Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model
Sugiura et al. Journal of Cardiothoracic Surgery
Tissue-engineered cardiac patch seeded with human induced pluripotent stem cell derived cardiomyocytes promoted the regeneration of host cardiomyocytes in a rat model
Tadahisa Sugiura 1 4
Narutoshi Hibino 1 4
Christopher K. Breuer 1 4
Toshiharu Shinoka 0 1 2 3 4
0 Department of Cardiothoracic Surgery, The Heart Center, Nationwide Children's Hospital , Columbus, OH , USA
1 Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital , Columbus, OH , USA
2 Cardiovascular Tissue Engineering Program, Department of Cardiothoracic Surgery, The Heart Center, Nationwide Children's Hospital , 700 Children's Drive, T2294, Columbus, OH 43205 , USA
3 Department of Cardiothoracic Surgery, The Heart Center, Nationwide Children's Hospital , Columbus, OH , USA
4 Tissue Engineering Program and Surgical Research, Nationwide Children's Hospital , Columbus, OH , USA
Background: Thousands of babies are born with congenital heart defects that require surgical repair involving a prosthetic implant. Lack of growth in prosthetic grafts is especially detrimental in pediatric surgery. Cell seeded biodegradable tissue engineered grafts are a novel solution to this problem. The purpose of the present study is to evaluate the feasibility of seeding human induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) onto a biodegradable cardiac patch. Methods: The hiPS-CMs were cultured on a biodegradable patch composed of a polyglycolic acid (PGA) and a 50:50 poly (l-lactic-co-ε-caprolactone) copolymer (PLCL) for 1 week. Male athymic rats were randomly divided into 2 groups of 10 animals each: 1. hiPS-CM seeded group, and 2. Unseeded group. After culture, the cardiac patch was implanted to repair a defect with a diameter of 2 mm created in the right ventricular outflow tract (RVOT) wall. Hearts were explanted at 4 (n = 2), 8 (n = 2), and 16 (n = 6) weeks after patch implantation. Explanted patches were assessed immunohistochemically. Results: Seeded patch explants did not stain positive for α-actinin (marker of cardiomyocytes) at the 4 week time point, suggesting that the cultured hiPS-CMs evacuated the patch in the early phase of tissue remodeling. However, after 16 weeks implantation, the area fraction of positively stained α-actinin cells was significantly higher in the seeded group than in the unseeded group (Seeded group: 6.1 ± 2.8% vs. Unseeded group: 0.95 ± 0.50%, p = 0.004), suggesting cell seeding promoted regenerative proliferation of host cardiomyocytes. Conclusions: Seeded hiPS-CMs were not present in the patch after 4 weeks. However, we surmise that they influenced the regeneration of host cardiomyocytes via a paracrine mechanism. Tissue-engineered hiPS-CMs seeded cardiac patches warrant further investigation for use in the repair of congenital heart diseases.
Tissue engineering; Induced pluripotent stem cell derived cardiomyocytes; Biodegradable cardiac patch; Congenital heart disease
Approximately 10,000 children undergo reconstructive
operations to repair complex congenital abnormalities
each year . Unfortunately, many pediatric patients
outgrow their implants much like they outgrow their shoes.
In pediatric cardiac surgery, currently used prosthetic
materials lack growth potential and they necessitate staged
repairs or re-operations. These additional surgical
procedures add additional morbidity because of increased
complexity due to the formation of significant pericardial
adhesions. Tissue engineering has emerged to solve this
issue by creating a living graft with growth potential.
Typically a tissue-engineered graft is made up of a
scaffold and seeded cells. As the scaffold degrades, neotissue
forms and a living, biocompatible tissue is created. Using
the classical tissue engineering paradigm, many cell types
have been considered as possibilities for seeding onto a
biodegradable scaffold, which provides sites for cell
attachment and space for neotissue formation . Induced
pluripotent stem (iPS) cells were first generated by nuclear
reprogramming of mouse fibroblasts in 2006 , and
human iPS (hiPS) cells were established in 2007 [4, 5]. Recent
studies have demonstrated various methods for the highly
efficient production of cardiomyocytes derived from hiPS
cells that maintained their typical electrophysiological
functions . Human iPS cells represent an unlimited source of
cardiomyocytes because of their great potential for
differentiation and are therefore one of the most promising
sources of cells for cardiac regeneration therapy [7, 8].
While hiPS cell derived cardiomyocytes (hiPS-CMs)
have been used for cardiac regeneration therapy, there
are currently significant limitations, which include a very
low percentage of engraftment success and cell survival.
Insufficient blood and oxygen delivery is one of the most
important causes of poor engraftment. To address this
challenge, we will test the feasibility of a cardiac patch,
seeded with hiPS-CMs on a biodegradable scaffold
composed of a polyglycolic acid (PGA) and a 50:50 poly
(l-lactic-co-ε-caprolactone) copolymer (PLCL), to grow into
neo-tissue. This cardiac patch was implanted onto a right
ventricular outflow tract (RVOT) wall defect created in an
immunocompromised rat heart, so that the patch will
receive blood supply directly from the luminal surface. The
purpose of this study is to demonstrate the feasibility and
application of this tissue-engineered patch in the repair of
a cardiac defect using rat RVOT reconstruction model.
The hiPS-CMs were obtained commercially (Cellular
Dynamics International (CDI), Madison, Wisconsin). To
perform immunofluorescent staining the hiPS-CMs were
cultured on a 4 well chamber mounted on glass slides
with a cover (Chember Slide System; nunc, NY, USA) at
a density of 1.0 × 105 cells for 1 week. Cell culture media
(iCell Cardiomyocytes Maintenance Medium: CDI) was
changed every 2 days. The hiPS-CMs were labeled with
a red fluorescent protein, which allowed us to track the
engraftment of the cells.
Preparation of tissue-engineered cardiac patch
A scaffold composed of a woven fabric of polyglycolic
acid (PGA) and a 50:50 poly(l-lactic-co-ε-caprolactone)
copolymer (PLCL) was constructed as previously
described . The scaffold is more than 80% porous and
0.6 to 0.7 mm in thickness. The hiPS-CMs were cultured
on the biodegradable patch with a diameter of 6 mm at
a density of 2.0 × 105 cells for 1 week. Cell culture media
was changed every 2 days.
Animal model and surgical implantation
All animals received humane care in compliance with
the National Institutes of Health (NIH) guideline for the
Care and Use of Laboratory Animals. The Institutional
Animal Care and Use Committee at Nationwide
Children’s Hospital approved the use of animals and all
procedures described in this study. Nude athymic rats
were purchased from Jackson Laboratories (Bar Harbor,
ME) and used for all experiments in the present study.
Adult male nude athymic rats weighing 230–300 g were
used for the right ventricular outflow tract (RVOT)
reconstruction procedure as previously described . Briefly,
anesthesia was obtained with ketamine (50 mg/kg, i.p) and
xylazine (5 mg/kg, i.p); it was maintained using isoflurane
(1.5%) in oxygen. The animals were intubated with a
16gauge catheter and respiration was maintained at 60
cycles/min with a tidal volume of 2.5 ml. The surgical
procedure was performed using aseptic techniques with
sterile instruments. First, the skin of the chest was
sterilized with a povidone-iodine solution and the heart was
exposed through a median sternotomy. A purse string
suture, with a diameter slightly larger than 6.0 mm, was
placed in the free wall of the RVOT with Prolene 7-0
polypropylene sutures (Ethicon, Somerville, NJ, USA). Both
ends of the suture were passed through a 22-gauge plastic
vascular cannulae, which was used as a tourniquet. The
tourniquet was tightened and the distended part of the
RVOT wall inside the purse string suture was resected. To
indicate that a transluminal defect had been created in the
RVOT, the tourniquet was briefly released to determine
whether massive bleeding occurred. Next, the cardiac
patch was sutured along the margin of the purse string
suture with 7-0 polypropylene over-and-over sutures to
cover the defect in the RVOT. After completion of
suturing, the tourniquet was released and the purse string
suture was removed. Fibrin glue (Evicel; Ethicon) was
applied to the patch as well as the suture line. After
expanding the lungs using positive end-expiratory pressure, the
sternum was closed parasternally with four interrupted
Prolene 5-0 polypropylene sutures (Ethicon). The muscle
layer and skin were closed with Vicryl 4-0 absorbable
sutures (Ethicon). The first 3 days after surgery,
buprenorphine (0.05 mg/kg) analgesia and cefuroxime (100 mg/kg)
antibiotic were administered twice a day subcutaneously.
Animals were randomly divided into 2 groups of 10
animals each: 1. hiPS-CM seeded group, and 2. Unseeded
group. Two animals in each group were sacrificed at 4
and 8 weeks postsurgery and 6 animals in each group
were sacrificed at 16 weeks postsurgery.
Histology and immunofluorescence
Explanted cardiac patches were fixed in 4%
paraformaldehyde, embedded in paraffin, sliced (5 μm thick
sections), and stained with Hematoxylin and Eosin
(H&E). H&E staining was used for cell counting. One
representative section from each explant at 16 weeks
after implantation was stained and imaged. All positively
stained nuclei were counted from high magnification
images. Collagen deposition was assessed with Picrosirius
red staining and images were obtained with polarized
light microscopy. Based on previous reports, we
correlated thick fibers (orange to yellow) with collagen I and
thin fibers (green) with collagen III .
Immunofluorescent staining for α-actinin as a marker of
cardiomyocytes was performed using rabbit anti-α-actinin
(sarcomeric) primary antibody (1:200, Abcam, Cambridge,
MA) followed by Alexa Fluor 488 anti-rabbit IgG
secondary antibody (1:300, Invitrogen, Carlsbad, CA).
Fluorescent images were obtained with an Olympus
IX51 microscope (Olympus, Tokyo, Japan).
Echocardiographic measurements were obtained
preoperatively and at 8 and 16 weeks postoperatively.
Animals underwent isoflurane anesthesia (2% isoflurane with
100% oxygen gas inhalation through a nose cone). When
the anesthesia plane was established, B-mode and
Mmode echocardiography was performed (Vevo
Visualsonics 770; Visualsonics, Toronto, ON, Canada). We
visualized left ventricle (LV) short axis view and right ventricle
(RV) outflow short axis view. RV and LV minimum and
maximum diameters were measured using M-mode
echocardiography and LV ejection fraction was calculated.
Numeric values are listed as mean ± standard deviation.
The number of experiments is shown in each case. Data
of continuous variables with normal distribution were
evaluated by student t test. P values less than 0.05
indicated statistical significance.
In both groups macroscopic post implantation images of
the cardiac patches showed fibrous adhesions on the
epicardial surface of the patches over the course of 16 weeks
(Fig. 2). There was no significant difference in
macroscopic findings between the groups.
Histology, immunofluorescent analysis
H&E staining showed cell infiltration within the scaffold
in both groups (Fig. 3), and nuclei were counted to
obtain the number of cells in the scaffold. There was no
statistical difference in the cell number between the
groups at 16 weeks after implantation (Unseeded group:
390 ± 71/HPF vs. Seeded group: 319 ± 30/HPF, p = 0.08
(HPF: high power field)) (Fig. 4a).
Fig. 1 Immunofluorescent images of hiPS-CMs. Representative immunofluorescent images of α-actinin merged with red fluorescent protein that
is originally expressed in hiPS-CMs and DAPI after 2 days in culture in a well
Fig. 2 Macroscopic images of the tissue-engineered hiPS-CMs seeded or unseeded cardiac patches. There were fibrous adhesions on the epicardial
surface of the patches over the course of 16 weeks in both groups (arrows indicate fibrous adhesions). There was no significant difference in
macroscopic findings between the groups
To evaluate the engraftment of implanted hiPS-CMs,
α-actinin staining was employed. Seeded patch explants
did not stain positive for α-actinin at the 4 and 8 week
time point, suggesting that the cultured hiPS-CMs
evacuated the patch in the early phase of tissue remodeling.
However, there were small islands of cells which stained
positive for α-actinin in the cardiac patch 16 weeks after
implantation. The area fraction of positively stained
αactinin cells was significantly higher in the seeded group
than in the unseeded group (Seeded group: 6.1 ± 2.8% vs.
Unseeded group: 0.95 ± 0.50%, p = 0.004), suggesting cell
seeding promoted regenerative proliferation of host
cardiomyocytes (Fig. 4b).
Visualization of Picrosirius red staining with polarized
light microscopy shows thick orange fibers and thin
green fibers which are correlated with collagen type I
and type III, respectively in both groups equally (Fig. 5).
Over the course of 16 weeks, the cardiac patch gradually
degraded and remodeled into collagenous tissue in both
the seeded and unseeded groups.
There was no statistical difference in RV maximum and
minimum diameters between the groups at each time
point (Fig. 6a, b). There was no aneurysmal change in either
group. There was no statistical difference between the
groups in LV maximum and minimum diameters and LV
ejection fraction at each time point (Fig. 6c, d, e). Either
unseeded or seeded cardiac patch implanted hearts showed no
functional or dimensional dysfunction at each time point.
Tissue-engineering in conjunction with therapeutic therapy,
is a novel approach for reconstruction of cardiac defects.
Many researchers believe that paracrine effects are the
major mechanism responsible for the therapeutic efficacy
of stem cell or progenitor cell therapy. These effects
classically refer to the ability of transplanted cells to release
various cardioprotective factors into damaged cardiac tissue for
attenuation of the remodeling process; in contrast, recent
reports suggest that cell transplantation upregulates various
cardioprotective factors in native cardiac tissue through
“crosstalk” between transplanted cells [12, 13]. In addition,
our group previously demonstrated, in a mouse model, that
bone marrow mononuclear cells seeded onto vascular
grafts disappeared in the early phase, but their initial
presence mediated for the appropriate vascular remodeling and
development via a paracrine mechanism . In the
present study, seeded hiPS-CMs were not present in the
patch after 4 weeks. However, α-actinin positive cells were
significantly greater in the seeded group than in the
unseeded group 16 weeks after implantation. Therefore, we
surmise that seeded hiPS-CMs might influence the
regeneration of host cardiomyocytes via a paracrine mechanism.
Fig. 3 Histological analysis of the grafts at 4, 8 and, 16 weeks after implantation. Hematoxylin and Eosin (H&E) staining demonstrated dense cellular
infiltration into the hiPS-CMs seeded or unseeded cardiac patches (a - f: high magnification images, g - l: low magnification images). Both seeded and
unseeded patches explants did not stain positive for α-actinin (green) at the 4 and 8 week time point (m, n, p, q). However, there were small islands of
cells which stained positive for α-actinin (green) in the cardiac patch 16 weeks after implantation (o, r, s). N native heart muscle, P patch
Fig. 4 Quantitative comparison of the cellular infiltration into the scaffold and α-actinin positive cell. a There was no statistical difference between
the groups in the cell number within the scaffolds at 16 weeks after implantation. b The area fraction of positively stained α-actinin cells was
significantly higher in the seeded group than in the unseeded group. *: p < 0.05
Fig. 5 Collagen deposition in the grafts at 4, 8 and, 16 weeks after implantation. Visualization of Picrosirius red staining with polarized light
microscopy showed thin (type III; green) to thick (type I; yellow) collagen fibers and scaffold fragments (white) in both groups. Over the course
of 16 weeks, the cardiac patch gradually degraded and remodeled into collagenous tissue in both the seeded and unseeded groups
Fig. 6 Echocardiographic analysis presurgery and 8 and 16 weeks postsurgery. a RV maximum diameter. b RV minimum diameter. c LV maximum
diameter. d LV minimum diameter. e LV ejection fraction. There was no statistical difference in RV maximum and minimum diameters between
the groups at each time point. There was no aneurysmal change in either group. There was no statistical difference in LV maximum and
minimum diameters and LV ejection fraction between the groups at each time point
Many studies performed on cardiac regeneration
therapy use stem cells such as hiPS-CM sheets [15, 16]
or stem cell injections . However, one of the most
critical issues for those studies is an insufficient blood
and oxygen supply resulting in poor engraftment of
transplanted hiPS-CMs. In this study, we applied a
RVOT reconstruction method to implant the hiPS-CM
seeded cardiac patches in conjunction with
incorporating a sufficient blood supply directly from the luminal
surface of the patch. In the tissue-engineering
paradigm, the scaffold provides a source of cell attachment
and initial structural integrity . Our PGA-PLCL patch
is porous and has a sponge like structure so that the
seeded hiPS-CMs can grow inside the patch, which
ensures adequate perfusion of nutrients and oxygen during
culture. Sufficient nutrient and oxygen delivery via
functional vasculature or perfusion is vital for the success of
cardiac stem cell therapy.
The mammalian heart’s capacity for self-renewal is
actively debated [18, 19]. Some studies suggest that new
cardiomyocytes regenerate at a very low rate [20–22]
and that they may be derived from the division of
preexisting cardiomyocytes in the mammalian heart. Other
studies suggest a high rate of stem cell activity with
continuous differentiation of progenitors to
There are some limitations in this study. First,
structural and functional immaturity of hiPS-CMs may result
in their death in the in vivo environment. Second, the
evacuation of the seeded hiPS-CMs may be due to the
interspecies difference. Finally, even though we showed
the cells which stained positive for α-actinin in the
cardiac patch 16 weeks after implantation, we were
unable to prove a mechanism for the regeneration of
In this study, the seeded hiPS-CMs were not present in
the patch in the early time point. However, they might
influence the regeneration of host cardiomyocytes.
Tissue-engineered hiPS-CMs seeded biodegradable
cardiac patches warrant further investigation for use in
the repair of heart diseases.
DAPI: 4′,6-diamidino-2-phenylindole; H&E: Hematoxylin and Eosin;
hiPS: Human induced pluripotent stem; hiPS-CMs: Human induced
pluripotent stem cell derived cardiomyocytes; HPF: High power field;
iPS: Induced pluripotent stem; LV: Left ventricle; NIH: National Institutes
of Health; PGA: Polyglycolic acid; PLCL: Poly (l-lactic-co-ε-caprolactone);
RV: Right ventricle; RVOT: Right ventricular outflow tract
Dr. Sugiura was the recipients of a Funding Award from Kanae Foundation
for the Promotion of Medical Science (Tokyo, Japan) and from Astellas
Foundation for Research on Metabolic Disorders (Tokyo, Japan) in 2013.
TS carried out the cell culture, surgery, acquisition of data, analysis and
interpretation of data, and writing of the manuscript. NH made substantial
contributions to conception and design. CKB was involved in revising the
manuscript critically for important intellectual content. TS has given final
approval of the version to be published. All authors have read and approved
the final manuscript.
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