Online Appendix for the following JACC article
TITLE: Aged Human Cells Rejuvenated by Cytokine Enhancement of Biomaterials for
Surgical Ventricular Restoration
AUTHORS: Kai Kang, MD, Lu Sun, MD, Yun Xiao, BSc, Shu-Hong Li, MD, MSc, Jun
Wu, MD, MSc, Jian Guo, MD, PHD, Shu-Ling Jiang, MD, Lei Yang, MD, Terrence M.
Yau, MD, MSc, Richard D. Weisel, MD, Milica Radisic, PHD, PEng, Ren-Ke Li, MD,
PHD
Supplemental Methods
Preparation of the collagen scaffolds
The collagen scaffolds were prepared as previously described (1,2). Briefly, scaffolds of
uniform size (2 cm × 2 cm × 2 mm) were cut from a sheet of Ultrafoam collagen sponge
(Davol) and immersed in a filtered solution of 24 mg/mL 1-ethyl-3-(3dimethylaminopropyl) carbodiimide HCl (EDC; Thermo Fisher Scientific) and 60
mg/mL N-hydroxysulfosuccinimide (Sulfo-NHS; G-Biosciences) in phosphate buffered
saline (PBS; Lonza) for 20 min. This EDC chemistry allows for immobilization of the
cytokines to the scaffold. To immobilize the cytokines, the scaffolds were immersed in a
mixed solution of 1 µg/mL VEGF (PeproTech) and 1 µg/mL bFGF (PeproTech) in PBS
for 2 h at room temperature, followed by washing 8× 5 min in fresh PBS. Control patches
were immersed in PBS. All patches were stored in PBS before cell seeding.
Tensile strength testing of the scaffolds
The tensile strength of the scaffolds (2 cm × 1 cm) was tested with the ElectroForce 5200
BioDynamic Test Instrument (Bose) using the 22 N load sensor. Displacement was
increased at a constant rate of 1 mm/min. The scaffolds (n=3/group) were loosely
mounted to the instrument at the beginning to prevent any initial stretch. Load and
displacement readings were recorded with WinTest software. Ultimate tensile strength is
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the stress at the breaking point, taken from the stress-strain curve. Young’s modulus was
obtained from the slope of the stress-strain curve, as previously reported (3).
Scanning electron microscopy (SEM) of the scaffolds
The scaffolds were imaged using environmental SEM. The scaffolds were prepared by
washing twice with distilled water to remove the PBS. They were then individually
placed into the specimen chamber of the microscope (Hitachi S-3400 N), and filter paper
was used to gently remove the excess water. The chamber was closed, the temperature
was decreased to −20°C, and the scaffolds were imaged under variable pressure mode at
70 Pa and 15 kV.
Quantification of cytokine immobilization and physical bonding to the scaffolds
ELISA was performed on freshly prepared collagen scaffolds using VEGF and bFGF
ELISA kits (Human VEGF Mini ELISA Development Kit and Human FGF-basic Mini
ELISA Development Kits, PeproTech), according to the manufacturer’s instructions.
Three groups were tested: scaffolds treated with EDC/Sulfo-NHS and immobilized with
both VEGF and bFGF (experimental group), scaffolds treated with EDC/Sulfo-NHS but
reacted with PBS during immobilization (control group), and scaffolds without
EDC/Sulfo-NHS treatment but reacted with both VEGF and bFGF (to show physical
bonding). After being prepared, the scaffolds were kept in PBS at 4°C overnight. The
supernatant was collected, and the scaffolds were digested with 0.276 mg/mL collagenase
type IA (Sigma Aldrich) for 1.5 h at 37°C, as previously described (4). The digestion
solution was further diluted in order to fall within the detection range of the ELISA kits.
Assessment of cytokine release from the scaffolds
To study the release of VEGF and bFGF from the scaffolds over time, the scaffolds
(n=3/group) were immersed in 1 mL PBS in 24-well plates for 28 days. The supernatant
was collected on day 1, 3, 7, 14, 21, and 28 and kept at −80°C for later analysis. After
collecting the supernatant on day 28, the scaffolds were digested with collagenase as
described above. All samples (diluted if necessary) were tested using the same ELISA
kits as described above.
Bone marrow collection
The collection of human bone marrow samples was approved by the Research Ethics
Board of the University Health Network. Each patient provided informed, written
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consent. Bone marrow aspirates were obtained from the sternum of eight patients
undergoing coronary bypass surgery at Toronto General Hospital. “Young” hMSCs were
isolated from patients aged ≤57 years (50.0±8.0 years, N=4); “old” hMSCs were isolated
from patients aged ≥66 years (74.5±7.4 years, N=4). All patients had coronary artery
disease and required CABG. Bone marrow aspirates were immediately mixed with
heparin and transported to the cell culture facility.
Cell isolation, culture, and seeding
Bone marrow mononuclear cells were separated by centrifugation with a Ficoll-Paque
gradient (1.077 g/mL density; GE Healthcare), seeded into 75 cm2 culture flasks in
Iscove’s modified Dulbecco’s medium (IMDM) containing 10% fetal bovine serum
(FBS) and antibiotics, and incubated at 37°C in 5% CO2. After 48 h, non-adherent cells
were removed by changing the medium. Thereafter, the medium was changed every 2–3
days, and adherent cells were cultured until they reached 80% confluence. The hMSCs
were harvested by trypsinization (0.25% trypsin with 0.02% EDTA) and passaged. Cells
in the third passage were employed for this study.
The cell seeding was performed as previously described (1,2). Briefly, the
scaffolds were incubated for 30 min in culture medium, dried on autoclaved Kimwipes,
and transferred to a 24-well plate. The hMSCs were trypsinized, centrifuged into a pellet
(0.5×106 cells/scaffold for MTT assay and 1.0×106 cells/scaffold for other assays and in
vivo studies), and resuspended in a volume of medium corresponding to 10 µL per
scaffold. Resuspended cells were evenly seeded onto the surface of the scaffold and
incubated for 40 min (37°C, 5% CO2) to allow for cell attachment, followed by the
addition of 1 mL fresh medium. The patches were cultured for 3 days before implantation
by SVR.
Evaluation of cell proliferation with MTT assay and BrdU staining
An MTT assay kit (Sigma) was used to assess the increase in the number of cells in the
scaffolds after 2 and 4 days of culture, according to the manufacturer’s instructions.
Scaffolds (n=4/group) were transferred to a 24-well plate and incubated with 500 µL
freshly prepared MTT working solution (1 mg/mL) at 37°C in 5% CO2 for 4 h. The
calibration curve for the MTT assay consisted of a blank scaffold (no cells) and scaffolds
with a known number of cells (0.2×106, 0.4×106, 0.8×106, 1.6×106). Scaffolds were
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transferred to another well and rinsed with 500 µL dimethyl sulfoxide (DMSO) for 10
min to ensure all of the formazan crystals had dissolved into solution. The MTT assay
was performed on the eluent of all scaffolds. Spectrophotometry was carried out at 620
nm using a plate reader (BioTech).
BrdU (5-bromo-2’-deoxyuridine) staining was used to confirm the number of
proliferating cells in the scaffolds. BrdU (10 µmol/mL; Sigma) was added to the medium
1 day after cell seeding. After culturing for 48 h, the scaffolds were snap-frozen, sliced (5
µm thickness), labeled with BrdU antibody (Abcam), and analyzed by fluorescent
microscopy (Nikon Eclipse TE200). A single blinded examiner randomly selected five
high-power fields (0.4 mm2, n=4/group). The percentage of cells positive for BrdU was
calculated and averaged.
Evaluation of cell differentiation with immunofluorescent staining and real-time PCR
Immunofluorescent staining was used to assess smooth muscle cell, fibroblast, and
endothelial cell transdifferentiation. Briefly, young or old hMSCs (1.0×106) were seeded
onto the scaffolds and cultured at 37°C in 5% CO2 for 4 days. The scaffolds were fixed
with 4% paraformaldehyde for 24 h and placed face down in cryomolds with a thin layer
of OCT compound (Tissue-Tek) at the bottom. The molds were then filled with OCT,
snap-frozen with dry ice, and cut into 5 µm thick slices for staining. Immunolabeling was
performed with antibodies against alpha smooth muscle actin (α-SMA; Santa Cruz) and
connexin 43 (Sigma). Quantification was performed as described for BrdU staining.
As collagen I and III are predominantly secreted by fibroblasts, the expression of
these genes (COL1A1 and COL3A1) was employed to indicate fibroblast differentiation.
The mRNA profile of collagen type I and III was assessed with real-time PCR. After 4
days of culture, scaffolds were snap-frozen and ground into powder, and total RNA was
extracted using the TRIzol method (Invitrogen). Reverse transcription was performed
using SuperScript III (Invitrogen) with 0.5 μg RNA as the template. Real-time PCR
amplification was performed using a GeneAmp PCR 9600 Thermocycler (Applied
Biosystems) for 40 cycles (each cycle: 95°C for 15 s, 55°C for 30 s, and 72°C for 30 s).
Relative expression was calculated by the comparative threshold cycle method and
expressed as 2-[(CT of Gene of Interest-CT of control) in disease-(CT of Gene of Interest-CT of control) in control condition].
We use human GAPDH as the control. All groups were compared with the Old group
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(control condition). The primer sequences of the regulated transcripts are summarized in
Supplemental Table 1.
Assessment of cell rejuvenation
The effect of cytokine enhancement on the rejuvenation of cells from old donors was
assessed by evaluating the expression of p16 (CDKN2A, cyclin-dependent kinase
inhibitor 2A), which encodes inhibitors of CDK4 (cyclin-dependent kinase 4). Increased
expression of the p16 gene has been demonstrated in older individuals, and that
expression reduced stem cell proliferation (5). The RGN gene encodes regucalcin
(senescence marker protein-30), which has been shown to be down-regulated in older
humans (6). Expression of p16 and RGN mRNA was determined by real-time PCR. The
procedure was the same as described above for collagen. The primers sequences are
summarized in Supplemental Table 1. Immunofluorescent staining and Western blot were
used to assess p16 protein expression. The procedure for immunofluorescent staining was
the same as described above. The mean intensity of p16 staining was measured with NISElements BR3.0 software. Western blot was performed with 10 μg of protein extracted
from the patch. We use the same p16 antibody (P16INK4a; Abcam) for both
immunofluorescent staining and Western blot.
Experimental animals
Adult, female, Sprague-Dawley rats (200–225 g) were used for the studies. All animal
procedures were performed in accordance with the Guide for the Care and Use of
Laboratory Animals (NIH, revised 1996) and were approved by the Animal Care
Committee of the University Health Network. To avoid immune rejection, all animals
were given 5 mg/kg cyclosporine A (Novartis) intraperitoneally each day beginning 3
days before the procedure until 28 days after patch implantation.
Surgical procedures
Animal procedures were performed as previously described (7,8). Briefly, all rats
underwent proximal left anterior descending coronary artery ligation to generate a
transmural MI. Two weeks later, echocardiography was used to screen the rats on the
basis of infarct size (Supplemental Fig. S1). Only those animals with an akinetic infarct
area greater than 25% but less than 35% of the left ventricular (LV) free wall were
selected for further investigation. The rats (n=8/group) were randomly assigned to one of
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the following six groups: Control=collagen patch; GF=collagen patch immobilized with
growth factors (VEGF and bFGF); Old=collagen patch seeded with old hMSCs;
Young=collagen patch seeded with young hMSCs; Old+GF=collagen patch immobilized
with growth factors and seeded with old hMSCs; Young+GF =collagen patch
immobilized with growth factors and seeded with young hMSCs.
Patch implantation was performed 14 days after coronary artery ligation. The
infarct region was resected, and the ventricle was repaired with one of the four patches,
as previously described (9,10). Briefly, through a left thoracotomy, a purse-string suture
was placed around the infarct area, and the tourniquet was snared down to prevent
bleeding during the repair. The transmural scar was removed and the defect was repaired.
The patches were trimmed to match the defect (7 mm diameter) and were oriented with
the cell-seeded surface on the endocardial side. The tourniquet was released, and the
purse-string stitch was removed after a continuous over-and-over stitch with 7-0
polypropylene to ensure hemostasis. Meticulous hemostasis was ensured prior to closure
of the thoracotomy.
Measurement of patch area and thickness
At 28 days after patch implantation, all animals were euthanized by injection of 10% KCl
into the heart. The heart was removed, and an intraventricular balloon was inserted
through the mitral valve and filled to 30 mmHg pressure for photography at a fixed
distance (73 cm). Patch area (n=5–8/group) was measured using computerized planimetry
(ImageJ software).
Next, the hearts were fixed with 4% paraformaldehyde for 24 h, followed by
sucrose at varying concentrations and durations (10% for 1 h, 20% for 1 h, and 30% for
24 h). The hearts were cut in half along the center of the patch and snap-frozen in molds
filled with OCT compound. Hearts were sliced into 5 µm thick sections and stained with
Masson’s trichrome. Patch thickness was quantified using computerized planimetry
(ImageJ software).
Assessment of cardiac function
LV function was evaluated by echocardiography (Sequoia C256 System, Siemens
Medical; 15 MHz linear array transducer) before MI (pre-ligation baseline), before patch
implantation (day 0), and 7, 14, and 28 days after patch implantation. M-mode and 2D
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images were obtained in the parasternal short-axis view at the level of the papillary
muscles. The measurements were performed by a single, blinded examiner. LV internal
diastolic dimension (LVIDd), internal systolic dimension (LVIDs), end-diastolic area
(LVEAd), and end-systolic area (LVEAs) were measured. Five consecutive cardiac
cycles were recorded and averaged. Percent ejection fraction (%EF), fractional shortening
(%FS), and fractional area change (%FAC) of the LV were calculated as follows: %EF =
(LVIDd)2 – (LVIDs)2 / (LVIDd)2  100; %FS = (LVIDd − LVIDs) / LVIDd 
100; %FAC = (LVEAd – LVEAs) / LVEAd  100. Additionally, cardiac function was
assessed at the end of the study (28 days after patch implantation) with a pressure-volume
catheter, as previously described (7).
Assessment of cell survival and vascular density by immunostaining
Cryosections of the specimens used to measure patch thickness were evaluated to assess
cell survival and vascular density. For cell survival, tissue sections were immunostained
with a monoclonal antibody against human mitochondria (Millipore). For vascular
density, sections were immunostained with antibodies against von Willebrand factorfactor VIII (vWF-FVIII; Dako) and α-SMA to evaluate capillary structures and mature
vessels, respectively. A single, blinded examiner randomly selected five high-power
fields (0.2 mm2, n=5–8/group). The number of positive cells was calculated and
averaged.
Assessment of cell phenotype by immunostaining
We used immunofluorescent staining to identify what cells were present in the patch area
following implantation. Cryosections of the specimens used to measure patch thickness
were evaluated for cell phenotype. To identify the patch area, tissue sections were
immunostained with a monoclonal antibody against human mitochondria (Millipore).
DAPI was used to stain nuclei. For the cell phenotype, sections were immunostained with
antibodies against CD45 (BD Pharmingen), vWF-FVIII, DDR2 (Santa Cruz), α-SMA,
and sarcomeric actinin (Sigma) and visualized with an Olympus Fluoview 2000 laser
scanning confocal microscope. The percentage of positive cells (among total number of
cells) was averaged for each marker.
Statistical analysis
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Statistical analysis was performed with GraphPad Prism 4 software. All data were
expressed as mean ± SD. Comparison of parameters among four groups was made with
one-way ANOVA (patch area, patch thickness, real-time PCR, immunofluorescent
staining, pressure and volume), two-way ANOVA (MTT assay, echocardiography), or
ANCOVA (preload recruitable stroke work and end-systolic pressure-volume
relationship). If the F test was significant (p<0.05), pairwise tests of individual group
means were carried out using the Newman-Keuls post-test, Bonferroni post-test, or
Duncan’s multiple range test. GraphPad Prism was used to test for equal variances. In
most instances, the variance was not significant. Accordingly, parametric statistics were
employed. When the variance was significant (e.g., Figure 6F), the non-parametric
Kruskal-Wallis test was used. A p value <0.05 was considered statistically significant.
References
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angiopoietin-1 for vascularization of engineered tissues. Biomaterials 2010;31:22641.
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8. Zhuo Y, Li SH, Chen MS, et al. Aging impairs the angiogenic response to ischemic
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Table 1. Primers and TaqMan probes used for real-time PCR.
Human gene
Forward primer
Reverse primer
TaqMan FAM-probe
Collagen I
5’-CCCCTGGAAAGA
ATGGAGATG-3’
5’-TCCAAACCACTG
AAACCTCTG-3’
5’-TTCCGGGCAAT
CCTCGAGCA-3’
Collagen III
5’-AAGTCAAGGAGA
AAGTGGTCG-3’
5’-CTCGTTCTCCAT
TCTTACCAGG-3’
5’-CATGGGCTTCCC
CGGTCCTAAA-3’
p16
5’-GATGTCGCACGG
TACCTG-3’
5’-TCTCTGGTTCTT
TCAATCGGG-3’
5’-CATGGTTACTGC
CTCTGGTGCCC-3’
RGN
5’-TGGATGCCTTTG
ACTATGACC-3’
5’-TTCCCCTCAGCA
TCAATACAC-3’
5’-AGCTTGTAAACA
CTTCTGCGGTTGGA-3’
5’-ACATCGCTCAGAC
ACCATG-3’
5’-TGTAGTTGAGGTC
AATGAAGGG-3’
5’-AAGGTCGGAGTCAACGG
ATTTGGTC-3’
GAPDH
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Supplemental Figure Legends
Supplemental Figure S1. Left ventricular akinetic and dyskinetic segments
At 14 days after left anterior artery ligation, the anterior free wall of the left ventricle
became thinner and akinetic, and some segments had paradoxical movement. (A, B)
Representative 2-dimensional echocardiographic images illustrating rat heart movement
in diastole (A) and systole (B). (C) Representative image of infarcted rat heart with left
ventricular akinetic and dyskinetic segments.
Supplemental Figure S2. Immunostaining of cells surviving in patch area
At 28 days after patch implantation, hearts were removed, snap-frozen, and sectioned.
Representative images (600×) of heart tissue sections stained with antibodies against
human mitochondria, CD45, von Willebrand factor (vWF), DDR2, smooth muscle actin
(SMA), and sarcomeric actinin (SARC). Arrows indicate positive cells. Nuclei are
stained blue with DAPI. The percentage of each cell type is presented for cells that were
either human mitochondrial antigen positive or negative.
Supplemental Figure S3. Factors that correlate with ejection fraction
Significant linear correlations were found between ejection fraction and the following
factors (all at 28 days after patch implantation): (A) Cell survival (expressed as the mean
number of human mitochondrial positive cells per 0.2 mm2 field). (B) Patch thickness
(expressed as the mean thickness of five random measurements of equal interval along
the patch). (C) Capillary density (expressed as the mean number vWF-FVIII positive
vessels per 0.4 mm2 field). (D) Arteriolar density (expressed as the mean number of αSMA positive vessels, with a diameter lager than 10 μm, per 0.4 mm2 field).
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