An organoid model to assay the role of CFTR in the human epididymis epithelium.
An organoid model to assay the role of CFTR in the human epididymis epithelium.
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DOI：
10.1007/s00441-020-03208-7
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发表时间：
2020-08
影响因子：
3.6
通讯作者：
Harris A
中科院分区：
文献类型：
--
作者：
Leir SH;Yin S;Kerschner JL;Xia S;Ahmadi S;Bear C;Harris A
来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，其致病机制仍不清楚。我们使用人附睾上皮细胞 (HEE) 类器官和极化 HEE 细胞培养物来测定人附睾中的 CF 跨膜电导调节因子 (CFTR)。从人附睾中建立了 3D HEE 类器官和膜插入物上的极化 2D HEE 细胞培养物。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 HEE 类器官培养物中存在关键的附睾细胞类型。 10 μM 的 FSK 诱导 HEE 类器官在 16 小时时肿胀 20%，而 5 和 10 μM CFTRinh172 处理显着减小了 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 则增加了 TER；此外，用特定的 siRNA 消除 CFTR 会显着增加 TER。 FSK 处理显着增加了 4-kDa 的通量，但没有增加 70kDa 葡聚糖的通量，表明 CFTR 的激活主要增强跨细胞扩散。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。这项研究有助于阐明 CFTR 依赖性细胞过程如何损害 CF 的生育能力。
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 hrs, 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 70kDa 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. This research facilitates progress in elucidating how CFTR-dependent cellular processes impair fertility in CF.
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影响因子：
4.1
作者：
Mutolo, Michael J.;Leir, Shih-Hsing;Harris, Ann
通讯作者：
Harris, Ann
影响因子：
3.6
作者：
Mandon, Marion;Hermo, Louis;Cyr, Daniel G.
通讯作者：
Cyr, Daniel G.
影响因子：
3.6
作者：
Dube, Evemie;Dufresne, Julie;Cyr, Daniel G.
通讯作者：
Cyr, Daniel G.
DOI：
10.1073/pnas.88.20.9262
发表时间：
1991-10-01
影响因子：
11.1
作者：
CRAWFORD, I;MALONEY, PC;HIGGINS, CF
通讯作者：
HIGGINS, CF
影响因子：
7.3
作者：
Melis N;Tauc M;Cougnon M;Bendahhou S;Giuliano S;Rubera I;Duranton C
通讯作者：
Duranton C