NEUROMESO

Exposure of mouse ESCs to a sequence of extrinsic signals, which recapitulates in vivo development, generates a bipotential neuromesodermal precursor (NMP) that can be directed to differentiate into either spinal cord or paraxial mesodermal tissue. To induce differentiation,mouse ES cells were plated on CellBINDSurface dishes (Corning) precoated with 0.1% gelatin (Sigma) at a density of 5x10^3 cells cm-2 in N2B27 medium. Cells were grown in N2B27 supplemented with 10ng/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 (NH) 100nM RA (Sigma) and 500nM SAG (Calbiochem) was added from D3-D5. Spinal cord identity (NP) was induced by the addition of 5nM CHIR 99021 (Axon) from D2 to D3 followed by 100nM RA, 500nM SAG from D3-D5. To induce mesodermal differentiation the cells were treated with CHIR99021 from D2-D5.

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