Dorsomorphin dihydrochloride

mESCs were submitted to cardiac differentiation using a protocol adapted from Chen et al. [18 (link)]. mESCs were dissociated by 0.25% trypsin-EDTA and cultured using the hanging drop (HD) method to form embryoid bodies (EBs). Approximately 600 cells in each 20 µL drop of differentiation medium [high glucose (4.5 g/L) Dulbecco’s Modified Eagle’s medium (DMEM; Gibco) supplemented with 20% (v/v) FBS, 50 U/mL penicillin-streptomycin (Gibco), 2 mM L-glutamine (Sigma-Aldrich), 0.1 mM β-mercaptoethanol (Gibco), 1% (v/v) nonessential amino acids (Gibco), 2 µM dorsomorphin dihydrochloride (Tocris Bioscience) and 1% dimethyl sulfoxide (DMSO; Sigma-Aldrich)] were plated on the lids of 100 mm plates (Corning) and cultured as HD for 2 days. After that, EBs were transferred to 60 mm plates (Corning) coated with poly 2-hydroxyethyl methacrylate (Sigma-Aldrich) and cultured in suspension using a medium with the same specification, except for dorsomorphin. On day 5, EBs were transferred to 0.1% (v/v) gelatin-coated dishes (35 mm; Corning) and cultured in differentiation medium, without dorsomorphin and DMSO for ten more days. In this step, the culture media was changed every 2 days. On day 15, the cardiomyocytes derived from the mESC (CM-mESC) were obtained.

Sodium iodoacetate (IAA), 2.4-dinitrophenol (DNP), adenosine 2′,3′-cyclic monophosphate (2′,3′-cAMP), adenosine 2′-monophosphate (2′-AMP), adenosine 3′-monophosphate (3′-AMP) were from Sigma–Aldrich (St. Louis, MO). Dorsomorphin dihydrochloride and MRS 1754 were from Tocris Bioscience (Bristol, UK). 8-Bromoadenosine-2′3′-cyclic monophosphate (8-Br-2′3′-cAMP) was from BIOLOG (Bremen, Germany).

For local treatment with chemicals or proteins, AG1X2-formate beads (for chemicals) or Affigel Blue beads (BIO-RAD, 1537302; for BMP4) were soaked in different concentrations of the desired protein or chemical overnight at 4 °C. Beads were washed in PCS before grafting. Dimethyl sulfoxide (DMSO, 0.2%) or BSA (0.1%) was used to dilute the chemicals or proteins, respectively, and for soaking the control beads. Final concentrations used for microbead-soaking: 50 ng/μl recombinant human BMP4 (R&D systems, 312-BP), 200 µM dorsomorphin dihydrochloride (Tocris, 3093), 2 µM ionomycin (Sigma, I9657). For chemical treatment to the whole embryo, the chemical was diluted first in PBS (1:10 v:v) and then in egg albumen (9:10 v:v), which was used to culture the embryos (under the vitelline membrane). For treatments with VIVIT (Tocris, 3930) and BAY 11-7821 (Tocris, 1744), embryos were first soaked in the chemical diluted in PCS for 1 h, prior to culture with albumen containing the same concentration of the chemical. Final concentrations used for treatment of whole embryos: 20 µM dorsomorphin, 200 µM flufenamic acid (Sigma, F9005), 2 µM ionomycin, 50 µM, nicardipine (Sigma, N7510), 20 µM U73122 (Sigma, U6756), 20 µM U73343 (Sigma, U6881), 12 µM VIVIT (Tocris, 3930), 12.5 µM BAY 11-7821 (Tocris, 1744).

The study was approved by the Ethical Committee of the CHA University Bundang CHA Hospital, Republic of Korea (application number: KNC12005). Human adult dermal fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in DMEM (WelGENE, Daegu, Korea) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine (Invitrogen) and 1x penicillin/streptomycin (P/S) (all from Invitrogen, Carlsbad, CA, USA).
Human iPS cells were cultured on vitronectin XF (Primorigen Biosciences, Madison, USA) coated culture dishes using our recently established xeno-free/feeder-free hPSC culture medium with minor modifications [17 (link)]. Briefly, the medium consisted of DMEM/F12, 15% KnockOut SR XenoFree CTS, 1x nonessential amino acids (NEAA), 1x GlutaMAX, 0.1 mM β-mercaptoethanol, 1x P/S (all from Invitrogen), 10 ng/mL basic fibroblast growth factor (bFGF) (CHA Biotech Co., Daejeon, Korea), 10 nM trichostatin A (TSA) (Sigma-Aldrich, St. Louis, MO, USA), 5 uM Gö6983 (Tocris, Ellisville, MO, USA), and 1 mM dorsomorphin dihydrochloride (Tocris).

Gelatin, putrescine, selenium, progesterone, apotransferrin, glucose and insulin were obtained from Sigma (Steinheim, Germany). Accutase was from PAA (Pasching, Austria). FGF-2 (basic fibroblast growth factor), noggin and sonic hedgehog were obtained from R&D Systems (Minneapolis, MN, USA). Y-27632, SB-43154 and dorsomorphin dihydrochloride were from Tocris Bioscience (Bristol, UK). MatrigelTM was from BD Biosciences (Massachusetts, USA). All cell culture reagents were from Gibco/Invitrogen (Darmstadt, Germany) unless otherwise specified.

Plasma LH was measured using a sensitive sandwich ELISA37 (link) with a theoretical detection range of whole blood mLH (in a 1:30 dilution) of 0.117–30 ng ml⁻¹. The intra- and interassay coefficients of variation were 6.05% and 4.29%, respectively. The GnRH antagonist cetrorelix acetate (Sigma, 0.5 mg Kg⁻¹) was injected i.p. 1 h before i.c.v. injection, while the ALK inhibitor (dorsomorphin dihydrochloride, Tocris, 100 μM) was injected intravenously 2 h before. Animals were killed by cervical dislocation and trunk blood was collected in sterile Eppendorf tubes and left on ice until centrifugation, plasma was frozen and stored at −80 °C until use. Plasma FSH and AMH levels were measured using, respectively, a commercial ELISA kit (Endocrine Technologies, Inc.) and a competitive ELISA kit (CUSABIO; #CSB-E13156m), following manufacturer's instructions.