Abstract
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.
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Acknowledgments
The authors thank Dr. J. Browne for the assistance, and the CWRU SOM Light Microscopy Core Facility (NIH Grant S10-OD024996 and S10RR021228) and the Cleveland Clinic Central Biorepository, PLMI, for providing some of the tissues for used in this study.
Funding
This research was supported by the Cystic Fibrosis Foundation (LEIR17G0), and the National Institutes of Health R01 HD068901 and R01 HL094585.
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All procedures performed in studies involving human participants were in accordance with the ethical standards of Case Western Reserve University research committee (CWRU IRB Protocol no. 2017-2099) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards.
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The video shows cumulative swelling of HEE organoids over 130 minutes at 37°C after the addition of CFTR agonist, using bright field microscopy. The assay was performed in 96 well plates coated with Matrigel and air-dried. Organoids were resuspended in HBSS solution (Wisent catalog: 311-513-CL; Ingredients: NaCl 136.9 mM, KCl 5.4 mM, NaHCO3 4.2 mM, CaCl2 1.3 mM, MgSO4 0.8 mM, KH2PO4 0.4 mM, Na2HPO4 0.3 mM, D-Glucose 5.5 mM; pH 7.25 ± 0.15, Osmolality 287 ± 15 mOsm/kg), and pipetted on these precoated plates. Thereafter, the swelling assay was performed, which showed absence of swelling for HEE organoids exposed to vehicle alone (DMSO), and presence of swelling upon addition of forskolin (10 μM) with VX-770 (1 μM), supporting the role for the cyclic AMP-activated CFTR chloride channel in this function. (MOV 646 kb)
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Leir, SH., Yin, S., Kerschner, J.L. et al. An organoid model to assay the role of CFTR in the human epididymis epithelium. Cell Tissue Res 381, 327–336 (2020). https://doi.org/10.1007/s00441-020-03208-7
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DOI: https://doi.org/10.1007/s00441-020-03208-7