Turkey Reproductive Tract Organoids Expressed Cell-specific Markers and Avian Influenza Virus Receptor Related Genes as in the Origin Tissues

Pitchaya Santativongchai¹; Sunantha Kosonsiriluk¹; Natalia Calixto Mancipe²; Kent M. Reed³; Kahina S. Boukherroub¹

1. Department of Animal Science, University of Minnesota, Saint Paul, MN, USA

2. Minnesota Supercomputing Institute, University of Minnesota, Minneapolis, MN, USA

3. Department of Veterinary and Biomedical Sciences, University of Minnesota, Saint Paul, MN, USA

Abstract Text:

The turkey reproductive tract serves as a notable entry point for pathogens including avian influenza virus (AIV). We recently developed turkey reproductive tract organoids as an alternative tool for investigating AIV infectivity in vitro. However, suitable cell-specific markers are lacking to validate the organoids as representative models of the reproductive tissues. Single-cell RNA sequencing (scRNA-seq) is used for characterizing cell populations in various tissues, without the limitations of commercial species-specific antibody availability. This study was designed to characterize cell populations in each region of turkey reproductive tract and identify gene expression markers to validate the corresponding organoids. Single cells were isolated from infundibulum (INF), magnum (MAG), isthmus (IST), uterus (UTR), uterovaginal junction (UVJ), and vagina (VAG) tissues of two turkey hens (33 weeks of age, 5th week of lay) by collagenase I and DNase I dissociation at 37ºC. Subsequently, approximately 10,000 cells of each region were captured and processed for scRNA-seq. The remaining cells were divided into two aliquots for RNA extraction or for culture into organoids within extracellular matrix (Matrigel) domes for bulk RNA sequencing. Computational analysis of scRNA-seq data identified 7 initial cell clusters, comprising epithelial cells, tubular gland cells, fibroblasts, two clusters of immune cells, and two clusters of proliferating cells of mixed origin, based on gene expression profiles. Additionally, in depth analysis on gene expression profiles within the epithelial cell cluster identified cell markers specific to each region of the tract. Bulk RNA sequencing data showed high expression of WFDC3, OVAL, VSNL1, and PSCA in the fully developed organoids (n=2) from INF, MAG, UTR, and UVJ, respectively, consistent with the markers presented in the single-cell data. Importantly, as in the respective tissues, the organoids expressed CMAS and ST3GAL4, which are reported to play an important role in endocytosis of influenza virus by affecting the sialic acid (SA) receptor synthesis. Follow up lectin histochemistry revealed SA α2,3-gal receptors (AIV receptors) presenting in the organoids and the epithelial cells of the corresponding tissues. Comparative bulk RNA sequencing of the organoid and origin tissue samples (n=4) is underway. This study generated the first single cell mapping of the turkey reproductive tract suggesting that turkey oviductal organoids could serve as in vitro models of the epithelial tissues. Further evaluation of AIV infectivity in the organoids is promising for studying AIV and developing preventive approaches in avian female reproductive system.