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 ng/mL bFGF (Cambridge Stem Cell Institute). Cells were routinely cultured on mitomycin C (M4287, Sigma-Aldrich) inactivated mouse embryonic fibroblast (MEF) feeder cells (Cambridge Stem Cell Institute) under 5% O2 and 5% CO2 at 37 °C. The medium was changed daily, and cells were passaged every 2–4 days by dissociation with Accutase (00-4555-56, Thermo Fisher Scientific) for 5 min. Marmoset PSCs were reset chemically using as previously described (Bergmann et al., 2022a). In short, conventional marmoset PSCs were seeded as clumps of 2–5 cells one day before resetting at 50,000 cells per well of a 12-well plate (1.3 × 104 cells per cm2) on MEFs. After 24 h, the medium was changed to PLAXA, which comprised N2B27 medium (NDiff, Y40002, Takara Bio) supplemented with 1% chemically defined lipids, 1 μM PD0325901 (Cambridge Stem Cell Institute), 10 ng/ml recombinant human LIF (Cambridge Stem Cell Institute), 50 μg/ml 1 L-ascorbic acid (Sigma-Aldrich), 2 μM XAV939 (SM38-200, Cell Guidance Systems) and 20 ng/ml activin A (Cambridge Stem Cell Institute). Throughout conversion, cells were passaged with Accutase (00-4555-56, Thermo Fisher Scientific) 1:1.5 every 3–4 days. Dome-shaped colonies first emerge at day 4–5 and naïve conversion is complete by day 9.  Naïve PSCs were prepared as previously described on MEFs and cultured to 60-70% confluency. Naïve PSC media was removed and PSCs were washed in PBS. Media was replaced with indicated culture conditions and differentiated for 5 days.  OKAE conditions were as previously described (Okae et al., 2018). Other conditions include OK XAV PD: advanced DMEM/F12 supplemented with 55 μM β-mercaptoethanol, 0.3% BSA, 1% ITS-X supplement, 50 ng/ml EGF, 2 μM XAV939, 1 μM PD0325901, 1 mM A83-01, 1 mM SB431542, 0.7 mM VPA and 1 mM Y27632 and PAVS: 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, 1 mM A83-01, 1 mM SB431542, 0.7 mM VPA and 1 mM Y27632. Both conditions were cultured on inactive MEFS unless otherwise specified. 5µg/ml collagen IV coating was generated by resuspending collagen in PBS and coating at 37°C for 2 hours. Additional molecules added during screening includes 2 μM XAV939, 20ng/mL BMP4, 0.5μM LDN193189 (LDN), 20ng/mL Activin A, 100ng/mL FGF2, 2 µM CHIR99021 (CHIR), 3 μM IWP2, 20 μM forskolin, 50 ng/ml EGF, 50 ng/mL HGF, 2 mM A83-01, and 2.5 µM PGE2. Media was changed every 24 hours.  After 4-5 days or at 90% confluency, epithelial colonies were washed with PBS and dissociated with TrypLE for 6-8 minutes or until cells balled up and formed 2-3 cell clumps. TrypLE was carefully removed to not disturb cells and cells were resuspended and replated in culture media supplemented with 10 µM Y-27 for 24 hours. cmTSCs were passaged 1:2 every 7 days or until 80% confluence was reached. Cells were cultured in 5% CO2 and 5% O2 at 37°C. Sequencing libraries were prepared according to an adapted version of the G&T protocol previously described (Clark et al., 2018; Macaulay et al., 2017). Samples were transferred to a 96-well plate and prepared for transcriptome and methylome sequencing, by physical separation of genomic DNA from mRNA. Genomic DNA was frozen and stored for processing. For each sample, RNA separation was performed using biotinylated oligo-dT30VN-tailed oligonucleotides (IDT) conjugated to Dynabeads Streptavidin C1 (65001, Invitrogen) in a buffer solution supplemented with RNase inhibitor (RNAsin; N2615, Promega). Naïve derived OK, OK XAV PD, and PAVS cmTSCs were transferred into individual tubes containing RLT buffer and immediately frozen in dry ice. Smart-seq2 library preparation was carried out in 96-well format as previously described (Picelli et al., 2014). Library quality was assessed using the High Sensitivity DNA Analysis Kit (Agilent, 5067-4626) on the 2100 Bioanalyzer system (Agilent).