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postgraduate thesis: Establishment of a three-dimensional co-culture model to investigate human endometrial gland development

TitleEstablishment of a three-dimensional co-culture model to investigate human endometrial gland development
Authors
Issue Date2024
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Li, X. [李欣桐]. (2024). Establishment of a three-dimensional co-culture model to investigate human endometrial gland development. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.
AbstractAs a key compartment of human endometrium, endometrial glands exert secretory functions in the menstrual cycles and early pregnancy. The development of endometrial glands via branching morphogenesis happens during postnatal period under the regulation of molecular factors and cell-cell/extracellular matrix (ECM) interactions. After puberty, endometrial glands proliferate, differentiate and shed in response to the cyclical hormonal changes during each menstrual cycle. Endometrial organoids (EOs) have been established as an in vitro model of human endometrial glands. In this study, a three-dimensional (3D) EO/human endometrial stromal cells (HESCs) co-culture model was established to investigate the development of human endometrial glands. The 3D co-culture model was established by hanging-drop co-culture of EOs and HESCs with subsequent co-culture in HESC-suspended Collagen-Matrigel-hydrogel. The co-cultured EOs showed development of a tubular gland structure with the expression of human endometrial glandular markers including Forkhead box protein A2 (FOXA2), Paired box 8 (PAX8) and E-Cadherin. The capacity of tubular gland development required physical contact with HESCs and the ECM environment of HESC-suspended Collagen-Matrigel-hydrogel, and was donor-specific. Functionally, the EO-derived tubular glands secreted proteins consistent with human endometrial-glandular origin including Progestagen associated endometrial protein (PAEP), Mucin-1 (MUC-1), Matrix metalloproteinases-3 (MMP-3) and Interleukin-6 (IL-6). The secretion of the EO-derived tubular glands stimulated invasion of extravillous trophoblasts (EVTs) via upregulation of Matrix metalloproteinases-2 (MMP-2) expression. To uncover the regulatory factors for EO-derived tubular gland development, bulk transcriptome and single-cell RNA sequencing analysis revealed differential gene expressions between EOs with and without tubular gland developmental capacity, and between EOs before and after tubular gland development, respectively. Reduced expression of WNT7B, and genetic knockdown of WNT7B expression in EOs by CRISPR-Cas9 induced their tubular gland development in the EO/HESC co-culture model. The interaction between transforming growth factor beta 1 (TGFβ1) and vitamin D receptor (VDR) was likely to regulate the expression of WNT7B in EOs via their interaction with HESCs. To mimic the hormonal environment in the proliferative phase of menstrual cycle for glandular development, EOs were pretreated with β-estradiol (E2) before co-culture. The treated EOs showed tubular gland development with E2 dose-dependent reduction in WNT7B expression. Similar finding was observed in mice, where intraperitoneal injection of E2 induced increased endometrial gland development and reduced endometrial glandular WNT7B expression. In clinical cases, women undergoing controlled ovarian stimulation for in vitro fertilization (IVF) treatment showed higher serum E2 concentration, along with lower endometrial glandular WNT7B expression compared to women with natural cycle. In conclusion, this study established a 3D EO/HESC co-culture model that induced the development of tubular glands from EOs that recapitulated the human endometrial glands morphologically, molecularly and functionally. WNT7B was identified as the intrinsic regulator for tubular gland development, which was likely to be extrinsically regulated by TGFβ1-VDR interaction between HESCs and EOs. E2 stimulated the development of tubular endometrial glands and downregulated endometrial glandular WNT7B expression in the in vitro EO model, an in vivo mouse model, and human endometrium undergoing controlled ovarian stimulation for IVF treatment.
DegreeDoctor of Philosophy
SubjectEndometrium
Cell culture
Dept/ProgramObstetrics and Gynaecology
Persistent Identifierhttp://hdl.handle.net/10722/352647

 

DC FieldValueLanguage
dc.contributor.authorLi, Xintong-
dc.contributor.author李欣桐-
dc.date.accessioned2024-12-19T09:26:57Z-
dc.date.available2024-12-19T09:26:57Z-
dc.date.issued2024-
dc.identifier.citationLi, X. [李欣桐]. (2024). Establishment of a three-dimensional co-culture model to investigate human endometrial gland development. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR.-
dc.identifier.urihttp://hdl.handle.net/10722/352647-
dc.description.abstractAs a key compartment of human endometrium, endometrial glands exert secretory functions in the menstrual cycles and early pregnancy. The development of endometrial glands via branching morphogenesis happens during postnatal period under the regulation of molecular factors and cell-cell/extracellular matrix (ECM) interactions. After puberty, endometrial glands proliferate, differentiate and shed in response to the cyclical hormonal changes during each menstrual cycle. Endometrial organoids (EOs) have been established as an in vitro model of human endometrial glands. In this study, a three-dimensional (3D) EO/human endometrial stromal cells (HESCs) co-culture model was established to investigate the development of human endometrial glands. The 3D co-culture model was established by hanging-drop co-culture of EOs and HESCs with subsequent co-culture in HESC-suspended Collagen-Matrigel-hydrogel. The co-cultured EOs showed development of a tubular gland structure with the expression of human endometrial glandular markers including Forkhead box protein A2 (FOXA2), Paired box 8 (PAX8) and E-Cadherin. The capacity of tubular gland development required physical contact with HESCs and the ECM environment of HESC-suspended Collagen-Matrigel-hydrogel, and was donor-specific. Functionally, the EO-derived tubular glands secreted proteins consistent with human endometrial-glandular origin including Progestagen associated endometrial protein (PAEP), Mucin-1 (MUC-1), Matrix metalloproteinases-3 (MMP-3) and Interleukin-6 (IL-6). The secretion of the EO-derived tubular glands stimulated invasion of extravillous trophoblasts (EVTs) via upregulation of Matrix metalloproteinases-2 (MMP-2) expression. To uncover the regulatory factors for EO-derived tubular gland development, bulk transcriptome and single-cell RNA sequencing analysis revealed differential gene expressions between EOs with and without tubular gland developmental capacity, and between EOs before and after tubular gland development, respectively. Reduced expression of WNT7B, and genetic knockdown of WNT7B expression in EOs by CRISPR-Cas9 induced their tubular gland development in the EO/HESC co-culture model. The interaction between transforming growth factor beta 1 (TGFβ1) and vitamin D receptor (VDR) was likely to regulate the expression of WNT7B in EOs via their interaction with HESCs. To mimic the hormonal environment in the proliferative phase of menstrual cycle for glandular development, EOs were pretreated with β-estradiol (E2) before co-culture. The treated EOs showed tubular gland development with E2 dose-dependent reduction in WNT7B expression. Similar finding was observed in mice, where intraperitoneal injection of E2 induced increased endometrial gland development and reduced endometrial glandular WNT7B expression. In clinical cases, women undergoing controlled ovarian stimulation for in vitro fertilization (IVF) treatment showed higher serum E2 concentration, along with lower endometrial glandular WNT7B expression compared to women with natural cycle. In conclusion, this study established a 3D EO/HESC co-culture model that induced the development of tubular glands from EOs that recapitulated the human endometrial glands morphologically, molecularly and functionally. WNT7B was identified as the intrinsic regulator for tubular gland development, which was likely to be extrinsically regulated by TGFβ1-VDR interaction between HESCs and EOs. E2 stimulated the development of tubular endometrial glands and downregulated endometrial glandular WNT7B expression in the in vitro EO model, an in vivo mouse model, and human endometrium undergoing controlled ovarian stimulation for IVF treatment.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsThis work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.-
dc.subject.lcshEndometrium-
dc.subject.lcshCell culture-
dc.titleEstablishment of a three-dimensional co-culture model to investigate human endometrial gland development-
dc.typePG_Thesis-
dc.description.thesisnameDoctor of Philosophy-
dc.description.thesislevelDoctoral-
dc.description.thesisdisciplineObstetrics and Gynaecology-
dc.description.naturepublished_or_final_version-
dc.date.hkucongregation2024-
dc.identifier.mmsid991044891407703414-

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