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>