Isolation of Endometrial Mesenchymal Stromal Cells

To obtain eMSC (CD140b⁺CD146⁺ cells), two separate positive magnetic bead selections were conducted [19 (link)]. After in vitro expansion, stromal cells were incubated with PE-conjugated anti-CD140b antibody (R&D Systems, Minneapolis, MN, USA) for 45 min at 4^oC and then with anti-mouse IgG1 coated magnetic microbeads (Miltenyi Biotec Inc.) for 15 min at 4^oC. The CD140b⁺ population was acquired by applying the cell suspensions to MS columns (Miltenyi Biotec Inc.) in a magnetic field. The CD140b⁺ stromal cells were cultured in fibronectin-coated plates containing growth medium at 37^oC in 5% CO₂ for one week to allow degradation of microbeads during cell expansion. When the plates reached 80% confluent, the cells were trypsinized and incubated with anti-CD146 antibody-coated microbeads (Miltenyi Biotec Inc.) for 15 min at 4^oC. CD140b⁺CD146⁺ eMSC were trapped in the column and flushed out for usage. The positive expression of CD140b and CD146 markers were confirmed by phenotypic study of eMSC [20 (link)]. Stromal cells at passage 1–4 were used in this study.