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Cells were fixed and immunostained with a standard protocol [15 (link)]. Antibodies used were: rabbit anti-Oct4 antibody (1/500; Santa Cruz; sc-9081), goat anti-Sox17 antibody (1/300; R&D; AF1924), mouse anti-Nkx6.1 (1/200; Developmental Studies Hybridoma Bank; F55A10), goat anti-Pdx1 (1/300; R&D; AF2419), mouse anti-Ngn3 (1/300; Developmental Studies Hybridoma Bank; F25A1B3), mouse anti-Insulin (1/600; Sigma-Aldrich; I2018), mouse anti-Glucagon (1/600; Sigma-Aldrich; G2654), rat anti-C-peptide (1/600; Developmental Studies Hybridoma Bank; GN-ID4), anti-rabbit IgG, Alexa Fluor 488 conjugated (1/600; Invitrogen; A21206), anti-goat IgG, Alexa Fluor 594 conjugated (1/600; Invitrogen; A11058), anti-mouse IgG, Alexa Fluor 488 conjugated (1/600; Invitrogen; A21202), anti-rat IgG, Alexa Fluor 594 conjugated (1/600; Invitrogen; A-11007). Nuclei were counterstained with DAPI (1/200; Dojindo; 340–07971) for 2D cultured cells and with TO-PRO3 (Thermo Fisher Scientific) for 3D cultured cells before specimens were mounted in Prolong Gold Antifade Reagent (Invitrogen). Immunostained specimens were examined with an inverted fluorescent microscope (Keyence, Japan) and confocal laser scanning microscope (OLYMPUS, Japan).

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.