P-797 Single-cell analysis sheds light on investigation of epithelial stem/progenitor cells in human endometrial organoid
Authors: Li J , Cao D , Chiu P C N , Lee C L 
Published: June 2023

Abstract

Study question
How to find endometrial epithelial stem cell (eeSC) in the human endometrial gland?

Summary answer
Endometrial organoid culture model combined with omics techniques can be a promising platform for investigating eeSC functions and endometrial gland development.

What is known already
Endometrial stem/progenitor cells are thought to be involved in endometrial regeneration, with endometrial stromal stem cells having already been extensively studied. On the other hand, study of human eeSC is challenging due to the lack of specific markers to isolate and examine their functional properties. In addition, the in vivo phenotype of primary endometrial epithelial cells cannot be maintained for a long duration in two-dimensional culture. The development of three-dimensional endometrial organoid model provides suitable alternatives for studying eeSC, as they can be tailored to be biomimetic and accurately recapitulate the native in vivo scenario of the glandular epithelial cells.

Study design, size, duration
This is a basic science study of endometrial organoids. Endometrial biopsies were performed on day 2 after the injection of human chorionic gonadotrophin in women undergoing in vitro fertilization treatment (IVF) but having no fresh embryo transfer in Queen Mary Hospital, The University of Hong Kong. Endometrial organoids were derived from human endometrial gland fragments. The established organoids were digested into single cells for droplet-based single-cell sequencing to investigate their cellular population and component.

Participants/materials, setting, methods
The filtered high-quality single-cell analysis data were clustered and assigned to specific cell types according to their expressions of classical markers. Potential stem cells cluster was identified by trajectory analysis, RNA velocity, CytoTRACE and literature search. The existence of specific eeSC markers was validated by immunofluorescence staining and flow cytometric analysis of primary endometrial tissues/endometrial organoids. The clonogenicity of the isolated eeSCs was determined by clonogenic assay.

Main results and the role of chance
The endometrial organoid exhibit morphology and marker expression pattern of human endometrial glands. They can also be expanded for long-term culture. The resemblance between in vitro generated organoid and the in vivo endometrial gland was further supported by single-cell RNA-seq analysis. Seurat clustering identifies 8 cell clusters in endometrial organoids. RNA velocity analysis revealed a potential eeSC cluster at the starting point of the differentiation trajectory from the immature cell cluster toward the most mature one. Besides, the eeSC cluster had relatively high expression of known stem-cell markers including Tumor-Associated Calcium Signal Transducer 2 (TACSTD2) and Aldehyde Dehydrogenase 1 Family Member A3 (ALDH1A3). Five genes including Intercellular Adhesion Molecule 1 (ICAM1), L1 Cell Adhesion Molecule (L1CAM), Annexin A2 (ANXA2), Mesothelin (MSLN), Integrin Subunit Alpha 3 (IGTA3) were subsequently identified as potential markers of the eeSC cluster. Further analysis by immunostaining demonstrated the expressions of CD54 and ANXA2 in ∼1% and ∼30% of the whole organoids cell suspensions, respectively. The expressions of CD54 and ANXA2 were also identified in the glandular tissue of human endometrium.

Limitations, reasons for caution
With basal out/apical in phenotype, the endometrial organoids are not exactly similar to the endometrium in vivo, hampering the exploration of their surface marker in cell interaction experiments. Like other organoid systems, it is inevitable for endometrial organoids to have heterogeneity in cells and inconsistency in expansion and biological performance.

Wider implications of the findings
Gene manipulation technology like CRISPR-Cas9 could be utilized to genetically alter the eeSc to study their biological properties. The construction of eeSC biobank will offer an opportunity for the development of personalized regenerative medicine with diagnosis, preventive intervention, and treatment approaches for gynecological diseases.

Trial registration number
Not applicableNot applicable