A six-min incubation with Gentle Cell Dissociation Reagent followed by complete GW 4064 AZD3759MedChemExpress AZD3759 solubility physical dispersions by repeated pipetting. Large cells (>70 m) were then collected by passing the suspension successively through a series of nylon cell strainers (70 m and 40 m, Fisher Scientific) to generate three cell size fractions (>70 m, 40?0 m, and <40 m). The captured cells on the strainer were recovered by inverting the strainer and rinsing with culture medium. Similarly, middle size cells (40?0 m) were obtained from the <70-m fraction by collection on a 40-m cell strainer after removal of <70-m cells. The three size fractions were used in all subsequent analyses. Portions of the size-fractioned cells were fixed in 4 (vol/vol) paraformaldehyde/PBS solution for 15 min and resuspended (5.0 ?102 and 1.0 ?105 cells/100 L PBS). The suspensions were loaded into a Shandon single cytofunnel and centrifuged for 5 min at 1,000 ?g in a Shandon CytoSpin 4 cytocentrifuge (Thermo Scientific). Derivation Primary Human PHTu and PHTd. Placental tissue samples were collected by the Obstetrical Specimen Procurement Unit at Magee-Womens Hospital of the University of Pittsburgh Medical Center. The work was performed under an exempt protocol approved by the Institutional Review Board (IRB) at the University of Pittsburgh. Under the protocol, patients provided written consent for the use of deidentified discarded tissues for research upon admittance to the hospital. Primary villous PHTs were derived and cultured according to published procedures (12, 49, 50) from three term human placentas (one female and two males). Multiple primary cultures were established from each placenta at a density of 3.5 ?105 cells/cm2 in DMEM supplemented with 10 (vol/vol) FBS and antibiotics under a 5 (vol/vol) CO2/air atmosphere at 37 . Triplicate cultures from each placenta were harvested at 9 h (PHTu) before syncytium formation and subsequently at 48 h (PHTd) when syncytium formation had occurred. The sieving technique used for the hESC-derived cells was thus unnecessary for the term placental STB. Total RNA was extracted from each sample (3 ?3 at 9 h and 48 h, respectively) to provide a total of 18 samples for RNA-seq analysis. Additional methods are described in SI Appendix, SI Materials and Methods. ACKNOWLEDGMENTS. We thank L. C. Schulz for her critical reading of the manuscript and her helpful comments; N. J. Bivens for RNA-seq; W. Spollen, C. Bottoms, and S. Givan for their sequence data analysis; Y. Tian for immunoassays; A. Jurkevich for technical assistance; M. Schauflinger, D. Grant, and T. A. White for sharing equipment; and D. F. Reith for his editorial assistance and administrative support. This study was supported by NIH Grant R01HD077108 (to T.E. and D.J.S.) and Grant R01HD067759 (to R.M.R.).7. Loke YW, King A (1995) Human Implantation: Cell Biology and Immunology (Cambridge Univ Press, Cambridge). 8. Miller RK, et al. (2005) Human placental explants in culture: Approaches and assessments. Placenta 26(6):439?48. 9. Sim CM, Sibley CP, Jones CJ, Turner MA, Greenwood SL (2001) The functional regeneration of syncytiotrophoblast in cultured explants of term placenta. Am J Physiol Regul Integr Comp Physiol 280(4):R1116 1122. 10. Wice B, Menton D, Geuze H, Schwartz AL (1990) Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res 186(2):306?16. 11. Orendi K, Gauster M, Moser G, Meiri H, Huppertz B (2010) The choriocarcinoma cell line BeW.A six-min incubation with Gentle Cell Dissociation Reagent followed by complete physical dispersions by repeated pipetting. Large cells (>70 m) were then collected by passing the suspension successively through a series of nylon cell strainers (70 m and 40 m, Fisher Scientific) to generate three cell size fractions (>70 m, 40?0 m, and <40 m). The captured cells on the strainer were recovered by inverting the strainer and rinsing with culture medium. Similarly, middle size cells (40?0 m) were obtained from the <70-m fraction by collection on a 40-m cell strainer after removal of <70-m cells. The three size fractions were used in all subsequent analyses. Portions of the size-fractioned cells were fixed in 4 (vol/vol) paraformaldehyde/PBS solution for 15 min and resuspended (5.0 ?102 and 1.0 ?105 cells/100 L PBS). The suspensions were loaded into a Shandon single cytofunnel and centrifuged for 5 min at 1,000 ?g in a Shandon CytoSpin 4 cytocentrifuge (Thermo Scientific). Derivation Primary Human PHTu and PHTd. Placental tissue samples were collected by the Obstetrical Specimen Procurement Unit at Magee-Womens Hospital of the University of Pittsburgh Medical Center. The work was performed under an exempt protocol approved by the Institutional Review Board (IRB) at the University of Pittsburgh. Under the protocol, patients provided written consent for the use of deidentified discarded tissues for research upon admittance to the hospital. Primary villous PHTs were derived and cultured according to published procedures (12, 49, 50) from three term human placentas (one female and two males). Multiple primary cultures were established from each placenta at a density of 3.5 ?105 cells/cm2 in DMEM supplemented with 10 (vol/vol) FBS and antibiotics under a 5 (vol/vol) CO2/air atmosphere at 37 . Triplicate cultures from each placenta were harvested at 9 h (PHTu) before syncytium formation and subsequently at 48 h (PHTd) when syncytium formation had occurred. The sieving technique used for the hESC-derived cells was thus unnecessary for the term placental STB. Total RNA was extracted from each sample (3 ?3 at 9 h and 48 h, respectively) to provide a total of 18 samples for RNA-seq analysis. Additional methods are described in SI Appendix, SI Materials and Methods. ACKNOWLEDGMENTS. We thank L. C. Schulz for her critical reading of the manuscript and her helpful comments; N. J. Bivens for RNA-seq; W. Spollen, C. Bottoms, and S. Givan for their sequence data analysis; Y. Tian for immunoassays; A. Jurkevich for technical assistance; M. Schauflinger, D. Grant, and T. A. White for sharing equipment; and D. F. Reith for his editorial assistance and administrative support. This study was supported by NIH Grant R01HD077108 (to T.E. and D.J.S.) and Grant R01HD067759 (to R.M.R.).7. Loke YW, King A (1995) Human Implantation: Cell Biology and Immunology (Cambridge Univ Press, Cambridge). 8. Miller RK, et al. (2005) Human placental explants in culture: Approaches and assessments. Placenta 26(6):439?48. 9. Sim CM, Sibley CP, Jones CJ, Turner MA, Greenwood SL (2001) The functional regeneration of syncytiotrophoblast in cultured explants of term placenta. Am J Physiol Regul Integr Comp Physiol 280(4):R1116 1122. 10. Wice B, Menton D, Geuze H, Schwartz AL (1990) Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res 186(2):306?16. 11. Orendi K, Gauster M, Moser G, Meiri H, Huppertz B (2010) The choriocarcinoma cell line BeW.
