Animals

Eight- to ten-week-old Sprague Dawley female rats weighing 180–220 g were used in all experiments, and all rats were purchased from Shanghai SLAC Laboratory Animals. The rats had free access to water and food and were maintained in a feeding room on a 12 h light and 12 h dark regimen with an average temperature of 22 °C and 70% to 80% relative humidity. All the procedures were approved by the Institutional Animal Care and Use Committee at the First Affiliated Hospital of USTC (code no. 2022-N(A)−119). The work has been reported in line with the ARRIVE guidelines 2.0.

Collection of human endometrium and rat endometrium samples

Human endometrial samples were obtained from nonmenopausal women who underwent hysteroscopy for nonneoplastic disease from the Department of Obstetrics and Gynecology. All donors gave consent, and all procedures were approved by the Ethics Committee of First Affiliated Hospital of USTC (IRB code NO. 2023KY300). Patients are required to be free of hormone use for the previous 3 months before surgery. Endometrium samples were collected into Dulbecco’s modified Eagle’s medium (DMEM, BI, CAT#:01–051-1ACS) supplemented with 100 U/ml penicillin, 100 μg/mL streptomycin (Biosharp, CAT#: BL505A), and 10% fetal bovine serum (FBS, Sigma, CAT#: F7524). When removed from the human endometrium, it will be better treated within 6 h.

It has been reported that MSCs isolated from aged rats have a low proliferation and differentiation capacity [25]. Therefore, rat endometrial samples were obtained from 8- to 10-week-old Sprague Dawley female rats weighing 180–220 g. In brief, the rats were killed, and the uterus was removed immediately. The rat uterus was transferred into the culture medium and then used for cell isolation.

Isolation of endometrial mesenchymal stem cells (eMSC)

Human endometrial tissue and rat endometrial tissue were treated using the same method. First, the tissue was cut into 1 mm × 1 mm pieces and washed with phosphate-buffered saline (PBS, CAT#: BI, 02–024-1ACS) 2 times to completely remove the culture medium. Second, the tissue pieces were transferred to a 15 ml Falcon tube containing 1 ml enzyme mix: 0.4 ml collagenase IV (10 mg/mL, CAT#: Gibco, 17,104–019), 0.4 ml collagenase V (10 mg/mL, CAT#: Solarbio, C8170), 0.2 mL DNAse I (1 mg/mL, CAT: Biofrox, 1121MG010) and 3 ml PBS. Then, the tube was placed in a shaker at 37 °C for 40 min. At the end of digestion, the mixture was centrifuged at 800 revolutions per minute (RPM) for 5 min. The supernatant was discarded and mixed with red cell lysis solution (CAT#: Biosharp, BL503B) to destroy red blood cells. Finally, the mixture was dissolved in PBS and screened through a 70 µm sieve at 800 RPM for 5 min to centrifuge the mixture through the sieve. The pellet was resuspended in DMEM containing 10% FBS, 1% penicillin and streptomycin and cultured at 37 °C in a 5% CO2 atmosphere, with a medium change every 2–3 days.

Flow cytometry analysis

After 3 passages, cells were digested from the culture dish with Accutase, resuspended in FACS buffer, and counted. For H-eMSC, flow cytometry analysis was conducted using a Human MSC Analysis Kit (CAT#: BD Biosciences, 562,245) based on the manufacturer’s protocol. For rat endometrium-derived mesenchymal stem cells(R-eMSC). An SD rat MSC Analysis Kit (CAT#: Cyagen, RAXMX-0901) was used for cell surface marker identification. All results were analyzed using FlowJo, version 10, software.

Differentiation of H-eMSC and R-eMSC into adipocytes, osteocytes, and chondrocytes

To identify the ability of human endometrium-derived mesenchymal stem cells (H-eMSC) to differentiate into multiple mesenchymal lineages (adipogenic, osteogenic, and chondrogenic). We used a Human MSC Functional Identification kit (CAT #: R&D Systems, SC006) based on the manufacturer’s protocol. Finally, we employed oil red O, alizarin red, and toluidine blue staining to verify the successful differentiation of mesenchymal stem cells into adipocytes, osteoblasts, and chondrocytes.

To induce R-eMSC differentiation into adipocytes and osteocytes. We used the Adipogenic Differentiation Induction Kit (CAT#: Cyagen, RAXMX-90031) and Osteogenic Differentiation Induction Kit (CAT#: Cyagen, RAXMX-90021) according to the manufacturer’s instructions. Briefly, after 3 weeks of differentiation, adipocytes, osteocytes, and chondrocytes were identified by staining with o oil red O, alizarin red, and toluidine blue, respectively.

Determining the estrus period of rats

We used vaginal secretions from rats to determine whether they were in the estrus phase as previously described [26]. Briefly, a cotton swab dipped in normal saline was inserted into the vagina of a rat. The swab was scratched gently inside the vagina to obtain the cells. The cotton was gently rubbed across a slide to collect the cells, and then the cell composition was observed under a microscope. A large number of cornified epithelial cells showed that the rats were in the estrus phase.

Establishment of the rat intrauterine adhesion (IUA) model

Rats in estrus were selected based on vaginal smear analysis and anesthetized with Zoletil 50 (Virbac France) (0.1 ml/100 g) by muscle injection. The lower abdomen was disinfected with iodophor after shaving, and then the skin and muscle were cut across the midline to expose the uterus. A 3–5 mm longitudinal incision was made in the uterus, and tweezers were inserted into the uterus of the rats and rubbed 20 times in all directions. The uterine surface was washed with 10 ml saline, and the uterine horn was closed using absorbable sutures. A total of about 60 female rats were used in this experiment. All rats were randomly assigned to each group.

GFP labeling of cells

We labeled H-eMSC and R-eMSC with an rAAV vector expressing GFP (Tsingke, China). A total of 1 × 106 cells (P3) were incubated in 1 µl rAAV working solution at 37 °C for up to 1 day, washed with PBS, and cultured in fresh medium containing 10% FBS. After 4 days, GFP expression was analyzed using a fluorescence microscope.

Transplantation of H-eMSC and R-eMSC ex vivo

A female rat was sacrificed (stopped breathing after a cervical dislocation), and its uterus was removed and cut along the long axis of the rat uterus using surgical scissors. The endometrium was scratched with a surgical blade 20 times, and the uterus was washed using PBS. A total of 2 × 106 cells labeled with GFP were transferred to the surface of the rat endometrium. Some medium was added to the culture dish and cultured at 37 °C in a 5% CO2 atmosphere. Subsequently, the tissues were taken at different time points for direct observation and frozen sectioning. All ex vivo transplantation experiments were conducted at least three times.

Frozen section

The rat uterus (3–4 mm) was placed in a small sealed box, and OCT was performed (CAT: # biosharp, BL557A). The sealed box was frozen in liquid nitrogen for 10–20 s. Equilibrate at −20 °C for approximately 15 min to prevent cracking of the tissue block when sectioning. Section the block at a range of 10–15 µm and place it on adhesive slides. Finally, the slides were observed directly under a fluorescence microscope.

Immunofluorescence

First, the paraffin sections were deparaffinized using xylene and then subjected to antigen retrieval. Following the repair, the sections were sealed at room temperature for one hour before being incubated with antibodies (CD45, CAT#: Abcam, ab10558, USA. CD68, CAT#: CST, 97778S, USA) overnight. The next day, the sections were incubated with secondary antibodies (Jackson labs, USA) at room temperature, shielded from light, for one hour. The nuclei were subsequently stained with DAPI (CAT#: Biosharp, BS097, China) and observed and counted using a fluorescence microscope (ZESIS). All immunofluorescence experiments were conducted at least three times for reliability.

Transplantation of H-eMSC and R-eMSC in vivo

The rats were divided into 3 groups. One group was transplanted with GFP-labeled H-eMSC, one group was transplanted with H-GFP-eMSC and immunosuppressed with methotrexate (H-eMSC + MTX) (CAT#: T1485, TargetMol, USA) and the last group was transplanted with R- GFP-eMSC. The left uterus of the rat was used for IUA modeling, and the right was used for IUA + eMSC therapy. GFP-labeled cells (2 × 106 cells dissolved in 20 µl DMEM) were injected into the left uterus of each rat, and the same volume of DMEM was injected into the right uterus as a control.

In vivo fluorescence imaging

Cardiac perfusion with normal saline was performed after the rats were anesthetized, and the uterus was subjected to imaging with an in vivo imaging system (Perkin Elmer, IVIS Spectrum, USA). The images were obtained at 1 d, 3 d, and 10 d after transplantation.

Flow cytometry to identify GFP-expressing cells

After the uterus was removed, the endometrial tissue of rats was digested according to a previously described cell isolation method. Two groups of single-cell suspensions were obtained by digesting each side uterus of rats. Single cells were dissolved in FSCS buffer before analysis. Then, IUA modeling was used as a negative control, and IUA + eMSC therapy was used as the experimental group.

Hematoxylin and eosin (H&E) and MASSON staining

After the rat uterus was removed from the rat, paraformaldehyde was fixed at room temperature for at least 1 day and then embedded in paraffin. The sections were cut into 5–10 μm thick continuous sections. The HE and MASSON staining were used to assess the intimal thickness, number of glands, and degree of fibrosis consistent with previous methods [14]. Endometrial thickness was measured from the luminal epithelium to the smooth muscle layer with imaging. The average of the measurement results of two mutually perpendicular lines is taken.

Fertility test

Eight- to ten-week-old Sprague Dawley female rats weighing 180–220 g were used in the cell therapy experiment. Group 1 (3VS3) was the sham operation group on the left and the control (without any treatment). In group 2, the left uterus was the sham-operated side, and the right side was the IUA side (15VS15). In group 3, the left uterus was the sham-operated side, and the right side was the IUA + R-eMSC therapy side (15VS15). In group 4, the left uterus was the sham-operated side, and the right side was the IUA + auto-eMSC therapy side (15VS15). On the 10th day after treatment, the female rats were housed together with healthy male rats at a ratio of 2:1. Discovery of the female rat vaginal plug was considered day 0 of pregnancy. The pregnant rats were sacrificed on gestation days 14–18, and the number of embryos on both sides was counted.

Statistical analysis

Statistical analysis was performed using GraphPad Prism 7 (San Diego, CA, USA). Quantitative data are represented as the mean ± standard deviation obtained from a minimum of three independent experiments. Statistical significance was determined by Student’s t-test or one-way analysis of variance, and *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 were considered to indicate a statistically significant difference.