Human and marmoset trophoblast stem cells differ in signalling requirements and recapitulate divergent modes of trophoblast invasion
Human and marmoset trophoblast stem cells differ in signalling requirements and recapitulate divergent modes of trophoblast invasion
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 