2.1 Ethics for animal and human embryonic stem cell2.1.1 Animal experiments

The Seoul National University Hospital’s institutional animal care and use committee (IACUC) reviewed and approved all animal experiments [IACUC approval No. 20-0033-S1A3(1)].

2.1.2 Human embryonic stem cell experiments

The institutional review board (IRB) of the Institute of Reproductive Medicine and Population, Seoul National University Medical Research Center [IRB approval No.219932-202205-05-01-01] reviewed and approved human embryonic stem cell (hESC)- related experiments and the usage of the hESC line, SNUhES34.

2.2 Mouse and human endometrial cell culture

7-week-old C57BL/6 female mice (Koatech, Gyeonggi-do, Korea) were sacrificed by cervical dislocation. The uterus was isolated, and lipid pads were removed. Isolated uteri were mechanically dissected and dissociated using collagenase type I (5 mg/ml) for 90 min. Dissociated cells were filtered through a 70-um cell strainer (SPL Life Sciences, Gyeonggi-do, Korea) and centrifugated at 2000 rpm for 10 min. Collected cell pellets were resuspended in culture media and replated. Culture media constituted with DMEM/F12, supplemented with 10% fetal bovine serum (FBS), 1% Insulin-Transferrin-Selenium-X (ITS), and 100 units/ml of penicillin-100 ug/ml of streptomycin. The medium was changed every three days. When the cells reached approximately 90% confluence, they were passaged using 0.25% trypsin–EDTA.

The HEC-1-A cell line for human endometrial cells was purchased from ATCC (HTB-112, Manassas, VA, USA). The cells were grown in the same media as mouse endometrial cells. Invitrogen purchased all the reagents for mouse and human endometrial cell cultures.

2.3 Production of mouse embryos2.3.1 Superovulation

6-week-old C57BL/6 female mice were injected into the peritoneal cavity with 10 IU/0.1 ml of pregnant mare serum gonadotropin (PMSG, Prospec, East Brunswick, NJ, USA). At 48 h later, 10 IU/0.1 ml of human chorionic gonadotropin (hCG, Sigma-Aldrich, Saint Louis, MO, USA) was intraperitoneally administered. The mice were sacrificed by cervical dislocation at 20 h after hCG injection.

2.3.2 Preparation of sperm

A dense sperm mass was obtained from the epididymis of C57BL/6 mice 8–10 weeks old. The sperm in an HTF media (Cosmo Bio, Carlsbad, CA, USA) drop of 100 ul covered with mineral oil was incubated at 37 °C in 5% CO2 in humidified air for 1 h to obtain fertilization ability.

2.3.3 Oocyte collection

Oocytes were obtained from oviducts at 20 h after hCG administration. The oviducts were isolated and placed in a 100 ul of TYH medium (Cosmo Bio) droplet in a dish. The cumulus-oocyte complexes were isolated from the swollen ampulla and transferred to the TYH medium. Then, oocytes were incubated for about 1 h at 37 °C in 5% CO2 in a humidified air atmosphere.

2.3.4 In vitro fertilization (IVF)

A pre-incubated, capacitated sperm was gently added to the freshly ovulated oocytes for a final motile sperm concentration of 1 × 106/ml. The combined sperm oocytes were incubated for 5 h. Then, the oocyte was washed through several medium changes and incubated in 40 ul of mWM media (Cosmo Bio) drop covered with mineral oil. Development of 2 cell embryos was counted 24 h after the completion of in vitro fertilization. The image of embryos was recorded using the i-solution program (IMT i-Solution, Daejeon, Korea).

2.4 Human embryonic stem cells and embryoid bodies2.4.1 Human embryonic stem cell (hESC) culture

The human embryonic stem cell line, SNUhES34 (46, XX), was provided by the Institute of Reproductive Medicine and Population, Seoul National University. The hESC line was maintained in feeder-free condition. Five ug/ml of vitronectin (VTN) was pre-coated at room temperature (RT) for 1 h. Full-grown colonies were incubated with 0.5 mM of EDTA for 5 min, and the reaction stopped. The colonies were detached and split with gentle pipetting into small clumps. The clumps were distributed evenly to the VTN pre-coated dish. Essential 8 medium was used for the culture, and the media was exchanged daily.

2.4.2 Human embryoid body (hEB) formation

Undifferentiated hESCs were treated with collagenase Type IV (2 mg/ml) and incubated for 30 min at 37 °C. The dissociated colonies were dissociated into small clumps and incubated overnight in a suspension culture. hEBs were incubated for 48 h and used as a human embryo alternative model. All the reagents for hESCs and hEBs were purchased from Invitrogen.

2.5 Endometrium-3D gel mixture preparation

Mouse endometrium cells at a density of 3 × 107 cells/well were mixed with pre-warmed Bio-Gel (Medipab, Seoul, South Korea) at 37 °C in ratio 1:1, 2:1 and 3:1 (Cells with media: Bio-Gel). Four hundred ul/well of the mixture were seeded in 4 well plates (SPL) and gelated at 4 °C overnight, and Casting gel (Medipab) was added for secondary gelation at 37 °C for 3 h. After washing with media, the firmed gel mixture was covered with 200ul/well of culture media. The cell mixture with gel was treated with a firming buffer every two days to harden it extra, and the media was changed. It was cultured for 1, 3, 5, 7, and 9 days.

2.6 Cell survival assay

For viability measurement, 3D-embedded, cultured cells were treated with a dissolving solution for 30 min at RT. The CCK-8 component was added to the cells and incubated for 2 h at 37 °C, 5% CO2. The absorbance of treated cells was measured at 450 nm using a microplate reader device (Molecular Devices, San Jose, CA, USA).

2.7 Fluorescence-activated cell sorting (FACS)

Cells were dissociated into single cells by treating Accutase (Invitrogen) for 5 min at 37 °C. Dissociated cells were incubated with blocking solution (PBS with 3% BSA and 0.3% Triton X-100, All from Sigma-Aldrich) for 30 min at RT and washed with PBS. The samples were incubated with conjugated antibodies for 1 h at 37 °C. Then, the samples were washed with PBST three times and analyzed by FACS Calibur™ (BD Biosciences, Franklin Lakes, NJ, USA). IgG antibody was used as the control for the analysis. Antibodies used for the study are listed in Supplementary Table 1.

2.8 Immunostaining

The samples were fixed with 4% paraformaldehyde for 20 min at RT. The samples were washed with PBS and blocked for over 12 h using blocking soln. Primary antibodies were treated for 1 h at 37 with a ratio of 1:100. After three times of washing with PBST, secondary antibodies were treated for 1 h at 37 °C with a ratio of 1:200. And then, the samples were cleaned using PBST and mounted with Prolong Gold containing DAPI (Invitrogen). Antibodies used for the analysis are listed in Supplementary Table 2 and purchased from Abcam (Waltham, MA, USA) and Santa Cruz Biotechnology (Dallas, Texas, USA).

2.9 Statistics

Statistical analysis was performed using Prizm GraphPad version 9.0 (GraphPad Software, San Diego, CA, USA). Data are expressed as the mean ± standard errors and were analyzed by one-way ANOVA. P value less than 0.05 (P < 0.05) is considered statistically significant.