Pnu 74654

Feeder-free-adapted WA09 hESCs were seeded at a concentration of 1.0 × 10⁴ cells per cm² in BD Matrigel Basement Membrane Matrix (BD Bioscience) on coated six-well plates and cultured for 24 hr in human embryonic stem cell culture medium (STEMPRO hESC SFM; Life Technologies). The medium was then replaced with NPC differentiation medium (Dulbecco’s modified Eagle’s medium [DMEM]/F12, 20% Knockout Serum Replacement [KSR] [Life Technologies], 1× nonessential amino acids [NEAA; Life Technologies], 1× GlutaMAX [Life Technologies], and 0.1 mM 2-mercaptoethanol [Life Technologies]) supplemented with 20 ng/ml of midkine (MK; Millipore), and small molecules: 2 μM each of dorsomorphin (6-[4-(2-Piperidin-1-ylethoxy) phenyl]-3-pyridin-4-ylpyrazolo [1,5-a] pyrimidine; Sigma), A 83-01 (3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide; Tocris), and PNU-74654 (benzoic acid, 2-phenoxy-, 2-[(5-methyl-2-furanyl)methylene] hydrazide; Sigma). Medium was changed daily. The cells were split 1:3 on days 3 and 6 using Accutase (Life Technologies). hNPCs were harvested for transplantation 9 days after the start of the differentiation protocol. For methods describing gene expression arrays and generation of LUC+ cells, please see the Supplemental Experimental Procedures.

Recombinant human TGF-β2 was obtained from R&D Systems (Minneapolis, MN, USA). Inhibitors ICG-001 and PNU-74654 were purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA) and Calbiochem-EMD Millipore (Temecula, CA, USA), respectively. As to primary antibodies, fascin was from Millipore (Temecula, CA, USA), E-cadherin from BD Transduction Laboratories (Lexington, KY, USA), active β-catenin (clone 8E7) from Upstate (Lake Placid, NY, USA), MMP9 and GAPDH from Abcam (Cambridge, MA, USA), α-SMA fluorescein isothiocyanate (FITC) conjugated and unconjugated from Sigma-Aldrich Corp., Pan actin from Abcam. All secondary antibodies for immunofluorescence staining were purchased from Molecular Probes (Invitrogen, Eugene, OR, USA); secondary antibodies for Western blots were obtained from LI-COR Biosciences (Lincoln, NE, USA).

Cells from each cell line were transferred into 96-well plates containing the corresponding aforementioned medium with 5×10³ cells per well. Following incubation at 37°C for 3–5 h, cell adhesion was reached and 100 µl Dulbecco's modified Eagle's medium (Sigma-Aldrich; Merck KGaA) was added. Cells were cultured at 37°C with the different concentrations of leptin (0, 25, 50 and 100 mM; Sigma-Aldrich; Merck KGaA), and 10 µl cell counting kit-8 solution (Sigma-Aldrich; Merck KGaA) was added at 24, 48 72 and 96 h later. Following incubation for another 4 h, optical density values at 450 nm were measured using a microplate reader. For Wnt inhibitor PNU-74654 (20 µM; Sigma-Aldrich; Merck KGaA) treatment, 20 µM PNU-74654 and 100 mM leptin was added to the culture medium under the same conditions as aforementioned.