Wnt3a 5036 wn 010

Wnt3a-conditioned medium was harvested from Wnt3a expressing L-cells according to the manufacturer's instructions (ATCC). VSTM2A-conditioned medium and control medium were harvested from 293T cells transiently transfect with VSTM2A expression or empty control constructs. Briefly, 293T cells were seeded in 10 cm dishes and transfected with 16 ug of p3.1-VSTM2A-His/myc or p3.1 empty vector using Lipofectamine 2000 as directed by the manufacturer (Thermo Fisher Scientific). Conditioned medium was harvested 48 h after transfection. Recombinant human VSTM2A His-tag protein (10037-VT), LRP6 (1505-LR-025) and Wnt3a (5036-WN-010) were purchased from R&D.

Human embryonic stem cell (hESCs) lines H1 (WA01) and H9 (WA09), were purchased from the National Stem Cell Bank (NSCB; WiCell, Madison WI). Human NKX2-5(eGFP/w) ESCs were a gift from Professors A.G Elefanty and E.G. Stanley, Monash University [11 (link)]. Human induced pluripotent iPSK3 (K3) cells were a gift from Professor Stephan A. Duncan of this institution [12 (link)]. Human iPSC line 963 was provided by the Wanek Consortium for HLHS (Mayo Medical School and Children’s Hospital of Wisconsin). All experiments utilizing H1 cells were performed during passages 30–70. Activin-A (338-AC-005), Bmp4 (314-BP-010), and Wnt3a (5036-WN-010) were from R&D Systems. Fgf2 (bFgf) was from Invitrogen (PHG0026). The small MW organic inhibitors employed in these experiments were CHIR99021 (Stemgent 04-0004-2) and IWP2 (Tocris 3533). Primary antibodies employed for immunofluorescent staining included anti-Myosin Heavy Chain monoclonal (DSHB MF20); anti-Oct4 monoclonal (Chemicon MAB-4305) or rabbit polyclonal (Santa Cruz sc-9081); and anti-Sox17 goat polyclonal (R&D Systems AF1924). Antibodies for flow cytometry were monoclonal anti-cardiac Cardiac Troponin T (TNNT2; Thermo Scientific MS-295-R7) coupled with secondary goat anti-mouse 594 IgG1 (Invitrogen #A21125); Alexa Fluor 488-conjugated mouse myeloma IgG MOPC-21 (Thermo Scientific 557721) was used as an IgG1 isotype control.