Smoothened agonist sag

For motor neuron differentiation, cells were seeded at high density (5.2 × 10⁴ cell/cm²) on Matrigel (Corning, Flintshire, United Kingdom)-coated plates in IMDM 20% FBS and sequentially exposed to SB431542 hydrate (10 μM, Sigma-Aldrich, United States), LDN193189 (100 nM Stemgent, Cambridge, MA, United States), CHIR-99021 (3 μM, Tocris Bioscience, Bristol, United Kingdom), basic FGF (10 ng/ml Thermo Fisher Scientific, United States), ascorbic acid (10 µM Sigma-Aldrich, United States), RA (100 nM Sigma-Aldrich, United States), Smoothened agonist (SAG) (500 nM, Sigma-Aldrich, United States), DAPT (10 μM, Tocris Bioscience, Bristol, United Kingdom), BDNF (10 ng/ml, R&D system, United States), GDNF (20 ng/ml, R&D system, United States), CTNF (10 ng/mL R&D system, United States) β-mercaptoethanol (βMOH) (25 µM Sigma-Aldrich, United States), Forskolin (10 µM STEMCELL Technologies, Vancouver, British Columbia, Canada), IBMX (100 μM, R&D system, United States).
From day 4, the IMDM medium was gradually replaced with increasing concentrations of N2 medium (IMDM, 1% penicillin/streptomycin, 2 mM L-glutamine, N2 supplement 1:100, B27 w/o vitamin A (B27-) 1:50). At day 11, cells were detached and replated (1.3 × 10⁵cell/cm²) on polyornithine/laminin coated plates in Neurobasal medium (Thermo Fisher Scientific) supplemented with N2, B27 1:50. (Figure 1).

ES cells were differentiated into spinal motor neurons as previously described.^{33 ,34 (link)} Briefly, Hb9::GFP mouse embryonic stem cells were plated into ES cell medium (ES DMEM, ES FBS, glutamine, non-essential amino acids, nucleosides, 2-mercaptoethanol, LIF (Life Technologies)) at approximately 5 × 10⁵ cells per gelatinized T25 flask. After 24 hours the media was replaced, and on day 2 of culture, ES cells were trypsinized and placed in suspension culture in motor neuron media (Advanced-DMEM/F12, Neurobasal, and Knockout Serum Replacement (Life Technologies)) at 5 × 10⁵ cells per untreated 10 cm tissue culture dish. In suspension culture, the cells aggregated into embryoid bodies (EBs). Two days after initial seeding the EBs were split 1 : 4 and induced into motor neurons with 1 μM retinoic acid (RA) (Sigma) and 1 mM smoothened agonist (SAG) (Millipore). After 3 days of exposure to RA and SAG, the EBs displayed strong expression of Hb9 : GFP transgene.

Neurons were differentiated from iNPC’s as previously described⁴⁰ . Briefly, iNPCs are plated in a 6-well plate and cultured for 2 days in DMEM/F-12 medium with Glutamax supplemented with 1% NEAA, 2% B27 (Gibco) and 2.5 µM of DAPT (Tocris). On day 3, DAPT is removed and the medium is supplemented with 1 µM smoothened agonist (SAG; Millipore) and FGF8 (75 ng/ml; Peprotech) for additional 10 days. Neurons are replated at this stage. Subsequently SAG and FGF8 are withdrawn and replaced with BDNF (30 ng/ml; Peprotech), GDNF (30 ng/ml; Peprotech), TGF-b3 (2 mM; Peprotech) and dcAMP (2 mM, Sigma) for 15 days.

1321N1 astrocytoma cells were cultured in DMEM (Sigma-Aldrich) supplemented with 10% FBS (Gibco) and 5 U ml⁻¹ Penstrep (Lonza). Hb9-GFP mouse stem cells were cultured as described (Wichterle et al, 2002 (link)) and differentiated into motor neurons with 2 μM retinoic acid (Sigma-Aldrich) and 1 μM Smoothened Agonist (SAG) (Millipore) for 5 d. Embryoid bodies were then dissociated with papain. All cells were maintained in a 37°C incubator with 5% CO₂.

Mouse embryonic stem cells expressing GFP under the motor neuron (MN)-specific promoter HB9 (HBG3 cells; gift from Tom Jessell) were cultured on primary mouse embryonic fibroblasts (Millipore). For differentiation into MNs, cells were treated with trypsin and resuspended in DFK10 culture medium consisting of knockout DMEM/F12, 10% knockout serum replacement, 1% N2, 0.5% L-glutamine, 0.5% glucose (30% in water), and 0.0016% 2-mercaptoethanol. The cells were plated on non-adherent Petri dishes to allow formation of embryoid bodies. After 1 d of recovery, 2μM retinoic acid (Sigma) and 1μM Smoothened Agonist (SAG) (Millipore) were added to the medium every day for 5 days. Embryoid bodies were then dissociated with papain and sorted using the FACSAria™ III (BD Biosciences).