Self-renewing Endometrial Epithelial Organoids of the Human Uterus [single-cell RNA-seq data]

The inner lining of the human uterus, termed the endometrium, is replete with glands that are hypothesized to be important for establishment of pregnancy through interactions with other endometrial cells and the embryo. A three-dimensional culture system was utilized to establish and generate endometrial epithelial organoids from normal human endometrium that display long-term expandability, can be cryopreserved, and are hormone responsive. Here, single cell RNA-seq was used to create a high-resolution gene expression atlas of the endometrial epithelial organoids and to define responsiveness to reproductive hormones. In order to investigate the roles of uterine glands in first trimester human pregnancy, we used an established protocol of Matrigel and WNT-activating conditions to generate three-dimensional human uterine gland organoid (HUGO) cultures from endometrial biopsies. The HUGO expand long-term, are genetically stable, differentiate following treatment with reproductive hormones, and can be cryopreserved. After passage, HUGO were grown for 4 days in expansion medium and then treated with estrogen (E2) for 2 days and then either nothing (Control), E2 and medroxyprogesterone acetate (MPA) or E2+MPA+cAMP for a further 6 days (n=3 replicates per patient and treatment). Total RNA was extracted from the organoids using a standard TRIzol-based protocol and Direct-zol RNA MiniPrep Plus isolation kit (Zymo Research, R2070). RNA quality and concentration was determined by a Fragment Analyzer (Advanced Analytical Technologies). RNA library preparation and sequencing was conducted by Illumnia platform by Novogene, California.