title: Human and marmoset trophoblast stem cells differ in signalling requirements and recapitulate divergent modes of trophoblast invasion
design: Human and marmoset trophoblast stem cells differ in signalling requirements and recapitulate divergent modes of trophoblast invasion
library_construction_protocol: Newf4 marmoset cells were a gift from the Sasaki lab: https://www.sciencedirect.com/science/article/pii/S1873506121000982?via%3Dihub cells were encapsulated as previously described (Kleine-Brüggeney et al., 2019; Munger et al., 2022; Schindler et al., 2021). In short, microfluidic devices were designed with two inlets for aqueous low-melt agarose and cells and a continuous oil phase. Cells were resuspended at 1.5 × 106 cells/100 μL in PBS with 3% BSA. The cell suspension was mixed 1:1 with low-melting-point agarose (Lonza) solution at 37°C. HFE-7500 (Fluorochem) supplemented with surfactant (0.3%; Pico-Surf by Sphere Fluidics) was used as the continuous oil phase. Syringes (SGE Analytical Science) controlled by automated pumps (CETONI, neMYSIS) injected the agarose-cell suspension separately from the oil-surfactant solution into the microfluidic chips for emulsification. Agarose droplets left the microfluidic chip through the outlet and were collected on ice for polymerization. PAVS_TSC: Passage 3 trophectoderm-like cells derived from Newf4 marmoset pluripotent stem cells. Newf4 conventional marmoset pluripotent stem cells were reset to a naïve state in PLAXA, and differentiated in advanced DMEM/F12 supplemented with 0.3% BSA, 1% ITS-X supplement, 55 μM β-mercaptoethanol, 1% chemically defined lipids, 1x GlutaMAX (Thermo Fischer), 1 μM PD0325901, 0.5 µM A83-01, 1µM SB431542, 0.7 mM VPA and 1 µM Y27632. OKXAVPD_TSC: Passage 3 cytotrophoblast-like cells derived from Newf4 marmoset pluripotent stem cells. Newf4 conventional marmoset pluripotent stem cells were reset to a naïve state in PLAXA, and differentiated in advanced DMEM/F12 supplemented with 55 μM β-mercaptoethanol, 0.3% BSA, 1% ITS-X supplement, 1.5 mg/ml L-ascorbic acid, 50 ng/ml EGF, 2 μM XAV939, 1 μM PD0325901, 0.5 µM A83-01, 1 µM SB431542, 0.7 mM VPA and 1 µM Y27632. OKAE: Passage 3 extraembryonic-like cells derived from Newf4 marmoset pluripotent stem cells in human trophoblast culture conditions (Okae et al. 2018).   Marmoset PSCs were maintained in KSR/bFGF medium, which is comprised of Dulbecco's modified Eagle medium (DMEM)/F12 (21331, Gibco) supplemented with 20% Knockout Serum Replacement (KSR) (10828028, Thermo Fisher Scientific), 1% GlutaMAX (35050061, Thermo Fisher Scientific), 1% MEM non-essential amino acids (11140035, Thermo Fisher Scientific), 100 μM β-mercaptoethanol (21985023, Thermo Fisher Scientific) and 10 