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STEM_767_sm_suppinfofig1.eps14659KSupplementary Figure 1 Neurosphere formation by shi-NS/PCs. (A), (B): shi-NS/PCs formed neurospheres indistinguishable from those of wt-NS/PCs. Scale bar = 200 μm. (C): ATP assay of shi- and wt-NS/PCs. There was no significant difference in the proliferation rate between the wt- and shi-NS/PCs (n = 32 assays. Each type of NS/PC was derived from four embryos with eight cultures for each embryo)).
STEM_767_sm_suppinfofig2.eps1801KSupplementary Figure 2 Representative motor-evoked potential (MEP) recordings in the control and transplanted mice during the experiment. MEP waves were not detected in any group immediately after injury or transplantation. Although MEP waves were detected in the shi-NS/PC and wt-NS/PC groups 7 weeks after injury, no waves were detected in the control group. Compound motor action potentials (CMAPs) obtained after sciatic nerve stimulation were observed at all time points and in all groups.
STEM_767_sm_suppinfofig3.eps3995KSupplementary Figure 3 shi-NS/PCs in an ICR mouse strain background did not survive in the injured spinal cord of C57BL/6J hosts, resulting in no functional recovery. Contusive SCI was induced by IH impactor (60 kdyn) and NS/PCs (5 × 105 cells/mouse; labeled with Venus fluorescent protein lentivirally) were transplanted into the lesion site 9 days after injury. (A): Immunohistological analysis using an anti-GFP antibody revealed that no shi-NS/PCs survived in the injured spinal cord. (B): On the other hand, wt-NS/PCs survived in the injured spinal cords of C57BL/6J mice as well as NOD/SCID mice. Scale bar = 500 ?m. (C): Mean BMS scores for each group over the 42-day recovery period. Although there was no significant difference in the BMS scores among the three groups until 21 days after injury, at day 28 and thereafter, the wt-NS/PC group showed significantly improved BMS scores compared with the other two groups (control, n = 4; shi-NS/PC group, n = 6; wt-NS/PC group, n = 4).
STEM_767_sm_suppinfofig4.eps1520KSupplementary Figure 4 Quantification of trophic factor mRNA levels produced by neurospheres from both NS/PC genotypes. Trophic factor gene expression plots of the value 2(-Δ CT) with a three-fold difference are displayed, with shi-NS/PCs (y-axis) and wt-NS/PCs (x-axis). Sixty-four factors (see Supplementary Table 1 are dotted between the lines indicating a three-fold difference (n = 3 each). mRNAs for ciliary neurotrophic factor (CNTF), growth differentiation factor 11 (GDF11), platelet derived growth factor A (PDGFA) and vascular endothelial growth factor B (VEGFB) were expressed at relatively high levels by both the shi- and wt-NS/PC-derived neurospheres. Although every analysis of wt-NS/PC-derived neurospheres detected MBP mRNA expression, it was never detected in the analyses of shi-NS/PC-derived neurospheres, confirming their identity as MBP-deficient shiverer mutant cells.
STEM_767_sm_suppinfotab1.doc467KSupplementary Table 1. The primers used in this study are shown in this table. All primers were manufactured by and obtained from Applied Biosystems.
STEM_767_sm_suppinfo.doc77KSupporting Information

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