Be identified but was expressed by day 6 (SI Appendix, Fig. S

Be identified but was expressed by day 6 (SI Appendix, Fig. S2N). However, at day 6 its expression was only partially coincident with that of CGA in subzones of the developing syncytial patches as viewed both in the intact colonies (SI Appendix, Fig. S2 M, N, and P) and in the isolated >70-m fraction (SI Appendix, Fig. S2 Q ). Another antigen, -aminobutyric acid A receptor, Pi (GABRP) that has previously been used as a trophoblast marker (18, 19), had a particularly interesting expression pattern. Its zones of expression within BAP-treated colonies at day 4 included only small areas that were CGA-positive (SI Appendix, Fig. S2 A ). However, by day 6 there was more complete but not perfect coincidence of GABRP and CGA immunofluorescence (SI Appendix, Fig. S2 E ). Gene expression data for the gene encoding gamma aminobutyric acid A receptor pi (GABRP), CGA, and CGB5 (one of six CGB genes), measured by microarray mirrored that obtained by immunohistochemistry (SI Appendix, Fig. S2 U ). GABRP expression changed from very low at 24 h to high at 72 h and 120 h (SI Appendix, Fig. S2U). Whereas CGA expression appeared to lag behind that of GABRP slightly (SI Appendix, Fig. S2V), CGB5 transcripts were not detectable at all at 72 h, but were up-regulated by 120 h (SI Appendix, Fig. S2W). Transcriptome Profile of the Size-Fractionated, Differentiated Cells and Comparison with Initiating H1 hESCs. Three separate experi-ments were performed to generate RNA from size-fractionated,Yabe et al.ESC-derived cells. RNA-sequencing (RNA-seq) analysis was performed on each RNA preparation. In addition, RNA was isolated and analyzed in parallel from three different cultures of H1 ESCs that had been maintained in the same basal medium as had been used in the BAP treatments, except it lacked BMP4, A83-01, and PD173074 and had been supplemented with FGF2 (4 ng/mL). Previous studies have shown that culture in atmospheric TAPI-2 web oxygen (20 ) conditions for an extended period can lead to a limited amount of spontaneous differentiation in H1 cells (20). Such changes may have occurred in one of the three ESC H1 samples (ESCu) samples shown in Fig. 4A, which appeared as an outlier relative to the other two samples (first lane in Fig. 4A). Based on the most GW 4064 chemical information highly regulated genes among all of the samples, the analysis was able to distinguish the ESCs from the mononuclear <40-m fraction and the >70-m STB fractions isolated after 8-d BAP-driven differentiation of the H1 cells. The <40-m fraction and the >70-m STB fractions also clustered separately, albeit closely, from each other and from the samples obtained from term placentas. The close clustering of the the <40-m and >70-m fractions by principal component analysis (Fig. 5B) probably reflects the fact that a continuum exists in the phenotypes created during the differentiation process initiated by BAP rather than gross cross-contamination. Importantly, the expression of many trophoblast marker genes, including ones considered to be characteristic of STB, were highly enriched in the >70-m relative to the <40-m fraction (Fig. 4B; see below), whereas a few more were down-regulated. The RNA-seq transcriptome analysis provides further evidence that BAP-differentiated hESC is composed only of trophoblast. Of 63 trophoblast gene markers examined (18, 21), only one, CDH1 (encoding E-cadherin), was not up-regulated in the ESC >70fraction compared with the ESCu (Fig. 5A). It should be emphasized that CDH1 was expressed robustly in all.Be identified but was expressed by day 6 (SI Appendix, Fig. S2N). However, at day 6 its expression was only partially coincident with that of CGA in subzones of the developing syncytial patches as viewed both in the intact colonies (SI Appendix, Fig. S2 M, N, and P) and in the isolated >70-m fraction (SI Appendix, Fig. S2 Q ). Another antigen, -aminobutyric acid A receptor, Pi (GABRP) that has previously been used as a trophoblast marker (18, 19), had a particularly interesting expression pattern. Its zones of expression within BAP-treated colonies at day 4 included only small areas that were CGA-positive (SI Appendix, Fig. S2 A ). However, by day 6 there was more complete but not perfect coincidence of GABRP and CGA immunofluorescence (SI Appendix, Fig. S2 E ). Gene expression data for the gene encoding gamma aminobutyric acid A receptor pi (GABRP), CGA, and CGB5 (one of six CGB genes), measured by microarray mirrored that obtained by immunohistochemistry (SI Appendix, Fig. S2 U ). GABRP expression changed from very low at 24 h to high at 72 h and 120 h (SI Appendix, Fig. S2U). Whereas CGA expression appeared to lag behind that of GABRP slightly (SI Appendix, Fig. S2V), CGB5 transcripts were not detectable at all at 72 h, but were up-regulated by 120 h (SI Appendix, Fig. S2W). Transcriptome Profile of the Size-Fractionated, Differentiated Cells and Comparison with Initiating H1 hESCs. Three separate experi-ments were performed to generate RNA from size-fractionated,Yabe et al.ESC-derived cells. RNA-sequencing (RNA-seq) analysis was performed on each RNA preparation. In addition, RNA was isolated and analyzed in parallel from three different cultures of H1 ESCs that had been maintained in the same basal medium as had been used in the BAP treatments, except it lacked BMP4, A83-01, and PD173074 and had been supplemented with FGF2 (4 ng/mL). Previous studies have shown that culture in atmospheric oxygen (20 ) conditions for an extended period can lead to a limited amount of spontaneous differentiation in H1 cells (20). Such changes may have occurred in one of the three ESC H1 samples (ESCu) samples shown in Fig. 4A, which appeared as an outlier relative to the other two samples (first lane in Fig. 4A). Based on the most highly regulated genes among all of the samples, the analysis was able to distinguish the ESCs from the mononuclear <40-m fraction and the >70-m STB fractions isolated after 8-d BAP-driven differentiation of the H1 cells. The <40-m fraction and the >70-m STB fractions also clustered separately, albeit closely, from each other and from the samples obtained from term placentas. The close clustering of the the <40-m and >70-m fractions by principal component analysis (Fig. 5B) probably reflects the fact that a continuum exists in the phenotypes created during the differentiation process initiated by BAP rather than gross cross-contamination. Importantly, the expression of many trophoblast marker genes, including ones considered to be characteristic of STB, were highly enriched in the >70-m relative to the <40-m fraction (Fig. 4B; see below), whereas a few more were down-regulated. The RNA-seq transcriptome analysis provides further evidence that BAP-differentiated hESC is composed only of trophoblast. Of 63 trophoblast gene markers examined (18, 21), only one, CDH1 (encoding E-cadherin), was not up-regulated in the ESC >70fraction compared with the ESCu (Fig. 5A). It should be emphasized that CDH1 was expressed robustly in all.