In vitro and in vivo characterization of GDNF-hNSPCs. (A) Compared with Mock-hNSPCs (DAPI+, blue), many of GDNF-hNSPCs express GDNF (green). (B) An ELISA of GDNF in Mock- and GDNF-CM. (C, D) Representative images of Mock- and GDNF-hNSPCs stained for differentiation markers and quantification of the percentages of cells that expressed these markers. DAPI+ Cells (blue) express nestin (red), GFAP (green), Tuj1 (red), PDGFR (green), and O4 (red). (E, F) Representative images of CM-treated SH-SY5Y cells and quantification of average neurite length under different experimental conditions. (G) hNuc+ GDNF–hNSPCs (red) implanted into the injured spinal cord show robust engraftment and an extensive distribution throughout the lesion and adjacent areas. The boxed area is shown at high magnification in the right panel. The asterisk indicates the lesion epicenter and arrows indicate the cell transplantation sites rostral and caudal to the lesion epicenter. (H, I) Representative images of Mock- and GDNF-hNSPCs stained for differentiation markers in vivo and quantification of the percentages of cells that expressed these markers. Confocal images show that hNSPCs positive for the human cell markers Ku80, STEM101, and STEM121 colocalize with differentiation markers (green). (J) Engrafted STEM121+ GDNF-hNSPCs (red) express GDNF (green). Scale bars: 50 μm in A, D; 100 μm in E; 1 mm in G; and 30 μm in I, J. Data represent the means±SEM. *p<0.05 vs. Mock-hNSPCs in C, H; *p<0.05 vs. vehicle, ***p<0.001 vs. vehicle, ##p<0.01 vs. Mock-hNSPCs in F.|@|~(^,^)~|@|Grafted GDNF-hNSPCs enhance neurite outgrowth, axonal extension, and myelination in the injured spinal cord. (A) Multiple NF+ neuronal processes (green) extend over engrafted hNuc+ human cells (red). Compared with Mock-hNSPCs, GDNF-hNSPCs significantly enhance neurite extension and host axonal sprouting. Arrows indicate hNuc+ donor-derived cells (red) and arrowheads indicate NF+ neuronal fibers (green). (B) Under high magnification, many NF+ neuronal processes (red) are co-labeled with an anti-MBP antibody (green). Arrowheads indicate co-labeled myelinated neuronal fibers (yellow or orange). (C) The intensity of MBP staining in engrafted and adjacent areas is markedly higher in the GDNF-hNSPC-treated group than in the Mock-hNSPC-treated group. Arrows indicate hNuc+ donor-derived cells (red) and arrowheads indicate MBP+ myelinated neuronal fibers (green). (D) Representative images of LFB staining (blue) in the lesion and adjacent areas of the spinal cord. Dashed lines indicate demyelinated areas. (E) Quantification of the demyelinated volume in the different experimental groups. (F, G) Representative electron microscopic images of axially sectioned spinal cords (T8~T10) and quantification of G-ratio. (H, I) Representative images of Schwann/2E immunostaining (green) in the injured spinal cords and quantification of immunostaining density in the different experimental groups. Scale bars: 1 mm in A, C, D; 100 μm in B; 1 μm in F, and 500 μm in H. Data represent the means±SEM. *p<0.05 vs. vehicle, #p<0.01 vs. Mock-hNSPCs.|@|~(^,^)~|@|Grafted GDNF-hNSPCs protect the dCST, promote the formation of detour circuits, and reduce lesion volume and glia scar formation. (A) Representative images of BDA-labeled dCST fibers (red) at 6 mm rostral to the lesion epicenter in the different experimental groups. The boxed areas in the left panels are shown at high magnification in the right panels. (B) Quantification of the number of dCST fibers up to 6 mm rostral and caudal to the lesion epicenter. (C) Representative images of dCST collaterals (black, left panel) and FG-labeled PSNs (white, right panel) in the cervical enlargement of the spinal cord in the different experimental groups. The white dashed lines in the right panel indicate the margins between gray and white matter of the spinal cord. (D) Quantification of dCST collaterals and FG-labeled PSNs in the cervical enlargement. (E) Representative images of the contacts between dCST collaterals (black) and PSNs (brown) in the cervical enlargement. Arrowheads show closely located collateral fibers and PSNs. (F) Quantification of the number of contacts between dCST collaterals and PSNs. (G) Representative images of GFAP immunostaining in the lesion and adjacent areas of the spinal cord in the different experimental groups. Dashed lines indicate GFAP-negative areas. (H) Quantification of lesion volume, spared tissue volume, and glial scars. Scale bars: 100 μm in A, C; 40 μm in E; and 1 mm in G. Data represent the means±SEM. *p<0.05 vs. vehicle, ***p<0.001 vs. vehicle, #p<0.01 vs. Mock-hNSPCs.|@|~(^,^)~|@|Transplantation of GDNF-hNSPCs promotes motosensory recovery, reduces expression of voltage-gated sodium channels and NPY, and increases expression of GABA. (A) The BBB open-field walking scores before transplantation and at various time points post-transplantation. (B) Von Frey tests for mechanical allodynia in lesioned hindlimbs of rats with SCI before transplantation and at various time points post-transplantation. (C) qRT-PCR analysis of Nav1.3 and Nav1.9 expression in the T8~T10 segment of the spinal cord. (D, E) Representative images and quantification of NPY expression in lamina I of the dorsal horn in the T8 segment of the spinal cord. (F, G) Representative images and quantification of GABA expression in the T8~T10 segment of the spinal cord. (H) Many engrafted hNuc+ GDNF-hNSPCs (red, arrowheads) express GABA (green, arrowheads) adjacent endogenous GABA+ cells (green, arrows). Scale bars: 200 μm in D; 500 μm in F; and 50 μm in H. Data represent the means±SEM. *p<0.05 vs. vehicle, **p<0.01 vs. vehicle, ***p<0.001 vs. vehicle, #p<0.05 vs. Mock-hNSPCs, ##p<0.01 vs. Mock-hNSPCs in A, B. *p<0.05 vs. vehicle, #p<0.05 vs. Mock-hNSPCs in C, E, and G.
Exp Neurobiol 2019;28:679~696 https://doi.org/10.5607/en.2019.28.6.679
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