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. 2014 Feb 20;2(3):323–336. doi: 10.1016/j.stemcr.2014.01.006

Figure 1.

Figure 1

Generation of mESC-Derived Neuronal Networks

(i) mESCs were grown on mitomycin-inactivated mouse embryonic fibroblasts (MMC-MEFs) in the presence of leukemia inhibitory factor (LIF) for up to 5 days in vitro (5 div) followed by (ii) embryoid body (EB) formation in the absence of LIF. (iii) After 24 hr, free-floating EBs had developed and were replated in neural induction medium (NIM; for formulation, see Experimental Procedures). (iv) After 7 days, serum-free, floating cultures of EB-like aggregates (SFEB) had developed and were replated in NIM containing FGF2 and EGF. (v) After 14–21 div, nSFEBs were dissociated, stored in liquid nitrogen, or directly cultivated on PDL/laminin-coated MEAs in the presence of FGF2 in order to induce neuronal predetermination. (vi) FGF2 concentration was gradually decreased in the medium to induce terminal differentiation and neuronal network development. For a detailed description of neuronal composition and neuronal network development, see Illes et al. (2009).