Bone morphogenetic protein 4 (bmp4)

Immortalized mouse cDC1 cells (22 (link)) were cultured in IMDM medium supplemented with 10% heat-inactivated FBS, 100x Glutamax, 10 mM HEPES, 100 μM Pen/Strep, 50 μM β-mercaptoethanol at 37° and 5% CO2. PBS supplemented with 20 mM HEPES and 5 mM EDTA was used for cell passaging. For activation, 2.5 x 10⁵ cells were seeded per well in a 12-well plate and incubated for 24 hrs with 5 μg/ml LPS and 10 ng/ml IFN-γ (485-MI, R&D Systems). Where indicated, cells were also incubated with 50 ng/ml recombinant Activin-A, TGF-β (Invitrogen), or BMP4 (Biotechne), or with 10 μM SB-431542. During the last 3 hrs of incubation, Golgi Plug (BD 555029) was added to the cells. Cells were then detached using PBS supplemented with 1% FBS and 2 mM EDTA, washed and stained for surface markers H2Kb FITC (Biolegend, clone AF88.5) and IA/IE AlexaFluor700 (BioLegend, clone M5/114.15.2), followed by fixation and permeabilization using FoxP3 staining buffer set (eBioscience) before intracellular staining with Ki67 eFluor 450 (Invitrogen, clone SolA15) and CXCL9 AlexaFluor647 (eBioscience, clone MIG-2F5.5). After the staining, cells were collected in FACS buffer (2% FBS, 2 mM EDTA in PBS), and data were acquired using an LSRII SORP or an LSR Fortessa cytometer (Becton Dickinson, Franklin Lakes, NJ, USA).

Anterior, posterior and late PS cells, respectively, were derived from hPSC by adapting previously published protocols^{19 (link)}. In brief, hPSC were dissociated to single cells before being plated at 25,000 cm⁻² in Essential 8^TM medium (E8; Thermo Fisher Scientific) containing 10 µM Y27632 onto glass coverslips that were thinly coated with 1% Geltrex^TM solution. On day 1, the medium was replenished with fresh E8. On day 2, the medium was replaced with anterior PS, posterior PS, and late PS differentiation medium, respectively, with the following composition: (1) anterior PS differentiation medium: Essential 6^TM medium (E6; Thermo Fisher Scientific) supplemented with 20 ng ml⁻¹ FGF2 (Peprotech), 10 µM LY294002 (Tocris), 25 ng ml⁻¹ BMP4 (R&D Systems), and 50 ng ml⁻¹ Activin A (R&D Systems); (2) posterior PS differentiation medium: E6 supplemented with 20 ng ml⁻¹ FGF2, 10 µM LY294002, and 50 ng ml⁻¹ BMP4; (3) late PS differentiation medium: E6 supplemented with 20 ng ml⁻¹ FGF2, and 8 µM CHIR99021 (Tocris). All differentiation protocols were carried out for 48 h before downstream analyses.

mESCs were seeded at a density of 9000 cells/well of a 12-well cell culture plate. On the next morning, mESCs were washed (once with DMEM) and then differentiated into either anterior primitive streak (APS) by adding 100 ng/mL Activin A (Thermo Fisher Scientific, #PHC9561) + 2 µM CHIR99021 (Peprotech, #2520691) + 20 ng/mL FGF2 (Thermo Fisher Scientific, # 13256029) or mid primitive streak (MPS) (30 mg/mL Activin A + 40 ng/mL BMP4 (Thermo Fisher Scientific, # PHC9533) + 6 µM CHIR99021 + 20 ng/mL FGF2) for 24 h. Subsequently, the cells were further differentiated into definitive endoderm with 100 ng/mL Activin A + 250 nM DM3189 (Peprotech, # 1062443) and lateral mesoderm with 1 µM A-83-01 (Tocris, #2939) + 30 ng/mL BMP4, respectively, for another 48 h. For experiments during directed differentiation toward mesoderm lineage, cells were pre-treated 4 days with a chemical inhibitor of EED (5 µM EED226, Selleckchem, #S8496) before the start of differentiation, and treatment was continued during differentiation, with a change of medium every 2 days.

Differentiation to specific embryonic lineages was performed in CDM-PVA, which has the same composition as CDM-BSA, with polyvinyl alcohol (PVA, 1 mg/ml, Sigma) instead of BSA. For early mesoderm differentiation, cells were grown in CDM-PVA with FGF2 (20 ng/ml), LY294002 (10 μM, Sigma) and BMP4 (10 ng/ml, R&D) for 36 h then treated with CDM-PVA with FGF2 (20 ng/ml) and BMP4 (50 ng/ml) for 3.5 days to generate LM as previously described (Cheung et al., 2012 (link)). To generate epicardium, LM cells were differentiated in CDM-PVA with BMP4 (50 ng/ml), recombinant human WNT3A (25 ng/ml, R&D systems) and RA (4 μM, Sigma) at a seeding density of 2.5×10³/cm² for 10 days. To investigate the role of WNT pathway, LM cells were differentiated with IWP2 (2 μM, Tocris) along with BMP4 and RA. Subsequently, epicardial cells were re-suspended in CDM-PVA with PDGF-BB (10 ng/ml, Peprotech) and TGFβ1 (2 ng/ml, Peprotech), designated as PT for 12 days. Epicardial cells were differentiated with PT in the presence of Y27632 (2 μg/ml, Calbiochem) to study the role of RhoA/RhoK in EPI-SMC differentiation. To generate EPI-CFs, epicardial cells were differentiated in CDM-PVA with VEGF (50 ng/ml, Peprotech) and FGF2 (50 ng/ml) for 12 days. HESCs were grown in CDM-PVA with FGF2 (12 ng/ml) and SB431542 (10 μM, Tocris) for 7 days to generate neuroectoderm (Vallier et al., 2009 (link)).