Xav939

Undifferentiated RUES2 hESCs (Female line, Rockefeller University, NIH registry number 0013) were plated at 1.6x10⁵ cells/cm² on Matrigel (BD) coated plates and maintained in an undifferentiated state with mouse embryonic fibroblast (MEF) conditioned media containing 5 ng/mL hbFGF (Peprotech, 100-18B). Directed differentiations using a monolayer platform were performed based on previous reports [27 (link)] with a modified protocol. Undifferentiated hESCs were plated as single cells as described previously and upon reaching appropriate confluency, treated with the Wnt/β-catenin agonist CHIR-99021 (1 μM, Cayman chemical, 13122) for 24 hours. Cells were then exposed to Activin A (R&D SYSTEMS, 338-AC-050) (100 ng/mL) in RPMI/B27 medium (day 0). After 17 hours, media was changed to RPMI/B27 medium containing BMP4 (R&D SYSTEMS, 314-BP-050) (5 ng/mL) and CHIR-99021 (1 μM, Cayman chemical,13122). On day 3, media was changed to RPMI/B27 medium containing the Wnt/β-catenin antagonist XAV-939 (1 μM; Tocris, 3748). Media was then changed on day 5 to RPMI/B27 medium. From day 0 to day 5, the B27 supplement utilized did not contain insulin (Invitrogen, 0050129SA). From day 7–14 a B27 supplement with insulin was used (Invitrogen, 17504044). For assays assessing the onset and rate of beating, cultures were analyzed independently during differentiation, with each well counted as n = 1.

For the systematic small-molecular inhibitor screen, PM organoids were generated and differentiated until day 3 (PSM) of our protocol. On day 3, media was replaced with fresh media containing combinations of small-molecule inhibitors targeting the FGF, WNT, BMP, and TGF-β signaling pathways at indicated concentrations. For targeting the WNT pathway, we used C59 (Tocris, Cat# 5148), XAV939 (Tocris, Cat# 3748), and CHIR99021 (Sigma-Aldrich, Cat# SML1046). For inhibiting the FGF pathway, we used PD173074 (Sigma-Aldrich, Cat# P2499). For inhibiting the BMP pathway, we used LDN193189 (Stemgent, Cat# 04-0074). For inhibition of the TGF-β pathway, we used A-83-01 (Tocris, Cat# 2939). Media was changed daily. We analyzed three replicates per condition in the primary screen and five replicates per condition in the secondary screen.

hiPSC-CMs were generated from the control LUMC0099iCTRL04 hiPSC line, which was derived from human fibroblasts extracted through skin biopsies from of a Caucasian woman. The LUMC0099iCTRL04 line is registered in the Human Pluripotent Stem Cell Registry (Seltmann et al., 2016 (link)), which contains all details pertaining to its generation and characterization (hPSCreg, 2019 ). hiPSC clones showing stem cell morphology were characterized for pluripotency marker expression and differentiation potential to hiPSC-CMs in BPEL medium (Ng et al., 2008 (link)) containing activin-A, BMP4, and CHIR99021 (Devalla et al., 2016 (link)). After 3 days, this medium was replaced by BPEL medium containing XAV939 (Tocris Biosciences) for ventricular differentiation (Ng et al., 2008 (link); Devalla et al., 2016 (link)). To differentiate hiPSC-CMs to atrial-like hiPSC-CMs, 1 μM all-trans retinoic acid (RA) was added (Devalla et al., 2015 (link)). Twenty days after differentiation, hiPSC-CMs were dissociated with TrypLE Select (Life Technologies), and plated at a low density (≈7.5 × 10⁴ cells) on Matrigel coated coverslips in BPEL medium (Devalla et al., 2016 (link)).

The HS578T cell line was obtained from the American Type Culture Collection (Manassas, VA, USA). Low passages of the cell line were used to avoid loss of fidelity to the original cells. Stable cell lines were derived from PIK3CA‐H1047R mouse mammary tumors. Cell lines derived from recurrent H1047R mouse tumors (RDR‐C234 and RDR‐A677) were kindly provided by Jean Zhao from Dana‐Farber Cancer Institute (Liu et al., 2011). Sources of other reagents were as follows: XAV939 (Tocris, Minneapolis, MN, USA); S31‐201 (SelleckChem, Houston, TX, USA); linsitinib, OSI‐906 (LC Labs, Woburn, MA, USA); LY294002 (Sigma‐Aldrich, St. Louis, MO, USA), and GDC‐0941 (Genentech, San Francisco, CA, USA). Neu‐N (c‐erbB2, ERBB2, Her‐2) mice were derived from the colony of Guy et al. (1992), bred to homozygosity as verified by Southern blot analysis (Reilly et al., 2000), and maintained at Johns Hopkins University.

The hiPSCs were differentiated into cardiomyocytes as previously described (Campostrini et al., 2021 (link)). One day prior to differentiation (d-1), the hiPSCs were harvested using TrypLE Select and plated onto Matrigel (1:100, Corning)-coated wells in StemFlex™ Medium containing RevitaCell™ Supplement (1:200 dilution). On d0, the cells were refreshed with mBEL medium containing 5 μM CHIR99021 (Axon Medchem). On differentiation d2, the cells were refreshed with mBEL medium containing 5 μM of XAV939 (Tocris) and 0.25 μM IWP-L6 (AbMole). From differentiation d4 on, the cells were maintained in mBEL medium. The hiPSC-CMs were cryopreserved at differentiation d20 or d21 as previously described in a freezing medium comprising of 90% Knockout Serum Replacement (Gibco) and 10% DMSO (Brink et al., 2020 (link)). Subsequent thawing and seeding of the cells were performed as previously described (Brink et al., 2020 (link); Campostrini et al., 2021 (link)).