Dine Blue. (Scale bar, 20 m.) (I) Comparison of DNA content per

Dine Blue. (Scale bar, 20 m.) (I) Comparison of DNA content per particle in four different preparations of ESCu, ESCd <40, ESCd >40 to <70, and ESCd >70. Error bars indicate means ?SE (n = 3). *P < 0.05, significant difference from ESCu. (J) Enlarged view (Scale bar, 2 m.) of STB area showing Necrosulfonamide solubility microvilli on the cell surface.cellular material, many of which appeared to have aggregated together during the cytospin procedure (Fig. 2D), the 40-m to 70-m fraction also contained similar, but generally less extensive, sheets of cells (Fig. 2C). The intertwined sheets from both cell size fractions contained many nuclei. Semithin and thin buy GW0742 sections of cells in the >70-m (Fig. 2 H and J) and 40-m to 70-m fractions (Fig. 2G) indicated that they were composed largely but not entirely of syncyitium as defined by nuclei in a common cytoplasm. It was also evident that some surfaces of the syncytial areas were densely covered with microvilli (Fig. 2J). Finally, whereas the average DNA per cell in the H1 undifferentiated embryonic stem cells (ESCu, Fig. 2I) was about 3 pg, consistent with the presence of a single diploid nucleus, average values for the <40-m fraction were slightly higher. By contrast, the DNAE2600 | www.pnas.org/cgi/doi/10.1073/pnas.content per particle in the 40-m to 70-m and >70-m fractions was 48.8 ?14.5 pg and 110 ?11.7 pg, respectively. These data, along with the information presented in SI Appendix, Table S3 suggest that the number of nuclei per cellular particle in the two larger cell fractions was 16 and 37, respectively, and that the 40-m to 70-m and >70-m fractions together comprised about 0.167 of the cells and 5 of the total cellular DNA recovered. Yields of multicellular sheets from the same fractions from two different induced pluripotent stem cell (iPSC) lines derived from umbilical cord mesenchyme (MRucAi and MRuc3i) treated under similar BAP conditions were not greatly different (0.089 and 0.126 , respectively) from those achieved with H1 ESCs, although the experiments with iPSCs have only been performed once.Yabe et al.Fig. 3. Immunostained colonies at day 8 of differentiation (A, E , and I ) and identical staining of three size-fractionated groups of dissociated cells deposited on glass slides by the cytospin procedure (B , H, and L). In all cases, hESCs were BAP treated for 8 d. All specimens were counterstained with DAPI (A , G, H, K, and L). (Scale bars, 100 m.)Additional Visible Phenotypic Features of the STB Fraction. When stained in situ at day 8, the presumed sycytial areas within the BAP-treated colonies were positive for CGA (Fig. 3 A and I), CGB (Fig. 3 F and G), ERVW-1 (Fig. 3 J and K), and negative for HLA-G (Fig. 3 E and G). The staining for CGA often appeared to be perinuclear, consistent with a Golgi localization (Fig. 3I and SI Appendix, Fig. S2M), but the same presumed syncytial regions positive for CGA were also positive for ERVW-1, with staining for the latter more uniform than for CGA (Fig. 3J). Similarly, the sheets of cells present in the 40-m to 70-m (Fig. 3C) and the >70-m (Fig. 3D) fractions isolated from such cultures at day 8 were all CGA-positive and HLA-G negative. Some, but not all, expressed CGB (Fig. 2H). For the remainder of this paper, we concentrate mainly on the >70-m fraction, which appears to be largely syncytial and less contaminated with mononucleated cells than the 40-m to 70-m fraction. The second subunit of hCG, CGB, was rarely detected in day 4 cultures when CGA could first.Dine Blue. (Scale bar, 20 m.) (I) Comparison of DNA content per particle in four different preparations of ESCu, ESCd <40, ESCd >40 to <70, and ESCd >70. Error bars indicate means ?SE (n = 3). *P < 0.05, significant difference from ESCu. (J) Enlarged view (Scale bar, 2 m.) of STB area showing microvilli on the cell surface.cellular material, many of which appeared to have aggregated together during the cytospin procedure (Fig. 2D), the 40-m to 70-m fraction also contained similar, but generally less extensive, sheets of cells (Fig. 2C). The intertwined sheets from both cell size fractions contained many nuclei. Semithin and thin sections of cells in the >70-m (Fig. 2 H and J) and 40-m to 70-m fractions (Fig. 2G) indicated that they were composed largely but not entirely of syncyitium as defined by nuclei in a common cytoplasm. It was also evident that some surfaces of the syncytial areas were densely covered with microvilli (Fig. 2J). Finally, whereas the average DNA per cell in the H1 undifferentiated embryonic stem cells (ESCu, Fig. 2I) was about 3 pg, consistent with the presence of a single diploid nucleus, average values for the <40-m fraction were slightly higher. By contrast, the DNAE2600 | www.pnas.org/cgi/doi/10.1073/pnas.content per particle in the 40-m to 70-m and >70-m fractions was 48.8 ?14.5 pg and 110 ?11.7 pg, respectively. These data, along with the information presented in SI Appendix, Table S3 suggest that the number of nuclei per cellular particle in the two larger cell fractions was 16 and 37, respectively, and that the 40-m to 70-m and >70-m fractions together comprised about 0.167 of the cells and 5 of the total cellular DNA recovered. Yields of multicellular sheets from the same fractions from two different induced pluripotent stem cell (iPSC) lines derived from umbilical cord mesenchyme (MRucAi and MRuc3i) treated under similar BAP conditions were not greatly different (0.089 and 0.126 , respectively) from those achieved with H1 ESCs, although the experiments with iPSCs have only been performed once.Yabe et al.Fig. 3. Immunostained colonies at day 8 of differentiation (A, E , and I ) and identical staining of three size-fractionated groups of dissociated cells deposited on glass slides by the cytospin procedure (B , H, and L). In all cases, hESCs were BAP treated for 8 d. All specimens were counterstained with DAPI (A , G, H, K, and L). (Scale bars, 100 m.)Additional Visible Phenotypic Features of the STB Fraction. When stained in situ at day 8, the presumed sycytial areas within the BAP-treated colonies were positive for CGA (Fig. 3 A and I), CGB (Fig. 3 F and G), ERVW-1 (Fig. 3 J and K), and negative for HLA-G (Fig. 3 E and G). The staining for CGA often appeared to be perinuclear, consistent with a Golgi localization (Fig. 3I and SI Appendix, Fig. S2M), but the same presumed syncytial regions positive for CGA were also positive for ERVW-1, with staining for the latter more uniform than for CGA (Fig. 3J). Similarly, the sheets of cells present in the 40-m to 70-m (Fig. 3C) and the >70-m (Fig. 3D) fractions isolated from such cultures at day 8 were all CGA-positive and HLA-G negative. Some, but not all, expressed CGB (Fig. 2H). For the remainder of this paper, we concentrate mainly on the >70-m fraction, which appears to be largely syncytial and less contaminated with mononucleated cells than the 40-m to 70-m fraction. The second subunit of hCG, CGB, was rarely detected in day 4 cultures when CGA could first.