Neurotransmitter Differentiation of Grafted Human NSCs

<div><p>Photomicrographs (A–J) illustrate evidence of glutamatergic (A and B, G and H), GABAergic (C–F), and cholinergic (I and J) neurotransmission in NSC grafts. As in previous figures, confocal microscopy is used primarily to confirm the colocalization of two markers in the same cellular compartment along three planes of sectioning.</p> <p>(A and B) These sections, stained for HNu and the prevalent AMPA receptor epitope GluR2/3, show both cytoplasmic and synaptic staining by epifluorescence (A) or confocal (B) microscopy. Insets in (A) represent magnifications of indicated neurons in main image; top- and bottom-left insets show two medium-size HNu (+) cells with cytoplasmic immunoreactivity, whereas bottom-right inset illustrates a larger HNu (+) cell containing multiple GluR2/3 (+) boutons.</p> <p>(C and D) These sections are stained for HNu and the GABA-synthesizing enzyme GAD and visualized with epifluorescence (C) or confocal microscopy (D). Arrows in (C) indicate multiple HNu (+) cells with cytoplasmic GAD immunoreactivity.</p> <p>(E and F) Confocal microscopy of a field stained with both human Syn (red in single-channel image on top left, to label graft-derived terminals) and GAD (green in single-channel image on bottom left, to label GABAergic terminals) shows colocalization of the two proteins (yellow color in merged images in F) in multiple synaptic boutons. Nearly all graft-derived boutons are inhibitory (F).</p> <p>(G and H) These sections (G, epifluorescence; H, confocal) are stained for human Syn to label graft-derived terminals (red) and mixed VGLUT1/ VGLUT2 antibodies to label glutamatergic terminals in the field (green). Despite significant overlap and apposition of graft-derived and VGLUT1/2 (+) terminals (G), the two groups of terminals are separate (H).</p> <p>(I and J) These two sections were dually stained for: HNu and choline acetyltransferase (I and insert) epifluorescence; confocal microscopy (J); and show that some of the largest NSC-derived neurons express cholinergic phenotypes. These cells elaborate multiple primary dendrites (I and insert). (J) is the confocal image of the neuron in the inset.</p> <p>Scale bars: (A), (C), (G), (I) 20 μm; (B), (D–F), (H), (J) 10 μm.</p></div

Maturation of Human NSC-Derived Neurons Based on the Elaboration of Axons, Synapses, and Innervation by Host Neurons

<div><p>(A) This photograph was taken through the ventral horn of a HNu/70 kDa neurofilament protein stained section 3 mo postgrafting and shows bundles of human 70 kDa neurofilament protein (+) axons (indicated with white arrows) originating in HNu (+) grafts (one indicated with an asterisk on top right) and coursing together (red arrows on bottom left) toward the ventral white matter.</p> <p>(B) This photograph shows an NSC graft in the ventral horn of a human Syn-stained section three months postgrafting. The sharp colocalization of Syn (+) puncta with the graft region (boundaries demarcated with arrows) is due to the selectivity of the antibody for human, but not rat, Syn protein.</p> <p>(C and D) These images (C, epifluorescence; D, confocal) were taken from triple-stained sections with HNu (red), TUJ1 (blue), and the presynaptic marker Bsn (green). The Bsn antibody used here recognizes rat and mouse, but not human, protein. (C) depicts a dense field of rat Bsn (+) terminals in proximity to HNu and TUJ1 (+) profiles. Examples of contacts between rat terminals and NSC-derived neurons are shown with arrowheads in the inset, which is a magnification of the profile at the center of the main image. The very large number of such terminals on NSC-derived cell bodies is best illustrated with confocal microscopy (D).</p> <p>(E and F) These photographs (E, epifluorescence; F, confocal) were taken from sections stained with HNu (red), TUJ1 (blue), and mixed VGLUT1/VGLUT2 antibodies (green) and show the innervation of HNu and TUJ1 (+) cells by glutamatergic terminals putatively originating in the host.</p> <p>Scale bars: (A) 80 μm; (B) 20 μm; (C–F) 10 μm.</p></div

Innervation of Host Motor Neurons by Graft-Derived Nerve Cells as Shown on Sections Stained with Human Syn (Red) and TUJ1 (Green) and Studied with Epifluorescence or Confocal Microscopy

<div><p>Host motor neurons are depicted as large TUJ1 (+) cell bodies, and NSC-derived terminals are labeled with human Syn antibodies.</p> <p>(A) This epifluorescence image shows the site of the original graft (arrow in lower left) and two synaptic fields with host motor neuron pools marked as (1) and (2), with respectively higher and lower density of synaptic appositions. The low-density field (2) is further enlarged in the inset.</p> <p>(B) This confocal image shows, in great detail, a large number of somatic and dendritic terminals from graft-derived nerve cells on a host motor neuron.</p> <p>Scale bars: (A) 200 μm; (B) 20 μm.</p></div

Differentiation of Human NSCs into Neurons after Transplantation into the Lumbar Spinal Cord of Normal Adult Sprague-Dawley Rats

<div><p>Outlined areas in (A) and (C) are enlarged in (B) and (D). All images illustrate the neuronal differentiation of NSCs two months postgrafting based on dual-label immunofluorescence for HNu (red) and a neuronal marker (green, representing TUJ1 and NeuN in [A and B], and [C and D], respectively). The predominance of double-labeled profiles in both (B) and (D) (indicated with asterisks) matches the avid neuronal differentiation of human NSCs in nude rats as illustrated in <a href="http://www.plosmedicine.org/article/info:doi/10.1371/journal.pmed.0040039#pmed-0040039-g003" target="_blank">Figure 3</a>. Single HNu-labeled profiles (D) are shown with arrowheads</p> <p>Scale bars: (A and B) 20 μm; (C and D) 10 μm.</p></div

Differentiation of Grafted Human NSCs into Neurons and Glial Cells

<div><p>Photomicrographs (A–F) illustrate cases of neuronal (A and B), astrocytic (C and D), and oligodendrocytic (E and F) differentiation of HNu (+) cells by epifluorescence (A, C, and E) or confocal (B, D, and F) microscopy. (G) is a composite of bar graphs illustrating the general differences in fate choice between parenchymal (upper level) and meningeal (lower level) sites of NSC grafts. (H) provides further detail in differential fate choice among three parenchymal sites and the pia compared side-by-side.</p> <p>(A and B) These two sections are stained for HNu and TUJ1 and show the abundance of NSC-derived neurons within the parenchyma of the ventral horn by epifluorescence (A) and confocal microscopy (B). Both preparations are taken from animals killed three months postgrafting. Inset is a magnification of demarcated area in (A). Note the homogeneous appearance of TUJ1 (+) cells in the A inset. Confocal sections have been virtually resectioned at the <i>x</i> and <i>y</i> planes to confirm the identity of the double-stained structure.</p> <p>(C and D) These sections, dually stained for HNu and GFAP, illustrate the substantial astrocytic differentiation of NSCs located by the pia membrane by (Figure 3, continued) epifluorescence (C) and confocal microscopy (D). Inset is a magnification of demarcated area in (C), and representative astrocytes are indicated with arrows. Confocal sections have been processed as in (B).</p> <p>(E and F) Oligodendrocyte differentiation in ventral white matter based on APC immunoreactivity in the cytoplasm of cells with HNu (+) nuclei as shown with epifluorescence (E) and confocal microscopy (F). Blue nuclei represent DAPI counterstain. Arrow depicts a double-labeled cell. Arrowheads point to host oligodendrocytes (APC [+], HNu [−] cells). Confocal sections have been processed as in (B).</p> <p>(G) Bar graphs depicting the fate choices of NSC grafts in the parenchyma (including ventral and dorsal horn and ventral white matter, upper graphs) or the meninges (lower graphs) at three weeks (3w), three months (3m), and six months (6m) in different treatment groups (avulsion, red; HCA treatment, blue; sham, green). Neuronal fate is represented by numbers of TUJ1-labeled HNu (+) cells, and astrocytic fate is represented by numbers of GFAP-labeled HNu (+) cells. NSCs in a neural stem/precursor state are depicted here as nestin-and HNu double-labeled cells. Asterisks indicate critical post hoc differences between subgroups where ANOVA is significant (<i>p</i> ≤ 0.05).</p> <p>(H) These graphs provide further detail into the role of spinal microenvironment in the fate choice of grafted NSCs by differentiating among three parenchymal sites and the pia. Cell fates are represented by the same markers as in (G) Asterisks on top of brackets indicate important post hoc differences where ANOVA is significant (<i>p</i> ≤ 0.05).</p> <p>Scale bars: (A), (C), (E) 20 μm; (B), (D), (F) 10 μm.</p></div

In Vitro Differentiation of Human NSCs Used for Transplantation

<div><p>(A) The vast majority of cells express the NSC-specific marker nestin (red) immediately before grafting. The DNA dye DAPI (blue) was used to reveal all cells in culture.</p> <p>(B and C) At 14 days within the differentiation phase (i.e., after bFGF removal), ~ 50% of cells acquire MAP2 immunoreactivity and neuronal cytology, with characteristic processes (red, B). A smaller number of cells differentiate into GFAP (+) astrocytes (green, C).</p> <p>(D) Real-time RT-PCR data showing increased neurotrophic factor and NRG expression in the course of NSC differentiation in vitro. The number of days on top of the columns is the days NSCs have been in a phase of differentiation (after withdrawal of fibroblast growth factor). Results are expressed as fold increases compared to levels expressed at the proliferation phase (day 0), the latter values designated as 1. Data represent average ± standard deviation of triplicate measurements of a representative cell culture sample at a given time point. The experiment was repeated twice with different sets of cell samples and yielded very similar results.</p> <p>Scale bars: 50 μm.</p></div

Experimental Treatment Conditions.

<p>Treatment Summary Table. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091408#pone-0091408-t001" target="_blank">Table 1</a> summarizes the 4 treatment groups of randomly assigned stroke animals.</p

A representative image of hNSE staining in the striatum.

<p>Human graft cells were densely confined to the striatum (arrow) with small amounts of hNSE+fibers that extended dorsally and ventrally from the striatum (arrowheads). Scale bar = 0.5 mm [Iv; lateral ventricle, Str: striatum].</p

NSI-566RSC cell grafts attenuate stroke-induced neurologic impairments.

<p>Neurological function was assessed using a battery of neurological tests (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0091408#pone-0091408-g002" target="_blank">Figure 2</a>). All animals displayed normal neurological function at Baseline (i.e., prior to stroke). At day 7 post-stroke, all animals exhibited significant impairment in neurological function, indicating that all animals received successful stroke. At 14 days post-stroke onwards, dose-dependent and timing-dependent effects of the treatment were recognized, in that the improvement in neurological performance was in the order of high dose to zero dose as follows: 20,000 cells/µl (D) >10,000 cells/µl (C) >5,000 cells/µl (B)>vehicle infusion only (A). In addition, over time there was a trend of better improvement, with the most significant improvement seen at 56 days post-stroke. <b>*</b>significant <0.05 vs. other treatment groups within time point; <b>^#</b>significant <0.05 vs. other time points.</p

Human Neural Stem Cell Replacement Therapy for Amyotrophic Lateral Sclerosis by Spinal Transplantation

<div><h3>Background</h3><p>Mutation in the ubiquitously expressed cytoplasmic superoxide dismutase (SOD1) causes an inherited form of Amyotrophic Lateral Sclerosis (ALS). Mutant synthesis in motor neurons drives disease onset and early disease progression. Previous experimental studies have shown that spinal grafting of human fetal spinal neural stem cells (hNSCs) into the lumbar spinal cord of SOD1^G93A rats leads to a moderate therapeutical effect as evidenced by local α-motoneuron sparing and extension of lifespan. The aim of the present study was to analyze the degree of therapeutical effect of hNSCs once grafted into the lumbar spinal ventral horn in presymptomatic immunosuppressed SOD1^G93A rats and to assess the presence and functional integrity of the descending motor system in symptomatic SOD1^G93A animals.</p> <h3>Methods/Principal Findings</h3><p>Presymptomatic SOD1^G93A rats (60–65 days old) received spinal lumbar injections of hNSCs. After cell grafting, disease onset, disease progression and lifespan were analyzed. In separate symptomatic SOD1^G93A rats, the presence and functional conductivity of descending motor tracts (corticospinal and rubrospinal) was analyzed by spinal surface recording electrodes after electrical stimulation of the motor cortex. Silver impregnation of lumbar spinal cord sections and descending motor axon counting in plastic spinal cord sections were used to validate morphologically the integrity of descending motor tracts. Grafting of hNSCs into the lumbar spinal cord of SOD1^G93A rats protected α-motoneurons in the vicinity of grafted cells, provided transient functional improvement, but offered no protection to α-motoneuron pools distant from grafted lumbar segments. Analysis of motor-evoked potentials recorded from the thoracic spinal cord of symptomatic SOD1^G93A rats showed a near complete loss of descending motor tract conduction, corresponding to a significant (50–65%) loss of large caliber descending motor axons.</p> <h3>Conclusions/Significance</h3><p>These data demonstrate that in order to achieve a more clinically-adequate treatment, cell-replacement/gene therapy strategies will likely require both spinal and supraspinal targets.</p> </div