Generation Of Human Endometrial Assembloids And Mechanism

The uterus is an essential organ for human reproduction;its inner lining,the endometrium,is a highly dynamic tissue that undergoes cyclical phases of growth(the proliferative phase,Pro-phase),differentiation(the secretory phase,Sec-phase),degeneration(the menstrual phase),and regeneration in response to ovarian hormone changes.Abnormalities during endometrial remodeling and regeneration can cause infertility,recurrent pregnancy loss,endometrial tumors,inflammation,a thin endometrium,and endometriosis.Endometrial dysfunction impacts many women of childbearing age.Some defects impair endometrial receptivity during the secretory phase after ovulation,which can lead to infertility.Establishing a successful pregnancy depends upon implantation,a complex process involving interactions between the endometrium and the blastocyst.The window of implantation is described as a narrow time frame with maximal endometrial receptivity and receptivity enables the endometrium to provide an optimal environment for embryo development and placenta formation.However,the regulation of the implantation window and the mechanisms underlying implantation are still unclear.Furthermore,results obtained from animal models cannot be easily translated to the human context due to differences in hormonal regulation and the complexities of the human maternal-fetal interface.The lack of a reliable and representative model is the most significant hurdle to understanding human endometrial function and interaction between mother and fetal.The endometrium is a multicellular tissue comprising a monolayer of columnar epithelial cells that form the endometrial surface,known as the luminal epithelium(LE),and a structure known as the tubular glandular epithelium(GE)located beneath this.In addition to endometrial epithelial cells(En ECs),the underlying layer also contains many endometrial stromal cells(En SCs),which are important during implantation and can sometimes contribute to endometrial diseases,such as intrauterine adhesions.Human En SCs undergo dramatic morphological and functional differentiation during the window of implantation(WOI).The rapid development of organoid culture technology,which has recently led to advances in the understanding of intestinal and retinal organoid generation,presents a promising approach to study endometrial biology.Indeed,glandular epithelium organoids from human and mouse endometrium can be cultured for a long time and recapitulate several characteristics of uterine glands in vivo,but there are still several limitations.First,these organoids are simply consisted of one cell type without En SCs,and does not clarify the interaction and influence between the En ECs and En SCs.Second,they lack typical LE-like structures and endometrial anatomy,which makes it difficult to fully recapitulate the gene expression patterns and functions of the in vivo endometrium,especially during the WOI.It is therefore critical to develop a hormone-responsive endometrial model with complete luminal and glandular structures and a WOI gene signature.The air-liquid interface(ALI)culture method was initially used to culture epidermal and respiratory tract epithelia.With this culture model,the basal surface of the cells is submerged in liquid,while the apical surface is exposed to air,thus mimicking epithelial structures in vivo.Airway epithelial cells grown in this manner form a pseudostratified cell layer with tight connections,cilia,and mucin synthesis abilities,recapitulating the features of their in vivo epithelial counterparts.We generated a novel endometrial assembloid(ALI-En Ao)using ALI culture method.Following serial examinations,we found that a favorable En EC:En SC ratio and improve the extracellular matrix for En Ao culture so that its physical stiffness and physiological environment were more conducive to the growth and development of En ECs and En SCs.We used an ALI culture system for the En Ao cultures to replicate the in vivo context of epithelial structures covered by a mucus layer in the luminal endometrium.We used ALI culture method to simulate the special in vivo environment of epithelial structure in which the endometrial cavity is only covered by mucus layer,and finally formed an endometrial assembloid containing luminal epithelium,glandular epithelium and stromal cells,which accurately reproduced the cell composition,anatomical structure and menstrual cycle changes of the endometrium in vivo.To determine if the similarities between ALI-En Ao and endometrium in vivo extended to the molecular level,we conducted a transcriptomic analysis of the ALIEn Aos by single-cell RNA-seq(sc RNA-Seq).In summary,not only did our RNA-seq analyses show that the ALI-En Ao exhibited similarity to the in vivo endometrium in terms of cell composition and gene expression,but it also constitutes an atlas of endometrial cell populations and their corresponding transcriptomes.We proved the existence of LE-and GE-like cells in ALI-En Ao from the transcriptome,revealed the similarity with luminal/glandular distribution and gene expression pattern in vivo,and further verified the specific markers of luminal epithelium by staining.Finally,we compared single-cell transcriptomes of ALI-En Ao and submerged culture En Ao(SC-En Ao).Our data show that ALI culture improves cell composition,anatomical structure,and gene expression patterns in En Aos by upregulating genes associated with polarity,ciliogenesis,decidualization,secretion,WNT signaling,and the WOI.In conclusion,we have established a novel endometrial assembloid model by combining En ECs and En SCs and using an improved matrix and ALI culture method.ALI-En Aos exhibit intact En SCs and glandular and luminal epithelia,which recapitulates human endometrium anatomy,cell composition,hormone-induced menstrual cycle changes,gene expression profiles,and dynamic ciliogenesis.By incorporating En SCs with organoids derived from patients,this innovative model may enable modeling of different endometrial diseases that contribute to infertility and pregnancy failure,and may facilitate the development of personalized and precise treatment plans for Assisted Reproductive Technology.Furthermore,this model will also be valuable for studying the complex human maternal-fetal interaction.