Evaluations of disease progression in G93A mice through Kaplan-Meier analysis.

<p>(<b>A</b>) Time elapsed until animals lost 15% of their maximum body weight. Mice receiving 1×10⁶ cells pre-symptomatically (<i>Gr 3</i>) significantly () maintained body weight vs. Media (<i>Gr 4</i>) mice. A similar trend was observed in mice treated with 2.5×10⁶ cells (<i>Gr 1</i>) beginning at symptomatic disease stage. (<b>B</b>) Time elapsed until hindlimb extension scores deteriorated by 70% of the initial score. The <i>Gr 1</i> and <i>Gr 3</i> mice significantly () delayed decline of hindlimb extension compared to <i>Gr 4</i> mice. A significant difference was also detected between <i>Gr 3</i> and <i>Gr 2</i> mice receiving 1×10⁶ cells at pre-symptomatic or symptomatic stage of disease, respectively. (<b>C</b>) Time elapsed until muscle strength decreased by 70% from the maximum value. Mice from <i>Gr 3</i> significantly () delayed muscle strength losses vs. <i>Gr 4</i>. <i>Gr 1</i> mice tended to maintain muscle strength post-transplant. (<b>D</b>) Time elapsed until rotarod latency decreased by 70% of the maximum value. Only mice from <i>Gr 3</i> performed better on the rotarod than other cell-treated mice and tended towards significance () taking more time to decrease latency by over 70% of the maximum value compared to <i>Gr 4</i>.</p

Characteristics of microglial cells in the spinal cords of G93A mice.

<p>Microglial densities were measured in the cervical (<b>A</b>) and lumbar (<b>B</b>) ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Microglial densities were significantly (p<0.001) higher in Media-injected mice (<i>Gr 4</i>) compared to control mice (<i>Gr 5</i>) of the same ages. MNC hUCB cell administrations significantly (p<0.001) decreased the number of microglia in the spinal cord of G93A mice compared to Media mice. No significant differences were detected between the cell-treated groups. *p<0.05, **p<0.01, ***p<0.001. (<b>C</b>) Immunohistochemical staining of microglia in the lumbar spinal cord at 17 weeks of age. Microglial cells stained for anti-Iba-1 antibody were sparse in controls (<b>a</b>) and microgliosis was noted in Media-treated animals (<b>b</b>). MNC hUCB cells decreased microglial density in mice from <i>Gr 1</i> (<b>c</b>), <i>Gr 2</i> (<b>d</b>), and <i>Gr 3</i> (<b>e</b>). Morphological analysis of microglial cells determined numerous activated cells with large cell bodies and short processes in Media-injected mice, whereas ramified microglia were mostly observed in cell-treated animals, particularly in <i>Gr 1</i> and <i>Gr 3</i> mice and controls (inserts in a–e). Scale bar: a–e is 200 µm; in a–e inserts is 25 µm.</p

Distribution of MNC hUCB cells in the spinal cord of G93A mice.

<p>Administered MNC hUCB cells were identified immunohistochemically by a human-specific marker (HuNu) in the spinal cord of cell-treated mice at 17 weeks of age, 4 weeks (symptomatic) or 8 weeks (pre-symptomatic) post-transplant. In the total area of cervical (<b>A</b>) and lumbar (<b>B</b>) cervical spinal cord, HuNu positive MNC hUCB cells were found irrespective (p>0.05) of injected cell doses or time beginning treatment. In all cell-treated mice, more than 50% of the observed cells were in ventral horn gray matter. (<b>C</b>) Immunohistochemical staining of MNC hUCB cells in the lumbar spinal cord. MNC hUCB cells positive for HuNu (green, arrow) were detected in the lumbar spinal cord of mice receiving 2.5×10⁶ (<b>a</b>) or 1×10⁶ (<b>b</b>) cells symptomatically or 1×10⁶ cells pre-symptomatically (<b>c</b>). Cells were frequently observed inside the capillary lumen, but also in the spinal cord parenchyma. (<b>a′</b>), (<b>b′</b>), and (<b>c′</b>) are merged images with DAPI. Scale bar: a–c′ is 50 µm.</p

Characteristics of motor neuron survival in the spinal cord of G93A mice.

<p>Motor neuron counts were performed in the cervical (<b>A</b>) and lumbar (<b>B</b>) ventral horns of G93A mice at 17 weeks of age and at end-stage of disease. Mice receiving 2.5×10⁶ cells symptomatically (<i>Gr 1</i>) or 1×10⁶ cells pre-symptomatically (<i>Gr 3</i>) had significantly higher motor neuron densities than the Media group (<i>Gr 4</i>) at 17 weeks of age or at end-stage of disease. In both cervical and lumbar spinal cords, motor neuron densities between <i>Gr 2</i> (1×10⁶ cells, symptomatic) and Media-injected (<i>Gr 4</i>) mice showed no significant differences (p>0.05). *p<0.05, **p<0.01, ***p<0.001. (<b>C</b>) Immunohistochemical staining of motor neurons in the lumbar spinal cord of G93A mice at 17 weeks of age. Motor neuron staining for anti-choline acetyltransferase (anti-ChAT) antibody showed healthy motor neurons in controls (<b>a</b>) although only a few neurons survived in the Media-treated animals (<b>b</b>). Cell-treated mice with (<b>c</b>) 2.5×10⁶ cells symptomatically (<i>Gr 1</i>) and (<b>e</b>) 1×10⁶ cells pre-symptomatically (<i>Gr 3</i>) demonstrated higher motor neuron survival than (<b>d</b>) mice receiving 1×10⁶ cells symptomatically (<i>Gr 2</i>). Scale bar: a–e is 50 µm.</p

Effect of multiple MNC hUCB cell administrations on lifespan of G93A mice.

<p>(<b>A</b>) Kaplan-Meier survival curves for G93A mice receiving 2.5×10⁶ (<i>Gr 1</i>) or 1×10⁶ (<i>Gr 2</i>) cells at symptomatic disease stage and 1×10⁶ (<i>Gr 3</i>) cells pre-symptomatically. Control group was Media-injected mice (<i>Gr 4</i>). Significant () increases in survival were determined in mice receiving 1×10⁶ cells at pre-symptomatic stage (p = 0.0015) and 2.5×10⁶ cells at symptomatic stage (p = 0.0022) vs. the Media-injected group. Survival of the mouse group receiving 1×10⁶ cells pre-symptomatically tended towards significance compared to survival of mice receiving same cell dose at symptomatic stage (p = 0.0595). (<b>B</b>) Percentages of surviving mice within age ranges. Media-injected animals survived no longer than 19.5 weeks, whereas 30% of mice receiving 2.5×10⁶ (<i>Gr 1</i>, symptomatic) or 1×10⁶ cells (<i>Gr 3</i>, pre-symptomatic) and 14.3% mice administered with 1×10⁶ cells (<i>Gr 2</i>) at symptomatic stage survived more than 140 days and 10% of mice from <i>Gr 3</i> (1×10⁶ cells, pre-symptomatic) were alive for more than 150 days.</p

Distribution of MNC hUCB cells in the lung, liver, kidney and spleen of G93A mice.

<p>MNC hUCB cells immunohistochemically positive for HuNu (green, arrows) were detected in the lung, liver, kidney, and spleen of mice receiving 2.5×10⁶ (<b>a, d, g, j</b>) or 1×10⁶ (<b>b, e, h, k</b>) cells symptomatically or 1×10⁶ cells pre-symptomatically (<b>c, f, i, l</b>). In the liver, lung and kidney, few cells were identified. In the spleen, a high density of HuNu cells was determined in all cell-treated mice (<b>j–l</b>). Scale bar: a–i is 50 µm; j–l is 200 µm.</p