Influence of culture media on the derivation and phenotype of fetal-derived placental mesenchymal stem/stromal cells across gestation

Abstract

IntroductionDerivation of pure fetal placental mesenchymal stem/stromal cells (pMSCs) is key to understand their role in placental development. However, isolated pMSCs are often contaminated by maternal-derived decidual MSCs (dMSCs). EGM-2 medium promotes the derivation of term fetal pMSCs, but the extent of first-trimester maternal pMSC contamination remains unclear. Culture media can also affect MSC phenotype. Here, we examined the effects of culture media on maternal pMSC contamination and fetal pMSC phenotype across gestation. MethodspMSCs were derived from first-trimester or term placentae in advanced-DMEM/F12 medium or EGM-2 medium. Proportions of maternal (XX) and fetal (XY) cells in male pMSC cultures were determined by fluorescence in-situ hybridization. pMSC phenotype was analysed by flow cytometry, immunohistochemistry and Alamar blue proliferation assays. ResultsWhen derived in advanced-DMEM/F12, all first trimester pMSC isolates exhibited maternal contamination (>72% XX cells, n = 5), whilst 7/9 term pMSC isolates were >98% fetal. When derived in EGM-2, all first trimester (n = 4) and term (n = 9) pMSC isolates contained 95–100% fetal cells. Fetal pMSCs in EGM-2 proliferated 2-fold (first-trimester) or 4-fold (term) faster than those in advanced-DMEM/F12 (p < 0.05, n = 3). Fetal pMSCs in both media expressed the generic MSC marker profile (CD90+, CD105+, CD73+, CD31-, CD34-, CD144-). However, pMSCs transferred from EGM-2 to advanced-DMEM/F12 increased expression of smooth muscle cell markers calponin and α-smooth muscle actin, and decreased expression of the vascular cell marker VEGFR2 (n = 3). ConclusionsDeriving first-trimester pMSC in EGM-2 dramatically reduces maternal dMSC contamination. Media affects fetal pMSC phenotype, and careful consideration should be given to application specific culture conditions.