Miller et al., MS# DB-08-0244-R2
ONLINE-ONLY APPENDIX
SUPPLEMENTAL MATERIALS AND METHODS
Antibodies used in histology, cell separation and flow cytometry.
In tissue immunofluorescence studies the following antibodies were used: sheep anti-insulin (The
Binding Site), rabbit anti-synaptophysin (Dako), anti-H3 histone (Cell Signaling) and anti-galactosidase (Novus Biological), rat anti-CD45 (Pharmingen) or control IgGs (Zymed), followed
by Lissamine Rhodamine-donkey anti-sheep or anti rat Abs, or Cy5-donkey anti-rabbit Abs (The
Jackson Laboratory). For immuno-histochemistry, antibodies used were: rat anti-PECAM-1 (BD,
clone Mec13.3), rat anti-pan-endothelial antigen (BD, clone Meca-32) and anti-F480 (Caltag);
sheep anti-insulin (The Binding Site), rabbit anti-synaptophysin (Dako), anti-pAkt (Cell
Signaling), anti-betagalactosidase (Novus Biological); mouse anti-PML (MAB3738, Chemicon)
and anti-adiponectin (MAB3608, Chemicon); biotin-rat anti-CD45 (Pharmingen), goat antiadipsin (Santa Cruz Biotec) or control IgGs, followed by HRP-Fa(b)2 or Biotin- or fluorochromeconjugated donkey anti-rat, anti-sheep, or anti-rabbit antibodies or Streptavidin-Alkaline
phosphatase (The Jackson Laboratory).
For flow cytometric analysis of cells dissociated from the grafts, an anti-PECAM-1 Ab (BD,
clone Mec13.3) followed by an RPE-conjugated donkey anti-rat Ab (Jackson Labs) were used.
After blocking with rat IgGs, cells were incubated with a biotin-conjugated anti-CD45 Ab
(Pharmingen) followed by Cy-Chrome Streptavidin. Infiltrating leukocytes were stained with
RPE-anti-Gr1 (clone RB6-8C5), RPE-anti-CD3 (clone 145-2C11) (BD Pharmingen), FITC-antiF480, Cychrome anti-CD45R (Caltag) antibodies. For cell separation, cells dissociated from the
grafts were labeled with anti-PECAM or anti-F480 Abs followed by RPE-conjugated donkey
anti-rat Ab or RPE-streptavidin and anti-PE microbeads (Milteni Biotec).
Miller et al., MS# DB-08-0244-R2
Transmission electron microscopy.
Islet clusters, dissected from the grafts under a dissecting microscope, were fixed in 2%
glutaraldheide/0.1.mol/l sodium cacodylate buffer, pH 7.4. After post-fixing in osmium tetroxide,
islet samples were dehydrated and embedded in Epon 812. Ultrathin sections were mounted on
gold grids (Ted Pella), counter-stained with uranyl acetate and lead citrate, and examined at a
Jeol-1200 microscope (Jeol USA).
Insulin Tolerance Test and measurement of serum insulin.
Six weeks old Id1+/-Id3-/- and sex and age-matched WT mice were starved for 4 hours and
injected intraperitoneally with 0.75U/kg of insulin (Novolog, Novo Nordisk). Glycemia was
monitored every 10 minutes using a glucometer (Therasense). Serum insulin content was
measured by using a mouse insulin ELISA kit (ALPCO Diagnostics).
DNA microarray experiments.
Biotynilated cRNA was prepared using a modified Eberwine protocol (1), by which messenger
RNA is converted to cDNA, followed by an amplification/labeling step mediated by T7 DNA
polymerase. The cDNA and cRNA filter cartridges (Ambion) were used according to the
manufacturer's instructions for RT and IVT cleanup, respectively. For microarray analysis, the
Illumina Mouse 6 Sentrix Expression BeadChip was used (Illumina, San Diego). Hybridization
of labeled cRNA to the BeadChip, and washing and scanning were performed according to the
Illumina BeadStation 500x manual. Essentially the amplified, biotin-labeled human cRNA
samples were resuspended in a solution of Hyb E1 buffer (Illumina) and 25% (v/v) formamide at
a final concentration of 25 ng/µL. 1.5 µg of each cRNA were hybridized. Hybridization was
allowed to proceed at 55°C, for 18 hours after which, the bead array matrix was washed for 10
minutes with 1X High temperature buffer (Illumina), followed by a subsequent 10 minute wash in
Wash E1BC buffer. The arrays were then washed with 100% ethanol for 10 min to strip off any
Miller et al., MS# DB-08-0244-R2
remaining adhesive on the chip. A 2 minute E1BC wash was performed to remove residual
ethanol. The arrays were blocked for 5 minutes with 1% (w/v) casein-PBS, (Pierce). The array
signal was developed via 10 minutes incubation with Streptavidin-Cy3 at a final concentration of
1µg/mL solution of (GE Healthcare) in 1% casein-PBS blocking solution. The Mouse 6 Sentrix
Expression BeadChip was washed a final time in Wash E1BC buffer for five minutes and
subsequently dried via centrifugation for 4 minutes at a setting of 275 rcf. The arrays were
scanned on the Illumina BeadArray Reader, a confocal-type imaging system with 532 (cye3) nm
laser illumination. Image analysis and data extraction was carried out as previously reported (2).
The array images were registered using an algorithm described previously (3). Essentially the
bead signals were computed with weighted averages of pixel intensities, and local background is
subtracted. Sequence-type signal was calculated by averaging corresponding bead signals with
outliers removed (using median absolute deviation). Preliminary data analysis and QC was
carried out using the BeadStudio software (Illumina). All samples were normalized
simultaneously using a multiple-loess technique described in Sasik et al. (4).
ELISAs of serum adiponectin and IL10.
Blood samples were collected from BM-reconstituted Id1+/-Id3-/- and WT mice and serum
adiponectin was measured by using a mouse adiponectin ELISA kit (ALPCO Diagnostics). For
determination of IL10, equal amount of protein (3 g) obtained from cell lysates of F480+ cells
isolated from the grafts by positive selection on magnetic columns were tested in duplicate using
a mouse IL10 ELISA kit (Biolegend).
Supplemental References
1.
Eberwine J, Yeh H, Miyashiro K, et al. Analysis of gene expression in single live neurons.
Proc Natl Acad Sci USA. 1992; 89: 3010-3014.
Miller et al., MS# DB-08-0244-R2
2.
Fan JB, Yeakley JM, Bibikova M, et al. A versatile assay for high-throughput gene
expression profiling on universal array matrices. Genome Res. 2004; 14: 878-885.
3.
Galinsky VL. Automatic registration of microarray images. II. Hexagonal grid.
Bioinformatics. 2003; 19: 1832-1836.
4. Sasik R, Woelk CH, Corbeil J. Microarray truths and consequences J Mol Endocrinol. 2004;
33:1-9.
Miller et al., MS# DB-08-0244-R2
SUPPLEMENTAL FIGURE LEGENDS
Figure S1.
Phenotypic characterization of PECAM-1+ cells in islet grafts and control
tissues. Flow cytometric analysis of cell suspensions prepared by collagenase digestion of control
kidney perfused in vivo with ISB4-FITC (A, positive control tissue), kidney tissue not perfused
with ISB4-FITC (B, negative control tissue) or islet grafts from BM-reconstituted WT (C, pool of
n=2) or Id1+/-Id3-/- mice (D, pool of n=2) perfused in vivo with ISB4-FITC. Cell suspensions
were stained by two-color immunofluorescence for PECAM-1 and CD45. Two distinct cell
populations i.e. a PECAM-1LowCD45+ (gate R1) and a PECAM-1HighCD45- (gate R2) were
identified. Gate on PECAM-1LowCD45+ (R1) show no significant bound ISB4-FITC in any tissue
sample. In contrast, most PECAM-1HighCD45- (R2) dissociated from the islet grafts at either 1
week or 4 weeks post-transplantation display bound ISB4-FITC, indicating that this population
comprises bona fide endothelial cells lining functional blood vessels. This fraction of
ISB4+PECAM-1HighCD45- endothelial cells is higher at 4 weeks vs 1 week post-transplantation
and in BM-reconstituted Id1+/-Id3-/- vs WT mice, suggesting a different kinetic by which
developing blood vessels become blood-perfused in the two grafts. The yellow square in the dot
plots and the grey vertical lane in the hystogram plots indicate the upper limit of the background
levels of staining from IgG isotype controls. Percentages of positive cells are indicated in each
histogram plot.
Figure S2.
Ultrastructural morphology of islet grafts by transmission electron
microscopy. (A) Islet grafts from BM-reconstituted Id1+/-Id3-/- mice showing cross-sections of
capillary-like blood vessels (asterisks), lined by a fenestrated endothelium (ec, arrowheads) as
well as perivascular cells (pc). Occasional red blood cells are observed inside these capillaries
(rbc). Insets show a higher magnification of endothelial fenestrae (arrowheads). Islet -cells
closely associated with these blood vessels display typical electrondense insulin granules. (B)
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Islet grafts from BM-reconstituted WT mice showing cross-sections of larger capillary blood
vessels (asterisks). The endothelium of these blood vessels is also fenestrated (arrowheads), and
at times more loosely associated with the basal membrane. Insets show a higher magnification of
endothelial fenestrae (arrowheads). Images are representatives of at least 40 ultrathin sections
from n=2 grafts.
Figure S3.
Detection of BM-derived blood vessels and inflammatory cells in the islet
grafts of BM-reconstituted Id1+/-Id3-/- mice. Tissue sections of islet grafts from Id1+/-Id3-/mice reconstituted with -gal+ BM from ROSA26 mice at 1 week post transplantation, perfused
in vivo with ISB4-FITC and immunostained for -gal (blue, panels A, B and C) and either
PECAM-1 (red, panel A and B) or CD45 (red, panel C). Immunostainings with control IgGs are
shown in panel D. Images in A and B show areas of the grafts harboring many -gal+PECAM-1+
blood vessels (arrows) which have bound ISB4-FITC in vivo and are therefore functional vessels.
ISB4+-gal+PECAM-1+ blood vessels are surrounded by other -gal+ BM-derived cells. Staining
for CD45 demonstrate the most of these cells are leukocytes. ISB4+ vascular structures remain
negative for CD45 (panel C, arrows and dotted area).
Figure S4.
Validation experiments of selected genes differentially expressed by DNA
microarray analysis. (A) Grafts tissue sections stained for adipsin by immunohistochemistry
confirm down-regulation of the protein in BM-reconstituted Id1+/-Id3-/- mice. (B) Tissue
sections of the grafts stained by immunoperoxidase for adiponectin demonstrate localization of
adiponectin-specific staining in the stroma of the grafts, indicating that the up-regulated
expression of this gene detected in whole graft RNA samples of WT mice is likely to be
accounted for by the increased representation of connective tissue. (C) Densitometric analysis of
adipsin and S100A8 specific-protein bands detected by Western blotting in cell lysates of islet
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cell clusters dissected from the grafts, or from cell fractions enriched for ISB4 + or F480+ cells by
immunomagnetic cell separation. (D) ELISA of IL10 detected in protein extracts of CD31+ cells.
Figure S5.
Evidence for similar peripheral insulin sensitivity in Id1+/-Id3-/- and WT
mice. (A) Serum insulin levels measured by ELISA in Id1+/-Id3-/- (red squares) and WT (blue
diamonds) mice fed ad libitum overnight. (B) Insulin tolerance test. Blood glucose levels
measured in Id1+/-Id3-/- and WT mice after insulin challenge do not show a significantly
different response in the two experimental groups. Values are mean + SEM of measurements
from n=7 mice per experimental group. (C) Serum adiponectin detected by ELISA in diabetic
BM-reconstituted Id1+/-Id3-/- (red) and WT (blue) mice. Values are mean + SEM of
measurements from n=5 mice per experimental group.
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Miller et al., MS# DB-08-0244-R2