Activin A

The mouse ES cell lines, HM1 [67] (link) and BTBR10 [68] (link) were maintained in ES cell medium [69] (link) with 1000 U/ml LIF (Chemicon) on mitotically inactive primary mouse embryo fibroblasts. To initiate differentiation, ES cells were removed from feeders by dissociation using 0.05% trypsin and then plated onto tissue culture plates for two short successive periods (20–30 mins) to remove feeder layers. To induce differentiation, the cells were plated on CellBINDSurface dishes (Corning) precoated with 0.1% gelatin (Sigma) at a density of 5×10³ cells cm⁻² in ‘N2B27’ medium. This medium comprised Advanced Dulbecco's Modified Eagle Medium F12 (Gibco) and Neurobasal medium (Gibco) (1∶1), supplemented with 1×N2 (Gibco), 1×B27 (Gibco), 2 mM L-glutamine (Gibco), 40 µg/ml BSA (Sigma), 0.1 mM 2-mercaptoethanol. Cells were grown in N2B27 supplemented with 10 ng/ml bFgf (R&D) for 3 days (D1–D3) and then were transferred into serum free media without bFgf (D3–D5). To induce ventral hindbrain identity NPCs (N_H) 100 nM RA (Sigma) and 500 nM SAG (Calbiochem) was added from D3–D5. Spinal cord identity (N_P) was induced by the addition of 5 µM CHIR99021 (Axon) or 100 ng/ml Wnt3a (R&D) from D2 to D3 followed by 100 nM RA, 500 nM SAG from D3–D5. To induce mesodermal differentiation the cells were treated with CHIR99021 from D2–D5. To induce terminal differentiation, cells were trypsinised and plated as single-cell suspension on plates coated with Matrigel (BD Biosciences) at a density of 1×10⁵ cells cm⁻² in N2B27 medium supplemented with bFgf (10 ng/ml). The next day bFgf was removed and cells were left to differentiate for an additional 3 days.
The mouse EpiSC line R04-GFP [55] (link) was routinely maintained in N2B27 supplemented with Activin A (20 ng/ml; R&D Systems) and bFgf (10 ng/ml; Peprotech) as previously described [70] (link). For differentiation of EpiSCs into NM progenitors approximately 1500–2000 cells/cm² were plated on fibronectin (Sigma)-coated wells in N2B27 medium supplemented with CHIR99021 (3 µM; Signal Transduction Division, Dundee) and bFgf (20 ng/ml). For grafting experiments the initial plating density was 2500 cells/cm² and cells were plated on either fibronectin or gelatin.
Human ESC lines MasterShef 5 and 7 (a gift of Prof. Harry Moore, University of Sheffield) and a Sox2GFP reporter line (a gift of Dr Andrew Smith, University of Edinburgh) were cultured in Essential 8™ medium on Geltrex™-coated plates. For hNMP differentiation cells were pre-treated for 1 h with ROCK inhibitor Y-27632 (10 µM; Calbiochem), dissociated with accutase and plated at approximately 10,000 cells/cm² (Sox2-GFP hESCs) or 80,000 cells/cm² (MasterShef5 and 7 hESC lines) on fibronectin-coated wells in N2B27 medium supplemented with 3 µM CHIR99021/20 ng/ml bFgf and Y-27632 (10 µM). The medium was replaced the following day with fresh N2B27 containing the same components minus the ROCK inhibitor. For directed differentiation of hESCs, cultures were differentiated in the presence of CHIR99021/bFgf for 72 h as described above. For neural/spinal cord differentiation 72 h CHIR99021/bFgf-differentiated cells were treated with Accutase (Sigma) and transferred onto Geltrex (Life Technologies)-coated plates either in N2B27 alone or N2B27 supplemented with RA (0.1 µM; Sigma), SAG (0.5 µM; Calbiochem) and purmorphamine (1 µM; Calbiochem) for 48 h. For mesodermal differentiation 72 h CHIR99021/bFgf differentiated cells were cultured in N2B27 supplemented with CHIR99021 (3 µM) for a further 48 h. For dual SMAD inhibition Sox2-GFP hES cells were plated at 10,000 cells/cm² on Geltrex™-coated wells in N2B27 supplemented with LDN193189 (100 nM; Stemgent) and SB431542 (10 µM; Sigma). This was followed either by re-plating and culture in N2B27 or in N2B27/CHIR99021 (3 µM)/bFgf (20 ng/ml) for a further 48–72 h. All experiments involving hES cells have been approved by the UK Stem Cell Bank steering committee.

rhAA was purified as histidine-tagged (His-tag) fusion protein from rice cell culture medium using affinity chromatography on a Ni²⁺—NTA agarose (Qiagen, Hilden, Germany) column, following the manufacturer’s instructions. Briefly, 50 mL of a 7-day induced culture medium was centrifuged at 13,200 rpm at 4 °C for 20 min and filtrated through a 0.2 µm filter to remove debris. The column was washed out of the preservative, and 700 µL of Ni²⁺—NTA agarose resin was added into the open column. After equilibrating with 5 mL of 10 mM Imidazole, the prepared culture medium (added 1M Imidazole to the culture medium to obtain the final 10 mM) was loaded to the purification column at a flow rate of about 0.5 mL/min. The Ni²⁺—NTA agarose column was washed with 10 mL of 10 mM Imidazole. Binding proteins were eluted using 250 mM Imidazole in a total volume of 1 mL. Fractions were collected, and purity was visually assessed by SDS-PAGE and Western blot analysis.

A vector expressing human INHBA cDNA (#RC203226, OriGene) and control cDNA (#PS100001, OriGene) were introduced into CAFs using the lipofectamine 3000 reagent (#L3000015, Thermo Fisher Scientific) according to the transfection protocol. CAFs were cultured in McCoy’s 5A with 1% FBS for 24 hours before treatment with 20 ng/ml of recombinant human Activin A (#338-AC-010, R&D Systems) for 24–48 hours.