Isolation and culture of endMSCs:
1- Healthy pre-menopausal women, without infections or immune disorders and who are not undergoing hormonal therapy, can be eligible as potential menstrual fluid donors. Menstrual fluid can be collected by the use of a menstrual cup or in a sterile urine container on day 2 or 3 of the menstrual cycle.
2- Dilute blood samples in PBS. If endometrial tissue fragments are retrieved, mechanical disaggregation is required. Transfer the recovered tissue to a sterile Petri dish and use a scalpel blade to obtain smaller pieces. Add the disaggregated tissue to the diluted blood. Centrifuge at 450 × g for 10 minutes.
3- Discard the supernatants to remove the residues of cervical mucus and resuspend the cells in DMEM containing 10 % fetal bovine serum (FBS), 1% penicillin/streptomycin and 1 % glutamine. Seed the resuspended cells onto tissue culture flasks and incubate at 37 ˚C in 5 % CO2 atmosphere.
4- After 24 hours, remove non-adherent cells by washing with PBS. Once some colonies are formed, detach the adherent cells using PBS containing 0.25 % trypsin (v/v), seed them at a density of 5 000 cells/cm2, and expand to 80% confluency.
5- Change culture medium every three days.
1- Stain 2 × 105 endMSCs with the appropriate concentrations of monoclonal antibodies against CD29, CD31, CD34, CD44, CD45, CD49d, CD49f, CD56, CD73, CD90, CD105, and HLA-DR and incubate for 30 min at 4 ºC in the presence of PBS containing 2% FBS.
2- After incubation, wash the cells and resuspend in PBS.
3- Use isotype-matched antibodies as negative controls.
4- For flow cytometric analyses, acquire 105 events on a FACScalibur cytometer (BD Biosciences, CA, USA).
1- Culture cells with an 80 % of confluence in a differentiation media for 21 days (Stem Pro Adipogenesis, Chondrogenesis and Osteogenesis Differentiation Kits, Gibco, Thermo Fisher Scientific, MA, USA).
2- After 21 days, fix cells with a paraformaldehyde 4 % solution for 20 minutes.
3- Wash cells 3 times with PBS and stain differentiated cell:
- Adipogenic staining: incubate cells with isopropanol 60 % during 5 min, dry, stain with 3.5 mg/ml de Oil Red O in isopropanol 60 % for 5 min and, wash with distilled water.
- Chondrogenic staining: wash cells twice with HCl 0.1 M, stain with 10 ng/ml Alcian Blue 8GX (diluted in HCl 0.1 M) for 2 h and wash with HCl 0.1 M and distilled water.
- Osteogenic staining: stain with 20 mg/ml Alizarin Red S (pH = 4.1‐4.2) for 30 min and wash with distilled water.
4- Quantify cell differentiation by spectrophotometry determining the absorbance of the extracts at 490 nm (Oil Red O and Alizarin Red S staining) and at 600 nm (Alcian Blue 8GX).
5- Confirm the in vitro differentiations by microscopic examination at 20X magnification