Etzioni, N.; Frum, T.; Johnson, K.; Alvarez-Maldonado, A. P.; Yllescas-Lopez, H. M.; Bayer, D. E.; Xiao, Z.; Eiken, M. K.; Loebel, C.; Wu, J. H.; Tsai, Y.-H.; Wu, A.; Zhang, C. J.; Dame, M. K.; Gunuguntla, B.; Cuttitta, A. J.; Ho, H.; Tigani, D. J.; Sexton, J.; Dasuri, V. S.; Makogonov, N.; OConnell, A. E.; Spence, J. R.; Torres, D. F.

Background & AimsThe human esophagus undergoes a tightly regulated developmental program, transitioning from a simple columnar epithelium in early development to a mature stratified squamous tissue essential for adult barrier function. Here, we constructed a developmental cell atlas spanning early development to adulthood and leveraged it to generate physiologically relevant in vitro models. MethodsWe utilized single-cell RNA sequencing and spatial multiplex proteomics of human esophageal tissue from early development through adulthood. We established a feeder-supported 2D culture system and a Matrigel-free, suspension-based 3D esophagoid model in a 96-well format. To interrogate WNT2B function, we analyzed patient tissue harboring WNT2B loss-of-function mutations and performed WNT inhibition in esophagoids. ResultsSequencing profiling identified stage-specific epithelial populations: multiciliated and GPC3 basal cells were unique to early development; KRT14 basal and CRNN luminal cells were adult-specific; and COL17A1, LY6D, and KRT4 populations were shared across stages. Spatially organized WNT2B, KIT, and VWC2 mesenchymal subtypes were identified. The 2D system preserved both epithelial and mesenchymal compartments with transcriptional fidelity. Esophagoids exhibited basal-to-luminal stratification, mesenchymal compartmentalization, and required stromal interactions for formation. WNT2B repressed self-renewal of TP63 basal progenitors and inhibited proliferation, confirmed by pharmacologic inhibition of WNT in the in vitro esophagoids. ConclusionsWe present a stage-resolved atlas of human esophageal development and a scalable esophagoid platform recapitulating esophageal architecture. WNT2B regulates progenitor dynamics by restraining basal cell self-renewal. Esophagoids provide a physiologically relevant system for modeling esophageal development and disease. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/733451v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): [email protected]@f0133org.highwire.dtl.DTLVardef@117c9a9org.highwire.dtl.DTLVardef@b8852b_HPS_FORMAT_FIGEXP M_FIG C_FIG Key Findings and ImplicationsO_LIDevelopmental Atlas: The study presents a comprehensive transcriptional and structural atlas of the human esophageal epithelium, identifying conserved and stage-specific epithelial populations from early development to adulthood. Notably, stage-specific gene expression of multiciliated and GPC3 basal cells were unique to early development, while KRT14 basal and CRNN luminal cells were adult specific, with COL17A1+ (basal), LY6D+ (epibasal), and KRT4+ (middle), shared at all stages. C_LIO_LIMesenchymal Diversity: Spatial and transcriptional profiling revealed distinct mesenchymal subtypes, including WNT2B, KIT, and VWC2 populations, which are spatially organized and contribute to epithelial-mesenchymal signaling. These findings reinforce the role of stromal-epithelial interactions in esophageal development. C_LIO_LI2D Esophagus Cell Culture System: A feeder-supported 2D cell culture system was developed that retains both epithelial and mesenchymal populations, preserving transcriptional fidelity and enabling long-term expansion for mechanistic studies. C_LIO_LI3D Esophagoid Model: A suspension-based 3D organoid system was optimized using a 96-well format, enabling high-throughput generation of esophagoids with robust epithelial stratification and mesenchymal compartmentalization. These organoids recapitulate key features of the human esophagus, including basal-to-luminal organization, and require stromal interactions for formation. C_LIO_LIFunctional Role of WNT2B in esophagus development: Both in vivo and in vitro analyses demonstrated that WNT2B regulates epithelial progenitor dynamics and tissue architecture by repressing self-renewal of basally localized TP63+ cells and inhibiting proliferation. Loss-of-function models and WNT pathway modulation confirmed its role in epithelial-mesenchymal crosstalk and organoid integrity. C_LI