Multiple Intravenous Administrations of Human Umbilical Cord Blood Cells Benefit in a Mouse Model of ALS

Background: A promising therapeutic strategy for amyotrophic lateral sclerosis (ALS) is the use of cell-based therapies that can protect motor neurons and thereby retard disease progression. We recently showed that a single large dose (25x10(6) cells) of mononuclear cells from human umbilical cord blood (MNC hUCB) administered intravenously to pre-symptomatic G93A SOD1 mice is optimal in delaying disease progression and increasing lifespan. However, this single high cell dose is impractical for clinical use. The aim of the present pre-clinical translation study was therefore to evaluate the effects of multiple low dose systemic injections of MNC hUCB cell into G93A SOD1 mice at different disease stages. Methodology/Principal Findings: Mice received weekly intravenous injections of MNC hUCB or media. Symptomatic mice received 10(6) or 2.5x10(6) cells from 13 weeks of age. A third, pre-symptomatic, group received 10(6) cells from 9 weeks of age. Control groups were media-injected G93A and mice carrying the normal hSOD1 gene. Motor function tests and various assays determined cell effects. Administered cell distribution, motor neuron counts, and glial cell densities were analyzed in mouse spinal cords. Results showed that mice receiving 10(6) cells pre-symptomatically or 2.5x10(6) cells symptomatically significantly delayed functional deterioration, increased lifespan and had higher motor neuron counts than media mice. Astrocytes and microglia were significantly reduced in all cell-treated groups. Conclusions/Significance: These results demonstrate that multiple injections of MNC hUCB cells, even beginning at the symptomatic disease stage, could benefit disease outcomes by protecting motor neurons from inflammatory effectors. This multiple cell infusion approach may promote future clinical studies

A novel method for the development of latent fingerprints recovered from arson simulation

AbstractA diverse range of physical and chemical methods is available for the development of latent fingerprints. But fingerprints exposed to extreme conditions like fire or arson are generally perceived to have been damaged. Electromagnetic radiations, soot deposition and high temperatures are the forces generated in a fire, which may affect the fingerprints at the scene. Thus, the potential value of highly crucial evidence like fingerprints remains unutilized. This study was conducted to determine if fingerprints could be developed after being subjected to an arson/fire scene simulation. Fingerprints on nonporous surfaces were subjected to high temperatures, soot deposition and subsequently treated with water. A novel fluorescent and a pre-existing small particle reagent was investigated for the same. Zinc carbonate based fluorescent small particle reagent was capable of developing latent fingerprints exposed to a maximum temperature of 800°C

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

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

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

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