H. Sakata et al. / 1
Supplementary materials and methods
Western blot analysis
The NSCs were treated with cell lysis buffer (9803; Cell Signaling Technology) according to the
manufacturer’s instructions, and used as whole cell lysate samples. Protein concentrations were
determined by comparison with a known concentration of bovine serum albumin using a kit
(23225; Thermo Fisher Scientific). Equal amounts of the samples (10-20 μg) were loaded per
lane and analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis on a 10%
NuPAGE Bis-Tris gel (NP0303; Invitrogen) and then immunoblotted. The primary antibodies
were a 1:200 dilution of a mouse monoclonal anti-p-STAT3 antibody (Tyr705) (molecular
weight: ~ 91 kDa) (sc-8059; Santa Cruz Biotechnology), a 1:200 dilution of a mouse monoclonal
anti-STAT3 antibody (molecular weight: ~ 91 kDa) (sc-8019; Santa Cruz Biotechnology), a
1:1000 dilution of a rabbit polyclonal anti-SOD2 antibody (molecular weight: ~ 25 kDa)
(SOD-110; Enzo Life Sciences), a 1:1000 dilution of a rabbit polyclonal
anti-copper/zinc-superoxide dismutase antibody (molecular weight: ~ 19 kDa) (SOD-101; Enzo
Life Sciences), a 1:5000 dilution of a mouse monoclonal anti-IL-6Lα antibody (molecular
weight: ~ 80 kDa) (sc-374259; Santa Cruz Biotechnology), a 1:1000 dilution of a mouse
monoclonal anti-NF-κB antibody (molecular weight: ~ 65 kDa) (6956; Cell Signaling
Technology), and a 1:100,000 dilution of a mouse monoclonal anti-actin antibody (molecular
weight: ~ 42 kDa) (A5441; Sigma-Aldrich). After incubation with horseradish
peroxidase-conjugated anti-mouse IgG (7076; Cell Signaling Technology) or anti-rabbit IgG
(7074; Cell Signaling Technology), the antigen was detected by SuperSignal West Pico and/or
Femto substrates (1856135/1856189; Thermo Fisher Scientific). Images were scanned with a
H. Sakata et al. / 2
GS-700 imaging densitometer (Bio-Rad Laboratories) and the results were quantified using
MultiAnalyst software (Bio-Rad).
Immunofluorescent staining
For immunocytochemistry, the NSCs cultured on eight-well chamber slides (154941; Thermo
Fisher Scientific) were washed with PBS and fixed with 4% paraformaldehyde in PBS for
15 min. They were washed with PBS and incubated for 1 h in blocking solution (PBS containing
3% bovine serum albumin and 0.3% Triton X-100). For immunohistochemistry, the animals
were anesthetized and perfused with PBS followed by 4% paraformaldehyde in PBS, pH 7.4.
The brains were postfixed overnight in the same fixative at 4C. For cryosectioning, fixed tissues
were cryoprotected in 10% sucrose in PBS overnight at 4C, then in 20% sucrose in PBS
overnight at 4C, and embedded in Tissue-Tek O.C.T. compound (4583; Sakura Finetek USA,
Inc.). Cryostat sections (20 μm) were cut and affixed to glass slides (12-550-15; Thermo Fisher
Scientific). Cells or tissue sections were subsequently incubated overnight at 4C in an
appropriate mixture of primary antibodies. The following antibodies were used: rabbit anti-GFP
(1:100; G10362; Invitrogen), goat anti-GFP (1:100; LS-C67095; LifeSpan BioSciences), mouse
anti-nestin (1:100; AB353; Millipore), mouse anti-Sox2 (a NSC marker) (1:50; 4900; Cell
Signaling Technology), rabbit anti-β-tubulin (1:500; PRB-435P; Covance), mouse anti-GFAP
(1:100; AB360; Millipore), rabbit anti-NG2 (an oligodendrocytic marker) (1:100 AB5320;
Millipore), rabbit anti-SOD2 (1:50; ab13533; Abcam), mouse anti-p-STAT3 (Tyr705) (1:50;
4113; Cell Signaling Technology), rabbit anti-VEGF (1:50; 07-1376; Millipore) and rabbit
anti-Ki-67 (1:50; ab16667; Abcam). After three washes in PBS, cells or tissue sections were
incubated for 1 h with a 1:500 dilution of the following secondary antibodies: Alexa Fluor
H. Sakata et al. / 3
488-conjugated donkey anti-rabbit IgG (A21206), Alexa Fluor 594-conjugated donkey
anti-rabbit IgG (A21207), Alexa Fluor 488-conjugated donkey anti-goat IgG (A11055), Alexa
Fluor 594-conjugated donkey anti-mouse IgG (A21203), and Alexa Fluor 647-conjugated
donkey anti-mouse IgG (A31571; all from Invitrogen). After three washes in PBS, the samples
were covered with VECTASHIELD mounting medium with DAPI. The samples were examined
by confocal microscopy or fluorescence microscopy.
Assessment of cell death and cell viability in vitro
Cell death was quantified by a standard measurement of LDH release using a LDH-cytotoxicity
assay kit (K311-400; BioVision). Cell viability was assessed with a cell proliferation reagent
using a WST-1 assay kit (05015944001; Roche Diagnostics). For in situ labeling of DNA
fragmentation, the NSCs were prepared as described in the immunofluorescent staining section.
An in situ cell death detection kit, TMR red (12156792910; Roche Diagnostics), was used
according to the manufacturer’s instructions.
Detection of vascular endothelial growth factor
For the in vitro study, culture supernatants (incubation duration: 6 h) were collected for analysis
and fresh brain tissue was removed 2 days after transplantation (n = 4 per group). The
rectangular cuboid tissue block of the cortex, 1 mm on either side of the NSC-transplanted
regions, was dissected (width 2 mm  length 5 mm) and used as a sample. Protein was extracted
as described in the Western blot analysis section. Commercial VEGF ELISA kits (MMV00;
R&D Systems) were used to quantify the concentration of VEGF in each of the samples.
H. Sakata et al. / 4
Quantification of survival of the transplanted NSCs
The transplanted GFP-positive cells were counted using unbiased computational stereology as
described above (n = 8 per group). All GFP-positive cells were counted on six serial coronal
sections per brain (1 mm apart).
Assessment of NSC proliferation and differentiation profiles
The proportion of GFP-positive cells, also stained with a proliferation marker (Ki-67) (n = 4 per
group) or lineage-specific phenotype markers (β-tubulin and GFAP) (n = 8 per group), was
determined by confocal microscopy. Split panel and z axis analyses were used for all counting.
One hundred or more GFP-positive cells were scored for each marker per animal.
BVD analysis
BVD was assessed 14 days after transplantation as described previously (Horie et al., 2011),
with some modifications (n = 4 per group). Briefly, vessels were labeled by jugular vein
injection of DyLight 594-labeled Lycopersicon esculentum lectin (50 mL) (DL-1177; Vector
Laboratories) 30 min before the animals were killed. The tissue sections (40 μm) were prepared
as described in the immunofluorescent staining section. The images were captured by confocal
microscopy at these six coordinates: (1) A–P, +1.0; M–L, ±2.5; D–V, -0.5; (2) A–P, -0.5; M–L,
±3.0; D–V, -0.5; (3) A–P, -2.0; M–L, ±3.0; D–V, -0.5. BVD was measured using ImageJ
software to determine pixel number/image, and the average of BVD ratio
(ipsilateral/contralateral) was calculated.
Water content measurement
H. Sakata et al. / 5
Mouse brains were harvested 2 days after stroke and transplantation for determination of water
content in the cerebral tissue (n = 4 per group). Wet and dry weight of cerebral hemispheres
(ipsilateral and contralateral) was measured and the corresponding water content was calculated
by the formula water content = 100  (wet weight - dry weight)/wet weight.
H. Sakata et al. / 6
Supplementary figure legends
Supplementary Figure 1 Characterization of GFP-positive NSCs in vitro. The NSCs grown as
adherent cultures were examined by immunocytochemistry for GFP (green) and the NSC
markers nestin (A) and Sox2 (red) (B). Nearly all the GFP-positive cells colocalized with both
NSC markers. Scale bars: 50 μm. After culturing in differentiation medium containing 1 μM
retinoic acid and 1% fetal bovine serum for 5 days, GFP-positive cells (green) expressed the
neuronal marker β-tubulin (C), the astrocytic marker GFAP (D), and the oligodendrocytic
marker NG2 (red) (E). Nuclei were counterstained with DAPI (blue). Scale bars: 20 μm.
Supplementary Figure 2 Up-regulation of SOD2 with IL-6 preconditioning. (A) Western blot
analysis of whole cell lysate from cerebral endothelial cells (CECs), neurons, NSCs, and
SOD2
NSCs. Basal expression of SOD2 is higher in the NSCs than in the CECs and neurons. While
SOD2 expression in the CECs increased after ischemic reperfusion injury, SOD2 expression in
the neurons and NSCs decreased. β-actin was used as an internal control. (B) Western blot
analysis of whole cell lysate from NSCs revealed expression of IL-6Rα under normal conditions.
Expression of IL-6Rα was reduced after 8 h of OGD and 3 h of RO (n = 4). (C) Western blot
analysis of whole cell lysate from NSCs preconditioned with IL-10. This preconditioning did not
activate STAT3. In addition, SOD2 expression was similar at any concentration we tested.
(D) Western blot analysis of whole cell lysate from non-PCNSCs or PCNSCs after 8 h of OGD and
3 h of RO. IL-6 preconditioning significantly increased expression of SOD2 together with
p-STAT3 under normal conditions and after OGD and RO. (E) Western blot analysis of whole
cell lysate from non-PCNSCs or PCNSCs. IL-6 preconditioning significantly increased NF-κB
H. Sakata et al. / 7
expression. BMS-345541 significantly reduced NF-κB expression without changing SOD2 levels.
β-actin was used as an internal control. OD = optical density. *P < 0.05, †P < 0.01, ‡P < 0.001.
Supplementary Figure 3 NSC death in vitro. (A) LDH assay revealed increased NSC death in a
time-dependent manner with OGD (n = 4). Con = control. (B) NSCs analyzed by TUNEL
staining (red) and DAPI (blue) after 8 h of OGD and 24 h of RO. The cell-counting study
revealed a significant decrease in TUNEL-positivity in the PCNSCs and SOD2NSCs (n = 4). Scale
bar: 20 μm. IL-6 preconditioning and SOD2 overexpression significantly reduced the release of
LDH from the NSCs under the oxidative stimuli H2O2 (200 μM) (C) and
diethylenetriamine/nitric oxide (DETA/NO) (250 μM) (D) (n = 4). LDH (E) and WST-1 (F)
assays revealed that AG490 (20 nM) inhibited IL-6-induced cytoprotection in the NSCs after 8 h
of OGD and 24 h of RO (n = 4). (G) In vitro ELISA of the CM from NSCs. IL-6 preconditioning
significantly increased VEGF levels after 8 h of OGD and 6 h of RO. *P < 0.05, †P < 0.001.
Supplementary Figure 4 The effects of grafted NSCs on the host brain cells. (A) Schematic
diagram and fluorescent staining with DAPI (blue) and GFP (green) 1 h after transplantation
show the location of the graft and the ischemic lesion (*). Scale bar: 100 μm. (B) Quantification
of the number of TUNEL-positive host brain cells beside the graft 2 days after transplantation.
Transplantation of PCNSCs significantly reduced the number of TUNEL-positive host brain cells
compared with non-transplanted and non-PCNSC groups (n = 4). (C) Fluorescent staining with
DAPI (blue) and ED-1 (red) in brain sections 2 days after transplantation. The number of
ED-1-positive cells beside the graft was significantly decreased in the PCNSC group compared
with the non-transplanted and non-PCNSC groups (n = 4). Scale bar: 20 μm. *P < 0.05, †P < 0.01.
H. Sakata et al. / 8
Supplementary Figure 5 Proliferation capacity of NSCs. (A) Immunostaining images of the
NSCs 2 days after stroke and transplantation. The sections were stained with GFP (green) and
Ki-67 (red). IL-6 preconditioning did not change the percentage of Ki-67-positive grafted cells (n
= 4). Scale bar: 20 μm. (B) Fluorescent staining of the NSCs with Ki-67 (red) and DAPI (blue)
48 h after OGD in vitro. The percentage of Ki-67-positive cells was similar between the
non-PC
NSCs and PCNSCs (n = 4). Scale bar: 20 μm.
Supplementary Figure 6 IL-6-induced VEGF expression in vivo. (A) Fluorescent staining with
DAPI (blue), GFP (green), and VEGF (red) in brain sections 2 days after transplantation. IL-6
preconditioning increased the expression of VEGF in the NSCs. Scale bar: 10 μm. (B) Brain
water content 2 days after transplantation. No changes in the water content of the ischemic brains
were observed among the groups.
Supplementary Figure 7 Sub-acute delivery of NSCs. NSCs were transplanted into the brain
7 days after onset of stroke. (A) Fluorescent staining with DAPI (blue), TUNEL (red), and GFP
(green) 2 days after transplantation. Sub-acute delivery of NSCs (7 days after stroke) resulted in a
decreased number of TUNEL-positive grafted cells compared with acute delivery (6 h after
stroke). IL-6 preconditioning significantly reduced the number of TUNEL-positive grafted cells at
the sub-acute stage of stroke (n = 4). Scale bar: 20 μm. (B) Fluorescent staining with DAPI (blue)
and HEt (red) in brain sections 1 h after transplantation. HEt signals in the host brain cells beside
the graft were significantly reduced at the sub-acute stage of stroke compared with the acute stage
of stroke. Scale bar: 10 μm. (C) Representative images of lectin-perfused vessels and
H. Sakata et al. / 9
quantification of BVD in the peri-infarct cortex 14 days after transplantation (21 days after stroke).
BVD was significantly increased in the PCNSC group (n = 4). Scale bar: 100 μm. (D) Behavioral
performance using the rotarod test and mNSS. Transplantation of PCNSCs significantly enhanced
behavioral improvement as indicated by the rotarod test on Days 21 and 28 compared with the
non-transplanted control group (n = 8). Black bars denote non-transplanted control group; yellow
bars denote non-PCNSC group with control-siRNA transfection; blue bars denote PCNSC group with
control-siRNA transfection; red bars denote PCNSC group with STAT3-siRNA transfection. The
labels show P value compared with the non-transplanted control group. *P < 0.05, †P < 0.001.