Dasuri, V. S. | Search bioRxiv

bioRxiv2026

Suspension-Based Human Esophagoids Recapitulate WNT2B-Dependent Regulation of Esophageal Basal Progenitors

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): org.highwire.dtl.DTLVardef@1af5743org.highwire.dtl.DTLVardef@8a061dorg.highwire.dtl.DTLVardef@1975c3forg.highwire.dtl.DTLVardef@292ce9_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

developmental biology↗

bioRxiv2025

Loss of WNT2B Increases Progression from Dysplasia to Colorectal Cancer

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related deaths in the United States, and upregulation of the WNT pathway is a primary driver in most cases. However, the role of individual WNT proteins in the development of CRC remains poorly understood. Our previous studies demonstrated that WNT2B loss-of-function leads to severe intestinal enteropathy in humans and increases chemically-induced colitis in mice, suggesting a protective function in the colon. Therefore, we investigated how loss of WNT2B affects CRC development. We used azoxymethane (AOM)/dextran sodium sulfate (DSS) to model colitis-associated cancer (CAC) and AOM-induced mutagenesis to model sporadic CRC. We measured the number and size of tumors and performed histopathological and molecular analyses. We also analyzed the Cancer Genome Atlas to evaluate WNT2B expression in human colon cancer. In CAC and CRC mouse models, Wnt2b KO mice showed decreased survival and enhanced tumor burden. Moreover, Wnt2b KO mice had larger tumors and enhanced dysplasia, with a higher frequency of animals progressing from adenomas to adenocarcinomas compared to control littermates. Wnt2b KO animals frequently presented with intestinal bleeding and rectum prolapse, which resembles obstructive CRC. Furthermore, WNT2B expression was downregulated in human CRC samples compared to healthy controls, which predicted a significantly lower patient survival. These findings support the conclusion that WNT2B is required for maximal resistance against tumorigenesis and raise the possibility that selectively increasing WNT2B signaling may be a useful colon cancer prevention strategy. SignificanceWNT2B loss-of-function increases colon cancer tumorigenesis. Targeting WNT2B may represent a novel strategy for intestinal diseases with a high risk of neoplastic transformation, potentially decreasing the progression to cancer development.

cancer biology↗

bioRxiv2024

Cataloguing the postnatal small intestinal transcriptome during the period of susceptibility to necrotizing enterocolitis

In the first postnatal month, the developing mouse intestine shifts from an immature to a mature intestine that will sustain the organism throughout the lifespan. Here, we surveyed the mouse intestine in C57Bl/6 mice by bulk RNA-Seq to evaluate the changes in gene expression over time from the day of birth through 1 month of age in both the duodenum and ileum. Using trends identified in the RNA-Seq analyses, we further evaluated expression of epithelial and mesenchymal cell markers, epithelial regulators, and immune cell markers. We confirmed key changes with qRT-PCR and immunofluorescence. In addition, we compared some findings to humans using human intestinal biopsies and organoids. This dataset can serve as a reference for other groups considering the role of single molecules or molecular families in early intestinal and postnatal development, expanding the limited literature on postnatal gene expression in the developing intestine.

developmental biology↗