Construction of a Three-Dimensional Calcific Aortic Valve Disease Model Using Human iPSC-Derived Valvular Interstitial Cells.

Calcific aortic valve disease (CAVD) is a progressive and life-threatening condition characterized by fibrocalcific remodeling of the valve leaflets. Valvular interstitial cells (VICs) are central mediators of calcific aortic valve disease (CAVD), as their osteogenic trans-differentiation drives pathological matrix remodeling and calcium deposition within the valve leaflets. Human-induced pluripotent stem cell-derived valvular interstitial cells (hiVICs) represent a promising patient-specific platform for disease modeling. While primary VICs (pVICs) readily undergo mineralization under osteogenic stimulation, hiVICs fail to calcify in conventional two-dimensional (2D) cultures. Our data suggests that the inability of hiVICs to calcify in 2D culture is related to FOXO1 (Forkhead box protein O1) activity, which suppresses the osteogenic transcriptional program by inhibiting RUNX2 (Runt-related transcription factor 2). To address this limitation, we then developed a three-dimensional tissue ring construct using hiVICs. When cultured in osteogenic medium, these constructs exhibited robust calcification, as confirmed by Alizarin Red and Von Kossa staining. FOXO1 was also identified as a mediator of calcification in the tissue ring constructs. Metformin treatment restored FOXO1 expression and inhibited calcification, while AS1842856, a selective FOXO1 inhibitor, exacerbated tissue construct mineralization and led to a near-complete tissue collapse. In summary, we establish a functional 3D hiVIC-based model of CAVD enabling mechanistic investigation and pharmacological screening and identify FOXO1 as a critical regulator of osteogenic transition.