Establishment of an in vitro implantation model using a newly developed mouse endometrial organoid

Abstract

Implantation failure is a major cause of infertility, but its mechanisms remain unclear due to the lack of techniques for constructing organized endometrial structures and recapitulating the implantation process in vitro. Endometrial organoids have recently been developed, but they consist of only epithelial cells, and their apical surface faces inward, preventing blastocyst attachment. We developed an apical-out mouse endometrial organoid incorporating epithelial and stromal cells, and examined its ability to recapitulate implantation with mouse blastocysts. Mouse uteri were digested with collagenase and cultured in monolayers. The resulting aggregates were then transferred to low-attachment plates for 3D culture. After 7 days, self-organized aggregates contained E-cadherin-positive epithelial cells outside and vimentin-positive stromal cells inside. Mucin 1 signals were observed on the apical side of epithelial cells, confirming the apical-out orientation. Organoids were stimulated with sex steroid hormones and co-cultured with blastocysts. Time-lapse imaging revealed the four implantation steps: blastocyst attachment, epithelial invagination, entosis and invasion. Invaded cells expressed proliferin while surrounding stromal cells expressed cyclooxygenase 2, indicating trophoblast differentiation and decidualization. This novel organoid closely recapitulates the mouse endometrium and implantation process in vitro.

Keywords: Apical-out; Decidualization; Endometrial organoid; Implantation.

Fig. 1.

Aggregate formation under adherent co-culture of EECs and ESCs. (A) Schematic of the culture procedure. (B) Representative bright-field images of the adherent co-culture of EECs and ESCs. Spontaneous aggregation of EECs and ESCs was observed from day 2 (yellow arrowheads) and formed a large ring-like structure on day 3. They were dissociated into smaller aggregates and reseeded onto the same well (day 4). On day 7, these aggregates developed into a three-dimensional, omega-shaped structure, with only the bottoms attached to the dish. Scale bars: 1000 μm. (C) Schematic and 3D immunostaining images of the aggregates under adherent co-culture. Representative cross-sectional slices of the aggregates in x-y, x-z and y-z planes are shown. Images were taken by confocal microscopy. E-cadherin for EECs; vimentin for ESCs; DAPI for nuclei. Scale bars: 100 μm. The localization of EECs and ESCs is shown in the schematic; EECs are shown in green and ESCs are shown in gray. (D) Representative scanning electron microscopy (SEM) images of the aggregates under adherent co-culture. On day 5, EECs demonstrated by the smooth surface (green arrowheads) partially localized to the outside of the aggregates. On day 7, the aggregates were almost covered by EECs (green arrowheads), except for the site attached to the dish where ESCs remained exposed (red arrowheads). Scale bars: 50 μm.

Fig. 2.

Suspension culture facilitates the formation of endometrial organoids composed of EECs outside and ESCs inside. (A) Bright-field images of suspension cultures of aggregates. The picked-up aggregates were transferred to low-attachment U-bottom dishes for suspension culture. During the 3 days of suspension culture, a transparent layer covering the outer surface gradually thickened and eventually enveloped the entire aggregate. Scale bars: 100 μm. (B) Schematic and immunostaining images of the aggregates under suspension culture. (Left) Representative image of the immunostaining for frozen sections of the aggregates are shown: E-cadherin for EECs; vimentin for ESCs; DAPI for nuclei. Scale bars: 20 μm. Arrows indicate the measured height of EECs. (Right) Quantification of the cell height of EECs before (day 7) and after (day 10) suspension culture is shown. Data are mean±s.d. of 15 aggregates in each group. Each data point is indicated as a dot. *P<0.01 (two-sided Student's t-test). (C) Representative scanning electron microscopy image of the aggregate after suspension culture. All surfaces of the aggregate were covered with smooth EECs, with no exposed ESCs. Scale bar: 100 μm. (D) A representative HE-staining image for frozen section of the aggregates after suspension culture. Scale bar: 50 μm. (E) Representative immunostaining images for frozen section of the aggregates after suspension culture. E-cadherin for EECs; vimentin for ESCs; DAPI for nuclei. Scale bars: 50 μm. (F) Representative immunostaining images of a progesterone receptor in frozen sections of the aggregates after suspension culture. DAPI indicates nuclei. Scale bars: 50 μm.

Fig. 3.

Novel endometrial organoids exhibit apical-out polarity. (A) Representative immunostaining images of MUC1 in the mice endometrium (left) and our endometrial organoid (right). The dotted lines indicate the border between the epithelial and stromal layers. DAPI indicates nuclei. Scale bars: 50 μm. (B) Scanning electron microscopy revealed the presence of microvilli, which are characteristic of the apical surface, on the surface of the organoids. Large dome-shaped protrusions were also observed on the surface, which were similar to pinopodes (arrowheads). Scale bars: 50 μm (top); 5 μm (bottom).

Fig. 4.

Time-lapse imaging of an in vitro implantation model with a novel endometrial organoid. (A) Schematic of the in vitro implantation model. To induce a receptive state for blastocysts in the organoids, the organoids were treated with E₂, cAMP and MPA for 48 h. The organoids were then co-cultured with blastocysts obtained from GFP transgenic mice in low-attachment U-bottom dishes. The representative bright-field images of the in vitro implantation process are shown. Blastocysts (GFP) attached to the organoids ∼48 h after the initiation of the co-culture. By 96 h, the blastocysts invaded the inside of the organoids. Scale bars: 100 μm. (B) Time-lapse imaging of in vitro implantation. After hormone treatment, the organoids were labeled with a red fluorescent dye and co-cultured with blastocysts (green). The process of in vitro implantation was monitored using time-lapse imaging from 48 h to 96 h after the initiation of the co-culture. Blastocysts attached to the surface of the organoids (attachment). EECs then invaginated, forming a cavity enveloping the blastocyst (invagination), and EECs were endocytosed by GFP-positive blastocysts (entosis-like cell-in-cell invasion). Finally, blastocysts invaded the ESC layer beneath the EEC layer (invasion). Scale bars: 100 μm. (C) Time-lapse imaging of co-culture of blastocysts (green) and organoids (red) without hormone pretreatment. The organoids without hormone treatment were co-cultured with blastocysts. Blastocyst behavior was monitored using time-lapse imaging from 48 h to 96 h after the initiation of the co-culture. Blastocysts did not attach to the organoid. Scale bars: 100 μm.

Fig. 5.

Implantation responses occurred during in vitro implantation. (A) Representative immunostaining images of proliferin in frozen section of implantation-positive organoid. The nuclei are stained with DAPI. Arrowheads indicate the blastocyst-derived cells that are positive for proliferin with a large nucleus. Scale bars: 50 μm. (B) Representative immunostaining images of Cox2 in frozen sections of implantation-positive organoid. The nuclei were stained with DAPI. Arrowheads indicate the ESCs surrounding the invading blastocyst-derived cells that are positive for Cox2. Scale bars: 50 μm.