Functional properties of motoneurons derived from mouse embryonic stem cells

Abstract

The capacity of embryonic stem (ES) cells to form functional motoneurons (MNs) and appropriate connections with muscle was investigated in vitro. ES cells were obtained from a transgenic mouse line in which the gene for enhanced green fluorescent protein (eGFP) is expressed under the control of the promotor of the MN specific homeobox gene Hb9. ES cells were exposed to retinoic acid (RA) and sonic hedgehog agonist (Hh-Ag1.3) to stimulate differentiation into MNs marked by expression of eGFP and the cholinergic transmitter synthetic enzyme choline acetyltransferase. Whole-cell patch-clamp recordings were made from eGFP-labeled cells to investigate the development of functional characteristics of MNs. In voltage-clamp mode, currents, including EPSCs, were recorded in response to exogenous applications of GABA, glycine, and glutamate. EGFP-labeled neurons also express voltage-activated ion channels including fast-inactivating Na(+) channels, delayed rectifier and I(A)-type K(+) channels, and Ca(2+) channels. Current-clamp recordings demonstrated that eGFP-positive neurons generate repetitive trains of action potentials and that l-type Ca(2+) channels mediate sustained depolarizations. When cocultured with a muscle cell line, clustering of acetylcholine receptors on muscle fibers adjacent to developing axons was seen. Intracellular recordings of muscle fibers adjacent to eGFP-positive axons revealed endplate potentials that increased in amplitude and frequency after glutamate application and were sensitive to TTX and curare. In summary, our findings demonstrate that MNs derived from ES cells develop appropriate transmitter receptors, intrinsic properties necessary for appropriate patterns of action potential firing and functional synapses with muscle fibers.

Figure 3.

ES cell-derived MNs were identified by eGFP labeling and patched under infrared-differential interference contrast (IR-DIC) microsopy. Low-magnification (10×) image of an embryoid body visualized under IR-DIC microscopy (A) and under epifluorescence (B) shows eGFP labeling of ES cell-derived MNs. High magnification (40×) image of an ES cell-derived MN visualized under IR-DIC microscopy (C) and under epifluorescence (D) confirms eGFP labeling before attaching a whole-cell patch-clamp recording electrode (E). Scale bars: B, 50 μm; D, 10 μm.

Figure 1.

Embryoid bodies containing HBG3 ES cells express eGFP after treatment with RA and Hh-Ag1.3. ES cells suspended in DFK10 medium were treated with RA (1 μm) and Hh-Ag1.3 (1 μm) at day 0 to direct differentiation toward an MN phenotype. Hb9 upregulation was verified by eGFP expression under fluorescence microscopy. At 5 d in culture, the ES cell aggregates reached maximum size, evidenced under phase-contrast microscopy, and maximum eGFP expression. Scale bar, 200 μm.

Figure 2.

RA-Hh-Ag-treated ES cells express neuronal and cholinergic markers. Fluorescent image of RA-Hh-Ag 1.3-treated ES cells shows that they extend long eGFP⁺ neurites when cocultured on C2C12 myotubes (A, D, G). Many eGFP⁺ ES cells expressed the neuronal marker MAP2 (B, C, arrow). Occasionally eGFP^-, MAP2⁺ neurites were seen (B, C, arrowhead). After 3 d in coculture, the majority of the eGFP⁺ cells expressed ChAT (E, F, arrows), and the neurites expressed VAChT (H, I, arrow). A small proportion of ChAT⁺ cells were eGFP^- (E, F, arrowheads) and some eGFP⁺ cells were ChAT^- (E, F, open arrows). Note the dense clustering of eGFP⁺, ChAT⁺ neurons at the edge of the embryoid body (D-F, top left). Together, these results indicate that most treated ES cells developed a cholinergic phenotype. D-I, Obtained using confocal microscopy (D-F, projection thickness = 9.9 μm; G-I, projection thickness = 13.3 μm). Scale bars: A-C, 30 μm; D-F, 50 μm; G-I, 100 μm.

Figure 4.

ES cell-derived MNs express functional neurotransmitter receptors. Voltage-clamp recordings of membrane current (I_m) during application of GABA (100 μm) (Ai), glycine (GLY; 100 μm) (Bi), and glutamate (GLUT; 100 μm) (Ci, Ciii). Current-voltage relationships for GABA-, glycine-, and glutamate-induced currents are shown in Aii, Bii, and Cii, respectively. Voltage steps (200 msec duration) from a holding potential of -60 mV to a range of test potentials between -90 and +40 mV (10 mV increments) were performed before and during drug responses. Steady-state currents (the last 20 msec of pulses) were used to generate current-voltage plots under control conditions and during drug responses after steady state was reached. Dotted lines indicate membrane current under control conditions. A holding potential of -60 mV was used during all drug applications. After glutamate application in cells in coculture conditions for > 5 d, EPSCs were recorded in ES cell-derived MNs (Ciii). Note: delayed responses to drug applications reflect the time taken for the drug to wash into the recording chamber (typically 1 min).

Figure 5.

ES cell-derived MNs express voltage-activated Na⁺, K⁺, and Ca²⁺ currents. A, Fast-inactivating Na⁺ currents. Voltage-clamp recordings of membrane current (I_m) in response to depolarizing voltage steps demonstrating fast-inactivating outward currents (i) that are sensitive to TTX (0.5 mm) (ii). Voltage-current relationships for fast-inactivating currents are shown in iii (n = 35). B, Delayed rectifier K⁺ currents (I_K). Voltage-clamp recordings show persistent outward currents (i) activated by depolarizing steps from a holding potential of -40 mV that are sensitive to TEA (30 mm) (ii). Current-voltage relationships for these persistent outward currents are shown in iii (n = 24), along with the blocking effects of TEA (n = 3). C, Transient K⁺ currents (I_A). Voltage-clamp recordings of outward currents activated by depolarizing steps from -80 mV (i) or -40 mV (ii). The subtraction of responses to steps from these two holding potentials revealed a transient current (iii) that required a holding potential of -80 mV for activation. This transient current (iv) was reduced by 4-AP (4 mm) (v). The current-voltage relationship for this transient current is shown in vi (n = 19), along with the blocking effects of 4-AP (n = 5). D, Voltage-activated Ca²⁺ currents. Voltage-clamp recordings after elimination of Na⁺ and K⁺ currents show inward currents activated by depolarizing steps from a holding potential of -60 mV (i) or -80 mV (ii). These currents are sensitive to cadmium and nickel, respectively. Current-voltage relationships for Ca²⁺ currents elicited from a holding potential of -80 mV are plotted in iii, using peak inward current (filled diamonds) or current averaged during the last 20 msec of voltage steps (open squares; n = 9).

Figure 6.

ES cell-derived MNs develop appropriate firing properties. A, Current-clamp recordings of membrane voltage (Vm) in response to current (I) injection (1 sec duration) in ES cell-derived MNs showing single action potentials 1 d after plating (i) and repetitive firing 4 d after plating (ii). B, Plots of instantaneous firing frequency versus time during a single current pulse showing spike frequency adaptation (i) and steady-state firing frequency versus injected current (ii). C, Responses to short (10 msec) current pulses in control showing a single action potential (i) and in the presence of FPL-64176 (10 μm) (ii), showing the induction of a long-lasting depolarization and action potential doublet, which was blocked by nifedipine (20 μm) (iii).

Figure 7.

RA-Hh-Ag-treated ES cells induce acetylcholine receptor clustering on myotubes. Ai, Fluorescence image of eGFP⁺ RA-Hh-Ag1.3-treated ES cells after 2 d of coculture with C2C12 myotubes. Aii, α-BTX staining revealed acetylcholine receptor clustering on C2C12 muscle fibers (Aii, arrows). Aiii, Merged image of Ai and Aii revealed acetylcholine receptor clustering only in close proximity to eGFP⁺ axons and cell bodies. B, Acetylcholine receptor clustering was absent on myotubes several millimeters away from the embryoid body. C, Confocal image depicting eGFP⁺ axons colocalized with acetylcholine receptors (projection of 18 optical sections of 0.75 μm each). Imaging in the x-z orthogonal plane, in a line through the region of apparent colocalization, confirmed eGFP⁺ terminals in close proximity to ACh receptors. Scale bars: A, B, 100 μm; C, 10 μm.

Figure 8.

ES cell-derived MNs form functional connections with muscle cells. Representative postsynaptic intracellular sharp electrode recordings from C2C12 myotube cocultures (A, left) revealed a high number of MEPPs (i.e., amplitude <1.5 mV) and relatively few EPPs (i.e., amplitudes >1.5 mV) (A, right). Application of 100 μm glutamate caused an expected increase in the number of EPPs (B), whereas the addition of 5 μm TTX blocked most EPPs (C). The frequency histograms in A-C were generated from ≈90 sec recordings. Similar responses to glutamate application were recorded from chick myotubes cocultured with ES cell-derived MNs (D, left). EPPs were blocked entirely by 50 μm d-tubocurarine (D, right).