Cell isolation and propagation

The uterine horns were collected from one 8-week-old C57BL female mice (Wuhan University Center for Animal Experiment). The Animal Ethics Committee of Wuhan University Center for Animal Experiment approved this study. Cell isolation and culture procedures were carried out according to the previous studies with minor modifications [24, 25, 29, 30]. In brief, fresh murine uterus from one female mouse was dissected and the uterine horns were collected as illustrated in Fig. 1A. Uterine horns were minced into pieces and dispersed into single cells by digestion with collagenase (StemCell, Vancouver, BC, Canada) plus trypsin. The primary mouse endometrial epithelial cells were co-cultured with irradiated mouse fibroblast 3T3 cells (J2 strain) (YongTech, Shenzhen, China) in primary epithelial culture basic medium (PECBM). PECBM contains DMEM and nutrient F-12 Ham (3:1) (v/v) (Sigma-Aldrich), supplemented with 5% FBS (GIBCO), 2 nM triiodothyronine (Sigma), 0.5% insulin–transferrin–selenium reagent (Life Technologies), 5 μg/ml transferrin (Life Technologies), 10 ng/mL epidermal growth factor (Sigma), 0.4 μg/mL hydrocortisone (Sigma), 1 nM cholera toxin (List Biological Labs), 0.5 μg/mL amphotericin B (Fungizone; Bristol-Myers Squibb), 40 μg/mL gentamicin (Gentacin; Life Technologies), and 5 to 10 mol/L Y-27632 (Enzo Life Sciences). The cells were cultured at 37 °C in a humidified incubator, with 5% CO2. The passaging CR cells was trypsinized in two steps [31]. The initial trypsinization was for removing feeders, and another trypsinization was for detaching epithelial cells. The cell growth curve was plotted as accumulated population doublings versus time (days) [29].

Fig. 1
figure 1

Continued cultures and expansion of mouse endometrial epithelial cells (MEECs). A A schematic diagram to illustrate the isolation, expansion, and ALI 3D cultures of MEECs. B The morphology of MEECs in CR condition (upper panel) and regular condition (lower panel). The fresh murine uterus tissues were minced into pieces and dispersed into single cells. And then cells were plated in primary epithelial culture basic medium (PECBM) and co-cultured with irradiated mouse fibroblasts as described in “Materials and methods” section or DMEM supplemented with 10% FBS. The morphology of MEECs was photographed under the phase contrast microscope. Magnification 10 ×. MEECs were marked as “epi.” C The growth curve of MEECs. The cell growth curve was plotted as accumulated population doublings versus time (days). D Cell identification (Cell ID)—the short tandem repeat (STR) analysis of the MEECs. STR analysis showed that 10 STR loci of MEECs do not match any other cell lines registered in the database

Short tandem repeat (STR) analysis

Cellular genome DNA of MEECs was extracted with a commercial kit (Tiangen, China). Short tandem repeat (STR) analysis (DNA fingerprinting) was performed commercially as described previously [30, 32]. Co-amplification and three-color detection of 10 loci (9 STR loci and the X-chromosome-specific loci) were recognized.

DNA damage response analysis

Cells were treated with or without 0.5 nM actinomycin D (Act D) for 24 h. SDS polyacrylamide gels electrophoresis was conducted and then transferred electrophoretically on to a 0.2 µm polyvinylidenedifluoride (PVDF) membranes (Immobilon-NC, Millipore), specifically probed with following primary antibodies: mouse anti-p53 (1:1000, Santa Cruz Biotechnology, CA, USA, sc-126), mouse anti-p21 (1:200, Santa Cruz Biotechnology, sc-6246), and mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, sc-47778) 4 °C overnight, and then conjugated with the following secondary antibodies: m-IgGκ BP-HRP (1:1000, Santa Cruz Biotechnology, sc516102). Immunoblots were developed with a mixture of enhanced chemiluminescence reagents ECL (Beyotime Biotechnology, Shanghai, China) and digitally photographed in UV trans-illuminator (Bio-Rad Laboratories, CA, USA).

Soft agar assay

Soft agar assays with 4 × 104 cells in 0.3% low-melting point agarose were performed according to previous study [30]. Colony images were analyzed and captured with the EVOS flat screen microscope (Life Technologies Corp Bothell, WA, USA).

Hematoxylin–eosin (H&E) and Masson’s trichrome staining

Tissues or ALI 3D cultures were fixed in 4% paraformaldehyde, dehydrated in a series of ethanol dilutions, and paraffin-embedded. Paraffin blocks were cut into 4 μm sections thickness and mounted on the glass slides. The sections were deparaffinized and hydrated through xylene and graded alcohol series and rinse for 5 min in tap water. The sections were stained with hematoxylin and eosin (H&E) (Zhongshan Golden Bridge Company, Beijing, China) and Masson’s trichrome staining kit (Maixin biotech company, Fuzhou, China). Morphological observation of tissues and ALI 3D cultures was photographed under the EVOS visual imaging microscope (Life Technologies).

DAB staining

The DAB staining was performed using a commercial DAB Detection Kit (Maixin biotech company). ALI 3D cultures or tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm sections. The sections were deparaffinized and hydrated through xylene and graded alcohol series and rinsed for 5 min in tap water. Antigens were retrieved by heating samples in a microwave for 15 min in citric acid buffer. The primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, Chicago, IL, USA, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, Cambridge, UK, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321) were incubated, respectively, on the slides at 4 °C overnight, then detected with the reaction-amplified reagent for 20 min, and conjugated with high-sensibility enzyme conjugated lgG polymer. Reactants were visualized with the fresh-prepared DAB chromogenic solutions for 3 to 5 min. Hematoxylin somatic cell staining reagent was used to counter-stain nuclei. All the coverslips were mounted on the glass slides using anti-quenching Fluoroshield™ histology mounting medium (Sigma-Aldrich) and visualized under a fluorescence microscope (BX51TF, Olympus company, Tokyo, Japan) with magnification 40 ×.

Quantitative real-time RT-PCR

Total RNA was extracted from the tissue samples or cells using TRIzol Reagent (Life Technologies), and reverse transcription was performed using PrimeScript™II1st Strand cDNA Synthesis Kit (Takara Bio Inc., Japan). The levels of mRNA were quantitated using Toyobo Real-time PCR Master Mix (Toyobo, Japan) and analyzed in Bio-Rad Real-time PCR System (Bio-Rad) as described previously [33]. The primers for amplifying genes are shown in Table 1.

Table 1 List of PCR primers used in this study

Immunofluorescence assay

MEECs were grown to an appropriate density and fixed in 4% (w/v) paraformaldehyde for 15 min, permeabilized with 0.5% Triton-X-100 for 15 min, and blocked with 5% bovine serum albumin for one hour at room temperature. After blocking, cells were labeled with the primary antibodies (1:100, rabbit anti-EpCAM, Proteintech, 21050-1-AP; 1: 100, rabbit anti-Mucin1, Abcam, ab109185; 1:100, mouse anti-P63, Abcam, ab735; 1:100, rabbit anti-CD44, Proteintech, 15675-1-AP; 1:100, mouse anti-ERα, Santa Cruz Biotechnologies, sc-71064; 1:100, mouse anti-PR, Santa Cruz Biotechnologies, sc-398898; 1:100, rabbit anti-Vimentin, Abcam, ab137321), and the secondary antibodies (1:100, fluorescently labeled goat anti-mouse lgG-cy3, BA1031, Boster company, Wuhan, China) according to the manufacture’s protocol. DAPI (0.5 mg/ml, D3571, Thermo Fisher) was used to stain the nucleus. Then, the fluorescence was detected by Leica DM4000B fluorescence microscope.

Matrigel three-dimensional (3D) culture

Single-cell suspensions of epithelial cells and HeLa cells were dispersed in a specifically differentiation medium (keratinocyte growth medium, Life Technologies) containing 5% pre-cooling Matrigel (BD Biosciences, USA). Morphogenesis assays (DAPI staining) were performed after 7 days as previously described [30, 34, 35].

Estrogen response assay

MEECs were treated with 17β-estradiol at the concentrations of 1 nM, 10 nM, 100 nM for 24 h. Cell lysates were collected for western blotting assay. The protein samples were specifically probed with primary antibodies: mouse anti-ERα (1:1000, Santa Cruz Biotechnology, sc-71064), mouse anti-PR (1:1000, Santa Cruz Biotechnology, sc-398898), and mouse anti-β-actin (1:1000, Santa Cruz Biotechnology, sc-47778) 4 °C overnight and then conjugated with the following secondary antibodies: m-IgGκ BP-HRP (1:1000, Santa Cruz Biotechnology, sc516102). Immunoblots were colorated with a mixture of enhanced chemiluminescence reagents ECL A and B (Beyotime Biotechnology) at a ratio of 1:1 and digitally photographed in UV trans-illuminator (Bio-Rad Laboratories).

Air–liquid interface (ALI) 3D culture

ALI cultures were performed as described previously and illustrated in Fig. 1A [30, 36, 37]. Single-cell suspensions (1 ~ 2 × 105) of MEECs in 400μL growth medium (CELLnTEC Advanced Cell Systems AG, Switzerland) were dispersed into the Millicell PCF inserts (12 mm size, Millipore, Massachusetts, USA) which were placed into a 6-well plate. About 2 ml of growth medium was also dropped into the well (outside the inserts). The 6-well plate was cultured at 37 °C, 5% CO2. After 48 h, the growth medium was replaced with differentiation medium (CELLnTEC Advanced Cell Systems AG) inside and outside the inserts and incubated for 16 h to allow cells to form an intercellular adhesion structure. The fresh differentiation medium was changed every 2–3 days. The 3D cultures were differentiated approximately 14–19 days and harvested for histology experiments.

Murine model of intrauterine injury and cells transplantation

8-week-old C57BL female mice were obtained from Wuhan University Center for Animal Experiment. Animal housing and killing was in accordance with guidelines of Laboratory Animal Requirements of Environment and Housing Facilities (Chinese Version). The Animal Ethics Committee of Wuhan University Center for Animal Experiment approved this study. A total of 24 mice were randomly divided into 3 groups (n = 16 uterine horns/group): sham operated/control group, non-transplanted/injury group, and cells-transplanted group. After injection of 2% sodium pentobarbital (45 mg/kg intraperitoneally), a vertical incision was made in the abdominal wall and the uterus was exposed. A small incision was made in each uterine horn, and the horns were traumatized using 27-gauge needle inserted two-thirds of the way through the lumen and rotated and withdrawn 10 times [3, 11, 38]. For cells-transplanted group, MEECs (1 × 106) in 50 ul PBS were injected into uterine immediately after the uterine injury. For non-transplanted/injury group, 50 ul PBS was administered via intrauterine injection after injury. For the sham operated/control group, the uterine horns were left intact after exposure by an abdominal midline incision. The rectus fascia and skin of control group mice were sutured with 6–0 absorbable polyglycolic acid (PGA, Jinhuan CR631) in an interrupted fashion after PBS rinse of abdominal cavity. The uterine horns were collected at 14 days, 21 days, 30 days, and 45 days after uterus damage. The mice were killed by euthanasia with an overdose of sodium pentobarbital (200 mg/kg intraperitoneally) followed by cervical dislocation. Cardiac arrest and dilated pupils represented the death of mice before tissue sampling. The formalin-fixed paraffin-embedded tissues were sectioned longitudinally and stained with Masson’s trichrome staining. Photographs were taken under the EVOS visual imaging microscope (Life Technologies) with magnification 10 ×, 20 ×, and 40 ×. The percentage of fibrosis area (collagen area/total tissue area) was obtained by scanning the value of collagen area and total tissue area using Image J software. Numbers of glands in five different fields of vision were counted, and thickness of endometrium was measured in randomly chosen field of vision by Image J software.

Functional recovery of injured murine endometrium

The function of the regenerated and repaired endometrium was assessed by investigating whether mice were capable of maintaining embryos development to advanced gestation [3, 39]. Additional batch of 24 female mice (8-week-old C57BL) were used and randomized into sham operated/control group, non-transplanted/injury group, and cells-transplanted group (n = 16 uterine horns/group). The intrauterine injury and cells transplantation were performed as above procedures. After three estrous cycles (14 days), three groups of female mice were individually bred with C57BL male mice at a ratio of 1:1. The day of vaginal suppository observation was considered as gestation day 0, and then female mice were separated from male mice (only once pregnancy). Female mice were killed with an intraperitoneal injection of pentobarbital sodium (200 mg/kg) followed by cervical dislocation at gestation day 21. The uterine horns were checked for the embryos.

Statistical analysis

The experiments were performed in three independent tests of triplicates. Data were analyzed with GraphPad Prism 8.0. ANOVA and Dunn’s multiple comparisons test were used across three experimental groups’ comparison. All values were expressed as mean ± SD and considered significantly different when p value was < 0.05.

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