MNIM-EOH cells express mature neuronal/MN markers after transplanted into the injured spinal cord slice culture.

<p>Confocal microscopy analysis of MNIM-EOH cells (green) immunostained with anti-NeuN (red) (<b>A</b>), anti-NF-M (red) (<b>B</b>), and anti-ChAT (red) (<b>C</b>) antibodies. In the slices, 37 of 472 EGFP+ cells were NeuN+, 20 of 316 EGFP+ cells were NF-M+, and 14 of 469 EGFP+ cells were ChAT+. Colocalization of EGFP and NeuN, NF-M, or ChAT in a single cell was confirmed by z-axis stack analysis. At least 4 slices were analyzed. Scale bars: 50 µm.</p

MNIM-EOH cells can trigger AChR clustering and form functional connections with cocultured myotubes.

<p>(<b>A</b>) Fluorescence images of MNIM-EOH cells at 1 day after cocultured with C2C12 myotubes. α-BTX staining revealed AChR clustering on C2C12 muscle fibers. Confocal Z-stack imaging, in a line through the region of apparent colocalization, confirmed EGFP+ axons in close proximity to AChRs (arrows). Scale bars: 10 µm. (<b>B</b>) End-plate currents (EPCs) were recorded from myotubes located in close proximity to MNIM-EOH cells. EPCs were blocked from the same cell after application of 15 µM pancuronium. However, after washing out of pancuronium, EPCs could be recorded again. The bars represent mean±SEM. * <i>P</i><0.05. The significance was determined by Student’s <i>t</i> test.</p

Increased expression of mature neuronal/motoneuronal markers and cell-cycle arrest occur in MNIM-treated EOH

<p><b>cells.</b> (<b>A</b>) Immunocytochemistry analysis of neuronal and MN-specific markers in uninduced EOH cells, MNIM-EOH cells, and MNIM-EOH cells further cultured in growth medium for 2 days after complete induction. Each row represents NeuN (red), NF-M (red), ChAT (red), and Islet-1 (red) staining with EGFP (green). Scale bar: 50 µm. (<b>B</b>) Quantitative analysis of NeuN, NF-M, ChAT, and Islet-1 expression. Fifty to 150 cells per group were analyzed in randomly chosen fields. The bars represent mean±SEM of at least three independent experiments. * <i>P</i><0.05, ** <i>P</i><0.001 versus GM. † <i>P</i><0.05, †† <i>P</i><0.001 versus MNIM. The significance was determined by ANOVA followed by Fisher’s LSD <i>post hoc</i> test. (<b>C</b>) Immunocytochemistry of BrdU incorporation (red) and EGFP (green) in EOH cells cultured in GM, MNIM, and MNIM+GM. (<b>D</b>) Quantitative analysis of BrdU incorporation. Fifty to 150 cells per group were analyzed in randomly chosen fields. The bars represent mean±SEM of at least three independent experiments. * <i>P</i><0.05 versus GM. The significance was determined by ANOVA followed by Fisher’s LSD <i>post hoc</i> test.</p

Excitable properties of MNIM-treated EOH cells.

<p>Representative perforated patch clamp recordings from uninduced EOH cells (<b>A</b>) or MNIM-EOH cells (<b>B</b>). The cells were held at −60 mV and currents elicited by stepping from −90 to +30 mV in 10 mV steps. MNIM-EOH cells express robust outward K+ currents but very small inward Na+ currents (filled upright triangle). Current injection (200 pA, 100 ms) induced action potential in MNIM-EOH cells, not in uninduced EOH cells. (<b>C</b>) Resting membrane potential in uninduced EOH cells and MNIM-EOH cells. The bars represent mean ± SEM. * <i>P</i><0.05. The significance was determined by Student’s <i>t</i> test.</p

MN marker gene expression in hMSCs expressing Olig2 and Hb9 in the presence of MNIM.

<p>(<b>A</b>) Schematic illustration of the protocol used for MN induction of hMSCs. (<b>B</b>) RT-PCR analyses of hMSCs expressing EGFP (E), EGFP-Olig2 (EO), EGFP-Hb9 (EH), and EGFP-Olig2-Hb9 (EOH) with MNIM treatment were preformed. Quantification of NF-M (<b>C</b>), Islet-1 (<b>D</b>), and VAChT (<b>E</b>) PCR products. The mRNA level of a given gene was quantified by densitometry and normalized to the corresponding β-actin level. The bars represent mean±SEM of at least three independent experiments. *<i>P</i><0.05 versus uninduced E. The significance was determined by Mann-Whitney test with Fisher’s LSD <i>post hoc</i> test.</p

Quantitative analysis of cell morphology (% of total cells).

<p>The values are expressed as the mean±SEM.</p

Changes in cell morphology of hMSCs expressing Olig2-Hb9 after the induction.

<p>Bright-field images of uninduced EOH cells (<b>A</b>), EOH cells induced for 5 days (<b>B</b>), completely induced EOH cells (<b>C</b>), and induced EOH cells further cultured in growth medium for 2 days after complete induction (<b>E</b>). (<b>D</b>) A higher magnification of the corresponding box shown in (<b>C</b>). (<b>F</b>) GFP (green), Olig2 (blue) and Hb9 (red) were expressed in EOH cells. Scale bar: 100 µm.</p

Polymerase chain reaction primer pairs.

<p>Polymerase chain reaction primer pairs.</p

<i>Ex vivo</i> assessment of EOH cells or MNIM-EOH cells after transplantation into the spinal cord slice culture.

<p>Confocal images of uninduced EOH cells (green) (<b>A-C</b>) or MNIM-EOH cells (green) (<b>D-F</b>) at 2 days after transplantation into the ventral horn area (vh) of a spinal cord slice. (<b>B</b>) and (<b>E</b>) are a higher magnification of (<b>A</b>) and (<b>D</b>), respectively. (<b>C</b>) Confocal image of transplanted EOH cells with a flattened and symmetrical fibroblast-like morphology. (<b>F</b>) Confocal image of transplanted MNIM-EOH cells with a neuron-like morphology. Scale bars: 100 µm.</p