Figure 4Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. On days 1 and 5 after seeding, cell fusion was monitored by staining for phalloidin (F-actin) (green) and DAPI (blue). Scale bar, 50 μm.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (B) Trophoblast cell fusion was quantified on days 1, 3, and 5 post-seeding. Values are means + SEM, derived independently from human placentas of three pregnancies.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (C) Quantification of the human βhCG hormone, normally secreted by functional syncytiotrophoblast cells, in the medium of a representative trophoblast culture on post-delivery days 2, 3 and 5.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (D) Gene set enrichment analysis (GSEA) of published datasets from human primary trophoblast cultures on days 5 and 2 (n=4 trophoblast culture of day 5 and day 2). Gene sets related to pregnancy, aging, immune response, cytokine activity, MAPK, JAK-STAT, NF-κB and metalloproteinase activity were found to be enriched in day 5 cultures, whereas on the same day gene sets related to cell cycle, E2F targets, mitosis and DNA replication were downregulated. Statistical significance was determined by permutation testing with normalized enrichment score (NES)Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the cell cycle-related genes p16, p21 and CCNE1 Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the syncytiotrophoblast markers GCM1, CGA and CGB (F). Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. (G) Immunoblot analysis of the indicated proteins in human trophoblast cells, derived independently from two placentas, on days 2 and 5.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the SASP components CCL2, CCL5, CCL8, IL6, IL1β, TGF-β and PDG-FC (H) in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.Human term placentas were dissected and cytotrophoblast cells were extracted and seeded. Quantitative RT-PCR analysis of expression of the metalloproteases MMP2, MMP3 and MMP9 (I), in human primary trophoblast cultures on days 5 and 2. Data information: Quantitative RT-PCR values are expressed as means +SEM. Results were obtained from trophoblast cultures derived from human placentas of four independent pregnancies. All RT-PCR experiments were performed in triplicates and repeated at least three times. Statistical significance was determined by unpaired two-tailed Student's t-test (*) p < 0.05; (**) p < 0.01.