Culture step IV substrate modifications. (a) Experimental layout. (b) Axo-dendritic appearance of neurons on FN vs. LPO substrates. (c) Whole-cell patch-clamp recordings reveal repetitive firing behavior of LPO neurons and significantly increased Na⁺ currents and singular APs on FN substrates. Images reflect biocytin immunofluorescence of individual recorded cells. (d) Analysis of spontaneous synaptic activity demonstrating ubiquitous excitatory [combined α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)- and NMDA-mediated EPSCs (Left); isolated NMDA components (Center)] and inhibitory events (Right). Note that 2-amino-5-phosphonovaleric acid (APV) blocks NMDA-mediated burst-like activity (lower EPSC traces). (e) Quantification of substrate-specific neuronal subtype antigen profiles. (f–h) Fluorescence photomicrographs depict transmitter antigens (green) of phenotypes without (f) or with (g and h) differential neuronal fate choice in LPO vs. FN conditions. Note varicosities that may represent GABAergic boutons arising from a GAD65⁺ cell as marked by the arrow (g Left). (i) Semiquantitative RT-PCR demonstrates a selective increase of the transcription factors Nkx2.1 and Isl-1 on LPO substrates. ∗∗, P < 0.001. (Scale bars: in b and f–h, 50 μm; in c, 30 μm.)

Culture protocol. (a and b) In step I, pluripotent ES cells expand on embryonic fibroblasts (a) and GL (b). (c) Step II represents the first stage of differentiation, when ES cells aggregate for 3 days in suspension to form EBs. (d and e) Step III is a 7-day period in which ESNPs are selected in adhesive conditions. (d) One day after EB plating. (e) Last day of step III. (f) In step IV, selected cells differentiate spontaneously on a second substrate. In all experiments, neural progeny was evaluated at 5, 10, or 15 (±1) days after passage on substrate 2.

Culture step III substrate modifications. (a) Experimental layout. (b) Phase-contrast images of cells at the end of step III. Note the nearly pure ESNP populations on LPO (arrowheads point to ESNP islands in GL and FN conditions). (c and d) Developing neurons appear morphologically uniform after transfer of step-III cells onto LPO in step VI (c), but quantification of neuronal and glial phenotypes reveals that, in step III, LPO increases neuronal and GL induces glial fate decisions (d). (e) Quantification of nestin and NeuN expression reveals a similar developmental time course in all investigated cell populations. (f) Single-cell patch-clamp analysis of differentiating neurons demonstrates typical progression of functional properties. (g) Stereotypic development of neuronal membrane biophysics on LPO, regardless of substrate pretreatment in culture step III. ∗∗, P < 0.001. (Scale bars, 150 μm.)

Evaluating mechanisms for differential neuronal fate choice in culture step IV. (a) Ratio of propidium iodide (Pi)/Hoechst indicates levels of dead cells at certain time points of in vitro differentiation. (b) Evaluation of BrdU uptake indicates no substrate-specific selective proliferation of ChAT⁺ or GAD65⁺ precursors throughout culture step IV. (c and d) Developmental profile of Isl-1 expression in LPO vs. FN conditions (c) and examples of Isl-1⁺/βIII tubulin⁺ cells at 14-days differentiation (d). (d Inset) Isl-1⁺/ChAT⁺ neurons. (e and f) Similar profile and examples of Nkx2.1-expressing cells (arrow in f points to one of these cells). (f Inset) Nkx2.1⁺/ChAT⁺ neurons. (g) Blocking SHH pathways with cyclopamine (CYCL) in LPO conditions significantly decreases the quantity of Isl-1⁺, Nkx2.1⁺, ChAT⁺, and GAD65⁺ neurons compared with control levels [tomatidine (TOM), a structural homologue of cyclopamine] without SHH-inhibiting activity. ∗, P < 0.05. ∗∗; P < 0.01. (Scale bars: d and f, 50 μm; Insets, 25 μm.)