Mutant SOD1 alters cell morphology and viability.(A-C) Staining of G93A–GFP-transfected cells with the G93A-specific antibody shows an accumulation of G93A SOD1 cells in the soma and processes at 21 days after transfection.(D-G) Representative fluorescent micrograph of cells transfected with wild-type SOD1, the SOD1 mutants or the control vector after 21 days. (H,I) Average length of neurites on days 7 and 21 after transfection. The values at the top of the bars represent the relative percentage difference between motor neurons transfected with the respective mutants or wild-type SOD1 compared with those expressing the control GFP vector. The length of neurites at 7 days after transfection was reduced by 21.52% (F=3.49, P>0.05) in cells expressing wild-type SOD1, 24.31% (F=6.69, P>0.05) in cells expressing I113T SOD1, 39.63% (F=14.26, P<0.05) in cells expressing G93A SOD1 and by 46.73% (F=11.49, P<0.05) in cells expressing A4V SOD1. (J) Percentage cell survival from day 4 to day 7 after transfection. (K) Percentage cell survival from day 4 to day 21 after transfection; *P<0.05 and **P<0.01 compared with the control. All experiments were executed in triplicate by a blinded observer. Statistical analysis of the percentage of GFP-positive cells compared with wild-type cells shows significant differences with the G93A (F=13.10, P<0.05) and A4V (F=19.18, P<0.01) mutations. Abbreviation: hWT, human wild type. Bars, 10 μm (A-C); 20 μm (D-G).

Generation of SOD1-transgenic motor neurons from hESCs. (A) Wild-type SOD1 and mutant G93A, I113T and A4V SOD1 cassettes were cloned into the BamHI site of the E/Hb9-3.6kb:EGFP vector containing the HB9 enhancer linked to the h-globin gene basal promoter. (B-D) G93A–GFP-transfected motor neurons co-stained for ChAT. (E-G) I113T–GFP-transfected motor neurons co-stained for HB9. (H-K) A4V–GFP-transfected motor neurons co-stained for ChAT and HB9. (L-Q) Action potential properties of hESC-derived motor neurons fall into two distinct classes that are typical of developing motor neurons. Panels (L-N) represent less differentiated cells with neurites extending =100 μm. Panels (O-Q) represent more highly differentiated cells with extensive axonal arborizations. Hb9:EGFP-positive cells were whole-cell patched in the current-clamp mode. Short duration (0.5 milliseconds) current pulses of increasing amplitude evoke single action potentials with a distinct threshold (M,P). Longer duration (250 milliseconds) current pulses elicit single action potentials (red) when the amplitude of the injected current is near to the threshold [(N) 100 nA; (Q) 80 nA]. When the amplitude of the stimulus pulse is increased, the more mature hESC-derived motor neurons respond with repetitive firing (Q). (R) Representative trace of fluo-3 ΔF (~[Ca2+]i) versus time for a single cell. Bath application of tetrodotoxin (TTX, 1 μM) abolishes spontaneous Ca2+ oscillations. Abbreviations: P, promoter; IRES-2, internal ribosomal entry site 2. Bars, 10 μm (B-D); 20 μm (E-O).