Download Myocardial tissue engineering with cells derived from human

 SHORT COMMUNICATION
Myocardial Tissue Engineering With Cells Derived from Human Induced-Pluripotent
Stem Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold
Ling Gao1, Molly E. Kupfer2, Jangwook P. Jung2, Libang Yang2, Patrick Zhang2, Yong Da Sie3, Quyen
Tran3, Visar Ajeti3, Brian T. Freeman2, Vladimir G. Fast1, Paul J. Campagnola3, Brenda M. Ogle2*, Jianyi
Zhang1*
1
Department of Biomedical Engineering, School of Medicine, School of Engineering, University of
Alabama at Birmingham; 2Department of Biomedical Engineering, University of Minnesota – Twin
Cities, and; 3Department of Biomedical Engineering, University of Wisconsin – Madison, Madison.
Running title: 3D-Printed Scaffolds for Engineered Myocardium
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B.M.O. and J.Z. are both corresponding authors.
Subject Terms:
Myocardial Infarction
Cell Therapy
Stem Cells
Addresses correspondence to:
Dr. Jianyi Zhang
Department of Biomedical Engineering
University of Alabama at Birmingham
[email protected]
Dr. Brenda M. Ogle
Department of Biomedical Engineering
University of Minnesota – Twin Cities
[email protected]
In December 2016, the average time from submission to first decision for all original research papers submitted
to Circulation Research was 13.4 days.
DOI: 10.1161/CIRCRESAHA.116.310277 1
ABSTRACT
Rationale: Conventional three-dimensional (3D) printing techniques cannot produce structures of the size
at which individual cells interact.
Objective: Here, we used multiphoton-excited, 3-dimensional printing (MPE-3DP) to generate a nativelike, extracellular matrix (ECM) scaffold with submicron resolution, and then seeded the scaffold with
cardiomyocytes (CMs), smooth-muscle cells (SMCs), and endothelial cells (ECs) that had been
differentiated from human induced-pluripotent stem cells (iPSCs) to generate a human, iPSC-derived
cardiac muscle patch (hCMP), which was subsequently evaluated in a murine model of myocardial
infarction (MI).
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Methods and Results: The scaffold was seeded with ~50,000 human, iPSC-derived CMs, SMCs, and ECs
(in a 2:1:1 ratio) to generate the hCMP, which began generating calcium transients and beating
synchronously within 1 day of seeding; the speeds of contraction and relaxation and the peak amplitudes
of the calcium transients increased significantly over the next 7 days. When tested in mice with surgically
induced MI, measurements of cardiac function, infarct size, apoptosis, both vascular and arteriole density,
and cell proliferation at week 4 after treatment were significantly better in animals treated with the hCMPs
than in animals treated with cell-free scaffolds, and the rate of cell engraftment in hCMP-treated animals
was 24.5% at week 1 and 11.2% at week 4.
Conclusions: Thus, the novel MPE-3DP technique produces ECM-based scaffolds with exceptional
resolution and fidelity, and hCMPs fabricated with these scaffolds may significantly improve recovery from
ischemic myocardial injury.
Keywords:
Heart, myocardial infarction, tissue engineering.
Nonstandard Abbreviations and Acronyms:
hiPSCs
3D
ECM
MPLSM
MPE-3DP
CMs
ECs
SMCs
hCMP
CL
SMA
MI
HNA
GFP
hCD31
BP
human induced-pluripotent stem cells
three-dimensional
myocardial extracellular matrix
multi-photon laser scanning microscopy
multiphoton-excited three-dimensional printing
cardiomyocytes
endothelial cells
smooth muscle cells
hiPSC-derived cardiac muscle patch
cycle length
-smooth muscle actin
myocardial infarction
human-specific nuclear antigen
green fluorescent protein
human specific CD31
bovine pericardium
DOI: 10.1161/CIRCRESAHA.116.310277 2
INTRODUCTION
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Although recent large-animal studies have shown that recovery from myocardial injury can be
improved by injecting cardiac cells that have been differentiated from human induced-pluripotent stem cells
(hiPSCs), the structural support and synchronized contractile activity of a transplanted myocardial tissue
equivalent (MTE) could provide additional benefits. Tissue engineers are beginning to improve the
effectiveness of MTE engineering for regenerative myocardial therapies1-3 by developing techniques that
can enhance the engraftment, improve the MTE maturation4, 5 and long-term functional benefits.6, 7 These
efforts may be aided by incorporating some of the more complex features of the myocardial extracellular
matrix (ECM) into the tissue’s design. This level of complexity cannot be reproducibly achieved with
established manufacturing technologies, but a newer modality, 3D printing, has been successfully used to
build structures with defined geometries from heterogeneous materials8 and, consequently, may be used for
generating scaffolds that mimic the ECM of native cardiovascular tissues. However, key structural features
of the native ECM need to be identified and incorporated (with adequate resolution and reliability) into a
scaffold to promote cell function and limit detrimental effects. In addition, from the potential clinical
application perspective, a high level of quality control of the scaffold product is required, which can only
be achieved by a computer controlled 3D printing technology.
The position of crosslinks within a matrix of photoactive biological polymers can be controlled
with high resolution. Printers that utilize single-photon excitation coupled to a sequence of photomasks
can achieve approximately 30 micron resolution in x, y and approximately 50 micron resolution in z. A
more advanced technique, multiphoton excited (MPE) photochemistry, can restrict excitation (and,
consequently, the photochemical reaction) in three dimensions via a method that is analogous to multiphoton laser scanning microscopy (MPLSM).9-14 Notably, the resolution of the features (<1 μm) is
determined by the MPE point spread function and can therefore approximate the feature size of components
of the extracellular matrix.15 The technique can also be combined with rapid prototyping and computeraided design16 to fabricate essentially any 3D structure that can be drawn.
For the experiments described in this report, we used our novel technique, multiphoton-excited
three-dimensional printing (MPE-3DP), to generate a scaffold with a native-like cardiac ECM architecture
from a solution of a photoactive gelatin polymer and then seeded the scaffold with cardiac cells
(cardiomyocytes [CMs], endothelial cells [ECs], and smooth muscle cells [SMCs]) that had been
differentiated from human, cardiac-lineage, induced-pluripotent stem cells (hciPSCs)17 to generate an
hciPSC-derived cardiac muscle patch (hCMP). The hCMP was subsequently characterized via a series of
in vitro analyses and then tested in a murine model of ischemic myocardial injury.
METHODS
A detailed description of the experimental procedures used in this investigation is provided in the online
Data Supplement.
DOI: 10.1161/CIRCRESAHA.116.310277 3
RESULTS
Fabrication of an ECM scaffold based on templates derived from optical image stacks of murine
myocardium.
Our approach can be summarized in two steps: first, native, murine, adult myocardial tissue was
examined to determine the size and distribution of various ECM features, which were incorporated into a
3D template; then, the template was scanned and used to map the positions of crosslinks in a solution of a
photoactive polymer (Figure 1A). Importantly, our scanning technique, modulated raster scanning, maps
the template directly to the scaffold by monitoring the brightness of each point in the image, and it is
accurate to resolutions of less than 1 μm. To our knowledge, this is the first time modulated raster scanning
has ever been successfully used to control the fabrication of a tissue-engineered scaffold and, consequently,
our results are particularly relevant for applications that require the fibrillar and mesh-like structures present
in cardiac tissue.18
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We chose to base our template on the distribution of fibronectin in murine myocardium (Figure 1B
and 1C). Fibronectin is uniformly distributed around each CM, so it can be used to determine the dimensions
of each individual cell compartment to form a grid (here termed, Adult Simulate). The scaffold was
generated from a solution of gelatin methacrylate, which can be crosslinked into complex structures with
high efficiency, allows creation of complex structures with thickness of approximately 100 m (Online
Video I, II and III), is biologically inert when both crosslinked and degraded, and the denatured collagen
exposes cell binding sites (including Arg-Gly-Asp, RGD), which should readily adhere to the seeded cells
and support biochemical signaling via focal adhesions.19 Analysis of the native myocardium suggested that
CMs reside in channels that are approximately 15 μm by 100 μm which, when incorporated in the hCMP
scaffold (Figure 1D and 1E), yielded a robust structure with high reproducibility and exceptional fidelity in
both coverage area (≥95%) and intensity variation (≥85%).
Integration of hciPSC-derived cardiac cells in hCMPs.
The hciPSCs were reprogrammed from human cardiac fibroblasts and differentiated into hciPSCCMs, hciPSC-SMCs, and hciPSC-ECs; then, the differentiated cells were characterized via the expression
of lineage-specific markers (Online Figure I), and the hCMP was formed by seeding ~50,000 cells (in a
2:1:1 ratio of hciPSC-CMs, hciPSC-SMCs, and hciPSC-ECs, respectively) into the fabricated scaffold,
where they quickly became assimilated and occupied most of the free space (Figure 1D). The hciPSC-CMs
began generating calcium transients (Figure 2A) and beat synchronously across the entire hCMP within
one day of seeding (Online Video IV and V). Over the next seven days, the speeds of contraction and
relaxation, the peak amplitude of the calcium transients (Figure 2B and 2C), and the expression of several
genes required for contractile function (cTnT, cTnI, and MHC) and for generating calcium transients
(SERCA2RYR2, CASQ2) increased significantly (Online Figure II). Contractile and calcium-transient
gene expression was also greater in the hCMPs than in monolayers grown from equivalent populations of
hiPSC-derived cardiac cells on day 7. Optical mapping of transmembrane potentials (Figure 2D and 2E)
revealed macroscopically continuous action potential propagation in hCMPs, showing an excellent
functional electrophysiological communications between cells. Conduction velocity in hCMPs increased
linearly with the increase of pacing cycle length (CL), reaching 18.8±0.8 cm/s at 800 ms pacing CL (Figure
2F). Action potential durations, APD50 and APD80, were about 214±10.4 and 270±12.7 ms at 800 ms pacing
CL, separately, and also showed significant correlation with pacing rate (Figure 2G). Autofluorescence
images obtained via 2-photon microscopy on day 7 (Figure 2H) indicated that the cells had aligned with
the channels of the fabricated scaffold (Figure 2I) to form multinucleate cells with an aspect ratio (i.e., cell
length:width) of approximately 5.5:1 (Figure 2J), which is close to the characteristic ratio observed in native
CMs (7:1). Analysis of hCMPs stained for expression of the CM protein cTnI, -smooth muscle actin
DOI: 10.1161/CIRCRESAHA.116.310277 4
( SMA), and the endothelial marker CD31 (Figure 2K) indicated that the original 2:1:1 ratio of hciPSCCMs, -SMCs, and -ECs was largely retained (Figure 2L), with CMs comprising ~45% of the remaining
cells. The 7-day survival rate of hciPSC-CMs in the hCMP was similar to that observed when cells were
maintained on Matrigel-coated plates and significantly greater than the rate achieved by culturing the cells
with bovine pericardium or polyethylene glycol (Figure 2M).
In vivo evaluations of hCMP transplantation in a murine model of myocardial infarction.
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In vivo assessments of the hCMPs were performed in a murine model of myocardial infarction
(MI). MI was surgically induced as described previously,20 then, animals in the MI+hCMP group were
treated with two hCMPs, animals in the MI+Scaffold group were treated with two patches of scaffold
material without cells, and animals in the MI group received neither the hCMP nor the cell-free scaffold.
The hCMPs and scaffolds were positioned over the site of infarction (Figure 3A) and held in place by
covering them with a piece of decellularized bovine pericardium that was sutured to the heart to avoid
movement of hCMPs off the heart. The decellularized bovine pericardium above the hCMP has beneficial
effect of preventing the adhesion between the heart/hCMP and the chest. The Sham group underwent all
surgical procedures for MI induction except the ligation step and recovered without either experimental
treatment. Quantitative PCR measurements for expression of the human Y chromosome indicated that
24.5±2.6% of the transplanted cells remained engrafted in the hearts of MI+hCMP animals for at least one
week after transplantation (Figure 3B). By week 4, the engraftment rate had declined to 11.2±2.3%, which
is still much higher than the rate reported in previous studies of cell-based myocardial therapy.21, 22 In order
to assess the engraftment rate by counting the grafted human cells as evidenced by human-specific nuclear
antigen (HNA) positivity, we also used histologic assessment of consecutive sections of the heart that spans
the entire area covered by the engrafted patch, and counting the grafted human cells as evidenced by HNA
positivity.17, 23 The engraftment rate of the 7 hearts studied using the histology method is 13.6 ± 2.2%, which
is in agreement with the quantitative PCR (11.2 ± 2.3%). The ratio of the hciPSC-tri lineage cardiac cells
at week 1 and 4 was also examined. The engrafted hciPSC-CMs were identified by the expression of both
green fluorescent protein (GFP) and cTnI, engrafted hiPSC-ECs were identified by expression of human
specific CD31 (hCD31), and engrafted hiPSC-SMCs were identified by the expression of both GFP and
SMA. At week 1 post transplantation the ratio of hciPSC-CMs, hciPSC-SMCs, and hciPSC-ECs was 1.42:
0.93: 1; at week 4 the ratio was 0.67: 0.86: 1 for hciPSC-CMs, hciPSC-SMCs, and hciPSC-ECs,
respectively. Hematoxylin and Eosin (HE) staining (Figure 3C), and immunofluorescence analysis of HNA,
GFP, cTnI, SMA, and hCD31 expression (Figure 3C and 3D) showed evidence of all three transplanted
cell lineages in the treated region
Cardiac function was evaluated on day 28 after injury via echocardiographic assessments (Figure
4A) of left-ventricular ejection fraction and fractional shortening; both parameters were significantly
greater for animals in the MI+hCMP group than in MI+Scaffold or MI animals (Figure 4B and 4C), while
measurements in the MI+Scaffold and MI groups were similar. Infarcts were also significantly smaller,
while the thickness of the infarcted region of the myocardial wall was significantly greater, in MI+hCMP
hearts than in MI or MI+Scaffold hearts (Figure 4D-4F), and analyses of TUNEL-stained sections (Online
Figure IIIA and IIIB) and sections stained for the presence of CD31 and SMA (Online Figure IIIC-IIIE)
indicated that apoptotic cells were significantly less common, while vascular structures (including
arterioles) were significantly more common, at the border of the infarcted region in hCMP-treated animals
than in the corresponding regions of hearts from animals in the MI+Scaffold or MI groups. hCMP treatment
was also associated with significant increases in the number of cells that expressed the proliferation marker
Ki67 (Online Figure IV). Thus, the transplanted hCMPs appeared to improve recovery from myocardial
injury by, at least in part, reducing apoptosis, promoting angiogenesis, and increasing cell proliferation.
DOI: 10.1161/CIRCRESAHA.116.310277 5
DISCUSSION
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The fate specification of progenitor cells in the developing heart is critically dependent on the
spatial and temporal factors of the ECM where the progenitor cells reside. In human pluripotent stem cell
differentiation to endothelial cells, the rate and quality of derived endothelial cells are critically influenced
by the temporal factors in 3D ECM. 24 The myocardial ECM provides substrates for cell adhesion,
sequesters soluble factors, and serves as a conduit for mechanical signaling. Our recent efforts to
characterize the ECM of the developing heart have provided us with an extensive body of knowledge
regarding the nanoscale distribution of the ECM,25 and by combining this knowledge with MPE-3DP, we
were able to generate a scaffold that is structurally native-like.16 The technique couples MPE with
modulated raster scanning to induce crosslinks in a solution of a photoactive gelatin polymer, thereby
creating an ECM scaffold with exceptionally high resolution. Notably, construction was guided by a
template composed of features that had been identified in the ECM of a native adult murine heart, which
suggests that the technique may be able to replicate the unique architecture of the myocardial ECM in each
individual. Furthermore, when hCMPs were generated by seeding the scaffolds with hciPSC-derived
cardiac cells and transplanted into infarcted mouse hearts, the treatment was associated with significant
improvements in cardiac function, infarct size, apoptosis, vascular density, and cell proliferation. Thus, the
hCMPs produced for this report may represent an important step toward the clinical use of 3D-printing
technology.
Our novel technique is the first to achieve a resolution of 1 μm or less and, consequently, can
reliably control the thickness of the walls that separate adjacent channels. Channel-wall thickness may be
a particularly important parameter for myocardial tissues, because contractions in adjacent fibers must be
synchronized by signaling mechanisms that traverse the wall. Notably, the seeded cells quickly settled into
the engineered channels of the scaffolds generated for this report, and synchronized beating was observed
as early as one day after cell seeding, which suggests that individual cells interacted with the features of the
scaffold, and that inter-channel coupling mechanisms were quickly established.
The functional improvement associated with hCMP transplantation in the murine MI model
suggests that this goal may have been at least partially achieved. Thus, in future investigations we will use
optical mapping technology to determine whether transplanted hCMPs are electromechanically coupled to
native myocardial tissues.26
In conclusion, we have developed a method by which the principles of 3D printing and
photochemistry can be combined to generate an ECM scaffold with unprecedented resolution from a
template based on the architecture of native myocardial ECM. When hCMPs were generated by seeding
the scaffolds with hciPSC-derived cardiac cells, the scaffold promoted cell viability and electromechanical
coupling in vitro, and the hCMPs were associated with high levels of cell engraftment, as well as significant
improvements in cardiac function, infarct size, apoptosis, vascularity, and cell proliferation in a murine MI
model. Subsequent investigations will focus on methods for creating hCMPs of sufficient size for largeanimal studies and means to improve effectiveness of the hCMPs by incorporating mixtures of ECM
proteins into the scaffold.
SOURCES OF FUNDING
This work was supported by the following funding sources: National Science Foundation, Award CBET1445650; Lillehei Heart Institute, UMN, High Risk High Reward; Institute for Engineering and Medicine,
UMN, Pilot Grant, and NIH RO1 HL 99507, HL114120, HL 131017, UO1 134764.
DISCLOSURES
None.
DOI: 10.1161/CIRCRESAHA.116.310277 6
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FIGURE LEGENDS
Figure 1. hCMP fabrication via 3D-MPE. (A) The ECM and associated crosslinking solution are passed
through the optical interrogation path while the laser power and dwell time are modulated to deposit ECM
at each x, y location in each z plane. The submicron-scale features produced in the ECM scaffold are
displayed in the inset (scale bar = 1 μm). Three-dimensional structures can be generated by combining
multiple layers with the same or different ECM pattern. (B) Sections from the heart of an adult mouse were
immunofluorescently stained for the presence of fibronectin and scanned via MPE (scale bar = 200 μm);
then, (C) the distribution of fibronectin in the native tissue was simulated in a template. The simulated
channels (green, 100 μm × 15 μm) are shown overlaying the fibronectin pattern of the native tissue (red) in
the inset (scale bar = 100 μm). (D-E) The simulated template was used to determine the position of
crosslinks in a solution of gelatin methacrylate, thereby producing a native-like ECM scaffold (D); then,
the scaffold was seeded with hiPSC-derived CMs, ECs, and SMCs to generate the hCMPs (E). The
complete hCMP is shown in the larger image (scale bar = 400 μm), while the individual channels and
incorporated cells are visible in the inset (scale bar = 50 μm).
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Figure 2. In vitro assessments of the hCMP. (A) Calcium transients were recorded in hCMPs on day 1,
3, and 7 after cell seeding, and used to calculate the (B) peak amplitude (F/F0; n ≥ 50 cells per time point).
(C) Videos of the beating hCMPs were taken on day 1, 3, and 7 and evaluated with motion vector analysis
software to calculate the speeds of contraction and relaxation (n=4 hCMPs per time point). (D-G) Action
potential propagation in hCMP was measured on day 7 (n=4). (D) Representative isochronal map of
activation spread and (E) selected optical Vm traces recorded during pacing with cycle length (CL) of 800
and 300 ms. AT, activation time; Dependence of (F) conduction velocity (CV) and (G) action potential
duration (APD50 and APD80) on pacing CL. (H) hCMPs were stained with DAPI on day 7; then,
autofluorescence images were obtained via 2-photon microscopy (scale bar = 20 μm) and used to calculate
the cells’ (I) angle of alignment relative to the long axis of the engineered channel and (J) aspect ratio. (K)
On day 7, ECs, SMCs, and CMs were identified in the hCMPs via immunofluorescence staining for the
presence of CD31, -smooth-muscle actin (SMA), and cardiac troponin I (cTnI), respectively; then (L)
the proportion of each cell type was calculated (5 fields per hCMP and 4 hCMPs). (M) Known quantities
of hiPSC-CMs were seeded into Matrigel, polyethylene glycol (PEG), decellularized bovine pericardium,
or the engineered scaffold and cultured for 7 days; then, the cells were immunofluorescently stained for
cTnI expression, and cell survival was quantified as the ratio of the number of cTnI+ cells observed to the
number of seeded cells. **P<0.01.
Figure 3. hCMP engraft and survive after transplantation into the hearts of mice with MI. Myocardial
infarction was surgically induced in mice. Animals in the MI+hCMP group were treated with two hCMPs
(0.1 million cells total). (A) The representative image showed two transplanted hCMPs on the mouse heart.
(B) The engraftment rate was determined in animals from the MI+hCMP group at week 1 and 4 after injury
via quantitative PCR measurements of the human Y chromosome (n=4 hearts per time point). (C) The
representative HE staining image and human specific nuclear antigen (HNA) immunostaining image
showed engrafted hCMP on the epicardial surface of the heart at week 4 after transplantation. BP=bovine
pericardium. (D) Sections taken from the region of patch in MI+hCMP animals at week 4 were
immufluorescently stained for the presence of HNA, cTnI, green fluorescent protein (GFP), SMA, and
the human isoform of the endothelial marker CD31 (hCD31); nuclei were counterstained with DAPI (scale
bar = 50 m). (i-ii) Engrafted hciPSC-CMs were identified by the expression of both HNA and cTnI (i) or
both GFP and cTnI (ii); (iii) engrafted hiPSC-SMCs were identified by the expression of both GFP and
SMA; (iv) engrafted hiPSC-ECs were identified by the expression of hCD31.
DOI: 10.1161/CIRCRESAHA.116.310277 9
Figure 4. hCMP transplantation improves cardiac function and reduces infarct size after MI. (A-C)
Cardiac function was evaluated at week 1 and 4 via (A) echocardiographic assessments of (B) leftventicular ejection fraction and (C) fractional shortening. (D-F) Sections of hearts from animals in different
groups were (D) Masson’s trichrome–stained for histological assessments of (E) infarct size and (F) infarct
wall thickness. Infarct size was calculated as a percentage of the circumflexion length of the left ventricular
free wall, and infarct wall thickness was calculated as a percentage of the thickness of the septal wall.
*p<0.05. BP=bovine pericardium.
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DOI: 10.1161/CIRCRESAHA.116.310277 10
NOVELTY AND SIGNIFICANCE
What Is Known?

Cellular therapy for myocardial repair has shown some benefit in preclinical and clinical settings, but
limited cell retention and associated lack of robust electromechanical coupling remain the major
problems.

Greater benefits may be achieved if the transplanted cardiac cells are provided with structural support
such that synchronized contractile activity of the graft can be integrated to recipient heart.

One approach to generate a structural support is 3D printing, which has recently advanced to include
the printing of biological materials such cells and extracellular matrix proteins (ECM).
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What New Information Does This Article Contribute?

The article describes the implementation of multiphoton-excited three-dimensional printing (MPE3DP) to generate a scaffold with native-like cardiac ECM architecture. The scaffold can effectively
harbor stem cell-derived cardiac cell types and can be handled for effective transfer to the damaged
heart.

The cell-seeded scaffold (termed human cardiac muscle patch, hCMP) beats synchronously in a culture
dish prior to transplant and with extended culture time beats with increased speed and calcium handling
suggesting cardiomyocyte maturation. In vivo, measurements of cardiac function and infarct size were
significantly better in infarcted hearts treated with the hCMPs than in hearts treated with cell-free
scaffolds.

The mechanisms for therapeutic improvement include improved cell engraftment, increased cellular
proliferation and vascular supply with decreased apoptosis.
We demonstrate multiphoton-excited, 3D printing to generate a human cardiac muscle patch (hCMP) that
could be effectively populated with cardiac cell types because it mimics the structural dimensions and
protein composition of the native myocardium. In a mouse model of myocardial infarction, hCMP
transplantation results in higher levels of cell engraftment, cell proliferation and myocardial vascular
density, which are accompanied by a significant reduction of infarct size and improvement of left
ventricular function. This work pushes the technical limits of 3D printing for cardiac tissue equivalents
such that cells and extracellular matrix proteins could be manufactured precisely as designed.
DOI: 10.1161/CIRCRESAHA.116.310277 11
Figure-1
A
Adult simulate
E
hCMP
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D
Printed scaffold
C
Adult
B
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100
m
100
m
Figure-3
A
D
hCMP-1
i HNA
DAPI cTnI
HNA DAPI cTnI
hCMP-2
B
Engraftment rate (%)
DAPI
HNA
cTnI
HNA
DAPI
cTnI
32
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24
16
8
0
Week 1 Week 4
ii GFP
cTnI
GFP cTnI DAPI
C
hCMP
iii GFP
SMA
GFP SMA cTnI DAPI
BP
iv hCD31
iV
HNA DAPI
hCMP
BP
DAPI
hCD31 cTnI DAPI
Figure-4
A
MI
Ejection fraction (%)
Sham (5)
MI (7)
MI+Scaffold (7)
MI+hCMP (8)
100
80
MI+hCMP
C
Sham (5)
MI (7)
MI+Scaffold (7)
MI+hCMP (8)
*
*
60
40
20
0
Week 1
Week 4
60
45
*
*
30
15
0
Week 1
BP
D
MI
E
MI (7)
MI+Scaffold (7)
MI+hCMP (7)
75
60
45
30
15
0
*
*
MI+Scaffold
F
Week 4
hCMP
BP
MI+hCMP
MI (7)
MI+Scaffold (7)
MI+hCMP (7)
Percentage of infarct
thickness (%)
Sham
Percentage of Infarct
size (%)
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B
MI+Scaffold
Fractional shorting (%)
Sham
100
80
60
40
20
0
*
*
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Myocardial Tissue Engineering With Cells Derived from Human Induced-Pluripotent Stem
Cells and a Native-Like, High-Resolution, 3-Dimensionally Printed Scaffold
Ling Gao, Molly Kupfer, Jangwook Jung, Libang Yang, Patrick Zhang, Yong Sie, Quyen Tran, Visar
Ajeti, Brian Freeman, Vladimir Fast, Paul Campagnola, Brenda Ogle and Jianyi Zhang
Circ Res. published online January 9, 2017;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2017 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571
The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/early/2017/01/09/CIRCRESAHA.116.310277
Data Supplement (unedited) at:
http://circres.ahajournals.org/content/suppl/2017/01/09/CIRCRESAHA.116.310277.DC1
Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in
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CIRCRES/2016/310277 R2
Supplemental Material
Myocardial tissue engineering with cardiac cells derived from human
induced-pluripotent stem cells and a native-like, high-resolution, 3-dimensionally
printed scaffold
Ling Gao1, Molly E. Kupfer2, Jangwook P. Jung2, Libang Yang2, Patrick Zhang2, Yong Da
Sie3，Quyen Tran3, Visar Ajeti3, Brian T. Freeman2, Vladimir G. Fast1, Paul J.
Campagnola3, Brenda M. Ogle2*, Jianyi Zhang1*
1
Department of Biomedical Engineering, School of Medicine, School of Engineering,
University of Alabama at Birmingham;
2
Department of Biomedical Engineering,
University of Minnesota – Twin Cities, Minneapolis, MN 555455 USA;
3
Department of
Biomedical Engineering, University of Wisconsin – Madison, Madison, WI 53706 USA
Short title: Gao, 3D-printed scaffolds for engineered myocardium
*Addresses for correspondence
Dr. Jianyi Zhang, Department of Biomedical Engineering, School of Medicine, School of
Engineering,
University of
Alabama
at
Birmingham,
Birmingham,
AL 35233
205-934-8421, E-mail: [email protected]
Dr. Brenda M. Ogle, Department of Biomedical Engineering, College of Science and
Engineering, University of Minnesota – Twin Cities, Minneapolis, MN, 55455,
612-624-5948, E-mail: [email protected]
1
CIRCRES/2016/310277 R2
SUPPLEMENTARY METHODS
Creation of the ECM scaffold via 3D-MPE printing
Methacrylated
gelatin
(100
mg/mL)
was
mixed
with
sodium
4-[2-(4-morpholino)benzoyl-2-dimethylamino]-butylbenzenesulfonate (MBS) (1 mM) at 1%
v/v, and crosslinking was performed on a glass slide, which served as the non-specific
background. Two-photon excitation of the MBS photoactivator was induced with a 100 fs
Ti:sapphire laser (Mira, Coherent, Santa Clara, CA) at 780 nm. A 10x, 0.5 NA objective
lens was used, and the average power at the focus was kept constant (~100 mW at 78
MHz repetition rate). Upon UV absorption, MBS degrades to benzoyl and an
α-aminoalkyl radical, which then attack residues containing aromatic groups and free
amines1; then, the radical protein links to a second protein molecule, generating a
covalent bond.
Our custom-built multiphoton instrument, which has been described in detail previously, 2
can produce scaffolds with more complexity and in a greater variety of sizes than can be
achieved with a commercial laser-scanning microscope. The Ti:sapphire laser is coupled
to a upright microscope stand (Axioskop 2, Zeiss, Thornewood, NY), and scanning is
performed by combining a laser-scanning galvos (Cambridge Technolgoies, Bedford, MA)
with a motorized stage (x-y-z, Ludl Electronic Products Ltd, Hawthorne, NY). The
apparatus is controlled with LabVIEW software, and data is acquired with a
field-programmable gate array (FPGA) board (Virtex-II PCI-7831R, National Instruments,
Austin, TX).2 The laser power entering the optical train is regulated with a 10 kHz
electro-optic modulator (EOM) (Conoptics, Danbuty, CT), and the laser is rapidly
shuttered with a second, higher-speed EOM (maximum 100 MHz; Conoptics).
Parameters such as power, scanning area, the scan rate of the galvanometer, and
repetition of the scanning pattern (i.e., the number of scans per layer) are set via a
graphical user interface (GUI), while the TPEF of the entrapped residual photoactivator
2
CIRCRES/2016/310277 R2
serves as the online diagnostic signal for crosslinking and is read by the FPGA. The
microscope is also equipped with phase-contrast and two-photon fluorescence imaging
capabilities for characterizing the scaffold (e.g. immunofluorescence and quality control),
and the minimum size of the features in the crosslinked protein structure corresponds to
the two-photon excited point spread function (PSF). For example, when using 0.75 NA
and 780 nm two-photon excitation, the lateral and axial resolution are about 600 nm and
1.8 μm, respectively2, 3 MPE polymerization can also produce features with sub-micron
resolution, because of chemical nonlinearity in the free radical kinetics,3 but this does not
occur during the crosslinking of proteins.
The FPGA was incorporated into the system to exploit the parallelism of command
executions (80 MHz clock rate) and to avoid bottlenecks in communication between the
CPU and the hardware, which occurs through four first in, first out (FIFO) channels.
Two FIFO channels relay information from the main LabVIEW program to the FPGA for
control of the galvanometer mirrors and fast EOM shutter, while the other two record
information from the PMT to create an image of the fabrication; thus, communication
between the CPU and hardware occurs in near real-time. The source code of the
instrument control software is freely available at: http://campagnola.molbio.wisc.edu/.
The scanning approach (i.e., “modulated raster scanning”) combines the advantages of
both raster scanning and vector scanning.4 The galvanometers are raster scanned at
maximum speed (~40 kHz), while the laser is shuttered at an even greater rate (10
MHz-40kHz) with the fast EOM. This approach is more accurate than modulating the
scanning speed and/or step size of the galvanometers, because it is run over the whole
field of view at constant speed and with the same pulse energy and repetition rate. The
average power entering the fast-shuttering EOM is set by the slower EOM, which allows
the entire scanning process to be performed at constant peak power and, consequently,
3
CIRCRES/2016/310277 R2
the fractional “on-time” within each pixel defines the integrated exposure dose, which is a
linear process that directly correlates to the resulting protein concentration. Thus, we
achieve a linear map between the intensity in the original image data and the fabricated
structure.
Fabrication was based on 8-bit, 3D image files (.bmp or .tif) that were acquired with
sampling that satisfied the Nyquist criterion, which is crucial for ensuring that the tissue is
accurately represented and that the resolution (i.e., the sizes of the features) matches
that of the original image, as well as for optimizing the structural integrity of the fabricated
scaffold in 3D. Fabrication proceeded through the image stack via a Z-step pattern, and
upon completion, the fidelity of the structure to the image stack was measured by using
FIJI software to compare, pixel-by-pixel, the gray-scale intensity of the original (or
processed) image to the two-photon excited fluorescence image of the fabricated
construct.
Generation and characterization of hciPSC-derived cardiac cells
Recent
reports
from
our
laboratory
suggest
that
the
cells
derived
from
induced-pluripotent stem cells (iPSCs) may be more effective for treatment of myocardial
injury, and that the Ca2+ handling profile of iPSC-derived CMs is more cardiac-like, if the
iPSCs are reprogrammed from cardiac, rather than dermal, fibroblasts.5, 6 Thus, the CMs,
SMCs, and ECs used in this study were generated from human, cardiac-lineage, iPSCs
(hciPSCs), which had been reprogrammed from male, human, cardiac fibroblasts by
transfecting the cells with lentiviruses coding for OCT4, SOX2, KLF4, and C-MYC.5 After
reprogramming, the hciPSCs were engineered to constitutively express green
fluorescent protein (GFP), cultured in Matrigel-coated plate with human iPSC growth
medium, and regularly passaged every 6-7 days.
4
CIRCRES/2016/310277 R2
hciPSCs were differentiated into ECs and SMCs as described previously.7, 8 Briefly, the
undifferentiated cells were treated with a GSK-3β inhibitor and ascorbic acid to induce
mesoderm
differentiation.
Five
days
later,
cells
that
expressed
CD34
(i.e.,
hciPSC-derived vascular progenitor cells [hciPSC-VPCs]) were collected via magnetic
nanoparticle selection; then, the hciPSC-VCs were differentiated into ECs by culturing
them on fibronectin-coated flasks with EC-developmental medium (EGM2-MV; Lonza,
Basel, Switzerland) or into SMCs by culturing them on collagen IV-coated flasks with
SMC-developmental medium (SmGM-2; Lonza) containing PDGF-BB and TGF-β. ECs
were purified to >95% via flow-cytometry selection for both CD31 and CD144 expression
and then characterized via the expression of CD31, CD144, and von Willebrand factor-8
(vWF-8). SMCs were characterized via the expression of  smooth-muscle actin (SMA),
smooth-muscle 22 alpha (SM22), and calponin.
hciPSCs
were
differentiated
into
CMs
as
previously
reported. 9
Briefly,
the
undifferentiated cells were expanded on a Matrigel ®-coated dish for 4 days; then,
differentiation was induced on day 0 by culturing the cells with a GSK-3β inhibitor in
RPMI basal medium plus B27 without insulin (B27–). The cells were recovered 24 hours
later, cultured in RPMI basal medium plus B27– for 2 days, and then cultured in RPMI
basal medium plus B27– and a Wnt signaling inhibitor; beating cells usually appeared
about 8 days after differentiation was initiated. The hciPSC-CMs were purified to >95%
via metabolic selection10 and characterized via the expression of cardiac troponin I (cTnI),
cardiac troponin T (cTnT), α-sarcomeric actin (αSA), Connexin 43, and ventricular
myosin light chain-2 (MLC-2v).
Creation of the human iPSC-derived cardiac muscle patchs (hCMPs) and control
patches
The hCMPs (2 mm × 2 mm, 100 μm thick) were created by combining the
5
CIRCRES/2016/310277 R2
3D-MPE–fabricated scaffold with a 2:1:1 ratio of hciPSC-CMs, hciPSC-ECs, and
hciPSC-SMCs. A total of ~50,000 cells (i.e., 12,500 cells/mm 2) were seeded onto the
surface of the scaffold; then, the hCMPs were cultured in Dulbecco minimum essential
medium (DMEM) with 10% fetal calf serum for 1 week before in vitro analyses were
performed or for 1 day before in vivo transplantation. For the in-vitro experiments, control
patches were created by combining the same density and ratio of hciPSC-CMs, -ECs,
and -SMCs with a chemoselectively cross-linked polyethylene glycol (PEG) gel11 or with
decellularized bovine pericardium; the control patches were also cultured in DMEM with
10% fetal calf serum for 1 week.
Measurement of calcium transients
hCMPs were loaded with 5 µM Fluo-4 AM (Life Technologies, USA) and incubated at
37C and 5% CO2 for 30 min; then, the intercellular AM esters were de-esterified by
incubating the hCMPs with Tyrode’s salt solution at 37C and 5% CO2 for another 30 min.
Intracellular calcium transients were recorded with a Zeiss Cell Observer Spinning Disk
Confocal microscope equipped with a 20X (NA = 0.5) objective and an environmental
chamber (37C, 5% CO2) over 30 to 120 s. Movies were analyzed with FIJI software to
measure the fluorescence intensities for 2 to 8 regions of interest (F) and for 3 to 8
background regions (F0) per acquisition.
In vitro analysis of contractility
Videos of the beating hCMPs were taken at 24 fps; then, the speed of contraction and
relaxation, as well as the peak beat rate, of the hCMP was determined with motion vector
analysis software developed by Huebsch, et al.12
Optical mapping of activation
hCMPs were transferred to a perfusion chamber mounted on an inverted microscope
6
CIRCRES/2016/310277 R2
and perfused with Hank's balanced salt solution at 37oC. Preparations were stimulated
with 2-ms rectangular pulses delivered from a bipolar electrode. Cells were stained with
Vm-sensitive dye RH-237 (5 μM) for 5 min. To eliminate motion artifact from optical
recordings, cell contractions were inhibited by supplementing staining and perfusion
solutions with 10 μM of blebbistatin. Dye fluorescence was excited using an Hg/Xe arc
lamp and a 560/55-nm excitation filter and measured at >650 nm using a 16x16
photodiode array at spatial resolution of 110 µm per diode, as previously described.13
Optical signals were digitally filtered to increase the signal-to-noise ratio. Activation times
were measured at the 50% level of action potential amplitude and used to construct the
isochronal maps of activation spread. Conduction velocity was calculated at each
recording site from local activation times and averaged across the whole map using
custom-written data analysis software. Action potential durations were measured at 50%
and 80% levels of repolarization (APD50 and APD80, respectively) as intervals between
repolarization and activation times.
Cardiac monolayer culture
Cell suspensions containing a 2:1:1 ratio of hciPSC-CMs, -ECs, and -SMCs were
seeded on a gelatin coated plate at a density of 1.2x105 cells/cm2 and cultured in
DMEM with 10% fetal calf serum; the culture media was changed every 2 days.
Quantitative RT-PCR Analysis
Total RNA was extracted by using Qiashredder and RNeasy mini kits (Qiagen, USA) as
directed by the manufacturer's instructions; then, the RNA (1-2 μg per 20 μL reaction)
was reverse transcribed with SuperScript™ II Reverse Transcriptase (Thermo Scientific,
USA) and appropriate primers (Supplemental Table 1). Quantitative PCR was performed
with Maxima SYBR Green Master Mix (Thermo Scientific) on a Realplex2 Real-Time
7
CIRCRES/2016/310277 R2
PCR system (Eppendorf, USA). Measurements were calculated via the 2−∆Ct method
and normalized to glyceraldehyde phosphate dehydrogenase RNA levels.
Murine model of myocardial infarction and hCMP administration
All experimental procedures that involved animals were approved by the Institutional
Animal Care and Use Committee of the University of Minnesota, performed in
accordance with the Animal Use Guidelines of the University of Minnesota, and
consistent with the National Institutes of Health Guide for the Care and Use of
Laboratory Animals (NIH publication No 85-23). Twelve-week-old immunodeficient
NOD-scid/γc–/– mice (Jackson Laboratory) were anesthetized with an intraperitoneal
injection of sodium pentobarbital (35 mg/kg), intubated, and ventilated with a small
animal respirator (Harvard Apparatus); then, a left thoracotomy was performed to expose
the heart, and the left-anterior descending coronary artery was permanently ligated with
an 8.0 surgical silk suture. Fifteen minutes after ligation, animals in the MI+hCMP group
were treated with two hCMPs, and animals in the MI+Scaffold group were treated with
two 3D-MPE–printed scaffolds that lacked any cells. Both treatments were applied to the
epicardial surface over the infarcted area—the hCMP was oriented approximately
parallel to the alignment of the surface myocardium and held in place by covering them
with a piece of decellularized bovine pericardium that was sutured to the heart to avoid
movement of hCMPs off the heart and prevent adhesions between the heart and the rib
cage. Both the hCMP and the cell-free scaffold were withheld from animals in the MI
group, and animals in the Sham group underwent all surgical procedures for MI induction
except the ligation step and received neither of the experimental treatments. The chest
was closed in layers and the animals were allowed to recover.
Echocardiographic assessments of cardiac function
Echocardiographic measurements were obtained via the method recommended by the
8
CIRCRES/2016/310277 R2
American Society of Echocardiography and performed on a Vevo770 Imaging System
equipped with an RMV 707B transducer (15-45MH; VisualSonics Inc, Canada); both
conventional 2-dimensional images and M-Mode images of the heart in a parasternal
short axis view were acquired. Left ventricular (LV) internal diameters at end-diastole
(LVIDed) and end-systole (LVIDes) were determined from 8 consecutive images, and LV
ejection fractions (EF) and fractional shortening (FS) were calculated according to the
following equations:
EF=(LVIDed3−LVIDes3)/LVIDed3×100%；FS=(LVIDed−LVIDes)/LVIDed×100%.
Immunohistochemical evaluations
The hCMPs were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.2%
Triton X-100 for 15 min, and then blocked with 5% donkey serum for 30 min. Primary
antibodies (rabbit anti-cTnI [Abcam, USA], mouse anti-cTnT [Thermo Scientific, USA],
mouse anti-SMA [Sigma-Aldrich, USA], and goat anti-CD31 [Santa Cruz, USA]) were
diluted in the blocking solution, and the hCMPs were incubated at 4°C; on the following
day, the hCMPs were incubated with secondary antibodies for 1 h and then stained with
DAPI. The alignment of cells in the hCMP channels was evaluated via multiphoton
microscopy. ImageJ software was used to fit an ellipse to the outline of each cell, which
was visible because of cellular autofluorescence, and the ellipse was used to determine
the cell’s aspect ratio and angle relative to horizontal; then, the angle of orientation for
each cell was normalized to the average longitudinal angle of the channel walls.
Murine hearts were cut into halves from the middle of the infarct. One of the halves was
frozen at the optimal cutting temperature for cryo-sectioning, and the other half was
stored in 10% formalin for paraffin-embedded sectioning. Embedded tissues were cut
into 7-μm sections; then, the sections were fixed with 4% paraformaldehyde for 20 min at
room temperature, permeabilized in 0.1% Triton X-100 at 4ºC for 10 min, and blocked
9
CIRCRES/2016/310277 R2
with UltraV block (Thermo Scientific, USA) for 7 min. Primary antibodies (mouse
anti-human specifc nuclear antigen, HNA [Emdmillipore, USA], goat anti-GFP [Abcam,
USA], goat anti-CD31 [Santa Cruz, USA], mouse anti-human specific CD31 [hCD31,
Dako, USA], mouse anti-SMA [Sigma-Aldrich, USA], rabbit anti-cTnI [Abcam, USA],
mouse anti-cTnT [Abcam, USA], rabbit anti-Ki67 [Abcam, USA]) were added to the
UltraV block buffer, and the sections were incubated at 4ºC. On the following day,
secondary antibodies conjugated with fluorescent markers (Jackson ImmunoResearch
Lab, USA) were added; then, the sections were incubated for 1 h at room temperature,
stained with DAPI, washed, and examined under a confocal microscope (Zeiss 710,
USA).
Engrafted hciPSC-cardiovascular cells were identified by the expression of HNA or GFP,
engrafted hciPSC-CMs were identified by the expression of both GFP and cTnI, or both
HNA and cTnI, engrafted hiPSC-ECs were identified by hCD31 expression, and
engrafted hiPSC-SMCs were identified by the expression of both GFP and SMA.
Vascular density was evaluated by counting the number of vascular structures that were
positive for either CD31 or SMA expression; then, the images used for the CD31 and
SMA evaluations were superimposed, and arteriole density was evaluated by counting
the number of structures that expressed both CD31 and SMA. Cells and vessels were
counted in 5 fields per section, 5 sections per animal.
Engraftment rate assessment
Engraftment rates were evaluated via quantitative PCR (qPCR) assessments of the
human Y-chromosome as previous reported.14 Briefly, whole hearts of female mice were
collected and digested overnight at 56 oC with proteinase K; then, the total DNA was
isolated from the digested buffer with a QIAGEN DNA isolation kit. The number of cells in
each mouse heart was determined by comparing the number of cycles required for each
10
CIRCRES/2016/310277 R2
sample to a standard curve calculated from the DNA of known quantities of
undifferentiated hciPSCs, and the engraftment rate was calculated as the number of cells
in each animal divided by the number of cells administered (1x105). Analyses were
performed with the SYBR Green kit (Thermo Scientific, USA) on an Eppendorf Realplex2
PCR
system
(Eppendorf,
USA)
with
the
following
primers:
sense,
ATCAGCCTAGCCTGTCTT-CAGCAA; anti-sense, TTCACGACCAACAGCACAGCAATG.
To assess the engraftment rate by counting the grafted human cells as evidenced by HNA
positivity, we also used histologic assessment of consecutives sections of the heart that
spans the entire area covered by the engrafted patch, and counting the grafted human
cells as evidenced by HNA positivity.
5, 15
Hearts were frozen or paraffin-embedded and
transversely cut into 7-μm sections from base to apex. Total cell nuclei were stained with
4,6-diamidino-2-phenylindole (DAPI). Engrafted hciPSCs-derived cardiovascular cells
were identified by counting the human specific nuclear antigen (HNA)-positive cells in
every 15th serial section of the heart and then multiplying by 15 to obtain the total number
of engrafted hciPSC-cardiovascular cells per heart. The engraftment rate of transplanted
hciPSC-cardiovascular cells was calculated by dividing the total number of engrafted cells
by the number of cells administered (1x105) and multiplying by 100.
Infarct size and thickness measurements
Hearts were frozen or paraffin-embedded and cut into 7-μm sections; then, the sections
were stained with an Accustain Trichrome Stains (Masson) kit (Sigma-Aldrich, USA). The
infarct size was measured from Masson’s trichrome-stained sections as the percentage
of the surface area of the left ventricular anterior wall occupied by scar, and infarct
thickness was calculated as the ratio of the thicknesses of the fibrous region to the
thickness of the septal wall. For each heart, the infarct size was averaged from three
areas of the heart: just below the ligation suture, midway between the ligation suture and
11
CIRCRES/2016/310277 R2
the apex, and a section close to the apex.
Apoptosis
Sections cut from embedded hearts were TUNEL stained with an In-situ Cell Death
Detection Kit (Roche Applied Science, Germany) and viewed at 40x magnification as
previously reported.5 The number of TUNEL-positive cells and the total number of cells
were counted in 5 to 6 slides per animal and 5 fields per slide.
Statistical Analyses
Data are presented as mean ± standard error. Statistical significance (P<0.05) between
two experimental groups was determined via the Student’s two-tailed t-test, assuming
unequal variance. Comparisons among more than two experimental groups were
evaluated via one-way ANOVA followed by Tukey post-hoc analysis. All statistical
analyses were performed with SPSS software (version 13.0, SPSS, USA).
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to endothelial progenitor cells. Stem Cell Res. 2015;15:122-9.
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Xenotransplantation
of
Long
Term
Cultured
Mesenchymal Stem Cells. Stem Cells. 2006.
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Swine
Bone
Marrow-Derived
CIRCRES/2016/310277 R2
ONLINE FIGURE LEGENDS
Online
Figure
I.
Immunofluorescent
analysis
of
lineage-specific
marker
expression in differentiated hiPSC-derived cardiac cells. The lineage of the
differentiated hciPSC-CMs was confirmed via the expression of cardiac troponin I (cTnI),
cardiac troponin T (cTnT), ventricular myosin light chain 2 (MLC-2v), α-sarcomeric actin
(αSA), and the gap-junction protein connexin-43 (Con-43); the lineage of the
differentiated hciPSC-SMCs was confirmed via the expression of -smooth muscle actin
(SMA), SM22, and calponin; and the lineage of the differentiated hciPSC-ECs was
confirmed via the expression of CD31, CD144, and von Villebrand factor 8 (vWF-8).
Nuclei were counterstained with DAPI (scale bar = 50μm).
Online Figure II. Expression of contractile- and calcium-transient–related genes in
hCMPs and in monolayers of hiPSC-derived cardiac cells. Quantitative PCR
measurements of the expression of genes involved in (A) contractile activity (cTnT, cTnI,
atrial myosin light chain 2 [MLC2a], MLC2v, α myosin heavy chain [αMHC], βMHC), and
(B) generating calcium transients (sarcoplasmic reticulum Ca2+-ATPase 2SERCA2,
phospholamban [PLB], sodium/calcium exchanger 1 [NCX1], ryanodine receptor 2
[RYR2], calsequestrin 2 [CASQ2]) were performed in hCMPs and in monolayers
composed of equivalent populations of cells on day 1 and day 7 after manufacture.
Results were normalized to measurements obtained in monolayers on day 1. *P<0.05,
**P<0.01; n=3-4 hCMPs or monolayers per time point with 3 replicates per
measurement.
Online Figure III. hCMP transplantation reduces apoptosis and increases
angiogenesis after MI. (A) Apoptotic cells were identified in sections from the
border-zone of ischemia in the hearts of animals from the MI (top row), MI+Scaffold
(middle row), and MI+hCMP (bottom row) groups via TUNEL staining; CMs were
16
CIRCRES/2016/310277 R2
visualized via immunofluorescent staining for the presence of cTnI and nuclei were
counterstained with DAPI. (B) Apoptosis was quantified as the percentage of cells that
were positive for TUNEL staining. (C) Sections from the border-zone of ischemia in the
hearts of MI (top row), MI+Scaffold (middle row), and MI+hCMP (bottom row) animals
were immunofluorescently stained for the presence of the CD31, SMA, and cTnI, and
nuclei were counterstained with DAPI; then (D) vascular density was determined by
quantifying the expression of CD31, and (E) arteriole density was determined by
quantifying the co-expression of CD31 and SMA. **P<0.01, n = 5-6 in each group;
scale bar = 50 m.
Online Figure IV. hCMP transplantation promotes cell proliferation after MI. (A)
Proliferating cells were identified in sections from the border-zone of infarct in hearts on
day 28 after MI by staining for expression of the proliferation marker Ki67 (green). (B)
Proliferation was quantified as the number of Ki67+ cells per high-power field (HPF).
*P<0.05, **P<0.01, n=5-6 in each group. Scale bar = 100m.
17
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ONLINE VIDEO LEGENDS
Online Video I. MPE-3DP to fabricate adult simulate grid layer by layer. The video
shows layer-by-layer fabrication of a scaffold of dimensions 400 x 400 x 100 microns. This
is the same structure as was used for the in vitro experiments. For in vivo studies,
structures like the one shown here were tiled together to generate millimeter scale
structures. Fabrication of each layer required approximately 10 seconds. The fluorescent
contrast arises from the two-photon excitation of Rose Bengal entrapped in the matrix.
Online Video II. 360 view of 3D rendering of adult simulate grid. The video shows a
3D projection of the series of fabricated layers shown in the S1 video, with a 360 view of
the fabricated structure.
Online Video III. Animation depicting the MPE-3DP process. First, a solution
containing the polymer or protein of interest and associate photocrosslinking agent is
added to the reservoir. The reservoir sits upon a slide on the stage of a multiphoton
microscopy rig. The pulsed laser is actuated and raster scanned across the x-y plane of a
given field of view. The dwell time of the laser at a given focal volume is dictated by the
intensity of the digital template at a given x-y position. The longer the dwell time, the more
crosslinking occurs and therefore a more stable structural feature is formed. The scan
time is on the scale of microseconds for each focal volume, milliseconds for each x-y line
and seconds for the entire raster (i.e., x-y plane). The animation shows a significantly
slowed visual depiction of proteins of focal volumes undergoing polymerization on three
different z planes. Not shown in the video is the digital template, which dictates the final
form of the structure. As noted at the end of the animation, the final structure can be
manipulated for use in vitro and in vivo.
Online Video IV. Spontaneous and synchronous contractile activity in a hCMP on
18
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day 1 after fabrication (magnification: 100 x).
Online Video V. Spontaneous and synchronous contractile activity in a hCMP on
day 3 after fabrication (magnification: 100 x).
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Online Table I. Primers for quantitative RT-PCR
Standard
Gene
Gene product Forward
name
Reverse
Size
bp
GAPDH
GAPDH
TCGACAGTCAGCCGCATCTTCTTT ACCAAATCCGTTGACTCCGACCTT
94
TNNI3
cTNI
CCTCACTGACCCTCCAAACG
104
TNNT2
cTNT
TTCACCAAAGATCTGCTCCTCGCT TTATTACTGGTGTGGAGTGGGTGTGG 165
MYH6
ɑMHC
CTCCGTGAAGGGATAACCAGG
TTCACAGTCACCGTCTTCCC
248
MYH7
ßMHC
ACCAACCTGTCCAAGTTCCG
TCATTCAAGCCCTTCGTGCC
131
MYL2
MLC 2v
ACATCATCACCCACGGAGAAGAGA ATTGGAACATGGCCTCTGGATGGA
164
MYL7
MLC 2a
GGAGTTCAAAGAAGCCTTCAGC
AAAGAGCGTGAGGAAGACGG
178
ACTN2
-actinin 2
CTTCTACCACGCTTTTGCGG
CCATTCCAAAAGCTCACTCGC
136
ATP2A2
SERCA 2
TCACCTGTGAGAATTGACTGG
AGAAAGAGTGTGCAGCGGAT
149
CASQ2
calsequestrin 2 GTTGCCCGGGACAATACTGA
CTGTGACATTCACCACCCCA
142
PLN
Phospholamban ACAGCTGCCAAGGCTACCTA
GCTTTTGACGTGCTTGTTGA
191
SLC8A1
NCX 1
CTGGAATTCGAGCTCTCCAC
ACATCTGGAGCTCGAGGAAA
108
RYR2
RYR 2
TTGGAAGTGGACTCCAAGAAA
CGAAGACGAGATCCAGTTCC
141
20
GAGGTTCCCTAGCCGCATC
Online Figure I
hiPSC-CMs
hiPSC-SMCs
hiPSC-ECs
cTnI DAPI
aSMA DAPI
CD31 DAPI
cTnT MLC-2v DAPI
SM22 DAPI
CD144 DAPI
Con-43 aSA DAPI
Calponin DAPI
vWF-8 DAPI
Online Figure II
A
**
*
4
3
*
*
Monolayer, day 1
hCMP, day 1
Monolayer, day 7
hCMP, day 7
*
*
2
1
0
cTnT
cTnI
MLC2a
MLC2v
aMHC
aActinin2
**
**
B
4
bMHC
*
*
10
3
*
*
2
8
6
4
1
2
SERCA2a
PLB
NCX1
RYR2
0
CASQ2
Monolayer, day 1
hCMP, day 1
Monolayer, day 7
hCMP, day 7
Online Figure III
A
B
DAPI cTnI
MI
TUNEL cTnI
DAPI cTnI
MI
MI+Scaffold
MI+hCMP
TUNEL DAPI cTnI
TUNEL DAPI cTnI
Percentage
(TUNEL+ cells /Total cells)
TUNEL cTnI
**
48
**
36
24
12
0
MI+Scaffold
TUNEL cTnI
DAPI cTnI
TUNEL DAPI cTnI
MI+hCMP
C
CD31
B
aSMA
CD31 aSMA DAPI cTnI
Vascular density (mm-2)
D
**
1600
MI
MI+Scaffold
MI+hCMP
**
1200
MI
CD31
aSMA
CD31 aSMA DAPI cTnI
800
400
0
MI+Scaffold
CD31
MI+hCMP
aSMA
CD31 aSMA DAPI cTnI
Arteriole density (mm-2)
E
**
600
450
300
150
0
MI
MI+Scaffold
MI+hCMP
**
Online Figure IV
A
Ki67 DAPI
Ki67 DAPI
MI
MI+Scaffold
B
MI
MI+Scaffold
MI+hCMP
**
Ki67+ cells /HPF
20
15
10
5
0
*
Ki67 DAPI
MI+hCMP