Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Supplementary Materials
Supplementary Methods
NPC derivation and cultures
Mice were anesthetized by intraperitoneal injection of pentobarbital (120 mg/kg)
and killed by cervical dislocation. The brains were removed and placed in artificial
cerebrospinal fluid (aCSF) (124 mM NaCl, 5 mM KCl, 1.3 mM MgCl2 , 0.1 mM
CaCl2, 26 mM NaHCO3, and 10 mM D-glucose, pH 7.3) aerated with 95% O2/5%
CO2 at room temperature. The SVZ neural tissue – excluding the subependyma – was
isolated after coronal sectioning and cut into 1 mm3 pieces. Pieces were transferred
into 30 ml of aCSF containing 1.3 mg/ml trypsin, 0.67 mg/ml hyaluronidase, and 0.2
mg/ml kynurenic acid (all from Sigma) and incubated, under continuous oxygenation
and stirring, for 90 min at 32-34°C. Tissue sections were then rinsed in aCSF for 10
min, transferred to DMEM/F12 (Life Technologies) medium containing 0.7 mg/ml
ovomucoid (Sigma), and carefully triturated with a fire-polished Pasteur pipette. Cells
were collected by centrifugation and re-suspended in GF-free, chemically defined
DMEM/F12 medium containing 2 mM L-glutamine, 0.6% glucose, 9.6 mg/ml
putrescine, 6.3 ng/ml progesterone, 5.2 ng/ml sodium selenite, 0.025 mg/ml insulin,
0.1 mg/ml transferrin, and 2 g/ml heparin (Sigma). Cells were then cultured in
NeuroCult® Proliferation Kit (Stem Cell Technologies). The number of primary
spheres was counted after 7-12 days in vitro (DIV). For cell amplification, 8000
cells/cm2 were plated at each sub-culturing passage in untreated tissue culture flasks.
After 3-4 days (time estimated to obtain the doubling of cell number), neurospheres
were harvested, mechanically dissociated, counted and re-plated under the same
culture conditions. NPCs at passage number ≤ 20 were used in all experiments. For
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
NPC transplantation, single cell-dissociated NPCs were infected with 3 x 106 T.U./ml
of two different 3rd generation pCCLsin.PPT-hPGK lentiviral vectors (LV)
engineered either with the GFP with nucleus/cytoplasm localization (#277) or with a
farnesylated GFP (fGFP) with localization into the plasma membranes (#1514) (see
Supplementary Fig. 2 for LV maps). The choice of an additional LV carrying the
fGFP was made upon the need of specific membrane labelling for in vivo experiments
looking (eg at transplanted NPCs differentiating into myelin forming cells). Two days
after LV infection, NPCs were harvested, centrifuged at 200 x g for 12 minutes and
plated without further dissociation at a 1:1 ratio. After 3 passages of amplification in
vitro, FACS analysis was performed to verify the efficiency of the infection (see
after), as described (Pluchino et al., 2009; Pluchino et al., 2005).
Immunofluorescence
Immunofluorescence on NPCs in vitro was performed as described (Pluchino et
al., 2003). NPCs were fixed with 4% PFA 10’ at room temperature (r.t.), then rinsed
three times with PBS 1X, and then incubated for 60 min at r.t with a blocking solution
[PBS 1x + 10% normal goat serum (NGS, Sigma), 0,1% albumin bovine serum (BSA,
Sigma)] to avoid a-specific binding of the antibodies. For intracellular staining, the
same blocking solution as above, plus 0.1% Triton X-100, was used. Then fixed cells
were incubated for 2 further hours at r.t. with an appropriate primary antibody diluted
in PBS 1X. Cells were then washed thrice in PBS 1X and then incubated for 45
minutes with the appropriate secondary antibodies. The nuclei were stained with 4,6diamine-2-fenilindole (1 μg/ml, DAPI, Roche). Cells were then washed and mounted
with Fluorescent mounting medium (Dako).
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
FACS analysis on NPCs in vitro
NPCs were stained with fluorophore-conjugated rat anti-mouse 4 integrin (clone
PS/2, Abcam), rat anti-mouse CD44 (clone IM7, BD Biosciences) or rat anti-mouse
CXCR4 (clone 2B11/CXCR4, BD Biosciences) diluted in a solution of 2 μg/ml of
mouse IgG (as FcR blocking reagent) in PBS for 10 minutes at room temperature, in
the dark. The final incubation volume was of 30 μl/well. Cells were rinsed with PBS
as before and re-suspended with 200 μl of physiologic solution. FACS analyses were
carried out on a Cyan-ADP (Dako Cytomation) or FACSCanto® II flow cytometer
(BD) and data were analyzed using FlowJo (Treestar), FCS Express V3 (De Novo
Software) and CellQuest (BD Biosciences) softwares. At least 30.000 events were
acquired for each sample.
Assessment of locomotor function
The recovery of open-field locomotor performance was evaluated using the Basso
Mouse Scale (BMS), as described (Shechter et al., 2009). Mice were observed
individually for 4 minutes each in an open field by three investigators (M.D., S.S. and
G.S.) blinded to surgery and treatment. Hind limb motor function was recorded and
scored according to the BMS guidelines. For statistical analysis of the BMS score, the
mean of the left and right hind limb scores was performed to yield a single BMS score
per mouse.
Tissue processing and histopathology
1. Detection of transplanted NPCs
At one week after transplantation, the numbers of GFP+ cells were calculated on a
total of n= 56 GFP+ immunostained spinal cord segment-representative 30 μm-tick
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
axial sections (150 m apart) on n= 3 mice/treatment group; At seven weeks after
transplantation (52 dpi), the numbers of GFP+ cells were calculated on a total of n= 72
GFP+ immunostained spinal cord segment-representative 10 μm-tick axial sections
(100 m apart) on n= 4 mice/treatment group.
2. Quantification of lesion volumes
At one week after transplantation, the lesion volumes were calculated as contours
on a total of n= 28 GFAP immunostained spinal cord segment-representative 30 μmtick axial sections (300 m apart) per mouse; At seven weeks after transplantation (52
dpi), the lesion volumes were calculated as contours on a total of n= 72 GFAP
immunostained spinal cord segment-representative 10 μm-tick axial sections (100 m
apart) per mouse. Calculations were made on n= 6-12 mice/treatment group with 5X
or 10X magnifications and lesion volumes were expressed in mm3. The lesion volume
contours were traced at the boundaries between GFAP-positive and GFAP-negative
stainings modified from (Galvan et al., 2008).
3. Quantification of demyelination volumes
At one week after transplantation, the demyelination volumes lesion were
calculated as contours on a total of n= 28 Luxol Fast blue stained spinal cord
segment-representative 30 μm-tick axial sections (300 m apart) per mouse; At seven
weeks after transplantation (52 dpi), the demyelination volumes lesion were
calculated as contours on a total of n= 72 Luxol Fast blue stained spinal cord
segment-representative 10 μm-tick axial sections (100 m apart) per mouse.
Calculations were made on n= 4-10 mice/treatment group with 2.5 X or 10 X
magnifications and lesion volumes were expressed in mm3. The demyelination
volume contours were traced at the perimeter of the demyelinated areas modified
from (Brambilla et al., 2005).
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
4. Quantification of the inflammatory cells
The Iba1 volumes at the injury site were calculated at seven weeks after
transplantation only (52 dpi), as contours on a total of n= 10-23 Iba1+ immunostained
stained spinal cord segment-representative 10 μm-tick axial sections (100 m apart)
per mouse. In addition, we calculated Iba1 volumes at the level of the injury-spared
spinal cord tissue including the anterior, lateral and dorsal columns. These latter
volumes were calculated on a total of n= 9-11 Iba1+ immunostained stained spinal
cord segment-representative 10 μm-tick axial sections (600 m apart). Calculations
were made on n= 3 mice per treatment group with 5X or 40X magnifications and Iba1
volumes were expressed in mm3.
The numbers of B220+ cells were calculated at seven weeks after transplantation
only (52 dpi), on a total of n= 72 GFP+ immunostained spinal cord segmentrepresentative 10 μm-tick axial sections (100 m apart) on n= 4 mice/treatment group.
The numbers of CD3+ cells were calculated at seven weeks after transplantation
only (52 dpi), on a total of n= 72 GFP+ immunostained spinal cord segmentrepresentative 10 μm-tick axial sections (100 m apart) on n= 3 mice/treatment group.
Electron microscopy
Mice were perfused with 4 % paraformaldehyde 0.5 % glutaraldehyde and
postfixed o.n. in 4 % paraformaldehyde. Vibratome sections (50 m) were cut and
washed in 0.1 M phosphate buffer (PB), cryoprotected in 25% sucrose and freezethawed (3X) in ice-cold methyl-butane, as described (Pluchino et al., 2009). Sections
were blocked in 0.3% bovine serum albumin-C (BSA, Aurion) and incubated in
primary chicken anti-GFP antibody (Aves Labs, Tigard; 1:200) for 3 days at 4°C.
Sections were washed in PB, blocked in 0.5% BSA and 0.1% fish gelatin for 1 hour at
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
room temperature (RT), and then incubated in colloidal gold conjugated anti-chicken
secondary antibody (1:50; Ultrasmall, Aurion) for 24 hours. Sections were washed in
PB and 2% sodium acetate and silver enhancement was performed (Aurion). After
washing again in 2% sodium acetate, sections were immersed in 0.05% gold chloride
for 10 min at 4°C, washed in sodium thiosulfate, washed in PB and post-fixed in 2%
glutaraldehyde for 30 min. Sections were postfixed with 1% osmium and 7% glucose
and embedded in Durcupan resin (Fluka). Semithin sections (1.5 m) were cut with a
diamond knife and studied under the light microscope. Selected sections were reembedded for ultra-thin sectioning (70 nm). Images were obtained under a Fei
electron microscope (Tecnai Spirit G2, FEI) using a digital camera (Morada, Softimaging System, Soft Imaging System, Olympus).
Gene expression analysis
The Low-Density Array has n= 8 separate loading ports, for a total of 384 different
wells per card. Each 2 μl-well contains specific, user-defined primers and probes,
capable of detecting a single gene, each gene is evaluated in duplicate. The card is
designed so as to load two different samples (n= 4 separate loadings ports for each
sample) for a total of 94 genes and two housekeeping genes [glyceraldehydes-3phosphate dehydrogenase (GAPDH) and 18S, a mandatory control designed into each
array by the manufacturer]. A complete list of the mRNAs included in the 96b LowDensity Array is provided at the end of this paragraph.
The following treatment groups were considered for gene expression analysis:
Treatment group (TG) 1: SCI mice sacrificed at 7 dpi (n= 5);
TG 2: SCI mice sacrificed at 14 dpi (n= 5);
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
TG 3: SCI mice receiving sterile PBS 1X (Sham) at 7 dpi into a total of n= 4
injection sites (250 nl/site) bilaterally from midline at both the anterior aspect of T13
and the posterior aspect of T11 (n= 5) and sacrificed at 7 days post-treatment (dpt);
TG 4: SCI mice receiving single cell-dissociated 150 x 103 GFP+ NPCs at 7 dpi
into a total of n= 4 injection sites (250 nl/site), as above (n= 5) and sacrificed at 7
days post-treatment (dpt);
TG 5: SCI mice at 21 dpi (n= 5);
TG 6: SCI mice sacrificed at 28 dpi (n= 5);
TG 7: SCI mice receiving sterile PBS 1X at 21 dpi into a total of n= 4 injection
sites (250 nl/site), as above (n= 5) and sacrificed at 7 days post-treatment (dpt);
TG 8: SCI mice receiving single cell-dissociated 150 x 103 GFP+ NPCs at 21 dpi
into a total of n= 4 injection sites (250 nl/site), as above (n= 5) and sacrificed at 7
days post-treatment (dpt);
Comprehensive summary of the experimental design and treatment grouping is
provided in Supplementary Fig 1.
At the different time points, the spinal cord tissue samples were individually
homogenized in 1 ml of QIAzol Lysis Reagent (#79306, Qiagen) using a rotor-stator
homogenizer. Total RNA was isolated from homogenized tissue samples using
RNeasy Lipid Tissue Kit (#74804, Qiagen) including DNase digestion. At the end,
RNA samples were redissolved in 20 μl of RNase-free water and their concentrations
were determinated spectrophotometrically by A260 (Nanodrop-ND 1000, Thermo
Fisher Scientific). cDNA synthesis was performed using High capacity cDNA
Reverse Transcription kit
(#4374966, Applied Biosystems) according the
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
manufacturer’s instructions. cDNA were prepared using the same amount of RNA; 50
μl of water solution containing 0.85 μg of each sample were added to an equal volume
of 2 X TaqMan Universal PCR Master Mix (Applied Biosystems) and loaded into
each of four ports on the Taqman Low-Density Array (Applied Biosystems). The realtime RT-PCR amplifications were performed using an Applied Biosystems 7900HT
Sequence Detection system. Thermal cycler conditions were standardized as follow: 2
minutes at 50°C [Uracil-DNA glycosylase (UNG) activation], 10 minutes at 94.5°C
(AmpliTaq Gold DNA Polymerase activation), 30 seconds at 97°C, 1 minute at
59.7°C for 40 cycles. Data were collected with instrument spectral compensations by
the Applied Biosystems SDS 2.2.1 software and analysed using the threshold cycle
(CT) relative quantification method. The threshold cycle (CT) indicates the cycle
number at which the amount of amplified target reaches a fixed threshold. Genes with
CT ≥ 33 were eliminated for lack of reliability. The software provides the average
value of CT between the two replicated. The CT method uses the formula 2-ΔΔCT to
calculate the expression of normalized genes vs a calibrator sample. The C T values
were normalized for endogenous reference [ΔCT = CT (target gene) – CT (GAPDH)]
and compared with a calibrator using the ΔΔCT formula [ΔΔCT = ΔCT (sample) – ΔCT
(calibrator)]. As calibrator sample, a whole spinal cord obtained from an untreated,
age-, sex- and strain- matched mouse was used. The 2- ΔΔCT then corresponds to the
ratio of the expression of each gene vs the whole spinal cord. Data were analysed in
logarithmic scale using log with base 2. Log2 = 1 then corresponds to 2-fold increase
in expression, while 2 corresponds to 4-fold increase in expression, etc. On the other
hand, Log2 = -1 corresponds to 2-fold reduction, while -2 corresponds to -4-fold
reduction, etc. Average value of log22-
ΔΔC
T
between the 5 samples per treatment
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
group was taken as the specific level of expression of a given gene compared to the
whole spinal cord.
To compare the gene expression levels between two groups of mice, we plotted the
Log2 of the fold change versus the negative Log10 of the p value. For each gene the
fold change has been calculated using the comparative Ct method as a ratio between
2-ΔΔCT(case) and 2-ΔΔCt(control). The corresponding p value was calculated performing a
Student’s t-test between cases and controls.
List of mRNAs included in the 96b Low-Density Array
Cod AB
GENE
CATEGORY
Reference
none
18S
Housekeeping
None
Mm03058063_m1
Mash1/Aslc1
Axonal growth
(Sugimori et al.,
2007)
Mm02344032_s1
Basp1/CAP-23
Axonal growth
(Bomze et al.,
2001)
Mm00476090_m1
Brevican (Bcan)
Astrogliosis
(Seidenbecher et
al., 1995)
Mm00480516_m1
Ctip2/Bcl11b
Astrogliosis
(Arlotta et al.,
2005)
Mm01340178_m1
BMP-2
Astrogliosis
(Fuller et al.,
2007)
Mm00432102_m1
BMP-7
Astrogliosis
(Fuller et al.,
2007)
Mm00432142_m1
C1qa
Inflammation
(Galvan et al.,
2008)
Cod AB
GENE
CATEGORY
Reference
9
Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Mm00438045_m1
Caspase (Casp)-3
Inflammation
(Citron et al.,
2008)
Mm00802247_m1
Casp-8
Inflammation
(Casha et al.,
2001)
Mm00516563_m1
Casp-9
Inflammation
(Colak et al.,
2005)
Mm00441242_m1
CCL2/MCP-1
Inflammation
(Perrin et al.,
2005)
Mm00441258_m1
CCL3/MIP-1
Inflammation
(Perrin et al.,
2005)
Mm00432360_m1
Cyclin D1
Cell cycle/Myelination
(Lange et al.,
2009)
Mm00483213_m1
N-Cadherin (NCad)
Axonal growth
(Skaper et al.,
2001)
Mm00432448_m1
Cyclin-dependent
Cell cyle/Myelination
kinase inhibitor
(Bedelbaeva et
al.)
(Cdkn)1a/p21
Mm00438168_m1
Cdkn1b/p27
Cell Cyle/Myelination
(Crockett et al.,
2005)
Mm01257348_m1
Cdkn2a/p16
Cell cycle/Myelination
(Atanasoski et
al., 2006)
Mm00486943_m1
Cdkn2d/p19
Cell cycle/Myelination
(Atanasoski et
al., 2006)
Zfp91-Cntf-
CNTF
Cell cycle/Myelination
Mm00446373_m1
Mm99999059_m1
(Lang et al.,
2008)
GranulocyteMacrophage colony-
Inflammation
(Huang et al.,
2009)
stimulating factor
(GM-CSF)/CSF-2
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Cod AB
GENE
CATEGORY
Reference
Mm01266652_m1
CSFr-1/CD115
Inflammation
(Shechter et al.,
2009)
Mm00507256_m1
CSPG-4 (NG2)
Astrogliosis/Myelination
(Buss et al.,
2009)
Mm00445553_m1
CXCL12/Stromal Cell-
Inflammation
derived Factor (SDF)-
(Opatz et al.,
2009)
1
Mm00436450_m1
CXCL2/MIP-2
Inflammation
(Armstrong et
al., 2004)
Mm01212723_g1
Ephrin (Eph) a3
Axonal growth
(IrizarryRamirez et al.,
2005)
Mm00433013_m1
Eph a4
Axonal growth
(Fabes et al.,
2006)
Mm01215897_m1
Eph b2
Axonal growth
(Fabes et al.,
2006)
Mm00500404_m1
GAP-43
Axonal growth
(Schmidt, 2004)
Mm99999915_g1
GAPDH
Housekeeping
None
Mm00497305_m1
Glypican (Gpc)-1
Axonal growth
(Bloechlinger et
al., 2004)
Mm01612247_mH
MHC-I
Inflammation
(Zanon and
Oliveira, 2006)
Mm00516023_m1
ICAM-1
Inflammation
(Isaksson et al.,
2000)
Mm00439561_m1
IGF-1
Axonal growth
(Cheng et al.,
1998)
Mm00580426_m1
IGF-2
Axonal growth
(Dugas et al.,
2008)
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Cod AB
GENE
CATEGORY
Reference
Mm00434210_m1
IL-15
Inflammation
(Gomez-Nicola
et al., 2008)
Mm00439770_m1
Integrin (Itg) 4
Inflammation
(Kerfoot and
Kubes, 2002)
Mm00801807_m1
Lymphocyte function-
Inflammation
associated antigen
(Cruse et al.,
1996)
(LFA)-1
Mm00496902_m1
Jagged 1
Astrogliosis
(Morga et al.,
2009)
Mm00493049_m1
L1 neural cell adhesion
Axonal growth
molecule (NCAM)
Mm00439445_m1
Laminin (Lam)-1
(Skaper et al.,
2001)
Axonal growth
(Jones et al.,
2003)
Mm00550083_m1
Lam-2
Axonal growth
(Jones et al.,
2003)
Mm01190515_m1
Lam-4
Axonal growth
(Jones et al.,
2003)
Mm00801853_m1
Lam-B1
Axonal growth
(Jones et al.,
2003)
Mm00440235_m1
Itg 2/CD18
Inflammation
(Mazzone and
Ricevuti, 1995)
Mm00476035_s1
Atonal homolog
Axonal growth
(Atoh)-1/Math-1
Mm00439506_m1
MMP-2
(Miesegaes et
al., 2009)
Inflammation/Astrogliosis
(Pizzi and
Crowe, 2007)
Mm01168420_m1
MMP-7
Inflammation/Astrogliosis
(Buss et al.,
2007)
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Cod AB
GENE
CATEGORY
Reference
Mm00442991_m1
MMP-9
Inflammation/Astrogliosis
(Pizzi and
Crowe, 2007)
Mm00500554_m1
MMP-12
Inflammation/Astrogliosis
(Pizzi and
Crowe, 2007)
Mm00439491_m1
MMP-13
Inflammation/Astrogliosis
(Pizzi and
Crowe, 2007)
Mm00490659_m1
MMP-16
Inflammation/Astrogliosis
(Pizzi and
Crowe, 2007)
Mm00456190_m1
Myelin transcription
Myelination
factor (MTF)-1
Mm00456815_m1
NCAM-1
(Aguirre et al.,
2007)
Axonal growth
(Skaper et al.,
2001)
Mm00484007_m1
Neurocan
Astrogliosis
(Rauch et al.,
1992)
Mm00476361_m1
NfkB
Inflammation
(Brambilla et al.,
2009)
Mm00447558_m1
Nkx 2.1
Myelination
(Butt et al.,
2008)
Mm00839794_m1
Nkx 2.2
Myelination
(Watanabe et al.,
2004)
Mm01278279_m1
Nkx 6.2
Myelination
(Cai et al., 2010)
Mm00435175_m1
iNOS
Inflammation
(Conti et al.,
2007)
Mm01309898_m1
nNOS
Inflammation
(Conti et al.,
2007)
Mm00626552_m1
Neuregulin (Nrg)-1
Myelination
(Taveggia et al.,
2005)
Cod AB
GENE
CATEGORY
Reference
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Mm00440466_s1
Neurogenin (Ngn)-1
Axonal growh
(Sugimori et al.,
2007)
Mm00435372_m1
Neuropilin (Nrp)-1
Axonal growth
(Gavazzi, 2001)
Mm00500896_m1
Netrin (Ntn)-1
Axonal growth
(Manitt et al.,
2006)
Mm00435422_m1
TrkB
Axonal growth
(Li et al., 2009)
Mm00497537_s1
Olig1
Myelination
(Lu et al., 2001)
Mm01210556_m1
Olig2
Myelination
(Lu et al., 2001)
Mm00443081_m1
Pax 6
Axonal growth
(Sugimori et al.,
2007)
Mm00478484_m1
Phosphacan (Pcan)
Astrogliosis
(Davies et al.,
2004)
Mm00445861_m1
NOGO-A
Axonal growth
(Chen et al.,
2000)
Mm00441291_m1
L-Selectin
Inflammation
(Fee et al., 2003)
Mm00441295_m1
P-Selectin
Inflammation
(Kerfoot and
Kubes, 2002)
Mm00436469_m1
Semaphorin 3a
Axonal growth
(Kaneko et al.,
2007)
Mm00441325_m1
Semaphorin (Sema) 3f
Axonal growth
(Lindholm et al.,
2004)
Mm00441343_m1
Sema 4f
Axonal growth
(Lindholm et al.,
2004)
Mm00600697_m1
GLAST
Astrogliosis
(VeraPortocarrero et
al., 2002)
Mm01198620_m1
SLIT1
Axonal growth
(Yi et al., 2006)
Mm01249143_g1
Socs3
Astrogliosis
(Miao et al.,
2006)
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Cod AB
GENE
CATEGORY
Reference
Mm01300162_m1
Sox10
Myelination
(Stolt et al.,
2002)
Mm00488369_s1
Sox2
Myelination
(Talbott et al.,
2005)
Mm00448840_m1
Sox9
Astrogliosis
(Guillemot,
2007)
Mm01962902_s1
Sprr1a
Axonal growth
(Bonilla et al.,
2002)
Mm01285373_m1
SLIT-ROBO
Axonal growth
(Yi et al., 2006)
Mm01219775_m1
STAT-3
Astrogliosis
(Herrmann et al.,
2008)
Mm00839861_m1
STAT-5a
Astrogliosis
(Xu et al., 2009)
Mm00441818_m1
Tissue inhibitor of
Astrogliosis
(Buss et al.,
metalloproteinases
2007)
(TIMP)-1
Mm00441825_m1
TIMP-2
Astrogliosis
(Buss et al.,
2007)
Mm01224941_m1
TIMP-3
Astrogliosis
(Buss et al.,
2007)
Mm00442346_m1
Toll-like receptor
Inflammation
(TLR)-2
Mm00445273_m1
TLR-4
(Kigerl et al.,
2007)
Inflammation
(Kigerl et al.,
2007)
Mm00449197_m1
VCAM-1
Inflammation
(Mabon et al.,
2000)
Mm00490179_m1
Versican (Vcan)
Astrogliosis
(DoursZimmermann et
al., 2009)
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Cod AB
GENE
CATEGORY
Reference
Mm01300555_g1
Wnt1
Axonal growh
(Liu et al., 2008)
Mm00437341_m1
Wnt4
Axonal growth
(Liu et al., 2008)
Mm00437347_m1
Wnt5a
Axonal growth
(Liu et al., 2008)
Ex vivo FACS analyses and cellular sorting
The following monoclonal antibodies, in different combinations, were used:
Biotin-conjugated anti-CD206 (MRC1) (Biolegend); APC-conjugated Streptavidin
(BD Bioscience); FITC-conjugated anti-GR1 (BD PharMingen); PE-conjugated antiCD45 (BD Bioscience); APC-Cy7-conjugated anti-F4/80 (Biolegend); PE-Cy7conjugated anti-CD11c (BD Bioscience); PacificBlue-conjugated anti-CD11b
(Biolegend); FITC-conjugated anti-CD11b (BD PharMingen); PE-conjugated antiCD3 (BD PharMingen); APC-conjugated anti-CD19 (BD PharMingen); PacificBlueconjugated anti-CD45 (Biolegend).
We identified the following distinct myeloid cell subsets:
(i) CD45+/CD11b+ myeloid cells;
(ii) CD45+/F4/80+/CD11b+/GR1– macrophage-lineage cells;
(iii) CD45+/F4/80+/CD11b+/CD11c+/GR1-/CD206- inflammatory macrophages
(Pucci et al., 2009);
(iv)
CD45+/F4/80+/CD11b+/CD11c-/GR1-/CD206+
tissue
remodelling/pro-
angiogenic macrophages (Pucci et al., 2009); and
(v) CD45+/F4/80-/CD11b+/CD11c+/GR1-/CD206- dendritic cells (DCs).
We analyzed all samples by a flow cytometer equipped with 3 lasers (Canto II; BD
Biosciences). DIVA software was used for acquisition of events (BD Biosciences).
Fluorochrome compensation was performed manually based on single color-marked
samples and/or compensation beads (BD Biosciences) when appropriate. All gates
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
were set based on specific fluorescence minus one (FMO) control samples. Analysis
was performed by FCS Express software (De Novo Soft). The following hierarchical
gating strategy was employed: 1.) Exclusion of doublets on an area (FSC-A) vs peak
(FSC-H) plot; 2.) Exclusion of debris on a physical parameter plot (FSC-A vs SSCA); 3.) Dead cells were excluded by 7-aminoactinomycin-D (7AAD) staining; and 4.)
Phenotypic identification of subpopulations (eg combination of up to 6 markers). The
numbers of positive cells were calculated on a total of n= 24 mice/treatment group
from a total of n= 2 independent experiments.
We also sorted distinct CD45+ haematopoietic cell populations from the lesioned
cord segment between T11 and T13 at 14 days after injury, from mice treated sub
acutely with NPCs or PBS using a MoFlo apparatus (Dako). As above, spinal cord
segments (4 mm-long) were dissected out from individual mice, and tissues were
homogenized and reduced to single-cell suspensions by collagenase IV (2 mg/ml),
dispase (0.2 mg/ml) and DNase I (0.1 mg/ml) treatment. Before sorting, we positively
selected total leukocytes by magnetic sorting (using CD45 MicroBeads; Miltenyi).
Cells were then incubated with 5% rat serum and 5 μg/ml rat anti-mouse FcγIII/II
receptor (CD16/CD32) blocking antibodies (BD PharMingen), and then stained using
the following monoclonal antibodies: Biotin-conjugated anti-CD206 (MRC1)
(Biolegend); APC-conjugated Streptavidin (BD Bioscience); FITC-conjugated antiCD3 (BD PharMingen); PE-conjugated anti-CD45 (BD Bioscience); APC-Cy7conjugated anti-F4/80 (Biolegend); PE-Cy7-conjugated anti-CD11c (BD Bioscience).
Cells were also stained with 7-AAD to exclude non-viable cells from further analysis.
We sorted the following cell subsets:
(i) 7-AAD–/CD45+/F4/80+/CD206high/CD11c– cells (myeloid);
(ii) 7AAD–/CD45+/F4/80+/CD206low/–/CD11c+ cells (myeloid); and
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
(iii) 7AAD– CD45+F4/80–CD3+ cells (lymphoid).
All gates were set based on a specific fluorescence minus one (FMO) control.
After sorting, purity of the cells was always ≥ 90%. Cellular sorting was performed
on a total of n= 24-30 mice/treatment group from a total of n ≥ 2 independent
experiments.
RNA isolation, reverse transcription and qRT-PCR
The SDS 2.2.1 software was used to extract gene expression raw data (CT). To
determine gene expression we employed a linear regression model (Pucci et al 2009).
Briefly, we implemented in R (http://www.R-project.org) a multivariate regression
model to compute over the whole dataset and estimate the fold-change in gene
expression for each single target gene. This model jointly evaluates the role of
different variables of interest providing for: (i) statistical significance of the observed
differences in expression level across the whole set of experimental samples; (ii)
identifying the experimental variables that contribute to each individual measurement;
(iii) subtracting experimentally introduced biases to obtain a stringent estimate of the
actual biological differences (Pucci et al., 2009). We performed the following
analyses:
1)
Comparative
gene
expression
profile
of
sorted
CD11c–/CD206+
and
CD11c+/CD206– (calibrator) from PBS- and NPCs-treated mice. Being the CT the
outcome variable, the covariates include the sorted cell type (e.g., CD11c–/CD206+
and CD11c+/CD206– macrophages), the mouse (i.e., biological replicate), the
treatment (i.e. PBS or NPCs) and the gene (gene of interest). In this case, the multiple
regression formula reads as follows (see below for details):
CT = β0 + β1 · XMouse + β2 · XTreat + β3 · XGene•Celltype + ε.
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
2) Comparative gene expression profile of sorted CD11c–/CD206+, CD11c+/CD206–
and CD3+, obtained from PBS- and NPCs-treated mice. Briefly, being the CT the
outcome variable, the covariates include the treatment group (i.e. PBS or NPCs), the
mouse (i.e., biological replicate) and the gene (gene of interest). In this case, for each
sorted population, the multiple regression formula reads as follows (see below for
details):
CT = β0 + β1 · XMouse + β2 · XGene•Treatment + ε.
In the regression formulas above, CT is the threshold cycle, βi are the coefficients
calculated by the model that represents the impact of the respective qualitative
variable Xj, with j being each of the covariates, and ε the residual error. The
implemented model leads to a procedure equivalent to test the ΔΔC T. A detailed
explanation of this equivalence can be found in (Pucci et al., 2009). The advantage of
this procedure with respect to two-by-two t-test comparisons lies on the joint nature of
the modeling of all covariates, which allows minimizing type I errors (false positive
results). Estimation technique is based on Likelihood Ratio Test. The model is
implemented in R-statistical software (version 2.6.1; see http://www.R-project.org).
Significance level is chosen at α = 0.05.
The profiled genes were CCR2, CCR7, CD86, Cox2, CXCL10, IL1beta, MCH-II,
NOS2, TNFalpha, Arg1, CD163, IGF1, Lyve1, MMP9, Tie2 for the macrophage
subset samples, and CD4, CD25, FoxP3, IL-10, IL-4, TGFbeta, CD8, FasL,
GranzymeB, IFNgamma, IL-17, IL-1beta, Perforin1 and TNFalpha for the CD3+ T
cell samples.
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
Author contribution
M.C. and S.P. designed research; M.C., D.B., E.B., M.D., C.A.-C., S.S., G.S., F.P.,
performed research; M.D.P. contributed analytical tools; M.C., D.B., C.A.-C., F.P.,
J.M.G.-V., M.D.P. and S.P. analyzed the data; G.C. and G.M. discussed the data;
M.C., M.D.P, and S.P. wrote the paper.
Supplementary Figures
Supplementary Figure 1. Experimental Design.
Supplementary Figure 2. Quantification of biotinylated dextran amine (BDA)labelled corticospinal tract (CST). Data are mean percentages of labelled CST (
SEM) over healthy controls from n 3 SCI mice per group at 7 days post-injury. 7 and
21 dpi refer to the time of injury.
Supplementary Figure 3. In vitro characterization of NPCs. (A and B), Maps of
the #277.GFP and #1514.fGFP LVs, respectively. (C-F) Direct fluorescence of
#277.GFP and #1514.fGFP NPCs in vitro (C and D), and in vivo (E and F), after
transplantation in the intact mouse spinal cord. Nuclei in C-F are counterstained with
Dapi. Scale bars: C-F, 10 m; E, 100 m. (G) FACS analysis for the expression of
GFP, as well as of major NPC functional markers on #277.GFP and #1514.fGFP
NPCs. The black lines on the left panels and the green/red lines on the right panels are
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
positive cells. The grey areas are non-infected NPCs, which have been used as
negative control.
Supplementary Figure 4. Quantification of tissue injury at early time points.
Stereological quantification of volumes of GFAP (A; red solid) and demyelination (B;
grey solid). The solid orange in A and B is the central canal, while the solid
transparent blue in B is the frank injury volume. Volumes in A and B have been
calculated at 7 days after the treatment. Data are min to max volumes from n= 5 mice
per group. * p 0.05, compared to PBS-treated controls.
Supplementary Figure 5. Quantification of low dose NPC transplants. (A)
Quantification of transplanted GFP+ NPCs in vivo at seven weeks after the
transplantation of 75 x 103 NPCs. Data are absolute min to max numbers of GFP+
cells/mouse ( SEM) from n 3 mice/group. (B and C) Representative axial images
of the GFP staining (brown) for stereological quantifications in A from two
representative SCI mice transplanted at 7 (B) or 21 (C) days after SCI. The blue
staining in B and C is for Hematoxilin. In the 3D renderings, the red solid is GFAP,
the green dots are GFP and the solid orange is the central canal. Dashed lines refer to
representative axial images.
Supplementary Figure 6. Electron microscopy of transplanted NPCs labelled
with pre-embedding immunogold for GFP. (A) Detail of a long cytoplasmic
process of a transplanted NPC (N) containing abundant intermediate filaments and
being in close contact with two monocytes/macrophages (m). (B) Detail of the NPC-
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
monocyte/macrophage cell-to-cell contact in A (arrows). Scale bars: A, 2 m; B, 500
nm.
Supplementary Figure 7. Volcano plot [x axis = log2 (Fold change); y axis = -Log10
(p-value)] showing statistically significant differentially expressed genes between SCI
mice injected with PBS at either 7 dpi (blue dots) or 21 dpi (red dots), as compared to
control SCI mice at the very same time points. Vertical grey lines (x = -0.5 and x=0.5)
correspond to fold changes of 0.7 and 1.4 respectively. The horizontal dashed line (y
= 1.3) corresponds to a p value= 0.05. Data have been calculated from n= 5 individual
mice per treatment group.
Supplementary Figure 8. qRT-PCR on FACS-sorted cells from the spinal cord of
PBS- and NPC treated SCI mice. (A) Gene signature of ‘alternatively-activated’
(M2-like; CD206+/CD11c–) macrophages in PBS-treated SCI mice showing a
characteristic tissue remodelling/pro-angiogenic phenotype. Data are mean fold
change (over M1-like, CD206–/CD11c+ macrophages) (± SEM). Green bars are
markers of M1-like macrophages, whereas blue bars are markers of M2-like
macrophages. (B) Gene signature of SCI-infiltrating CD3+ cells in NPC-transplanted
SCI mice. Data are as mean fold change (over PBS-treated) (± S.E.M.). Data in A and
B have been measured at 14 days post-injury (7 days after sub acute treatment) from
n= 24-30 mice/treatment group and a total of n≥ 2 independent experiments. *p≤
0.05; **p≤ 0.005 and ***p≤ 0.001.
Supplementary Figure 9. Flow cytometry analysis of lymphoid cell subsets
present in the injured spinal cord at 14 days post-injury (7 days after sub acute
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
treatment). CD45+ hematopoietic cells isolated from injured spinal cord were stained
with 7-AAD to exclude non-viable cells from further analysis. T-cells are showed in
(A), while B-cells are showed in (B). All gates were set based on specific
fluorescence minus one (FMO) control samples. For each lymphoid cell subset,
quantitative data are shown on the left, while representative density plots are shown
on the right. White whiskers are SCI mice injected with PBS, while black whiskers
are SCI mice injected with 150 x 103 NPCs. Data are min to max % marker-positive
cells from n= 24 mice/treatment group and a total of n= 2 independent experiments.
(C-D) Stereological quantification of the total numbers of CD3+ (C) and B220+ (D)
cells (dark grey dots) in the severely contused spinal cord. Grey and black whiskers
are SCI mice injected with 75 x 103 and 150 x 103 NPCs, respectively. White
whiskers are SCI mice injected with PBS. The solid orange in C and D is the central
canal, while the solid transparent blue in C and D is the frank injury volume.
Numbers in C and D have been calculated at 56 days after the injury. Data are mean
to max absolute numbers of marker-positive cells from n= 3 mice per group. *p≤ 0.05
and **p≤ 0.005, as compared to PBS-treated controls.
Supplementary Tables
Supplementary Table 1. mRNA expression data from TaqMan® Array Micro
Fluidic Card on lesioned cord segment of mice either non-injected, PBS injected or
NPC injected at 14 dpi or 28 dpi. For each gene the table reports fold change and pvalue for PBS injected versus non-injected mice and for NPC injected versus PBS
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Cusimano et al. BRAIN-2011-00594.R3
NPCs instruct professional phagocytes in the injured cord
injected mice. The fold change has been calculated using the comparative Ct method
as a ratio between 2-ΔΔCT(target) and 2-ΔΔCt(control). The corresponding p-value has been
calculated performing a Student’s t-test between target and control. The table also
reports log2(Fold change) and -Log10(p-value), which have been used to build the
volcano plots.
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