Endometrial Organoids: A New Model for the Research of Endometrial-Related Diseases†

Gu ZY, Zhiyue Gu, Shuangzheng Jia, Song Liu, Jinhua Leng, Leng JH
Biology of reproduction · 2020 · vol. 103(5) , pp. 918–926 · doi:10.1093/biolre/ioaa124 · PMID:32697306 · PMC7609820 · W3044207631
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Endometrial organoids offer a stable, patient-specific model for studying endometrial diseases and drug screening, overcoming limitations of cell lines and animal models despite ongoing challenges with co-culture and cancer applications.

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This paper reviews how endometrial organoids (EOs)—3D self-organizing cultures derived mainly from adult endometrial stem/progenitor cells—can model endometrium biology and related diseases, addressing limitations of animal models and conventional cell lines. It summarizes early mouse and human organoid studies, including hormone-responsive EOs across endometrial stages, and reports that organoid culture efficiency and morphology vary by disease type; for endometriosis, it describes the development of patient-matched ectopic- and eutopic-endometrium organoids that recapitulate disease-associated phenotypes and genetics. The paper also highlights analytical advances such as combining bulk and single-cell RNA sequencing to build a high-resolution gene expression atlas, while noting that medium composition and reagent requirements differ by species and that success rates can be variable (e.g., higher in healthy/EuEOs than in cancer-derived EOs). This paper is centrally about endometriosis — it includes detailed discussion of establishing endometriosis patient–matched ectopic and eutopic endometrial organoids and using them to recapitulate endometriosis-associated features.

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Abstract

An ideal research model plays a vital role in studying the pathogenesis of a disease. At present, the most widely used endometrial disease models are cell lines and animal models. As a novel studying model, organoids have already been applied for the study of various diseases, such as disorders related to the liver, small intestine, colon, and pancreas, and have been extended to the endometrium. After a long period of exploration by predecessors, endometrial organoids (EOs) technology has gradually matured and maintained genetic and phenotypic stability after long-term expansion. Compared with cell lines and animal models, EOs have high stability and patient specificity. These not only effectively and veritably reflects the pathophysiology of a disease, but also can be used in preclinical drug screening, combined with patient derived xenografts (PDXs). Indeed, there are still many limitations for EOs. For example, the co-culture system of EOs with stromal cells, immune cell, or vascular cells is not mature, and endometrial cancer organoids have a lower success rate, which should be improved in the future. The investigators predict that EOs will play a significant role in the study of endometrium-related diseases.
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Eos

Boretto and colleagues found the number of proliferating cells (Ki67+) in mouse EOs increased after exposure to estrogen (E2), but not to progestogen (P4) [ 18 ]. Furthermore, several relevant genes appeared specific to regulation after treatment with E2 or P4, such as Egf, Igf1 , Lf (increased by E2) and Alox15, Lif, and Prss28 (upregulated by P4) [ 18 ]. Mouse EOs also responded to ovarian hormones in vivo. When treated by ovarian hormones, mouse EOs transplanted under the kidney capsule of mouse could expand and assemble into an organized structure with glandular-type protuberances [ 18 ]. Same as in vivo, human EOs showed physiological, cycle-mimicking responses to hormones [ 18 , 19 , 21 ], which was usually not observed for endometrial epithelia to response to hormones in 2D culture system. Boretto et al. detected that the Ki67+ cell proportion in human EOs increased with the E2 treatment, but decreased with the addition of P4 (while reducing the E2 concentration to half) [ 18 ], which was similar to the situation in vivo [ 26–28 ]. In the level of protein and mRNA, the E2 treatment also upregulated the OLFM4, an intestinal stem cell marker [ 29 ], which was negative without E2 exposure [ 19 , 21 ]. In addition to E2 and P4, Turco et al. further explored the effect of stimulation of cAMP, a second messenger, placental hormones (hCG and hPL) as well as PRL [ 19 ]. Among them, cAMP contributed decidualization of endometrial stromal cells in vivo [ 30 ]. After exposure to E2 followed by P4, the relevant genes 17βHSD2, PAEP, SPP1, LIF, IGFBP4, IGFBP5 , and CYCLIN A1 all increased in hormonally treated organoids by microarray analysis, and 17βHSD2, PAEP, SPP1, LIF were further confirmed by qRT-PCR [ 19 ]. In addition, after treatment of E2 followed by P4, the expression of both ERα and PR of treated organoids were higher than those untreated in most, but not all organoids [ 19 ]. However, Fitzgerald et al. confirmed that after E2 stimulation alone for 2 days, EOs exhibited an upregulation in ERα and PR, and after exposure to E2 + P4 for another 6 days, the decrease and even absence of ERα and PR appeared [ 21 ]. The difference might be correlated with concentration of hormones and the duration of P4 exposure in two studies. In vivo, the ERα and PR of the luminal epithelium (LE) and glandule epithelium (GE) would decline or disappear between the proliferative to the secretory phase of the menstrual cycle, especially after mid-secretory stage [ 31 ]. The addition of cAMP had upregulated the expression of differentiation markers PAEP (glycodelin) and SPP1 (osteopontin) at gene and protein level [ 19 ]. It is noteworthy that signals from decidualized stroma, PRL and signals from the placenta, hCG and hPL can further stimulated the differentiation of human EOs, synthesizing abundant PAEP and SPP1, producing more cilia, and downregulating the progenitor cells marker SOX9 [ 19 ]. Fitzgerald et al. further explored the influence of hormones on cell types of human EOs. In combining organoid and single-cell analysis, Fitzgerald et al. found 5 cell types, including ciliated, epithelial, proliferative, stem, and unciliated cells, in both control and E2-treated day six EOs [ 20 ]. In addition to these five cell types, there were also secretory cells in group of control, E2 + MPA(medroxyprogestone), and E2 + MPA + cAMP treated day 12 EOs [ 20 ]. It noteworthy that after exposure to hormones, regardless of whether E2 or MPA, the proportion of stem cells decreased. Prolonged EOs culture and the addition of progesterone promoted the proportion of secretory cells [ 20 ]. In addition, the proportion of ciliated cells significantly increased following the E2 treatment, which indicated that cilia were one of the indicators of epithelial cell differentiation. This was consistent with the study conducted by Haider et al., in which E2 signaling induced ciliogenesis in the endometrium and organoids [ 23 ]. Combining E2 stimulation and NOTCH pathway inhibition greatly increased the ciliogenesis. However, inhibiting the NOTCH signaling alone was insufficient, that is, E2 plays a decisive role in cilia cell formation [ 23 ]. On the contrary, the proportion of proliferative type cells did not significantly change with the hormone intervention, and the cAMP had little effect on the cell variety in EOs [ 20 ]. The results of this research gave the investigators an idea of which types of epithelial cells were most responsive to hormone stimulation. In addition, via scRNA-seq, they further identified in which cell OLFM4 and PAEP (the expressions of these genes were increased mostly after exposure to E2 and P4, respectively) has a significantly increased expression. OLFM4 predominantly upregulated in the stem and ciliated cells, and PAEP was predominantly upregulated in the ciliated, proliferative, secretory, and stem cell types [ 21 ]. Furthermore, the types and quantities of certain important receptors (such as COL1A2, IL1RN, SPP1, and TGM2) and ligands (such as CALM3, FN1, TIMP2) were also altered in the organoid with the hormone treatment, reflecting that the communication between different cell types changed under the stimulation of hormones [ 20 ]. Due to the hormone reactivity of EOs, researchers have considered that EOs plays a great auxiliary role in the study of abnormal pregnancy and hormone-related endometrial diseases. For example, if something goes wrong on either side of the secretory transformation of the glandular epithelium or the decidualization of the stromal cells, the pregnancy may fail or cause complications, including preeclampsia and fetal growth restriction [ 32 , 33 ]. These endometrial changes are all correlated to hormone stimulation.

The

Although EOs contain a lot of advantages, these also encounter some limitations at present. First, the improvement of ECM is also needed. At present, the main ECM in the EO culture is Matrigel purified from Engelbreth-Holm-Swarm mouse sarcoma [ 55 ]. Although it is effective in promoting organoid growth and self-organization, it may not completely resemble the human endometrial niche for animal-derived and ill-defined characteristics. In order to overcome these limitations, a number of synthetic and chemically defined hydrogels and extracellular matrix hydrogel derived from decellularized tissues and microstructured collagen scaffolds have been developed for 3D cell culture, as well as for organoids cultures [ 56–58 ]. In addition, collagen scaffolds and stomal cell induced-scaffolds via agarose 3D Petri Dishes have also been used in the co-culture of endometrial epithelial and stromal cells, indicating that the other types culture materials, rather than the Matrigel, are also feasible in the EO culture [ 37–39 ]. The advantages of EOs as an ideal model for the study of pregnant and gynecological diseases. Second, the ECO efficiency was lower than other types of EOs, the same phenomenon happened in prostate, pancreas and colorectal cancers [ 16 ], and the purity of tumor organoids was also a challenge [ 16 ]. It might be possible to improve the efficiency of organoid culture by adjusting the composition of the medium, and selecting the metastatic lesions [ 16 , 20 ]. Therefore, the efficiency of organoid formation and the underlying mechanism needs to be further explored. Third, the present EO culture mainly pays attention to epithelial cells, but focuses less on other type of cells, such as stromal cells, vascular endothelial cells and immune cells, which are also critical for disease development. As described above, there have been studies that achieved the co-culture of endometrial epithelial cells and stromal cells. The EOs were cultured in new kinds of ECMs, rather than Matrigel. One of the ECMs was 3D porous collagen scaffolds, while the other ECMs were provided by stromal cells using agarose 3D Petri Dishes [ 38 , 39 ]. Therefore, it can be observed that the exploration of new culture ECMs and co-culture methods promoted each other. However, these co-culture models are not mature and convincing enough, since these were not identified as to whether these could be both phenotypically and genotypically stably cultured in the long term. In addition, pregnant diseases and endometrial diseases are closely correlated to immune system abnormalities and vasculature dysfunction. For example, in terms of pregnancy, epithelium, decidualized stromal cells, resident immune cells, vasculature and placenta trophoblast communicate together to facilitate pregnancy establishment, and any kind of cell dysfunction would affect the final outcome. In order to explore the pathogenesis, the role of immune cells and vasculature must be considered. In studies on scaffold-free EOs via agarose 3D Petri Dishes, by detecting the typical surface markers, no immune cells were virtually present [ 38 ]. And there is currently no report on the role of the vasculature in EOs. Therefore, EOs should improve the protocol of co-culture of epithelial cells and stromal cells, striving to explore the technology of co-culture with immune cells, as well as endothelial cells. Lastly, it has to be mentioned that there are still remain challenges for EO clinical applications in the future, such as how to establish and improve the EOs biobanking for drug screening efficiently, and how to develop an efficient evaluation system to judge the drug reactivity, in order to be truly effective in patients.

Discussion

The uterus is an important organ, and its main function is to conceive a fetus. In addition, the uterus is closely associated with many gynecological diseases, such as endometrial hyperplasia, endometrial cancer and endometriosis. On the base of the culture media of organoids derived from other organs, a great deal of researchers explored and summarized the culture medium suitable for EOs, which was an R-spondin1-based culture method, and slightly different in media composition between various types of endometrium. However, several key components were all contained in the culture medium ( Table 2 , the specific key ingredients and its concrete functions). Intriguingly, Forskolin, a cAMP activation, could slow the cell differentiation in liver organoids [ 13 ], but has a contradictory phenomenon in EOs. Turco et al. compared this to the exposure to E2 + P4, and the treatment with E2 + P4 + cAMP induced a more differentiated phenotype [ 19 ], which might be associated with the ovarian sexual hormone responsiveness. The efficiency of EO formation varies depending on the nature of the endometrium, with the healthy endometrium in the secretory phase being highly efficient, and the successful rate of EOs derived from the EC being lower [ 19 , 20 ]. For the colony forming ability, Turco et al. reported that the successful rate of EOs was inefficient (2–4%), which might be correlated to the stem cell nature of the organoid. A majority of studies have attempted to identify the markers of endometrial epithelial stem/progenitor cells, such as SSEA-1, SOX9, N-cadherin, LGR5 [ 59–62 ]. Epithelial cells with progenitor nature were considered to be located in the basalis layer of the premenopausal endometrium [ 63 , 64 ], but these did not only existed in the basalis. Hapangama et al. discovered that the functionalis endometrium at the secretory phase from women with endometriosis comprised of basalis-like cells that expressed SSEA1 or SOX9, and the percentages of which were higher than that of the non-endometriosis group [ 62 ]. Therefore, in future studies, the investigators would figure out the proportion of stem cells in the functionalis and basalis layer, and determine how these vary in vivo at different stages in the menstrual cycle, which are beneficial for the original EO culture. In fact, the influence of endometrial epithelial cells on the colony-forming of organoids is complicated, Turco et al. established the human EOs from SOX9 negative cells [ 19 ]. The same situation occurred in other organoids. Sato T et al. sorted Lgr5 hi cells, Lgr5 low cells and Lgr5 -ve cells from mouse intestinal adenoma organoids, and found that although Lgr5 hi cells has the highest organoid-forming efficiency, Lgr5 low cells and Lgr5 -ve cells also formed organoids with considerable efficiency [ 14 ]. Slightly different, Huch et al. reported that in mouse pancreas organoids culture, the clone forming ability of Lgr5+ cells was significantly higher than that of Lgr5-cells, which were 16 and 1.6%, respectively [ 15 ]. Notably, the organoids derived from Lgr5- cells could result in Lgr5+ mouse organoids, liver, or pancreas [ 14 , 15 ]. Therefore, the ability of stem cells to establish organoids may be relatively stronger, but cells that do not temporally possess a stem cell nature can also form organoids with a relatively lower efficiency. More detailed mechanisms need further exploration. As discussed above, as an ideal model for the study of pregnant and gynecological diseases, EOs have the following advantages ( Figure 2 ): (1) mimic the endometrium of phenotypic characteristics, such as the expression of several classical epithelial markers (E-cadherin, PanCK, Muc1, and EPCAM) and ERα, PR, the formation of mucus, microvilli, cilia, and glandular polarization, which was supported by the presence of laminin at the basolateral side, and of microvilli and cilia directed towards the lumen; (2) maintain the characteristics of the original endometrium in term of genetics, for instance, both healthy EOs and disease-related EOs reveal disease-associated traits at the transcriptomic and genetic level, and some mutations found in original tissues also presented with the corresponding organoids; (3) the response to hormones through phenotype and genetic changes, not only E2, P4 but also cAMP, hCG, hPL, and PRL, can influence the cell types, morphology, genetics, and cell product; (4) long-term expansion, cryopreservation, and thawing can maintain the stability of phenotypes and genetics, which solves the problem of insufficient original specimens and offers the possibility for long-term stable use (such as some prolonged drugs screening experiments). Taken together, EOs are expected to be an ideal pregnant and gynecological disease model in the future.

Phenotypic

Mouse EOs expressed epithelial markers pancytokeratin (PanCK) and E-cadherin, as well as estrogen receptor α (ERα), via immunohistochemical analysis [ 18 ]. Others such as mucus (analyzed by PAS staining) and apical microvilli (reviewed by transmission electron microscopy, TEM) were also detected in mouse EOs, resembling the mouse endometrial epithelium in vivo [ 18 ]. A series of researchers had conducted in-depth research on human EOs [ 18 , 19 , 21 ]. Among them, Turco et al. gave the most detailed description of the specific cell surface markers and cellular typical structures. First, Turco et al. identified the EOs glandular epithelial nature by hierarchical clustering analysis. The EOs expressed E-cadherin, PanCK, Muc1and EPCAM, which reflected the epithelial and glandular nature, and several nuclear markers (FOXA2, SOX17 and PAX8) [ 19 ]. The epithelial polarity was intact by presenting EPCAM, and showing Laminin at the basolateral membrane. Similar to mouse EOs, TEM revealed a microvillous, pseudostratified columnar epithelium supported by the basement membrane, and PAS staining had endometrial glandular secretions glycogen [ 19 ]. Analogously, epithelial nature (E-cadherin, PanCK), ERα and progesterone receptor (PR), mucus, cell polarization as well as secretory vacuoles, were also observed in EcEOs [ 20 ]. In addition to expressing EC-related markers in ECOs, Boretto et al. further found that they could also distinguish type I and type II EC, according to the expression of ERα and PR in ECOs [ 20 ]. As it is already known, type I EC is estrogen-dependent, while type II EC is estrogen-independent [ 24 , 25 ]. Apart from phenotypic characteristics, EOs could also closely resemble the endometrium at the genetic level. The transcriptomic and genetic analyses of EOs reveal disease-associated traits and mutational landscape of the primary tumor. For instance, several important genes associated with signal pathway, hormonal response and adhesion/invasion factors differed among healthy EOs, EcEOs, and EuEOs. Compared with healthy EOs, the differential gene of EcEOs derived from diverse stages were further analyzed by RT − qPCR. LEF1, WNT11, LGR6, LIFR, SOX9, MMP2, IL-1β , and IL-8, which appeared to be upregulated, especially LGR6 in the higher stages III − IV, MMP2 in stages II and IV, IL-1β, and IL-8 in stages I − II EcEOs, while the progesterone-regulated gene PAEP was downregulated [ 20 ]. It is noteworthy that LGR6 was one of the top regulated genes in EcEOs, when compared to EuEOs from individual endometriotic patients. As for EC, abundant somatic copy number alteration in primary tumors was reserved in the corresponding ECOs [ 20 ]. It was added that the gene signature of decidual EOs were fully similar to non-pregnant EOs [ 19 ]. All of these disease-derived EOs could recapitulate the disease phenotype (histological and molecular features) in vivo, and some differential gene expression levels between gland samples and organoids might be associated with their different microenvironments [ 19 ]. The genetic stable of organoids had also been observed in other organ-derived organoids, such as the pancreas, liver and prostate organoids [ 13 , 15 , 16 ].

Con Culture

Organoids have advantages in pathogenesis research and drug screening, but classical EOs culture conditions (artificial extracellular matrix, Matrigel and EOs expansion medium) favor epithelial cells, and contaminated stromal cells are lost during subsequent passage. Several researches that studied organoids derived from other organs have already attempted to tackle the problem of co-culture, such as live organoids, co-culturing hPSC-derived hepatic endodermal cells with human endothelial, and human mesenchymal stem cells, thereby contributing to more closely resembling the liver cytoarchitecture [ 34 ]; Chakrabarti et al. successfully co-cultured the mouse-derived gastric cancer organoids with autologous immune cells used for the study of PD-L1/PD-1 interactions within the tumor microenvironment in vitro [ 35 ]. Mouse ASC-derived pancreatic islet organoids were co-cultured with endothelial cell, and this improved the ability of long-term expansion [ 36 ]. Murphy and Wiwatpanit et al. was the first to attempt to generate scaffold-free multicellular EOs, which were responsive to sex hormones, and contained epithelial and stromal cells [ 37 ]. They also applied this model to subsequent studies correlated to polycystic ovary syndrome [ 38 ]. Unlike the EOs summarized above, they combined the stromal and epithelial cells (1:3 ratio by volume) with 1.5% agarose 3D Petri Dishes using stromal cells as scaffolds, and cultured these in MammoCult growth medium [ 38 ]. It was noteworthy that in that culture system, similar to the native endometrial tissue, stromal cells provided the scaffold support for epithelial cells. In addition, 3D porous collagen scaffolds also made co-culture of EOs and stromal cells came true. Abbas et al. detected that stromal cells-only or EOs-only cultured on scaffolds could express the characteristics of corresponding cells and hormone reactivity, respectively, which was the basis of co-culture [ 39 ]. It should be noted that original EOs (established in Matrigel) and primary stromal cells were developed via classical culture methods, and that these were sequentially transferred to the scaffold [ 39 ]. Stromal cells were cultured in scaffolds with advanced DMEM/F-12 supplemented with 10%(v/v) FBS, glutamine and antibiotics, and co-cultured with EOs after 2 days. After washing several times, the medium was changed to EOs expansion medium [ 19 , 39 ]. Lastly, after 10 days of co-culture, they verified the existence of stromal cells and epithelial cells by immunofluorescence and flow cytometry [ 39 ]. The porous collagen scaffold-based model of the endometrium including both epithelial and stromal cells provided a new field for studies on endometrial related diseases. Therefore, the co-culture of epithelial cells and stromal cells will further mimic the endometrium in vivo via considering stromal–epithelial interactions. Indeed, these co-culture models also faced some challenges as to whether it could be cultured over a long period of time and maintain both phenotypic and genotypic stability simultaneously.

Introduction

As the inner lining of the uterus, the endometrium contains epithelial and stromal cells, and undergoes periodic changes in growth, differentiation, and degeneration under ovarian hormones [ 1–3 ]. The endometrium is associated with a number of gynecological problems, embracing infertility, endometrial hyperplasia, endometrial cancer, and endometriosis [ 3 ]. All of these endometrial diseases seriously affect a women’s health, such as endometrial cancer (EC), which is the fourth most common type of female cancer, and the endometriosis influences approximately 6–10% of women of reproductive age [ 4 , 5 ]. The main obstacle of studying these diseases is the lack of ideal models. Existing research models have obvious shortcomings. Rodents do not have menstruation and cannot faithfully mimic the human situation. Primates, although have spontaneous menstruation, are high cost and ethically limited. Primary cells cannot maintain long-term culture in vitro, and there are transformed phenotypes in immortalized or carcinoma-derived endometrial cell lines [ 6–11 ]. Therefore, a reliable endometrial model is the key to study endometrium-related diseases, and endometrial organoids (EOs) are expected to conquer these problems. As a kind of 3D culture system, organoids are self-organizing and genetically stable, which contain progenitor/stem cells and differentiated cells that resemble the original tissues [ 12 ]. Organoids derived from other tissues, such as the gastrointestinal tract, liver, pancreas, prostate, and fallopian tube, have already been established [ 13–17 ]. Boretto et al. and Turco et al. were the first scholars to report the study of EOs, including organoids derived from mouse and human endometrium [ 18 , 19 ]. On these bases, Boretto et al. and Fitzgerald et al. further studied the characteristics of human EOs [ 20 , 21 ], involving organoids from normal and abnormal endometrium. These researches described the specific composition of the EOs culture medium, the phenotypic (typical surface markers and structures) and genetic characteristics, and the responsiveness to hormones, and there were several similarities and differences in these studies. As an ideal study model, EOs have the potential to be used in pathophysiological research, drug screening for new therapy or drug testing for personalized medicine, regenerative medicine, and the EOs biobank of endometrium-related disease.

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