Ression in the two types of STB, there were a few striking differences. In particular, SLC28A2, SLC13A4, SLC7A2, SLC35F2, and SLC38A3 were much more strongly SB 202190 custom synthesis expressed in ESCd >70 than in PHTd, whereas the reverse was true for SLC40A1, SLC44A2, SLC43A2, and SLC38A9. The inferred roles of the protein products of these genes is shown in SI Appendix, Table S6. Of the 81 known ATP-binding cassette (ABC) transporter genes, 25 were significantly expressed above background in both the ESCd >70 and PHTd (SI Appendix, Fig. S4B). Again there were some differences between the two kinds of STB. For example, transcripts for ABCG2, ABCA7, ABCD3, and ABCB4 were higher in ESCd >70 than the PHTd. Overall, however, the profiles were quite similar. A brief summary of the likely functionsPNAS | Published online April 5, 2016 | EDEVELOPMENTAL BIOLOGYFig. 5. Fold-change differences in relative expressions of known trophoblast gene markers in comparisons of different cell fractions: (A) >70-m size fraction (ESCd >70) from BAP-differentiated ESCs relative to control ESCs that had been cultured in parallel (ESCu); (B) >70-m size fraction (ESCd >70) from BAPdifferentiated ESCs relative to the <40-m fraction (ESCd <40); (C) >70-m size fraction (ESCd >70) from BAP-differentiated ESCs relative to STB generated from cultured cytotrophoblast isolated from term placentas (PHTd, C). CEP-37440MedChemExpress CEP-37440 Sixty-two transcripts of selected trophoblast markers are compared in A and B, whereas only 61 genes are shown in C. The missing gene in C is MRGPRX1 whose expression in PHTd was undetectable. Gray horizontal bars indicate where differences in gene expression are not significant (q > 0.05).SEE COMMENTARYPNAS PLUSRelative Expression of Genes for Pregnancy-Specific Glycoproteins and L Galectins in ESCd >70 and STB Generated from PHTd. Thepregnancy-specific glycoprotein (PSG) family of genes is expressed abundantly in the human placenta and can be detected in maternal serum, with concentrations rising as placental size increases (28). Interestingly, the PSG genes were expressed almost exclusively by PHTd, with only PSG4 transcripts detected above background in ESCd >70 (SI Appendix, Fig. S6D). Again, these data suggest that ESCd >70 collected at day 8 after BAP exposure represent STB from early stages of pregnancy. They further confirm the distinctiveness of ESCd >70 and PHTd. Certain members of a family of genes whose protein products encode -galactoside binding lectins are known to be highly expressed in placentas. The concentration of at least one of these antigens (LGALS13) rises in maternal serum as pregnancy progresses (29, 30). Transcripts for seven members of this large gene family were detectable in ESCd >70 and PHTd cells (SI Appendix, Fig. S6D), and, as with PSG members, expression was much higher in the latter than in the former.Fig. 6. RNA-seq ased gene expression values (FPKM) for CGB, CGB5, and CGB8 (A), CGA (B), and CGB7 and LHB (C) in different cell fractions. Asterisks indicate statistically significant differences (q < 0.0005) for expression in ESCd >70 (n = 3) relative to expression values in any other cell fraction. (D) Schematic diagram of the 58-kb region CGB/LHB gene loci at chromosome 19q13.3 based on the Ensembl database (51).of the protein products of the differentially expressed ABC genes are listed with the SLC in SI Appendix, Table S6.Potential for Steroid Metabolism in ESCd >70 and STB Generated from PHTd. Placental STB is actively involved in steroid metab-Metal.Ression in the two types of STB, there were a few striking differences. In particular, SLC28A2, SLC13A4, SLC7A2, SLC35F2, and SLC38A3 were much more strongly expressed in ESCd >70 than in PHTd, whereas the reverse was true for SLC40A1, SLC44A2, SLC43A2, and SLC38A9. The inferred roles of the protein products of these genes is shown in SI Appendix, Table S6. Of the 81 known ATP-binding cassette (ABC) transporter genes, 25 were significantly expressed above background in both the ESCd >70 and PHTd (SI Appendix, Fig. S4B). Again there were some differences between the two kinds of STB. For example, transcripts for ABCG2, ABCA7, ABCD3, and ABCB4 were higher in ESCd >70 than the PHTd. Overall, however, the profiles were quite similar. A brief summary of the likely functionsPNAS | Published online April 5, 2016 | EDEVELOPMENTAL BIOLOGYFig. 5. Fold-change differences in relative expressions of known trophoblast gene markers in comparisons of different cell fractions: (A) >70-m size fraction (ESCd >70) from BAP-differentiated ESCs relative to control ESCs that had been cultured in parallel (ESCu); (B) >70-m size fraction (ESCd >70) from BAPdifferentiated ESCs relative to the <40-m fraction (ESCd <40); (C) >70-m size fraction (ESCd >70) from BAP-differentiated ESCs relative to STB generated from cultured cytotrophoblast isolated from term placentas (PHTd, C). Sixty-two transcripts of selected trophoblast markers are compared in A and B, whereas only 61 genes are shown in C. The missing gene in C is MRGPRX1 whose expression in PHTd was undetectable. Gray horizontal bars indicate where differences in gene expression are not significant (q > 0.05).SEE COMMENTARYPNAS PLUSRelative Expression of Genes for Pregnancy-Specific Glycoproteins and L Galectins in ESCd >70 and STB Generated from PHTd. Thepregnancy-specific glycoprotein (PSG) family of genes is expressed abundantly in the human placenta and can be detected in maternal serum, with concentrations rising as placental size increases (28). Interestingly, the PSG genes were expressed almost exclusively by PHTd, with only PSG4 transcripts detected above background in ESCd >70 (SI Appendix, Fig. S6D). Again, these data suggest that ESCd >70 collected at day 8 after BAP exposure represent STB from early stages of pregnancy. They further confirm the distinctiveness of ESCd >70 and PHTd. Certain members of a family of genes whose protein products encode -galactoside binding lectins are known to be highly expressed in placentas. The concentration of at least one of these antigens (LGALS13) rises in maternal serum as pregnancy progresses (29, 30). Transcripts for seven members of this large gene family were detectable in ESCd >70 and PHTd cells (SI Appendix, Fig. S6D), and, as with PSG members, expression was much higher in the latter than in the former.Fig. 6. RNA-seq ased gene expression values (FPKM) for CGB, CGB5, and CGB8 (A), CGA (B), and CGB7 and LHB (C) in different cell fractions. Asterisks indicate statistically significant differences (q < 0.0005) for expression in ESCd >70 (n = 3) relative to expression values in any other cell fraction. (D) Schematic diagram of the 58-kb region CGB/LHB gene loci at chromosome 19q13.3 based on the Ensembl database (51).of the protein products of the differentially expressed ABC genes are listed with the SLC in SI Appendix, Table S6.Potential for Steroid Metabolism in ESCd >70 and STB Generated from PHTd. Placental STB is actively involved in steroid metab-Metal.