Modeling corticotroph deficiency with pituitary organoids supports the functional role ofNFKB2in 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 byNFKB2heterozygous 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 byNfkb2-deficientLym1mice, which have normal pituitary functions.
Purpose
The aim of this study was to create a human disease model to define the role ofNFKB2in 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 homozygousTBX19K146R/K146Rmissense pathogenic variant in hiPSC, an allele found in patients with congenital isolated ACTHD. We then used the same method to produceNFKB2D865G/D865Gmutant 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 thisin vitromodel recapitulates corticotroph cell differentiation.TBX19K146R/K146Rorganoids conserved early expression ofHESX1, but had significantly decreasedPITX1,TBX19,LHX3,andPOMCtranscription. NFKB2D865G/D865Gorganoids also had dramatically reduced corticotrophs. Furthermore,NFKB2D865G/D865Gsignificantly 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 toTBX19orNFKB2mutations. TheNFKB2variant 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.
Related articles
Related articles are currently not available for this article.