Clostridioides difficile（C. difficile）毒素A（TcdA）和B（TcdB）通过破坏上皮屏障功能部分引起抗生素相关性结肠炎。精确的体外模型是检测早期毒性动力学、研究疾病病因和开发新治疗方案的必要条件。癌细胞系和器官样本的特性本质上限制了这些努力。我们开发了差异化的人结肠上皮（hCE）成年干细胞衍生的单层膜，并在上皮单层的顶部/底部方面调查毒素的影响，并评估泄漏上皮屏障是否增强了毒性。单细胞RNA测序（scRNAseq）将C. difficile相关基因映射到人类谱系。转录组学将hCE与Caco-2比较，指导体外干细胞分化的时机，揭示了TcdA的转录反应。经过荧光渗透试验和跨表皮电阻（TEER）测量细胞毒性。在双氯芬酸诱导的泄漏肠模型中评估了TcdA/B毒性的贡献。scRNAseq证明了广泛而不定的毒素受体表达。体内吸收性结肠细胞显示出增加的毒素受体、Rho GTPase和细胞连接基因表达。先进的TcdA毒性通常会降低细胞因子/趋化因子并增加紧密连接和死亡受体基因。分化的Caco-2细胞仍不成熟，而hCE单层与体内成熟结肠细胞类似。基底暴露于TcdA/B引起的毒性和细胞凋亡比顶部暴露更明显。受毒素暴露的顶部增强了双氯芬酸的作用。顶部/底部毒性是分离的，基底毒素暴露后快速发生，而上皮毒素暴露后的毒性和规模增加。泄漏接头增强了TcdB上皮暴露的毒性。hCE单层代表了评估微生物毒素对肠道上皮的影响的生理相关和敏感的系统。

Clostridioides difficile (C. difficile) toxins A (TcdA) and B (TcdB) cause antibiotic-associated colitis in part by disrupting epithelial barrier function. Accurate in vitro models are necessary to detect early toxicity kinetics, investigate disease etiology, and develop preclinical models for new therapies. Properties of cancer cell lines and organoids inherently limit these efforts. We developed adult stem cell-derived monolayers of differentiated human colonic epithelium (hCE) with barrier function, investigated the impact of toxins on apical/basal aspects of monolayers, and evaluated whether a leaky epithelial barrier enhances toxicity. Single-cell RNA-sequencing (scRNAseq) mapped C. difficile-relevant genes to human lineages. Transcriptomics compared hCE to Caco-2, informed timing of in vitro stem cell differentiation, and revealed transcriptional responses to TcdA. Transepithelial electrical resistance (TEER) and fluorescent permeability assays measured cytotoxicity. Contribution of TcdA/B toxicity was evaluated in a diclofenac-induced leaky gut model. scRNAseq demonstrated broad and variable toxin receptor expression. Absorptive colonocytes in vivo displayed increased toxin receptor, Rho GTPase, and cell junction gene expression. Advanced TcdA toxicity generally decreased cytokine/chemokine and increased tight junction and death receptor genes. Differentiated Caco-2 cells remained immature whereas hCE monolayers were similar to mature colonocytes in vivo. Basal exposure of TcdA/B caused greater toxicity and apoptosis than apical exposure. Apical exposure to toxins was enhanced by diclofenac. Apical/basal toxicities are uncoupled with more rapid onset and increased magnitude post-basal toxin exposure. Leaky junctions enhance toxicity of apical TcdB exposure. hCE monolayers represent a physiologically relevant and sensitive system to evaluate the impact of microbial toxins on gut epithelium.