKB2 variant studied induced a significant decrease in corticotroph differentiation, confirming the causative role of NFKB2 in isolated or syndromic ACTHD and demonstrating for the first time a direct functional role of NFKB2 in human pituitary development.
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