Clonally derived neurosphere efficiently contributes to spinal cord regeneration and reconstitutes the whole complement of spinal cord cell types. Clonally.

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Clonally derived neurosphere efficiently contributes to spinal cord regeneration and reconstitutes the whole complement of spinal cord cell types. Clonally derived neurosphere efficiently contributes to spinal cord regeneration and reconstitutes the whole complement of spinal cord cell types. (A) GFP+ cells cultured in microwells of an AggreWell 400 plate at clonal density with GFP− white feeder cells. Arrow indicates a single EGFP+ cell in the microwell, imaged on the first day of the culturing procedure. (B) Clonal neurosphere that arose from the single EGFP+ cell indicated by the arrow in A after 34 d of culturing. (C) A clonal GFP+ neurosphere was engrafted into the injured spinal cord of the nontransgenic host. After 1 wk of healing the tail was amputated close to the implant, maintaining GFP+ cells within the 500-μm zone behind the amputation plane. (D–F) Outgrowth of the GFP+ spinal cord from the implanted clonal neurosphere monitored at 7, 24, and 54 dpa. (G–J) Cross-sections of the regenerated spinal cord from F at a caudal level. In this portion of the regenerate the full lateral half of the spinal cord was reconstituted from GFP+ cells. Cross-sections were analyzed by immunofluorescence for expression of cell type-specific markers. (G) Anti-GFAP staining shows GFP+ cells have formed GFAP+ radial glia. (H) Anti–βIII-tubulin staining shows GFP+/βIII-tubulin+ neuronal cells. (I) Cross-section immunostained for Pax6 shows that implanted GFP+ cells express Pax6 in the normal, lateral spinal cord domain. (J) Cross-section immunostained for Pax7 shows that implanted GFP+ cells in the dorsal domain express Pax7. (K and L) Shh in situ hybridization shows clonally derived Shh+/GFP+ floor plate cells (arrows). (M and N) Msx1 in situ hybridization shows Msx1+/GFP+ roof plate cell indicated by arrow. (O) βIII-Tubulin/MBP double-immunostaining of the cross-section. βIII-Tubulin was labeled with Cy5 secondary antibody and is shown in yellow. (P–R) Enlarged views of boxed area in O, which shows the axonal layer of the spinal cord containing MBP+/GFP+ myelin (arrowheads). In G–J, green indicates GFP+ cells; red indicates cell type-specific immunostaining; blue indicates Hoechst staining. In K–M, green indicates GFP, and blue indicates Hoechst staining. In O–R, yellow indicates βIII-tubulin; red indicates MBP; green indicates GFP; and blue indicates Hoechst staining. (Scale bars: A and B, 50 μm; C–F, 1 mm; G–J, 25 μm; K–N, 50 μm; O, 20 μm; P–R, 5 μm.)‏ Levan Mchedlishvili et al. PNAS 2012;109:34:E2258-E2266 ©2012 by National Academy of Sciences