Supplemental Figure Legends
Figure S1. Schematic illustration of the generation of chimeric mice
(A) First reconstitution: All recipients were old female mice reconstituted with bone marrow
(BM) cells from either young or old male GFP mice. This generated young chimeras (Ym) and
old chimeras (Om), respectively. Eight weeks after the first reconstitution, the animals were used
for studies or secondary reconstitution. (B) Second reconstitution: The chimeric mice underwent
a second irradiation, with the heart carefully protected with a lead shield. The second
reconstitution replaced only peripheral BM stem/progenitor cells with old BM stem/progenitor
cells; therefore, the cardiac BM stem/progenitor cells remained either young (Yc) or old (Oc).
This generated cardiac micro-chimeric mice. Young micro-chimeras (Om-Yc) retained the
integrated young BM cells in the heart, whereas old micro-chimeras (Om-Oc) had only old BM
cells in the heart and periphery (and so served as controls).
Figure S2. Number of progenitor cells in chimeric mice one year after reconstitution
Old female mice (12–13 months) were reconstituted with young or old GFP (green) BM cells,
generating young chimeras (Ym) and old chimeras (Om), respectively. One year later, GFP+
cells in the BM (A), spleen (B), and heart (C) of the reconstituted mice were quantified by flow
cytometry. The number of GFP+ cells in all three organs was significantly higher in the Ym
group compared with the Om group. *p<0.05, **p<0.01.
Figure S3. Number and function of progenitor cells after second reconstitution
Three days after the second reconstitution, progenitor cells of young micro-chimeras (Om-Yc)
and old micro-chimeras (Om-Oc) were assessed. (A) The number of colony- or blast-forming
units for granulocytes/macrophages (CFU-GM),
granulocytes/erythrocytes/megakaryocytes/macrophages (CFU-GEMM), erythroblasts (BFU-E),
and fibroblasts (CFU-F) in the BM were measured (using semisolid medium). On Day 7 after
culture, there were no significant differences between the Om-Yc and Om-Oc groups
(n=6/group). (B) The number of angiogenic cells (KDR+ and CD34+) and stem cells (cKit+ and
CD34+) in the blood was quantified by flow cytometry. There were no significant differences
between the Om-Yc and Om-Oc groups (n=3/group).
Figure S4. Differentiation markers of progenitor cells after second reconstitution
MI was induced 3 days after the second reconstitution by coronary artery ligation.
Immunolabelling of myocardial sections 14 days post-MI assessed progenitor cell differentiation
in young micro-chimeras (Om-Yc) and old micro-chimeras (Om-Oc). Representative confocal
images show the cells are CD34− (A), cKit+ (B), Tie2+ (C), CD44+ (D), cardiac isoform αMHC−
(E), and DDR2− (F).
Figure S5. Schematic illustration of rejuvenation of the aged cardiac-resident BM-derived
progenitor cell pool and improved cardiac function after MI
Cardiac progenitor cells are uncommitted cells retained from mesodermal differentiation of the
heart and/or progenitor cells originating from the BM that take up permanent residence over time
in the adult heart. The number and function of these progenitor cells are depleted as an individual
ages. The dysfunctional progenitor cells contribute to a reduced capacity to repair injury or
regenerate tissue. Reconstitution of the aged BM with young marrow cells (top left panel)
rejuvenated the old cardiac-resident BM-derived progenitor cell pool, in part by repopulating it
2
with young cardiac-resident BM-derived progenitor cells (top right panel). After a coronary
occlusion (bottom panel), a rejuvenated myocardial progenitor cell pool (A) provides paracrine
support and guides vascular remodelling without these cells committing to differentiation,
though they may support/stimulate other cardiac or extracardiac stem/progenitor cells. Similar
events may occur with the aged stem cell pool (B); however, cardiac remodelling and
dysfunction may not be prevented because the cellular response is insufficient to achieve cardiac
repair.
3
Supplemental Methods
Real-time PCR for measurement of Y chromosomes
To estimate the number of male donor bone marrow (BM) cells in the hearts of female recipients
after reconstitution (n=5/group) or 3 days after myocardial infarction (MI; n=5/group), we used
real-time PCR. A standard curve was generated using BM mononuclear cells (MNCs) from male
C57BL/6 mice: 10 million cells were resuspended in 1 mL of ice-cold PBS, and genomic DNA
was extracted using a Qiagen Genomic DNA extraction kit, according to the manufacturer’s
instructions. A serial dilution was performed on the standard to produce a standard curve with
105, 104, 103, 102, and 101 Y chromosome copies.
Real-time PCR was used to determine the number of Y chromosomes (male cells) in the
old female recipient hearts as a measure of transplanted cell incorporation. Hearts were cleared
completely of blood, and tissues were snap-frozen in liquid nitrogen and homogenized to
powder. Genomic DNA was extracted using the Qiagen Genomic DNA extraction kit.
Y chromosomes were detected using the TaqMan method. Briefly, 1 μg of DNA was used
as a template for quantitative real-time PCR of Y chromosome SRY genes using the ABI PRISM
7900HT Sequencing Detection System (Applied Biosystems). Specific primers, designed based
on the GenBank sequences of the sex-determining region of the Y chromosome of the mouse
(GeneID: 21674), were used at 100 nM. The cycling conditions were 10 min at 95°C (for
activation of polymerase), 40 cycles of 30 s at 95°C (for denaturing), 1 min at 59°C (for
annealing and extension).
Fluorescent in situ hybridization
A Cy3 Y chromosome probe kit (Cambio) was used (following manufacturer’s instructions) to
detect Y chromosomes from the male donor cells in the female hearts (n=3/group). Briefly, fixed
4
sections (2% paraformaldehyde) were digested (pepsin, 37°C/10 min), dehydrated (graded
alcohol), denatured with probe (60°C/10 min), hybridized (37°C/16 h), washed in 50%
formamide and 2× SSC buffer (37°C/5 min), and then counterstained with DAPI.
Angiogenic cell assays
MNCs were isolated from the BM and spleen of chimeric mice (n=4/group) by density gradient
centrifugation (Histopaque-1083, Sigma). MNCs (2×106/cm2) were plated on fibronectin-coated
culture slides (BD Biosciences) and cultured in Endothelial Cell Basal Medium-2 (EBM-2;
Lonza) supplemented with endothelial growth medium SingleQuots and 20% FBS. Four days
later, the cells were incubated for 4 h with 10 µg/mL of 1,1'-dioctadecyl-3,3,3',3'tetramethylindocarbocyanine–labelled acetylated low-density lipoprotein (Di-acLDL;
Biomedical Technologies, Inc.). The cells were fixed in 2% paraformaldehyde and then
counterstained with FITC-labelled lectin (Sigma). Angiogenic cells (positive for both Di-acLDL
and lectin) were identified and quantified in five randomly selected high-power fields.
CFU assays
MNCs were isolated from BM (n=3–4 chimeric mice/group; n=4–9 secondary chimeric
mice/group) as described above for angiogenic cell assays. Cells were cultured in MesenCult
medium (Stem Cell Technologies) for 7 days. Giemsa solution (EMD chemicals) was used to
stain and quantify adherent colonies (CFU-F). For CFU-F, CFU-GM, CFU-GEMM, and BFU-E,
colonies were counted visually under the microscope in a blinded manner.
To quantify CFU from chimeric mouse hearts (n=4/group), the myocardium was perfused
with phosphate buffer for 30 min by using a Langendorff system (to remove possible
contamination of circulating stem cells) and then digested with 0.1% collagenase type II
5
(Worthington) at 37°C for 30 min. Two million cells were plated in duplicate in MethoCult
methocellulose medium for 10 days and then counted.
Induction of MI
At 8 weeks after primary reconstitution (n=11/group) or 3 days after secondary reconstitution
(n=5/group), mice were intubated and ventilated with 2% isoflurane. Through a thoracotomy, the
left coronary artery was permanently ligated.
Flow cytometry
MNCs were isolated from blood and labelled with both FITC-conjugated anti-mouse CD34 and
PE-conjugated anti-mouse KDR (VEGFR2) or cKit antibodies (all from eBiosciences). Dual
positive cells were identified as angiogenic or progenitor cells by flow cytometry (n=4/group).
To quantify progenitor cells in the myocardium, hearts (n=5 mice/group) were first
perfused for 30 min with PBS by using a Langendorff system to remove possible contamination
by circulating cells. The tissue was then digested with 0.1% collagenase type II (Worthington) at
37°C for 30 min. The cells were stained with PE-conjugated anti-mouse cKit antibody
(eBiosciences). More than 20,000 cells were counted, with manufacturer-supplied isotype
controls or non-transgenic (non-Tg) cells used to establish gating.
Cardiac function measurement
Cardiac function was measured with echocardiography at different time points before and after
MI and with a pressure-volume catheter at the end of the study.
For echocardiography, mice were sedated with 2% isoflurane and echocardiographic
images were recorded. Fractional area contraction (FAC) was calculated using the following
equation: FAC (%) = [(end-diastolic area – end-systolic area) / end-diastolic area] × 100.
6
For pressure-volume analysis (at 28 days after MI in chimeric mice or 14 days after MI in
secondary chimeric mice), animals were first anesthetized with 2% isoflurane and ventilated.
Next, a pressure and conductance catheter (1.4F, Millar Instruments, Inc.) was introduced into
the left ventricle through the right carotid artery and pressure-volume loops were obtained during
brief apnea and before and after inferior vena cava occlusion.
Morphology and immunohistochemistry
After the functional analysis was complete (Day 14 or 28 after MI), the hearts were arrested,
perfused with 10% formalin, and fixed for 1 day in 10% formalin. Scar tissue in the left
ventricular free wall was identified with Masson’s trichrome staining. The hearts were cut into 1
mm thick sections and photographed to measure scar area and scar thickness (n=6/group). Scar
area was calculated for each section by multiplying the scar length by the section thickness. Total
area was calculated as the sum of scar areas from each section. Scar thickness was estimated by
calculating the average of three measurements of scar thickness in each section. Heart slices
were embedded in paraffin and sliced into 5 μm thick sections. The tissue slides were stained
with Masson’s trichrome and hematoxylin and eosin (H&E) according to the manufacturers’
instructions.
Integration of transplanted BM stem cells into the heart was evaluated 12 weeks after
reconstitution (n=6/group) or, for experiments that involved an MI, after functional analysis on
Day 14 (n=6/group). Fixed heart tissue was sliced into 4–5 μm thick sections. The sections were
incubated with a primary antibody (Alexa 488-conjugated rabbit anti-GFP [Molecular Probes] or
goat anti-GFP [Abcam]) and one of the following antibodies: mouse anti-sarcomeric actinin
(Sigma), mouse anti-myosin heavy chain (Santa Cruz), Cy3-conjugated anti-smooth muscle actin
(Sigma), rabbit anti-human von Willebrand factor (Dako), rat anti-mouse-CD45, -CD34, or -
7
cKit, PE-conjugated rat anti-mouse Tie2, rat anti-mouse CD44 (all from BD Biosciences), mouse
anti-fibronectin (Sigma), or goat anti-DDR2 (Santa Cruz). The sections were incubated with an
appropriate secondary antibody, and the nuclei were identified with DAPI staining (Sigma). Five
randomly selected fields were acquired from each of the remote (non-infarcted), border, and scar
(infarct) areas using an Olympus Fluoview 2000 laser scanning confocal microscope.
BrdU labelling
To determine whether the GFP+ cell population proliferated after MI, BrdU (50 mg/kg; Sigma)
was administered to mice by intraperitoneal injection for 3 consecutive days. Coronary artery
ligation was performed 1 day later. Animals were sacrificed 3 days after ligation and hearts were
obtained. Frozen tissue sections (10 µm) were stained for BrdU incorporation. Briefly,
cryosections were fixed in 2% paraformaldehyde. Tissue sections were first permeabilized with
0.5% Triton X-100 and subsequently treated with 2N HCl for 30 min to expose the antigens.
Sections were incubated with rat anti-BrdU (1:50; Abcam) for 2 h in a humidified atmosphere.
After incubation with the primary antibody, the slides were washed three times in PBS (pH 7.4)
and incubated with an Alexa 568-conjugated goat anti-rat secondary antibody for 1 h at room
temperature. Tissue sections were subsequently stained with the anti-GFP antibody. The nuclei
were identified with DAPI staining. Labelled nuclei cells were quantified in 10 independent
fields by microscopic analysis (NiKon), with the BrdU-labelling index expressed as the ratio of
BrdU+ cells or BrdU+/GFP+ cells to total nuclei.
Quantification of myocardial cytokine levels
Myocardial tissue from the infarct regions was collected and frozen (n=5 mice/group). The tissue
was homogenized in lysis buffer (20 mM Tris [pH 7.4], 150 mM NaCl, 1 mM EDTA, 1 mM
EGTA, 1% Triton, 2.5 mM Na pyrophosphate, 1 mM β-glycerolphosphate, 1 mM Na3VO4, 1
8
μg/mL leupeptin, 1 μg/mL pepstatin, 1 mM phenylmethylsulfonyl fluoride) and incubated at 4°C
for 1 h. The samples were centrifuged at 10,000 × g for 10 min, and the supernatant was
collected. VEGF and stem cell factor (SCF) levels were measured using an ELISA kit (R&D
Systems, following manufacturer’s instructions) and normalized to pg/mg total protein.
Immunoblotting and RT-PCR
We used Western blot to detect phosphorylated Akt (serine-473) in the infarcted myocardium
(n=5/group). Briefly, heart tissue homogenates were prepared and separated (using 10% SDSPAGE) and then transferred to a polyvinylidene fluoride (PVDF) membrane. The samples were
blocked (5% milk for 1 h), incubated with rabbit phosphorylated anti-Ser-473 Akt primary
antibody (Cell Signaling), washed (3× 5 min in TBS-0.05% Tween), visualized by
chemiluminescence and analyzed using densitometry (AlphaImager 2200).
We measured gene expression of angiopoietin-1 (n=5/group) and angiopoietin-2
(n=4/group) using RT-PCR (SuperScript III, Invitrogen). Primer pairs were specific for
angiopoietin-1 (sense: 5’-CAGTGGCTGCAAAAACTTGA-3’; antisense: 5’TCTGCACAGTCTCGAAATGG-3’) and angiopoietin-2 (sense: 5’GAACCAGACAGCAGCACAAA-3’; antisense: 5’-AGTTGGGGAAGGTCAGTGTG-3’).
Image analysis of immunohistochemical data
We used Image J software to measure the factor VIII+ (n=5/group) and SMA+ (n=5/group) pixel
areas (expressed as percentage of total possible pixels in 0.2 mm2). In SMA-stained sections,
only SMA+ blood vessels (≥30 µm) were identified and quantified.
9