Immunochistochemical staining of MNC hUCB cells in the lumbar spinal cord of G93A mice administered with different cell doses.

<p>MNC hUCB cells were found in the lumbar spinal cord of mice receiving A), B) 10×10⁶; C), D) 25×10⁶; and E), F) 50×10⁶ cells by anti-human nuclei staining (green, asterisks). Merged images are with DAPI. Some a), b), c), d), e), f) MNC hUCB cells expressed Nestin (red, asterisks). Cells in images a, b, c, d, e, f are same in images A, B, C, D, E, F. Scale bar: A–e is 25 µm.</p

Effect of MNC hUCB cell administration on lifespan of G93A mice.

<p>A) Kaplan-Meier survival curves for G93A mice receiving 10×10⁶, 25×10⁶, and 50×10⁶ MNC hUCB cells. Control groups were Media and CsA. Significant (four pointed star) increase in survival between cell transplanted and Media-injected or CsA-injected groups was determined in mice receiving 25×10⁶ cells (χ² = 4.134, p = 0.04). B) Some animals (33.3%) from the group receiving 25×10⁶ cells were alive at 20 wks of age, when only 21.4% of the group receiving 10×10⁶ cells and 7.7% of the group receiving 50×10⁶ cells survived. G93A mice administered with 25×10⁶ cells survived until 25 wks of age (22 wks of age–25%, 23 wks of age–16.7%, 25 wks of age–8.3%). Media-injected or CsA-injected mice survived no longer than 20 weeks of age.</p

Effect of MNC hUCB cell administration on hematological response in the peripheral blood from G93A mice.

<p>A) Many MNC hUCB cells were found in the peripheral blood of mice receiving all three cell doses (A-10×10⁶, B-25×10⁶, C-50×10⁶ cells). Administered human cells were verified by anti-human nuclei staining (green, asterisks). The nuclei in a, b, and c are stained with DAPI, same images as A, B, and C. Scale bar: A-c is 25 µm. B) White blood cell (WBC) counts demonstrated significantly reduced numbers of WBC cells in Media (p<0.05) and CsA-injected (p<0.05) G93A mice compared to C57BL/6 mice. All mice treated with MNC hUCB cells showed slightly increased WBC counts. C) WBC differential analysis showed a low percentage of neutrophils in control hTgn and C57BL/6 mice. A significant increase of neutrophils (almost 4–5 fold) was found in Media (p<0.01) and CsA (p<0.001) vs. hTgn or C57BL/6 mice. Decreased neutrophils in the G93A mice administered with MNC hUCB cells were most significantly pronounced in the 25×10⁶ cell group (p<0.05) compared to CsA-injected mice. The lymphocyte percentage was significantly decreased in Media (p<0.001) and CsA (p<0.001) groups vs. hTgn. Lymphocyte percentages in mice treated with 10×10⁶ and 50×10⁶ cells did not differ from Media mice. In mice receiving 25×10⁶ cells, a significant increase of lymphocytes (p<0.05) vs. CsA-injected mice was determined. Lines indicated significant differences between mouse groups.</p

Characteristics of disease progression in G93A mice.

<p>A) body weight, B) extension reflex, and C) rotorod test of G93A mice administered with three different cell doses, Media-injected, CsA-injected and control hTgn mice. At 16 wks and 20 wks of age, mice receiving 25×10⁶ cells better maintained their body weight, extended hindlimbs, and stayed longer on rotorod than other cell transplant or control groups. The four pointed stars in A, B, and C indicates significant difference: <i>in body weight</i>–16 wks of age: 25×10⁶ cells vs. 50×10⁶ cells (p<0.05); 20 wks of age: 25×10⁶ cells vs. CsA (p<0.05); <i>in extension reflex</i>–25×10⁶ cells vs. Media or 50×10⁶ cells (p<0.05); 20 wks of age-25×10⁶ cells vs. CsA or 10×10⁶ cells (p<0. 01); <i>in rotorod</i>–14 wks of age: 25×10⁶ cells vs. Media (p<0.05); in 20 wks of age-25×10⁶ cells vs. CsA (p<0.05) or 10×10⁶ cells (p< 0.01).</p

Effect of MNC hUCB cell administration on microglial cell density in the spinal cord of G93A mice.

<p>Microglial cell density in the ventral cervical (A) and lumbar (B) horns of all G93A, treated with different cell doses or non-treated (Media, CsA), mice was significantly (p<0.001) increased vs. C57BL/6 animals. Cell density in mice injected with CsA was significantly lower in the cervical spinal cord compared to Media (p<0.01) and 10×10⁶ cells (p<0.001) groups of mice and in the lumbar spinal cord compared to Media (p<0.001), 25×10⁶ cells (p<0.01) and 50×10⁶ cells (p<0.001) groups. A) In the cervical spinal cord, mice receiving 10×10⁶ MNC hUCB cells showed significantly (p<0.001) higher microglial cell density vs. mice with 25×10⁶ and 50×10⁶ cell doses. Animals from these last two groups had microglia of similar appearance and significantly (p<0.01 and p<0.05, respectively) reduced numbers of cells compared to the Media group. B) In the lumbar spinal cord, a significant decrease of microglial cell density was observed in mice receiving 10×10⁶ and 25×10⁶ cells vs. Media mice. Density of microglial cells in mice receiving cell transplants correlated positively with cell dosage. Mice with 10×10⁶ cells had a significantly lower density of microglia than mice administered with 25×10⁶ (p<0.05) and 50×10⁶ (p<0.001) cell doses and cell density was significantly (p<0.05) reduced in mice with 25×10⁶ cells vs. 50×10⁶ cells. Lines indicated significant differences between mouse groups.</p

Human Th1/Th2 cytokines in plasma from G93A mice administered with different MNC hUCB cell doses.

<p>FAST Quant microspot assay for human Th1/Th2 Cytokines (IL-1 β, TNF α, INF γ, IL-2, IL-4, IL-5, IL-6, IL10, and IL-13) in plasma from G93A mice receiving three different cell doses was performed by Schleicher & Schuell BioScience, Inc. (Keene, NH, USA). Plasma samples from hUCB (n = 10) were used as controls. Quantitative results of cytokines were presented as pg/mL. The 50% of mice receiving 25×10⁶ cells showed evidence of human IL-4 and IL-13 (Th2). In 62.5% of mice with 50×10⁶ cell treatment, all human cytokines were detected except for IL-5, IL-5, and IL-10 (Th2). There were no human cytokines detected in mice treated with 10×10⁶ cell dose. The hUCB plasmas themselves, mostly contained Th2 cytokines (IL-4, IL-10, and IL-13).</p

Immunochistochemical staining of MNC hUCB cells in the cervical spinal cord of G93A mice administered with different cell doses.

<p>MNC hUCB cells were found in the cervical spinal cord of mice receiving A) 10×10⁶; B), C) 25×10⁶; and D), E) 50×10⁶ cells by anti-human nuclei staining (green, asterisks). Merged images are with DAPI. Some a), b), c), d), and e) MNC hUCB cells were Nestin positive (red, asterisks). Cells in images a, b, c, d, and e are same in images A, B, C, D, E. cc–central canal. Scale bar: A–e is 25 µm.</p

Cytokine profile in the spleen of G93A mice administered with different MNC hUCB cell doses.

<p>The RNase protection assay was used to determine the mRNA expression of proinflammatory cytokines (IL-1 α, IL-1 β, TNF α, TNF β, and IL-2) and anti-inflammatory cytokine IL-10 in the spleen of G93A mice administered with different MNC hUCB cell doses. Control groups were Media, CsA, hTgn, and C57BL/6 mice. The mRNA expression presented as the optical density (OD) values obtained from each band normalizing against the OD obtained from the L32, a house-keeping gene, band. Lines indicated significant differences (p<0.001, p<0.01, and p<0.05) between mouse groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002494#s2" target="_blank">Results</a>).</p

Cytokine profile in the lumbar spinal cord, brainstem, and motor cortex of G93A mice administered with different MNC hUCB cell doses.

<p>The RNase protection assay was used to determine the mRNA expression of proinflammatory cytokines IL-1 α, IL-1 β, TNF α, and TNF β in the lumbar spinal cord (left column), brainstem (center column), and motor cortex (right column) of G93A mice administered with different MNC hUCB cell doses. Control groups were Media, CsA, hTgn, and C57BL/6 mice. The mRNA expression presented as the optical density (OD) values obtained from each band normalizing against the OD obtained from the L32, a house-keeping gene, band. Lines indicated significant differences (p<0.001, p<0.01, and p<0.05) between mouse groups (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002494#s2" target="_blank">Results</a>).</p