Organoid cultures derived from primary human tissues facilitate the study of disease processes and the development of new therapeutics. Most men with cystic fibrosis (CF) are infertile due to defects in the epididymis and vas deferens; however, the causative mechanisms are still unclear. We used human epididymis epithelial cell (HEE) organoids and polarized HEE cell cultures to assay the CF transmembrane conductance regulator (CFTR) in the human epididymis. 3D HEE organoids and polarized 2D HEE cell cultures on membrane inserts were established from human caput epididymis. Single-cell RNA sequencing (scRNA-seq) was performed to map cell type–specific gene expression in the organoids. Using forskolin (FSK) to activate CFTR and inhibitor CFTRinh172 to block its activity, we assessed how CFTR contributes to organoid swelling and epithelial barrier function. The scRNA-seq data showed key caput epididymis cell types present in HEE organoid cultures. FSK at 10 μM induced HEE organoid swelling by 20% at 16 h, while 5 and 10 μM CFTRinh172 treatment significantly reduced HEE organoid size. In transepithelial resistance (TER) measurements, FSK reduced TER, while inhibition of CFTR increased TER; also, depletion of CFTR with specific siRNAs significantly increased TER. FSK treatment significantly increased the flux of 4-kDa but not 70-kDa dextran, suggesting activation of CFTR mainly enhances transcellular diffusion. We have demonstrated that CFTR contributes to the maintenance of HEE cell TER and that cultured HEE organoids are a useful model to investigate human epididymis function. These results facilitate progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.

中文翻译：

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用于测定 CFTR 在人附睾上皮中作用的类器官模型

源自原代人体组织的类器官培养物有助于疾病过程的研究和新疗法的开发。大多数患有囊性纤维化 (CF) 的男性由于附睾和输精管缺陷而无法生育；然而，致病机制仍不清楚。我们使用人类附睾上皮细胞 (HEE) 类器官和极化的 HEE 细胞培养物来测定人类附睾中的 CF 跨膜电导调节剂 (CFTR)。3D HEE 类器官和膜插入物上的极化 2D HEE 细胞培养物是从人类附睾头上建立的。进行单细胞 RNA 测序 (scRNA-seq) 以绘制类器官中细胞类型特异性基因表达的图谱。使用毛喉素 (FSK) 激活 CFTR 并使用抑制剂 CFTRinh172 阻断其活性，我们评估了 CFTR 如何促进类器官肿胀和上皮屏障功能。scRNA-seq 数据显示了 HEE 类器官培养物中存在的关键附睾头细胞类型。10 μM 的 FSK 在 16 小时时诱导 HEE 类器官肿胀 20%，而 5 和 10 μM C​​FTRinh172 处理显着降低了 HEE 类器官的大小。在跨上皮电阻 (TER) 测量中，FSK 降低了 TER，而抑制 CFTR 增加了 TER；此外，用特定 siRNA 消耗 CFTR 显着增加了 TER。FSK 处理显着增加了 4-kDa 但不是 70-kDa 葡聚糖的通量，表明 CFTR 的激活主要增强了跨细胞扩散。我们已经证明 CFTR 有助于维持 HEE 细胞 TER，并且培养的 HEE 类器官是研究人类附睾功能的有用模型。