Rhactivina

To initiate pancreatic differentiation, the cells were dissociated using TrypLE Express to single cells and seeded at 150,000 cell/cm² onto 1:30 dilution of growth factor reduced Matrigel (BD Biosciences) in DMEM/F12 in E8-MEF conditional media with 10 uM Y27632. Two days following seeding the differentiation was started. Day 1 cells were exposed to RPMI +3 uM CHIR-99021 (Stemgent) +100 ng/ml rhActivinA (R&D Systems). Day 2–3: +100 ng/ml rhActivinA. Day 4–5: +50 ng/ml FGF7 (Peprotech). Day 6–9: DMEM +50 ng/ml FGF7 + 2 μM RA (Sigma) +0.25 μM SANT-1 (Sigma) +100 ng/ml rhNoggin (R&D Systems).
To generate neural progenitor cells, H1 hPSCs were cultured on Matrigel coated plates in E8 media for two days before induction to the neural program for five days DMEM/F12:Neurobasal media at 1:1 ratio +1xB27 + 1×N2 + 2mM Glutamax (all Invitrogen).
Prior to cardiac differentiation, hPSCs were passaged onto Matrigel coated plates. At 70–95% confluence cells were exposed to rhActivinA and WNT3A for 1 day (R&D Systems) in Advanced RPMI (Invitrogen) supplemented with 2% KOSR (Gibco), ascorbic acid (Sigma), NEAA (Gibco), BSA (Gibco) and thioglycerol (Sigma). For the next 2 days, cells were treated with ActivinA, WNT3A, BMP4, and transferrin; at day 4 with BMP4 and transferrin; from day 6 onwards, with basal differentiation medium only.

For cell stimulation with rhBMP6 (S. Vukicevic, University of Zagreb, Croatia), rhBMP9 (PeproTech, Hamburg, Germany), rhTGFβ-1 (PeproTech), and rhActivin-A (R&D Systems), growth factors were reconstituted and stored according to manufacturer instructions. For small-molecule–based inhibition (SMKI), K02288 (Alex Bullock, Oxford, UK) [120 (link)] and SB-431542 (Sigma-Aldrich) [47 (link)] were added to cells 1 h prior to ligand stimulation with indicated concentrations unless stated otherwise. For long-term Rho-kinase inhibition, Y-27632 (Stemcell Technologies) was added at the indicated concentration at time of cell-seeding and 12–48 h later.

To evaluate the BMP and TGFβ pathways, we stimulated fibroblasts with rhBMP2, rhTGFβ1 (PeproTech) or rhActivinA (R&D Systems) reconstituted in PBS for three time points (three biological replicates per sample). At day 1, 2 × 10⁵ fibroblasts were seeded in 6-well plates in 2 mL DMEM, 10% FCS, 1% l-Glutamine, and 1% pen/strep. The next day, media was changed to starvation media (DMEM without serum, 1% l-Glutamine and 1% pen/strep) and cells were starved for five hours. Next, cells were stimulated with BMP2 (5 nM), TGFβ1 (0.2 nM) or Activin (3 nM) in PBS for 0, 30, and 60 min. After stimulation, cells were washed with 1× PBS and added 1× SDS Laemmle buffer. Scraped protein lysates were boiled at 95 °C for 5 min and froze at −20 °C. At day 3, we analyzed SMAD1/5 and SMAD2/3 phosphorylation via western blot.