(Guyette Et Al., 2015) Bioengineering Human Myocardium On Native Extracellular Matrix PDF

Bioengineering Human Myocardium on Native Extracellular Matrix
Jacques P. Guyette,1,2, Jonathan M Charest1, Robert W Mills3, Bernhard J. Jank1,2, Philipp T. Moser1,2,
Sarah E. Gilpin1,2, Joshua R. Gershlak4, Tatsuya Okamoto1, Gabriel Gonzalez1,2, David J. Milan3,5, Glenn
R. Gaudette4, Harald C. Ott1,2,6,7
1
Center for Regenerative Medicine, Massachusetts General Hospital; 2Harvard Medical School;
3
Cardiovascular Research Center, Massachusetts General Hospital; 4Department of Biomedical
Engineering, Worcester Polytechnic Institute; 5Division of Cardiology, Massachusetts General Hospital;
6
Division of Thoracic Surgery, Department of Surgery, Massachusetts General Hospital, and;7Harvard
Stem Cell Institute.
Running title: Human Myocardium on Native Extracellular Matrix
Subject Terms:
Cell Biology/Structural Biology
Contractile Function
Address correspondence to:

Dr. Harald C. Ott
Assistant Professor in Surgery
Harvard Medical School
Division of Thoracic Surgery
Department of Surgery
Massachusetts General Hospital
55 Fruit Street, Blake 15
Boston, MA 02114
hott@partners.org
In September 2015, the average time from submission to first decision for all original research papers
submitted to Circulation Research was 12.75 days.
DOI: 10.1161/CIRCRESAHA.115.306874 1
Downloaded from http://circres.ahajournals.org/ at CENTRAL MICHIGAN UNIVERSITY on October 27, 2015

ABSTRACT
Rationale: More than 25 million individuals suffer from heart failure worldwide, with nearly 4,000 patients
currently awaiting heart transplantation in the United States. Donor organ shortage and allograft rejection
remain major limitations with only about 2,500 hearts transplanted each year. As a theoretical alternative
to allotransplantation, patient-derived bioartificial myocardium could provide functional support and
ultimately impact the treatment of heart failure.
Objective: The objective of this study is to translate previous work to human scale and clinically relevant
cells, for the bioengineering of functional myocardial tissue based on the combination of human cardiac
matrix and human iPS-derived cardiac myocytes.
Methods and Results: To provide a clinically relevant tissue scaffold, we translated perfusion-
decellularization to human scale and obtained biocompatible human acellular cardiac scaffolds with
preserved extracellular matrix composition, architecture, and perfusable coronary vasculature. We then
repopulated this native human cardiac matrix with cardiac myocytes derived from non-transgenic human
induced pluripotent stem cells (iPSCs) and generated tissues of increasing three-dimensional complexity.
We maintained such cardiac tissue constructs in culture for 120 days to demonstrate definitive sarcomeric
structure, cell and matrix deformation, contractile force, and electrical conduction. To show that functional
myocardial tissue of human scale can be built on this platform, we then partially recellularized human whole
heart scaffolds with human iPSC-derived cardiac myocytes. Under biomimetic culture, the seeded
constructs developed force-generating human myocardial tissue, showed electrical conductivity, left
ventricular pressure development, and metabolic function.
Conclusions: Native cardiac extracellular matrix scaffolds maintain matrix components and structure to
support the seeding and engraftment of human iPS-derived cardiac myocytes, and enable the bioengineering
of functional human myocardial-like tissue of multiple complexities.
Keywords:
Pressure-controlled perfusion decellularization, iPS-derived cardiac myocytes, human myocardial tissue,
extracellular matrix, cardiac regeneration, stem cell, cardiac differentiation
Nonstandard Abbreviations and Acronyms:
ECM – extracellular matrix MFI – mean fluorescence intensity

DCD – donation after cardiac death HDM – high density mapping
DBD – donation after brain death TTRX – time to X% relaxation
iPSC – induced pluripotent stem cell µCT – micro-computed tomography
BJ-RiPS – BJ fibroblast RNA-induced pluripotent stem
SDS – sodium dodecyl sulfate
(cells)
µOCT – micro-optical coherence tomography HLA – human leukocyte antigen
APD80 – action potential duration at 80%
DOI: 10.1161/CIRCRESAHA.115.306874 2

INTRODUCTION
More than 5 million people in the United States live with heart failure (HF), with more than 800,000
new cases diagnosed each year1. Patients suffer from poor quality of life, and require frequent interventions,
leading to greater than 1 million hospital discharges and a cost of more than $39 billion per year2. While
artificial mechanical support can be offered to a small group of patients, heart transplantation remains the
only curative treatment option for end-stage HF. Donor organ shortage is its major limitation, as nearly
1,500 heart transplant candidates will endure extended waitlist periods while facing 5-year mortality rates
of approximately 50%2, 3. While heart transplant recipients benefit from significantly improved short-term
survival rates, long-term survival remains limited due to chronic rejection and adverse effects from life-
long immunosuppression4. The engineering of bioartificial myocardium, and ultimately of bioartificial
hearts, from patient-derived cells could theoretically solve both of these problems. While many hurdles
remain, the creation of bioartificial functional human myocardium from adult-derived, genetically non-
altered cells is the first vertical step towards this goal, and can provide new pathways for repair and
replacement of myocardium in HF patients.
The onset of HF is preceded by myocardial necrosis, fibrosis, and extracellular matrix (ECM)
remodeling, resulting in progressive loss of architecture and contractile function1, 2, 5. Partial reverse
remodeling and myocardial augmentation has been accomplished by direct delivery of contractile cardiac
myocytes and passive biomaterials6-9. Following similar principles, different scaffold materials and cells
have been combined to generate tissue-engineered myocardium. In both approaches contractile graft
thickness is limited due to poor diffusion and neovascularization that fails to meet the demand of cardiac
myocyte metabolism10-12. Graft architecture and electromechanical integration are constrained by non-
physiologic scaffold properties, and the altered properties of scarred myocardium. Successful clinical
application of myocardial regeneration will depend on the ability to establish physiologic tissue
architecture, and immediate graft perfusion upon implantation. Native human heart matrix provides the
physiologic micro and macro architecture, ECM composition, and the perfusable hierarchical vascular bed,
serving as a foundational scaffold to engineer bioartificial myocardium13, 14.
By translating our previous work to human scale15, we demonstrate successful application of whole-
organ perfusion decellularization to human hearts, and generate acellular cardiac scaffolds with preserved
ECM material properties, structure, patent coronary conduits, with an immunologic profile that induces a
constructive remodeling response. By comparing brain death and donation after cardiac death donors, we
validate this new regenerative platform from more than 5,500 currently unused donor hearts per year3. By
deriving cardiac myocytes from non-transgenic adult derived iPSC at clinical scale, we provide the
necessary building blocks for clinically relevant myocardial regeneration. By repopulating native human
heart matrix in sections, fibers, and biomimetic whole organ culture, we show the potential for creating
functional human myocardial-like tissue from an autologous source to augment or replace lost myocardial
function, and serve as a patient-specific development platform.
METHODS
All methods and associated references are included in the online data supplement.
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RESULTS
Perfusion decellularization of human cadaveric hearts.
In a first set of experiments, we scaled perfusion decellularization to donated human hearts that were
recovered by the New England Organ Bank and deemed unusable for clinical transplantation. Between
2012 and 2015, 73human non-transplantable hearts were recovered for research after specific consent was
obtained. Donor records (n = 73; 5 patient records unavailable) indicate a distribution of 36 males versus
32 females, 29 cardiac death donors (DCD) versus 39 brain death donors (DBD), an average age of 52.43
± 14.29 years, an average height of 170.96 ± 9.53 cm, an average weight of 81.54 ± 20.53 kg, and an
average body mass index of 27.76 ± 6.09 kg/m2. Heart identification numbers, corresponding donor data,
and associated experimental tests are outlined in Supplemental Table I. We applied aseptic pressure-
controlled antegrade coronary flow to 40 hearts in a custom organ chamber (60 mm Hg, average cold
ischemia time of 455 ± 184 minutes; Supplemental Fig. I). Hearts were perfusion-decellularized with 1%
SDS (168 hrs), deionized H2O (24 hrs), 1% Triton-X100 (24 hrs), and PBS washes (168 hrs) following a
standardized protocol (Fig. 1A)16. Decellularized hearts contained 346.17 ± 27.92 ng/mg wet tissue of
residual double-stranded DNA (dsDNA), which could be removed by treatment with endonuclease (Fig.
1B). Subsequent perfusion of decellularized whole-hearts with 25 U/mL of endonuclease at room
temperature for 24 hrs demonstrated a 99.05% reduction in dsDNA (3.27 ± 3.12 ng/mg wet tissue, p <
0.05), with thresholds meeting established criteria for decellularized biomaterials17. Biochemical analysis
of perfusion-decellularized hearts from both DCD and DBD donors indicated a high retention of insoluble
collagen, moderate decrease of sulfated-glycosaminoglycans, and lower concentrations of α-elastin and
soluble collagen (Fig. 1C, Supplemental Fig. II). Importantly, we did not detect measureable amounts of
residual SDS in decellularized hearts (< 0.02% w/v extract; below detection limit)18.
Proteomics analysis indicated an 89.14% reduction of the cadaveric cardiac proteome (967 proteins),
with the identification of 105 unique proteins in decellularized hearts (Fig. 1D). Matrisome proteins were
identified by querying our cadaveric and decellularized lists of unique proteins with the UniProt database
for the subcellular location search terms “extracellular matrix” and “basement membrane”, exclusively and
combined, as defined cellular component terms by the Gene Ontology Consortium19, 20. Acellular heart
scaffolds showed a marked retention of 36 matrisome proteins following the perfusion decellularization
process (Fig. 1D). Normalized relative abundances of uniquely identified matrisomal proteins in both
cadaveric and decellularized human myocardium were further categorized into ECM-protein families (Fig.
1E). The four largest ECM-protein families are conserved across cadaveric and decellularized cardiac
matrix (collagens, laminins, fibrillins, and proteoglycans), with the largest relative contributions to the
decellularized scaffolds being fibrillin-1, collagen IV, heparin sulfate, and laminin gamma-1. Whole lists
of unique proteins for both cadaveric and decellularized myocardium are provided (Supplemental Tables
II, III). Histological evaluation confirmed the retention of collagens (I, III, and IV), laminin, and fibronectin
within acellular human heart scaffolds (Fig. 1F, Supplemental Fig. III). Cardiac matrix fiber composition
and architecture were maintained, showing vacant spacing between fibers with a loss of nuclei and the
cardiac myocyte marker myosin heavy chain. Insoluble adipose tissue matrix and lipid molecules remain
on the epicardial surface after decellularization (Fig. 1A), but further histological analysis confirmed the
absence of cells and preservation of matrix structure (Supplemental Fig. IV). In evaluation of how the
decellularization process affects the material properties of the cardiac matrix scaffold, equibiaxial
mechanical testing of transmural samples displayed similar moduli between cadaveric and decellularized
samples, along both the base/apex axis (376.3 ± 191.7 vs. 370.8 ± 150.9 kPa) and the septum/free-wall axis
(581.4 ± 325.5 vs. 451.4 ± 257.8 kPa; Fig. 1G). Anisotropic elastic behavior was measured by an
“anisotropy ratio” comparing the difference and the sum of the two orthogonal moduli within a tissue
sample. A value of zero indicates perfect biaxial isotropy while a value approaching one indicates a high
degree of anisotropy. Cadaveric and decellularized samples exhibited measurable anisotropic ratios (0.314
DOI: 10.1161/CIRCRESAHA.115.306874 4

± 0.196 and 0.167 ± 0.158; Fig. 1H) that were statistically different from zero (p = 0.0027 and p = 0.0086),
but not statistically different from each other (p = 0.0984).
Immunogenic profile of decellularized human cardiac matrix.
Taking a critical step towards the potential use of our human cardiac scaffolds in clinical applications,
we assessed the immunogenic profile of perfusion-decellularized myocardium after subcutaneous
implantation in Sprague-Dawley rats for two weeks. Comparisons were made versus cadaveric human
myocardium, as well as decellularized porcine myocardium that was perfusion-decellularized in the same
manner as donated human hearts described above16. Histological evaluation of explanted material by
immunofluorescence showed a considerable number of CD68+ mononuclear cells in all three groups. There
appeared to be a more discernable M1-macrophage proinflammatory response by cadaveric human and
decellularized porcine myocardium, while decellularized human myocardium presented with a more
definitive M2-macrophage immunomodulatory response (Fig. 2A). A CellProfiler image processing
pipeline and CellProfiler Analyst were used to quantify macrophage phenotype across images
(Supplemental Fig. V)21, 22. From all cells counted in each high-power field (number of fields/group: 28 ±
1; number of cells/field: 1,152.5 ± 294.2), cadaveric human tissue contained 70.49 ± 1.72% CD68+ cells,
while decellularized human matrix contained 79.63 ± 3.22%, and decellularized porcine matrix contained
66.44 ± 6.17% CD68+ cells (Fig. 2B). Co-staining with CD80 demonstrated the presence of M1
macrophage phenotype in cadaveric human tissue (63.13 ± 9.79%), decellularized human matrix (52.11 ±
7.05%), and decellularized porcine matrix (67.73 ± 3.01%) from all cells counted per field. Co-staining for
CD163 on adjacent sections revealed that cadaveric tissue implants elicited only 38.30 ± 7.46% M2+ cells,
while decellularized human matrix implants elicited 69.54 ± 8.44% and decellularized porcine matrix
elicited 53.52 ± 6.80% M2+ immunomodulatory cells (p < 0.01). A CD163/M2+ macrophage response has
previously been shown as an indication of reconstructive remodeling described in decellularized matrices23.
During tissue explantation at the 2-week time point, blood samples were also collected to analyze whole
blood and serum. Whole blood analysis did not indicate a statistical difference between any groups with
respect to total white blood cell components (Supplemental Fig. VI). Sera were also tested against human
leukocyte antigen (HLA) single antigen beads, where experimental groups were normalized to the sham
group, and antibody reactivity to beads with specific HLA alleles was determined by mean fluorescence
intensity (MFI) using HLA Fusion software (version 3.0; One Lambda). Implanted decellularized human
matrix showed reactivity to only 2 HLA beads, yielding values of <100 MFI that are well below the positive
cut-off value of 500 MFI (Fig. 2C). Implanted cadaveric myocardium showed reactivity to >50 HLA beads,
yielding values that ranged between 5000 and 7900 MFI, and showing high reactivity against patient-
specific HLA types attributed to the implanted cadaveric myocardium (Fig. 2C). The single antigen bead
assay confirmed our qualitative assessment (by immunofluorescence) that the decellularization process
removes HLA from the cardiac matrix as cells are removed (Fig. 2D).
Decellularized coronary vasculature.
In assessment of acellular coronary vasculature, DCD hearts had slightly lower coronary flow (Fig. 3A)
and higher vascular resistance (Fig. 3B) compared to DBD hearts during detergent perfusion, but achieved
similar flow and resistance by the end PBS washes. This initial difference did not reach statistical
significance. Histological evaluation by pentachrome stain showed decellularized vasculature retained
structural blood vessel features, with preservation of the intima, media, adventitia, and elastic laminas (Fig.
3C). Scanning electron microscopy further confirmed that the decellularization process removed all
endothelial cells, without evidence of basement membrane disruption (Fig. 3D,E). Cardiac computed
tomography scans revealed conservation of the conductance arteries of the coronary tree, showing structure
and patent lumens stemming from the left and right main coronaries (Fig. 3F). Angiography of acellular
hearts from patients presenting with coronary artery disease confirmed the preservation of perfusable
coronary vessels, but revealed that the decellularization process does not resolve luminal narrowing related
DOI: 10.1161/CIRCRESAHA.115.306874 5

to atherosclerotic plaques (Supplemental Fig. VII). To visualize perfusion of microvasculature within

decellularized hearts, we cannulated and directly perfused epicardial conductance arteries with resuspended
0.2 µm-diameter fluorescent microspheres, and tracked microsphere movement through mid-myocardial
and endocardial regions using micro-optical coherence tomography (µOCT). Using a Z-projection tool to
generate a running average from the acquired video sequences, we confirmed bead-flow through distinct
pathways or microvascular channels (Supplemental Video I).
To evaluate the preservation of microvasculature, we perfused cadaveric human hearts and

decellularized human hearts with radio-opaque silicone, and performed a systematic comparison of full-
thickness left ventricular samples by micro-computed tomography (µCT). Reconstructions of high-
resolution µCT images were made on full-thickness samples from the epicardial surface to the
endocardium, revealing that the decellularized cardiac matrix retains an intact vascular hierarchy, with
branching extensions with diameters on the order of ~10 µm (Fig. 3G). Analyzing two-dimensional µCT
images from the epi-, mid-, and endocardial regions for the number of vessels normalized to tissue area
found similar vessel densities and trends between the cadaveric and decellularized hearts (Fig. 3H). In the
subepicardial region, no statistical difference in vascular density was found between cadaveric myocardium
(15.94 ± 3.03 vessels/mm2) and decellularized matrix (13.44 ± 1.38). Likewise, both mid-myocardial and
subendocardial regions showed no statistical difference in vascular densities between cadaveric
myocardium (25.93 ± 0.40 and 20.64 ± 3.52, respectively) and decellularized matrix (22.44 ± 2.04 and
18.30 ± 3.6, respectively). Further analysis of the µCT images was done to measure the minor axis of vessel
cross-sections as an indication of vessel diameter, which confirmed similar vessel-diameter distribution
between the cadaveric and decellularized samples in all three myocardial sub-regions (Fig. 3I,J).
In vitro cardiac differentiation of BJ RiPS.
To examine the translational value of our approach, we sought to generate a clinically relevant number
of human cardiac myocytes from a non-transgenic cell source. We therefore obtained and expanded human
BJ fibroblast RNA-induced pluripotent stem cells (BJ RiPS, clone 1.1)24. To induce cardiac differentiation,
we applied temporal modulation of canonical Wnt/β-catenin signaling to BJ RiPS cultured in monolayer25.
We then mapped gene expression profiles of 15 markers by RT-PCR at different developmental stages
(days 0, 3, 5, 7, 14, 30, 60, 120, and 180) to monitor and define the profile of BJ RiPS through stages of
cardiac differentiation and maintenance, and to define checkpoints for quality control required at a clinically
relevant scale (Fig. 4A). Prior to the onset of differentiation at day 0, BJ RiPS exhibited definitive
expression of classic pluripotency genes OCT4, SOX2, and NANOG. Treatment with Gsk3 inhibitor
CHIR99021 for 24 hours markedly decreased OCT4, SOX2, and NANOG expression by day 3, while
expression of genes typically found in mesendoderm and mesodermal tissues were elicited (MIXL1, T,
ISL1, and GATA4). Starting on day 3, 48-hour treatment with Wnt inhibitor IWP4 decreased MIXL1, T,
and ISL1 by day 5, while essential early-stage cardiac genes emerged (GATA4, NKX2.5, MEF2C, and
TBX5). By day 7 through day 14 in cardiac maintenance media, definitive late-stage cardiac genes became
expressed (MYH6, MYH7, MYL2, and TNNT2), as large areas of differentiated cardiac myocytes began
spontaneously contracting by day 14 (Supplemental Video II). Early- and late-stage cardiac markers tended
to increase by day 30, and expression persisted through days 60, 120, and 180 post-differentiation. By day
180, most cardiac genes appeared to taper, which is consistent with observations of decreased contractility
within culture plates. In conjunction with increased cardiac gene expression, BJ RiPS-derived cardiac
myocytes isolated between days 30 and 60 post-differentiation showed positive cardiac protein expression
and structure for sarcomeric α-actinin, cardiac troponin T, and myosin heavy chain by immunofluorescence
(Fig. 4B). Cardiac protein expression in day 30-60 cardiac myocytes was further confirmed by flow
cytometry, yielding high populations positive for sarcomeric α-actinin and cardiac troponin T (Fig. 4C),
achieving differentiation efficiencies of >85%. As day 30-60 cardiac myocytes appeared to be most robust
for cardiac gene and protein expression, cells within this time frame were enzymatically dispersed and
isolated for seeding experiments.
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Recellularization of myocardial slices and fibers.
To test whether perfusion-decellularized human cardiac matrix can support engraftment of cardiac
myocytes and enable myocardial tissue formation, we reseeded matrix sections (200 µm) with human iPS-
derived cardiac myocytes in non-perfused two-dimensional culture (Fig. 5A). Acellular human heart matrix
(Fig. 5Ai) supported the attachment, viability, and function of human cardiac myocytes (Fig. 5Aii) by
evidence of spontaneously contracting slices within 4-7 days of culture. Serving as an early milestone for
creating force-generating cardiac tissue, functional myocardial slices were robust and could be maintained
for 120 days in culture. To characterize the mechanical function, we first used high spatial-resolution
imaging (high density mapping, HDM)26 to determine the area deformation of small defined regions of
interest (Supplemental Fig. VIII). By independently analyzing matrix-seeded cardiac myocytes, as well as
the underlying cardiac ECM within the same region (20 µm below the slice surface), we confirmed stable
cell-matrix interaction and quantified the degree to which cell contractility translated to matrix deformation.
In spontaneously contracting slices, cardiac myocytes achieved 0.59 ± 0.26% area strain at a natural
frequency of 1.36 ± 0.32 Hz, which translated to 0.47 ± 0.17% area strain of the ECM with an average
natural frequency of 1.38 ± 0.43 Hz (Fig. 5B). Functional myocardial slices could be electrically paced at
1 Hz or 2 Hz, and data for both cells and matrix could be captured at consistent dominant frequencies (Fig.
5B). Area strain of myocardial matrix could be significantly increased to 1.55 ± 0.55% when paced at 1 Hz,
and decreased to 1.02 ± 0.31% when paced at 2 Hz. The technique to measure in vitro area strain is
concurrent with the in vivo regional cardiac functional metric of systolic area contraction27. Regenerated
human myocardial slices achieved strain below normal adult myocardium (~20%), however, the integration
of human cardiac myocytes with human heart matrix formed mechanically active tissue on the same order
of magnitude as compliant ECM cardiac patches tested in vivo (~4%)27.
Moving to a more physiologic three-dimensional model, we reseeded non-perfused cardiac fibers (15
mm length, 2.5 mm diameter) cut from the left ventricular free wall of decellularized cardiac scaffolds (Fig.
5C). Reseeded cardiac fibers supported the attachment, viability, and function of human cardiac myocytes
by evidence of spontaneously contracting tissue within 7-10 days, which could be maintained for 60+ days
in culture (Supplemental Video III). Using micro-optical coherence tomography (µOCT) that allows for
imaging contractility 250 µm within cardiac fibers with high spatial and temporal resolution (Fig 5C,
Supplemental Video IV)28, we then applied HDM and post-processing analysis for strain analysis. Areas
100-200 µm within spontaneously contracting cardiac fibers achieved 8.32 ± 0.74% strain (Fig. 5D) at a
frequency of 1.49 ± 0.06 Hz (Fig. 5E).
We measured force generation on myocardial slices by measuring deflections of contractile nodes using
a custom setup for adhered slices (Supplemental Fig. IX,A). Force could be measured in spontaneously
contracting cardiac tissues, and was modulated by electrical pacing (Fig. 5F). Spontaneously beating areas
within unstimulated slices generated 14.93±3.85 µN of contractile force, which was decreased to 10.30 ±
2.81 µN when electrically stimulated at 2 Hz. Force generation in human myocardial slices was orders of
magnitude higher than single human iPS-derived cardiac myocytes29, 30, achieving 33.8% of native murine
embryonic heart tissue (53.0±15.5 µN)31, suggesting multicellular contractile tissue formation.
Using a more traditional setup to measure contractile force in muscle tissue (Supplemental Fig. IX,B),
spontaneously contracting cardiac fibers generated 102.7 ± 76.3 µN of force, while electrical pacing at 2
Hz could increase force generation to 124.1 ± 94.7 µN (Fig. 5G) by recruiting more recellularized areas to
contract in synchrony as a syncytium. Force curves measured from both myocardial slices and cardiac fibers
were further analyzed to assess amplitude, period, time to 50% relaxation (TTR50) , and time to 90%
relaxation (TTR90) under different pacing conditions (Fig. 5H, Supplemental Fig. X). Cardiac fibers
generated contractile force at an order of magnitude higher than myocardial slices, while decreasing trends
for period, TTR50, and TTR90 were similar for both constructs (Fig. 5H). Captured frequencies of both slices
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and fibers effectively correlated with pacing frequencies (Supplemental Fig. XI). Loading fiber-seeded
cardiac myocytes with voltage-sensitive di-8-ANEPPS dye, cardiac fibers were tested in a custom pacing
and imaging chamber to perform voltage mapping of cardiac electrical activity. Fibers paced with 10 V at
1 Hz generated electrical conductivity that could be mapped across large surface-areas, with a conduction
velocity of 32 ± 56 cm/s (Fig. 5I). Action potentials at multiple points within conduction areas were assessed
based on relative fluorescence intensity signal, with an average action potential duration at 80% (APD80)
of 405 ± 116 ms. Immunofluorescent staining of reseeded fibers for cardiac markers sarcomeric α-actinin,
cardiac troponin T, and myosin heavy chain showed large areas of cardiac myocytes with a striated
phenotype, and a high degree of alignment (Fig. 5J). Further analysis by transmission electron microscopy
(TEM) confirmed cardiac myocyte elongation and myocardial tissue formation, demonstrated by aligned
myofibrillogenesis (Fig. 5K, left panel) and definitive sarcomeric structures (right panel).
Whole-heart experiments.
Whole-heart recellularization experiments were carried out using a custom human heart bioreactor
capable of providing coronary perfusion and LV wall mechanical stimulation (Fig. 6A). Mechanical
stimulation was achieved by oscillating the pressure inside a balloon placed within the LV to determined
targets, thus imparting strain to the LV wall (Fig. 6B). Validation of strain delivery with oscillating balloon
pressure was performed using HDM on the presumed reseeding area of the LV wall of a decellularized
porcine heart under mechanical stimulation in our bioreactor (Fig. 6C, Supplemental Video V).
We reseeded whole acellular human hearts with ~500 million human BJ RiPS-derived cardiac
myocytes by making 5 intramyocardial injections into the basal anterior and mid anterior segments of the
left ventricle (using ~500 µL/injection, for total volume of 2.5 mLs), between the left anterior descending
and left circumflex coronary arteries (Fig. 6D). To improve cell retention, we adopted a technique in which
mattress sutures were tied at each injection point before needle extraction to minimize cell loss (Fig. 6E)32.
LV balloons were implanted through the mitral valve post cell delivery to prevent balloon puncture, and
then pre-filled to fully occupy the ventricular cavity. Recellularized human hearts were then mounted in the
human heart bioreactor to provide antegrade perfusion to the main coronary arteries and cultured for 14
days, with matrix strain stimulation starting on day 7 of culture (Fig. 6F, Supplemental Video VI).
Metabolic function was monitored during culture by sampling perfusate from the coronary sinus to
compare against baseline media (Table 1). Over consecutive 48-hour culture periods for 10 days, no
significant differences were found for measures of pH, pO2, pCO2, HCO3, TCO2, or percent O2 saturation.
However, a significant decrease of glucose suggested consumption (baseline: 186.33 ± 1.53 mg/dL, culture:
155.75 ± 6.70 mg/dL, p < 0.01). Conversely, significant lactate production was detected (baseline: 0
mmol/L, culture: 2.44 ± 0.19 mmol/L, p < 0.01).
After 14 days in culture, we performed electromechanical functional analysis on the regenerated

myocardium, using a bench-top perfusion setup. Electrical stimulation was delivered through cardiac leads
placed within the anterolateral wall of the left ventricle (0.8 Hz, 10-80 V, 5 ms). Stimulated myocardium
generated visible contractions (Supplemental Video VII), with recordable repolarizations (Fig. 7A). Using
a force transducer to measure force based on deflections of contracting regions on the epicardial surface,
continuous electrical stimulation at 0.8 Hz could generate regional contractions measuring ~350 µN (Fig.
7B). By introducing a pressure transducer into the LV balloon and pre-filling the balloon to an isovolumetric
pressure of 20 mmHg, stimulated myocardial contractions generated developed pressures of 2.4 ± 0.1
mmHg, with a dP/dt max of 139.7 ± 5.7 mmHg/s, a dP/dt min of 41.6 ± 3.5 mmHg/s, and a calculated tau
of 47.3 ± 10.6 ms (Fig. 7C). Using an 8x8-pin plaque electrode array with pins spaced 2.54 mm apart,
spontaneous depolarizations could be detected across the recellularized myocardium with action potential
durations of 322 ms (Fig. 7D).
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Histological analysis revealed dense regions of engrafted iPS-derived cardiac myocytes in the depth of
the left ventricular wall, indicated by large areas of myosin heavy chain (MHC) positive cells in successive
sections throughout recellularized volumes (Fig. 7E). By linear interpolation of the areas of MHC-positive
signal in tissue sections across reseeded regions, we detected a tissue repopulation of up to 50% within the
target region of 5 cm3 (Fig. 7F, Supplemental Table I). Coronary perfusion during whole heart culture
provided sufficient nutrient and oxygen supply to maintain >90% viability after 14 days in culture (Fig.
7G). Trichrome staining of recellularized areas confirmed engraftment, with reseeded cells integrating with
cardiac matrix (Fig. 7H). Recellularized regions of whole-heart scaffolds contained large areas of cardiac
myocytes that expressed sarcomeric α-actinin, cardiac troponin T, and myosin heavy chain by
immunofluorescence (Fig. 7I). Reseeded cardiac myocytes within biomimetically stimulated hearts
appeared to demonstrate a range of maturity, where some cardiac myocytes showed rounded immature
sarcomere formation, while other cardiac myocytes displayed elongated and striated phenotypes.
DISCUSSION
Loss of viable myocardium leads to architectural and functional decline, and ultimately results in the
development of heart failure. Bioengineered myocardium generated from patient derived cells could
provide an alternative to allo-transplantation and mechanical support. Recent reports on the native
extracellular matrix platform and robust protocols for directed differentiation of human pluripotent stem
cells provide key building blocks that could be combined towards that goal. Herein, we describe
regeneration of functional human myocardial tissue constructs based on decellularized human cardiac ECM
and human iPS-derived cardiac myocytes, and report four technical milestones that are paramount for
bringing this technology closer to clinical relevance: the generation of biocompatible acellular whole organ
scaffolds from discarded human donor hearts by perfusion decellularization, which elicit a more modulatory
immunological response; the directed differentiation of adult derived, non-transgenic iPSC to generate
cardiac myocytes at a clinical scale; the engraftment of human iPS derived cardiac myocytes on native
ECM for the development of functionally contractile tissue; and the up-scaling to biomimetic organ culture
to generate functional myocardial tissue in the context of a human heart.
Perfusion decellularization has been applied to hearts from several species15, 33-35. Through
collaboration with the New England Organ Bank and thanks to the generous gift of organ donors and their
families, we had access to hearts not used for transplantation. We followed a previously published detergent
based perfusion decellularization protocol to generate scaffolds of reproducible characteristics15, 36, 37. In
our studies, the increased warm ischemia time and potentially incomplete heparinization of DCD donors
did not have any detectable detrimental effect on the decellularization process or the resulting scaffold when
compared to hearts from DBD donors. This finding is of importance, given that approximately 1,600 DCD
donor hearts are currently available per year in the US3, 38, and this number is expected to increase39-41.
While human donors represent a more heterogeneous pool compared to well-characterized porcine donor
herds, current organ donation, allocation, and procurement infrastructures are well established, and donors
undergo extensive screening to ensure freedom from pathogens. Furthermore, differences in anatomy and
physiology between porcine and human hearts (e.g. shape, pulmonary venous anatomy) may prove to be
relevant in regenerated myocardial grafts42, 43. From a regulatory perspective, perfusion decellularized
human heart matrix could be a readily available and safe first step on a pathway towards clinical translation.
As has been reported in other human organs44, decellularization of hearts with structural disease and
atherosclerotic lesions yielded scaffolds with corresponding matrix defects. In line with prior work15, 36, 45,
we found preservation of a hierarchical network of macro- and microvascular channels after
decellularization, providing the foundation for the formation of metabolically active myocardium. Our
analysis revealed preserved vascular density and binned diameter distribution in decellularized myocardium
DOI: 10.1161/CIRCRESAHA.115.306874 9

compared to cadaveric controls and data reported in human hearts and animal models46, 47. Using high
resolution µCT imaging (6 µm), this finding extended to comparable lower-boundary density and
distribution of small-diameter vessels (<80 µm) in decellularized and cadaveric hearts. Though there was
no statistical difference found in vessel density across regions, decellularized hearts tended to demonstrate
slightly lower densities, visibly noticeable in the µCT of mid- and endocardial regions. This presents a
technical limitation, as the loss of endothelium and tissue-turgor make the recruitment of deeper vascular
conduits more challenging. For culture of recellularized hearts, perfusion pressure must be increased
towards the higher-end of physiological range in order to ensure media flow to reseeded regions. Active
and passive mechanical properties of native myocardium are a result of mutually orthogonal fiber directions
and play a key role in translating mechanical function from cells, to tissue, and to organ.48. In human hearts,
perfusion decellularization did not affect the anisotropic behavior of myocardium upon passive mechanical
testing, indicating the preservation of the native fiber framework within the myocardial matrix49. Artificial
scaffolds designed to reproduce some of the anisotropic behavior of myocardial tissue have been
repopulated with cardiac myocytes to create engineered myocardial tissue grafts with anisotropic
properties50, 51. However, reproduction of the multidimensional complexity of native cardiac fiber
architecture in a synthetic whole organ scaffold has not been accomplished to date. Based on our results we
concluded that perfusion decellularization of whole human hearts captures a structural blueprint that could
guide the formation of electrically and mechanically orthotropic myocardium.
In the present study, perfusion decellularization of human hearts resulted in acellular scaffolds
consisting of major extracellular matrix components such as collagens, elastin, and glycosaminoglycans,
which is in line with similar data published in other organ systems and species15, 36, 37, and decellularized
human heart sections52. Quantitative analyses of extracellular matrix components were similar in DCD and
DBD hearts, with preservation of insoluble collagen and glycosaminoglycans, and reduction of soluble
collagen and elastin. This is consistent with a loss of mainly immature collagen, while more mature,
crosslinked, high-strength collagen is preserved53. Histological and immunohistochemical analysis showed
absence of nuclei and cytoplasmic components such as myosin heavy chain. Further compositional analysis
using mass spectrometry-based label-free proteomics revealed an 89.14% reduction of total protein content
to 105 proteins and confirmed preservation of 36 matrisome proteins, including important structural
proteins such as fibrillin-1, collagen IV, collagen VI, and laminin. The relevance of the observed low level
of intracellular cardiac peptides within the cardiac scaffolds for recellularization and transplantation has yet
to be determined.The potential clinical value of any organ matrix depends on complete removal of cell
surface xeno- or allo-antigens, and substantial reduction of residual double stranded DNA. Perfusion
decellularized human heart matrices contained less DNA than the currently accepted standard for biomesh
implants, and could therefore be compatible with clinical transplantation17. We further confirmed absence
of human leukocyte antigens by immunostaining of decellularized hearts, and the absence of antibody
induction in an immunocompetent animal model in vivo. While we did not observe the generation of panel
reactive antibodies, decellularized human heart matrixes promoted a CD163 heavy macrophage response,
consistent with reconstructive remodeling described in non-crosslinked, decellularized matrix implants23,
54-56
. Notably, decellularized porcine myocardium showed a similar immunogenic profile as decellularized
human myocardium by histology, although we did not examine the humoral response to porcine matrix.
These early results hold promise from a device perspective, since in vivo remodeling and graft homeostasis
will depend on the absence of chronic inflammation and the promotion of a reconstructive response57.
Since induced pluripotent stem cells were first described, the generation of human iPS-derived cardiac
myocytes holds immediate promise for cardiac applications58, 59. In cardiac regeneration, techniques have
been developed for the delivery of human iPS-derived cardiac myocytes to improve cardiac function post-
infarct7, 60, provide cardioprotection61, and enhance homing and survival in infarcted myocardium62. These
approaches build on the extensive body of literature on ES derived cardiac myocytes, which have been
recently reported to mediate myogenesis in a primate model of cardiac repair32. In parallel, the field of
myocardial tissue engineering has provided a solid foundation for protocols to repopulate various scaffold
DOI: 10.1161/CIRCRESAHA.115.306874 10

materials with human iPS-derived cardiac myocytes and create functional tissue7, 34, 63-66. Our intent was to
combine the techniques of directed differentiation and the principles of myocardial engineering to generate
human myocardial grafts of clinical relevance, for applications in high-throughput screening and as a
foundational basis for creating human myocardial grafts of clinically relevant scale.
With potential translation in mind, we chose to derive cardiac myocytes from a non-genetically altered
pluripotent cell source that could be generated from patient-derived somatic cells via repeated
administration of synthetic messenger RNAs67, 68.. This method is characterized by lack of vector
integration, high efficiency, and low rate of chromosomal abnormalities, and has been validated by several
human reprogramming laboratories69. In order to generate cardiac myocytes at a clinically relevant scale,
we adapted a directed differentiation protocol based on temporal modulation of canonical Wnt/β-catenin
signaling to BJ RiPS cultured in monolayer25. This method yielded 5x108 cardiac myocytes with reasonable
use of resources and workload in the setting of an academic laboratory. In vitro differentiation of pluripotent
stem cells recapitulates the sequential stages of embryonic cardiac development70. Therefore, with Wnt/β-
catenin signaling25, we identified definitive stages for the development of precardiac mesoderm (day 3),
cardiac mesoderm (day 5), heart field specific progenitors (day 7), and iPS-derived cardiac myocytes (days
14, 30, 60, 120, and 180). While embryoid body methods have been applied to this cell line previously71,
we show that this protocol is highly reproducible for the cardiac differentiation of BJ RiPS, and
demonstrates a facile method to generate a clinical number of cardiac myocytes with a high efficiency.
Using three-dimensional models of increasing complexity we were then able to document the formation
of mechanically and electrically active myocardial tissue. In slice experiments, we first confirmed
biocompatibility, cell-mediated matrix deformation, and force generation. The formation of contractile
tissue on myocardial slices is evidence of reestablishment of cell-cell adhesions, as well as cell matrix
coupling, which are essential features of the myocardial microenvironment72. Despite being a stationary
model, the observed development of strain and force generation in cardiac slices further confirms that native
ECM provides a physiologic microenvironment known to be beneficial to pluripotent cell-derived cardiac
myocytes and myofibers13, 73. In a second set of experiments, we scaled native ECM recellularization to
myofibers of 15 mm in length and 2.5 mm in diameter. An improvement to stationary slices, but still non-
perfused, the resulting tissue showed improved cell engraftment and orientation, increased strain and force
generation, electrical conductivity, and improved fiber microarchitecture. This is in line with the
observation that physiologic substrate stiffness improves contractility of neonatal and iPSC derived cardiac
myocytes74, 75. The observed improvement in dynamic electrical and mechanical tissue function in fibers
may further be related to improved rigidity matching and resulting cardiac myocytes-tissue synchronization
within the native cardiac ECM76.
Generation of tissue constructs of clinical value, either as whole heart grafts or myocardial implants
ultimately requires regeneration of human myocardium on a whole organ scale 32, 63, 77, 78. We therefore
attempted tissue assembly in whole human heart matrices similar to prior reports in rat and mouse hearts15,
79
. Depending on its size, nearly one billion cardiac myocytes are lost due to a myocardial infarction (MI)80,
while several billion cardiac myocytes would be required to completely repopulate an acellular human
heart. To examine the clinical feasibility of myocardial regeneration, we attempted to deliver ~500 million
cardiac myocytes to a highly vascularized ~5 cm3 volume of the left ventricle, between the left anterior
descending and left circumflex coronary arteries. Efficient cell delivery with minimal matrix damage could
be achieved via five injections to multiple depths within the ventricular wall over a square epicardial region
(Fig. 6E).
To enable tissue development, we designed and built a biomimetic bioreactor system to accommodate
whole heart scaffolds, largely based on reported isolated working heart models, and the well documented
role of mechanical stimulation in the formation of myocardial tissue constructs81-84. Media perfusion via the
native coronary vascular channels provided sufficient nutrient and oxygen supply to enable the formation
DOI: 10.1161/CIRCRESAHA.115.306874 11

of myocardial tissue within the context of the whole heart scaffold. After a culture period of 14 days,
repopulated myocardial segments were metabolically active, provided contractile function, and responded
to electrical stimulation. Myocardial tissue was immature, showing a range of rounded to elongated
sarcomeric phenotypes, which corresponds to reported morphologic features of developing myocardium85,
86
and the in vivo fate of pluripotent cell derived cardiac myocytes87. This endpoint was chosen based on
the need to analyze cell viability and tissue formation, however, full maturation will likely require longer
culture times and improved biomimetic regimens.
While we believe that the present data set proves the translational value of the human acellular matrix
platform and the ability to generate myocardial tissue of clinically relevant origin and platform, the present
experiments did not aim to achieve full recellularization of the myocardium of an entire human heart.
Several challenges lie ahead in bringing this technology to clinical scale and applications. Full
recellularization of the myocardial matrix and valve structures are necessary for generating sustainable
ventricular pump function. Achieving this necessitates improved recellularization techniques to enhance
cell coverage without damaging the matrix (e.g. uni-directional or bi-directional vascular techniques).
Additionally, biomimetic culture conditions (i.e. static or dynamic stretch, electrical stimulation, media
components) must be optimized to elicit the maturity of reseeded cardiac myocytes and drive tissue
development. Considerations should potentially be made to donor matrix age and composition, for the
promotion of improved cell-matrix integration. A larger-scale immunological study is needed for a more
complete understanding host-response to cardiac matrices over time, as well as to understand the
modulatory or reconstructive effects on the matrix. Importantly, techniques to achieve and test for complete
re-endothelialization are critical in clinical application for the maintenance of blood-flow throughout the
organ (i.e. vascular recruitment) and to prevent matrix-induced blood coagulation. The present study
provides a foundational toolset to generate grafts of human scale for therapeutic applications, and multiple
model platforms to generate patient derived human myocardium of different scales for analytical purposes.
Undoubtedly, many hurdles towards the regeneration of whole heart grafts remain, including the need for
complete re-endothelialization, repopulation of the valvular apparatus, and re-establishment of a functional
conduction system.
ACKNOWLEDGMENTS
We thank the New England Organ Bank and all of the explant surgeons for their continued support and
coordination involved in organ procurement. We further thank the Program in Membrane Biology
Microscopy Core at MGH, as well as Ann Tisdale at the MEEI, for technical support with scanning electron
microscopy (supported by NIH grants DK43351 and DK57521). We thank Jeremy Song, MD for his
contributions to decellularization process development. Additionally, we thank Wenpo Chuang, MD and
Roger Ng, MD for their contributions to cardiac angiography. We thank TuKiet Lam, PhD and his team at
the MS & Proteomics Resource at Yale School of Medicine for support with proteomics analysis, as well
as James Titus and Chris Owen at MGH for support and use of fluoroscopy suites. We further thank John
Favreau, PhD for program support involved with HDM functional analysis. We thank Patricia Della Pelle,
Gertraude Warren, Jed Barger, and Susan Saidman, PhD for their help with immunogenic profiling and
HLA testing. For vascular analysis, we thank Brian Ghoshhajra, MD in the Department of Radiology at
MGH. Further, we thank Daniel Brooks, Mathew Scott, and Adriana Martinez-Betancourt in the Center for
Advanced Orthopedic Studies at Beth Israel Deaconess Medical Center for their assistance with micro-
computed tomography of microvasculature. In addition, we would like to thank Megan Scanlan for her
assistance with transmission electron microscopy on cardiac fibers. Lastly, we want to thank Kengyeh Chu,
PhD and Joseph Gardecki, PhD of the Wellman Center for Photomedicine at MGH, for their assistance
with micro-optical coherence tomography on cardiac fibers and microvasculature.
DOI: 10.1161/CIRCRESAHA.115.306874 12

SOURCES OF FUNDING
The present study was supported by the US National Institutes of Health (NIH) Director’s New Innovator
DP2 OD008749-01, and by the National Heart Lung and Blood Institute R21 HL108663-2 and R01
HL115282.
DISCLOSURES
HC Ott is founder and stockholder of IVIVA Medical Inc. This relationship did not affect the content or
conclusions contained in this manuscript.
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FIGURE LEGENDS
Figure 1: Biological and mechanical characterization of decellularized human myocardium.
A. A human heart before (i), during (ii), and after (iii) pressure-controlled perfusion decellularization.
B. DNA content of decellularized myocardium pre- and post-perfusion with nuclease (n = 3 hearts).
Normalized to tissue wet weight. Error bars are standard deviation. Asterisk (*) indicates p < 0.05
compared to pre-perfusion with nuclease.
C. Biochemical analysis for insoluble collagen (hydroxyproline), soluble collagen, α-elastin, and
sulfated glycosaminoglycans (s-GAGs) on normal cadaveric human myocardium (C, n = 5 hearts),
decellularized human cardiac matrix from hearts donated after brain death (B, n = 4), and
decellularized human cardiac matrix from hearts donated after cardiac death (D, n = 4). Three tissue
samples were analyzed per heart per component and the average of those three was used as the
representative value for the component in that heart. Normalized to tissue wet weight. Error bars
are standard deviation. Asterisks (*) indicate p < 0.05 compared to cadaveric condition.
D. Number of unique proteins identified through proteomics analysis in cadaveric (C) and
decellularized (D) human myocardium. Total (black) represents all unique proteins identified.
Matrisome (white) represents proteins in the total known to be part of the extracellular matrix
and/or basement membrane. Other (grey) represents non-matrisome proteins in the total.
E. Left: Pie charts of major components (collagens, laminins, fibrillins, and proteoglycans) in the
cadaveric and decellularized matrisome based on normalized relative abundance. Right: Stacked
bar charts illustrating further breakdown of the major components into specific identified proteins.
F. Immunohistochemical staining of decellularized human myocardium for collagens I, III, IV,
laminin, fibronectin, and myosin heavy chain (MHC). Scale bar, 250 μm, except MHC, 100 μm.
G. Biaxial mechanical testing of cadaveric (C, n = 8 hearts) and decellularized (D, n = 10 hearts)
human myocardium. B/A, base to apex axis. S/FW, septum to free-wall axis. Error bars are standard
deviation.
H. Anisotropy observed in biaxial mechanical testing of cadaveric (C, n = 8 hearts) and decellularized
(D, n = 10) human myocardium. Error bars are standard deviation.
Figure 2: Immunological characterization of decellularized myocardium.

A. Staining of macrophage markers CD68, CD80, and CD163 in human cadaveric (C), human
decellularized (D), and porcine decellularized (P) myocardium tissue samples after two weeks
implantation in a rat model. Scale bars, 100 µm.
B. Quantification of CD68+, CD68+/CD80+, and CD68+/CD163+ cells in human cadaveric (C, n =
3), human decellularized (D, n = 3), and porcine decellularized (P, n = 3) explants as a percentage
of total identified cells in the field (3-5 fields averaged per explant) after two weeks implantation
in a rat model. Error bars are standard deviation. Asterisk (*) indicates p < 0.05 between groups
using an ANOVA with Tukey’s post-hoc test.
C. HLA single antigen bead reactivity of implanted decellularized (i) and cadaveric (ii) human matrix,
with mean fluorescence intensity (MFI) plotted against beads with specific HLA alleles (generated
using HLA Fusion software, version 3.0). Asterisks (*) indicate beads presenting donor-specific
antigens. MFI values annotated for increased readability.
D. Immunofluorescence detection of HLA in cadaveric and decellularized human myocardium. Scale
bar, 100 μm.
DOI: 10.1161/CIRCRESAHA.115.306874 19

Figure 3: Characterization of vasculature in decellularized human myocardium.

A. Mean flow rates of each solution under constant pressure perfusion for non-DCD (n = 3 hearts) and
DCD (n = 4) populations. Error bars are SEM.
B. Mean vascular resistance calculated under constant pressure perfusion for non-DCD (n = 3 hearts)
and DCD (n = 4) populations. Error bars are SEM.
C. Pentachrome staining of decellularized LAD coronary artery. Adventitia (a), tunica media (m),
tunica intima (i), exterior elastic lamina (ext), interior elastic lamina (int). Scale bar, 500 μm. Color
indications: elastic fibers – black, collagen – yellow, fibrin – red, mucins – blue to green.
D. Scanning electron micrograph of cadaveric LAD coronary artery. Dotted white square indicates
region of higher magnification. Scale bar, 500 μm (left) and 20 μm (right).
E. Scanning electron micrograph of decellularized LAD coronary artery. Dotted white square
indicates region of higher magnification. Scale bar, 500 µm (left) and 20 µm (right).
F. Cardiac CT of a decellularized human heart. Left panel: long axis view. Right panel: posterior view.
LCA, left coronary artery. LAD, left anterior descending coronary artery. OM, obtuse marginal
coronary artery. LCX, left circumflex coronary artery. RCA, right coronary artery. AM: acute
marginal coronary artery.
G. Three-dimensional reconstruction of microvasculature in a left ventricular segment of
decellularized human heart. Scale bar, 1 mm.
H. Vessel density in epicardium, mid-myocardium, and endocardium of cadaveric (C, n = 3) and
decellularized (D, n = 7) heart samples.
I. Distribution of vessel sizes in the epicardium, mid-myocardium, and endocardium of cadaveric
human heart samples (n = 3).
J. Distribution of vessel sizes in the epicardium, mid-myocardium, and endocardium of decellularized
human heart samples (n = 7).
Figure 4: Generation of human iPS-derived cardiac myocytes.

A. Timeline of human iPSC differentiation into late stage cardiac myocytes (days 0, 3, 5, 7, 14, 30,
60, 120, and 180) with corresponding gene activation as measured by PCR (n = 3). Error bars are
standard deviation. Flow chart relates culture time with media, supplement, and cell type.
B. Immunofluorescence staining on differentiated cells isolated between days 30-60 for cardiac
markers α-actinin, troponin T, and myosin heavy chain (MHC). Scale bars, 50 µm.
C. Flow cytometry analysis of a representative culture of human iPS-derived cardiac myocytes
positive for α-actinin (left) and troponin T (right), isolated between days 30-60 post-differentiation.
Figure 5: Generation of functional tissue constructs based on decellularized human myocardium.

A. Cardiac matrix slices: decellularized human cardiac matrix slice (200 μm) in a culture well (i),
seeded with iPS-derived cardiac myocytes (ii) for 120-day culture. Ruler ticks are 1 mm.
B. Area strain of matrix-seeded iPS-derived cardiac myocytes and underlying cardiac matrix,
comparing spontaneous contraction (U, n = 5 beating areas) with electrical stimulation at 1 Hz (n
= 4) and 2 Hz (n = 4). Error bars are standard deviation. Black and blue asterisks (*) indicate p <
0.05 compared to unstimulated cells and matrix, respectively.
C. Left: Macro image of a reseeded cardiac fiber. Scale bar, 5 mm. Right: Representative µOCT still-
frame, depicting a longitudinal cross-section within a reseeded cardiac fiber. Red shading indicates
example HDM region of interest. Scale bar, 100 μm.
D. HDM computed area strain under unpaced conditions (n = 4 fibers). Error bar indicates standard
deviation.
E. HDM-captured frequencies under unpaced conditions (n = 4 fibers). Error bar indicates standard
deviation.
F. Representative force curves of a single contractile area within a myocardial slice, depicting force
modulation under unpaced (solid line) or paced conditions (dashed line).
DOI: 10.1161/CIRCRESAHA.115.306874 20

G. Representative force curves of a single reseeded cardiac fiber, depicting force modulation under
unpaced (solid line) or paced conditions (dashed line).
H. Mean amplitudes, periods, times to 50% relaxation (TTR50), and times to 90% relaxation (TTR90)
of captured force-curves of slices (gray squares; n = 9, 9, 9, & 3 for unpaced, 1 Hz, 2 Hz, and 3 Hz
respectively) and fibers (red triangles; n = 4, 3, 4, & 2 for unpaced, 1.5 Hz, 2 Hz, and 2.5 Hz
respectively) under paced and unpaced (U) conditions. Error bars are standard deviation.
I. Conductivity map (left) and example action potential (right) from a reseeded cardiac fiber.
J. Immunofluorescence staining on reseeded cardiac fibers for cardiac markers α-actinin, troponin T,
and myosin heavy chain (MHC). Scale bars, 25 µm.
K. Transmission electron micrographs of recellularized cardiac fibers. Dashed black box indicates
region of higher magnification. Left panel: red arrows indicate Z-bands, mt indicates regions of
mitochondria. Right panel: Z, I, A, H, & mt indicate Z-bands, I-bands, A-bands, H-bands, and
mitochondria respectively. Scale bars, 2 μm and 500 nm for left and right panels respectively.
Figure 6: Human heart bioreactor.

A. Schematic layout of the human heart bioreactor. Arrows indicate fluid flow directions.
B. Diagram of mechanical stimulation in the human heart bioreactor. ε, strain; P, pressure; Q,
volumetric flow; MV, mitral valve.
C. Representative LV-pressure vs. matrix strain curve during mechanical stimulation in the human
heart bioreactor (3 cycles plotted, starting with blue and ending with red), determined by HDM
analysis on the epicardial surface of the heart (inset) through biomimetic cardiac cycles.
D. Recellularization of human acellular cardiac scaffolds by intramyocardial injection between the left
anterior descending artery and the left circumflex artery.
E. Recellularized human acellular cardiac scaffold inside of the bioreactor organ chamber during
biomimetic culture. Black arrows indicate intramyocardial injection locations. Red arrows indicate
media perfusion into main coronary arteries. Blue arrows indicate fluid flow into left ventricle
balloon during mechanical stimulation.
F. Example pressure (top) and flow rate (bottom) traces in the coronary arteries (red) and left ventricle
(blue) during mechanical stimulation in the human heart bioreactor.
Figure 7: Repopulation of decellularized human myocardium in whole hearts with human iPS-
derived cardiac myocytes.
A. Electrocardiogram of a recellularized acellular cardiac scaffold during electrical stimulation
(pacing).
B. Force trace of recellularized myocardium under electrical stimulation at 0.8 Hz.
C. Left ventricular pressure development in a recellularized human acellular cardiac scaffold under
electrical stimulation at 0.8 Hz including corresponding mean ± SD peak pressure (P), max dP/dt,
min dP/dt, and tau (n = 6 pressure pulses).
D. Conductivity map (top) and action potentials (bottom) taken from the epicardial surface of a
reseeded human heart. Heat map legend labels indicate millisecond intervals.
E. Schematic of seeding volume estimation based on myosin heavy chain (MHC, red) staining images.
Scale bar, 2 mm. LAD, left anterior descending coronary artery. LCX, left circumflex artery.
F. Cell coverage from base to apex of recellularized human cardiac scaffolds. Error bars are standard
deviation of measurements taken from 2 adjacent tissue sections.
G. Fluorescent TUNEL of recellularized human cardiac scaffolds. Scale bar, 200 μm.
H. Trichrome staining of recellularized human cardiac scaffolds. Scale bar, 200 μm.
I. Immunofluorescent staining on recellularized human myocardium for cardiac markers α-actinin,
troponin T, and myosin heavy chain (MHC). Scale bars, 50 μm.
DOI: 10.1161/CIRCRESAHA.115.306874 21

TABLE 1. Metabolic measurements during whole-heart culture.
48-hr Media
Baseline Changes
Measurement Units Mean SD Mean SD p-value
pH - 7.32 0.08 7.26 0.05 0.3895
pCO2 mmHg 30.00 7.26 31.75 4.85 0.7397
pO2 mmHg 164.33 22.28 187.75 38.53 0.3600
HCO3 mmol/L 15.10 0.90 14.13 0.32 0.1941
TCO2 mmol/L 16.00 1.00 15.25 0.50 0.3257
sO2% % 99.00 0.00 99.25 0.50 0.3910
Lac mmol/L 0.00 0.00 2.44 0.19 0.0001
Glu mg/dL 186.33 1.53 155.75 6.70 0.0018
DOI: 10.1161/CIRCRESAHA.115.306874 22

NOVELTY AND SIGNIFICANCE
What Is Known?
 Currently, heart transplantation remains the only curative option for replacing necrotic cardiac tissue
and sustaining longer-term survival in patients with heart failure.
 The creation of bioengineered myocardium, either in the form of a cardiac tissue graft or a bioartificial
heart, theoretically holds the promise to improve contractile function to the injured heart.
What New Information Does This Article Contribute?
 Human hearts were decellularized using aseptic pressure-controlled perfusion to create acellular cardiac
scaffolds with preserved extracellular matrix components, architecture, material properties, and
microvasculature.
 Characterization of the acellular human scaffolds with thorough proteomics, microvasculature, and
immunogenicity analyses.
 Human cardiac myocytes were derived from a non-transgenic induced pluripotent stem cell line, to
generate cardiac myocytes at a clinically relevant scale, and create force-generating human myocardial-
like tissue slices, fibers, and ventricular segments on whole-heart scaffolds.
In a first step towards the creation of bioartificial myocardium, we sought to engineer myocardial tissue
that combines acellular human cardiac matrix and clinically relevant human iPS-derived cardiac myocytes.
We used a new pressure-controlled perfusion system to aseptically decellularize human hearts. The
resulting acellular human cardiac scaffolds were void of cellular material, while maintaining matrisomal
content and extracellular matrix structure. Immunogenicity studies confirmed the removal of human
leukocyte antigens, while also eliciting a reconstructive remodeling response when implanted in vivo.
Casting analysis determined that acellular scaffolds contained a preserved microvasculature, with a vessel
distribution similar to cadaveric hearts. Using a directed cardiac differentiation protocol on a non-transgenic
induced pluripotent stem (iPS) cell line, we generated a clinically relevant number of human cardiac
myocytes. We then reseeded acellular human cardiac matrix with human iPS-derived cardiac myocytes to
create force-generating myocardial-like tissues of increasing three-dimensional complexity. Recellularized
cardiac slices and fibers could be maintained for 120 days in culture, showing increased sarcomeric
structure and electromechanical function. We then upscaled these techniques to partially recellularize intact
hearts, to show that functional myocardial tissue could be built on a whole-heart platform.
DOI: 10.1161/CIRCRESAHA.115.306874 23
Figure 1
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CIRCRES/2015/306874D/R2
SUPPLEMENTAL MATERIAL

JP Guyette1,2, JM Charest1, RW Mills3, BJ Jank1,2, PT Moser1,2, SE Gilpin1,2, JR Gershlak4, T Okamoto1,
G Gonzalez1,2, D Milan3,5, GR Gaudette4, HC Ott1,2,6,7
METHODS
Human Heart Preparation and Decellularization. In collaboration with the New England Organ Bank
(NEOB), donated human hearts that were not suitable for transplantation were retrieved for research after
consent (n = 67; 5 patient records unavailable). Documented records from recovered hearts indicate there
were 32 male donors, 30 female donors, an average age of 53 ± 14 years, and an average body mass index
of 27.78 ± 6.25. Human hearts were recovered in standard fashion and delivered to Massachusetts
General Hospital, with an average cold ischemia time of 455 ± 184 minutes. All human tissue
experiments were performed in accordance with institutional guidelines and approved by the Partners
Human Tissues Research Committee at the MGH. Informed research consent was required by all human
donors, which was obtained by the NEOB. Hearts were randomly assigned to experimental groups as they
were received; 38 hearts were designated for pressure- controlled perfusion decellularization and 29
hearts were maintained to satisfy the control group. Twenty-four hearts were retrieved from donation after
cardiac death (DCD) donors, and the remaining hearts were recovered from donation after brain death
(non-DCD) donors. Upon delivery, the custom decellularization chamber was assembled and the aorta
was cannulated in a biological safety cabinet, using sterile gloves and aseptic technique. The heart was
placed in a sterilized organ decellularization chamber, and the aortic cannula connected to an in-chamber
perfusion line of the pressure-controlled pump system.1 Anterograde coronary perfusion was applied for a
constant pressure of 60 mmHg at room temperature, using a feedback control unit to regulate and record
pressure and flow. Hearts were aseptically decellularized with the following succession of solutions:
phosphate buffered saline with 1 U/mL heparin (1 hour); 1% sodium dodecyl sulfate (SDS) in deionized
water (168 hours); deionized water (DI H2O, 24 hours); 1% Triton-X in deionized water (24 hours);
phosphate buffered saline (168 hours). Decellularization was visually confirmed by an opaque appearance
of the cardiac ECM, accompanied with a loss of tissue turgor and rigidity (Fig. 1A). Analysis of recorded
data indicated trends towards increased coronary flow and subsequent decreased vascular resistance over
the course of the decellularization process (Fig 2A,B), but differences were not statistically significant. In
addition, a comparison of decellularizing DCD and non-DCD hearts showed that DCD hearts appeared to
have lower coronary flow and higher vascular resistance, however, differences were not statistically
significant (Fig. 2A,B). Following decellularization, hearts were decannulated and stored in PBS with
antibiotics/antimycotics at 4°C.
SDS, DNA, GAG, Collagen, and Elastin Quantification. Biochemical analysis of human heart samples
(cadaveric and decellularized) was performed to determine the presence of extracellular matrix proteins
(insoluble collagen, soluble collagen, elastin, and glycosaminoglycans) and DNA. Hydroxyproline
content as a measure of insoluble collagen was quantified using the Hydroxyproline Assay Kit, as per the
manufacturer’s instructions (Sigma-Aldrich, St. Louis, MO). Soluble collagen, alpha-elastin, and sulfated
glycosaminoglycans were quantified using the Sircol, Fastin, and Blyscan assay kits, respectively
(Biocolor, UK). For DNA quantification, tissue samples were digested overnight at 55 °C in 310 µL of a
solution containing 10 mM Tris (pH = 8.0), 5 mM EDTA, 0.1 M NaCl, 1% SDS, and 650 µg/mL
Proteinase-K (Life Technologies, Carlsbad, CA). Double-stranded DNA (dsDNA) was then isolated
1
CIRCRES/2015/306874D/R2
using a phenol/chloroform extraction and alcohol precipitation, and quantified using the Quant-iT
PicoGreen dsDNA kit (Life Technologies). All biochemical data is presented as the total mass of the
component extracted normalized to tissue sample wet weight in Figure 1, and normalized to dry weight
(lyophilized tissue) in Supplemental Figure II2. The mean dry weight of all tissue samples was 2.7 mg.
For each component (except DNA), biochemical analysis was carried out for three locations (anterior free
wall, septum, and apex) on each heart. An analysis of variance revealed no difference in component
content between locations. Thus, for each component the values for the three locations on a given heart
were averaged to obtain a representative value for that heart. DNA values were obtained from one septum
tissue sample per heart. For endonuclease treatment of acellular cardiac matrices, biopsies were taken
before and after whole-organ perfusion with 1 L of Pierce Universal Nuclease (25 U/mL) for 24 hours.
All samples were processed and analyzed for dsDNA quantification using the Quant-iT PicoGreen
dsDNA kit, as described above.
LC-MS/MS Proteomic Matrix Analysis. Both cadaveric and decellularized heart tissue samples were first
homogenized and sonicated in radioimmunoprecipitation assay (RIPA) buffer, and spun down to collect
supernatant. A chloroform/methanol/water protein precipitation was performed, and washed twice. The
protein pellet was solubilized in Rapigest, treated with DTT/IAN, and digested with Lys-C/Trypsin.
Samples were quantified using a Nanodrop and run on a microcapillary tandem mass spectrometry (LC-
MS/MS) Q-Exactive Plus system, with long gradient for label-free quantification. Acquired proteomics
data was processed using Progenesis QI software. Two distinct areas of the left ventricle in 3 different
hearts were analyzed for each group, for a total of 12 samples (6 cadaveric myocardium, 6 decellularized
myocardium). The subcellular location of each identified human protein was then determined using the
Universal Protein Knowledge Base (UniProtKB, www.uniprot.org, accessed on Feb. 21st, 2015).3 All
identified proteins (proteome) whose subcellular location included either the extracellular matrix or
basement membrane were designated as part of the matrisome.4
Biaxial Mechanical Testing of Myocardium. Two-dimensional (2D) tensile testing was performed on
cadaveric and decellularized heart samples. Full-thickness samples of approximately 25 x 25 mm were
cut from the anterior wall of the left ventricle. Sample width, length, and thickness were measured using a
micrometer. Four graphite dots were glued to the surface of each specimen for tracking displacement.
Two sutures (2-0 silk) were placed approximately 2 mm from the edge in each side of the sample and tied
creating a loop approximately 5 cm in length. The sutures were looped around pulleys on a custom planar
biaxial test device. Room temperature PBS was used to float the samples in the testing chamber and the
tare load was set when the sutures were under tension and the load increased slightly (~0.2 kPa). Loads
were applied bi-axially (1:1 ratio of x-load to y-load) under stress control up to 20 kPa. The forces along
the axes were measured with torque transducers and the movement of the graphite particles was measured
using a CCD camera. The stress was calculated as the force acting on the cross-sectional area and the
strain was calculated as the change in length divided by the original length at the tare load. For each
sample, moduli were calculated as the slope of the linear region of the stress-strain curve. To quantify the
degree of anisotropic behavior exhibited by the moduli of the tissue samples, an anisotropy ratio was
calculated for each tissue sample; where, anisotropy ratio = |Ex – Ey| / (Ex + Ey). Thus a ratio close to
zero would indicate little or no anisotropy and a ratio close to one would indicate a high degree of
anisotropy.
Immunogenic Profiling of Decellularized Human Cardiac Scaffolds. Overview: Twelve rats were
randomly divided into 4 equal groups of 3 each. Cardiac material was implanted subcutaneously in 3
experimental groups, while the sham operated group underwent the procedure for the subcutaneous
pocket but did not receive an implant. The cardiac materials implanted were decellularized human heart,
cadaveric human heart, or decellularized porcine heart. All animals were sacrificed at 2 weeks after
implantation. At the time of sacrifice, blood samples were taken from each rat for whole blood and serum
analysis. In addition, the explanted tissues were examined using immunohistochemical methods for cell
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surface markers representative macrophage profiles (i.e. M1, M2). All procedures were performed in
accordance with the National Institutes of Health guidelines for care and use of laboratory animals, and
with approval of the Institutional Animal Care and Use Committee at the Massachusetts General Hospital.
Animals and Surgical Procedure: Male, Sprague-Dawley rats weighing 300 to 500 g were purchased
from Charles River Laboratory (Wilmington, MA). Each animal was maintained on a diet of dry food and
housed individually in an environment maintained at 68-76° C for 24 h a day, with a light/dark cycle of
12/12 hours. During the surgical procedure, a 1.0 cm2 subcutaneous pocket was created along the sagittal
plane of the dorsal surface of each animal (between the two scapulae), as previously described. 5, 6 All
animals were survived for 2 weeks, at which point they were sacrificed by exsanguination during blood
collection analysis. Cardiac Material: Three types of cardiac material were prepared: cadaveric human
myocardium, decellularized human cardiac matrix, and decellularized porcine cardiac matrix. Pieces of
cardiac material from fresh cadaveric myocardium or freshly decellularized cardiac matrix were cut to 0.5
cm3 volumes. Cadaveric and decellularized human cardiac material was obtained through our
collaboration with the New England Organ Bank, as described above. Decellularized human cardiac
matrix was prepared as described above. Cadaveric and decellularized rat cardiac material was harvested
from Sprague-Dawley rats weighing 300 to 500 g were purchased from Charles River Laboratory
(Wilmington, MA). Decellularized porcine cardiac matrix was prepared as previously described.1 Whole
Blood Analysis: Whole blood was collected from each rat in Vacutainer® tubes (BD Biosciences,
Franklin Lakes, NJ), spun down as per manufacturer recommendation, and stored at 4°C until analysis.
Samples were run on a HESKA HemaTrue Analyzer (HESKA, Loveland, Colorado), and values from rats
in each group were averaged for each component. Immunohistological Analysis: Implanted materials
were harvested with an equal amount of adjacent fascia tissue. The specimens were fixed in 10% neutral
buffered formalin and embedded in paraffin. Serial sections of the embedded tissue specimens were then
cut at 5 µm. The tissue sections were stained with hematoxylin and eosin, and representative sections
were prepared for immunohistochemical staining by deparaffinization with xylene and rehydration
through a graded ethanol series. A heat-mediated antigen retrieval technique that included a 20-min boil
in 0.01 M citrate buffer, pH 7.0 was used. Once cooled, 3 separate washes of phosphate buffered saline
(PBS) were applied (5 min each). Primary antibody dilutions were made with blocking buffer and were as
follows: mouse anti-rat CD68 (cat#: MCA341GA; AbD Serotec, Raleigh, NC) at 1:50 dilution in PBS,
rabbit anti-CD80 (cat#: bs-2211R; Bioss, Inc., Woburn, MA) at 1:100 dilution, and rabbit anti-rat CD163
(cat#: bs-2527R; Bioss, Inc.) at 1:100 dilution. Secondary antibodies were applied for 1 hour at room
temperature (25°C). Secondary antibody dilutions were made with blocking buffer, as follows: 1:400 anti-
mouse 488 and 1:400 anti-rabbit 594 (both from Invitrogen, Eugene, OR). A CellProfiler image analysis
pipeline was used to quantify macrophage phenotype across images (Supplemental Fig. V).7 HLA
Analysis: Sera was collected from each rat in Vacutainer® tubes (BD and Company, Franklin Lakes, NJ),
spun down as per manufacturer recommendation, and stored at 4°C until analysis. Sera was run against
Panel Reactive Antibody (PRA) single-antigen beads, using clinical test kits for LABScreen PRA Class I
and II (cat#’s: LS1PRAS and LS2PRAS; One Lambda, of Thermo Fisher Scientific, Canoga Park, CA).
The secondary antibody used to detect antigen-reactive rat antibodies was R-Phycoerythrin1-conjugated
AffiniPure, goat anit-rat IgG (cat #: 112-116-071; Jackson ImmunoResearch Laboratories, Inc, West
Grove, PA). Samples were run through a Luminex IgG-assay, using a LABScan100TM Luminex analyzer,
equipped with xPONENT software (Life Technologies, Grand Island, NY). The LABScan100TM Luminex
required a 100-event minimum per sample to determine a successful test read-out. Using HLA Fusion
Software (version 3.0; One Lambda, of Thermo Fisher Scientific, Canoga Park, CA) for post-processing,
experimental samples were analyzed using the sham group as a reference, and histograms we generated
by the software by plotting mean fluorescence intensity (MFI) attributed to beads with specific HLA
alleles (Fig. 1Ki,ii). HLA Immunohistochemistry: Freshly prepared samples were fixed in 10% neutral
buffered formalin, embedded in paraffin, and sectioned at 5 µm. Sections were deparaffinized with xylene
and rehydration through a graded ethanol series, and then treated for 30 minutes in citrate buffer at 95°C
for antigen retrieval. Primary antibodies were allowed to attach overnight at 4 °C. Primary antibody
dilution was made with blocking buffer as follows: 1:100 anti-HLA 1 ABC [EMR8-5] (cat#: ab70328;
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Abcam, Cambridge, MA). Secondary antibody was applied for 1 hour at room temperature (25°C).
Secondary antibody dilution was made with blocking buffer, as follows: 1:400 anti-mouse 594
(Invitrogen, Eugene, OR). Images were recorded using a Nikon Eclipse TE200 microscope (Nikon,
Melville, NY).
Histology and Immunocytochemistry. For immunohistochemistry, tissue samples were fixed in 5%

formalin, paraffin-embedded, and sectioned following standard protocols. Briefly, 5 µm sections were
deparaffinized, and treated for 30 minutes in citrate buffer at 95°C for antigen retrieval. Primary
antibodies were allowed to attach overnight at 4 °C. Primary antibody dilutions were made with blocking
buffer, as follows: 1:50 anti-laminin G1 (cat#: sc-13144; Santa Cruz Biotechnology, Dallas, TX), 1:50
anti-elastin (cat#: sc-58756; Santa Cruz Biotechnology), 1:50 anti-fibronectin (cat#: sc-59826; Santa Cruz
Biotechnology), 1:100 anti-collagen I (cat#: ab6308; Abcam, Cambridge, MA), 1:300 anti-collagen III
(cat#: ab6310; Abcam), 1:200 anti-MHC (cat#: ab15, anti-heavy chain cardiac myosin antibody [3-48];
Abcam), 1:50 anti-collagen IV (cat#: LS-C79592; Lifespan Biosciences, Seattle, WA). Anti-mouse
conjugated to HRP was added at 1:100 for 30 min, and subsequently developed with 3,3’-
diaminobenzidine (Dako, Carpenteria, CA) until good staining intensity was observed. Images were
recorded using a Nikon Eclipse TE200 microscope (Nikon, Melville, NY).
For cardiac fiber preparations, recellularized fibers were first washed with PBS and then fixed with
5% formalin for 24 hours at 4°C prior to paraffin embedding. For whole-heart preparations, recellularized
scaffolds were perfusion-fixed with recirculating 5% formalin through the left main coronary artery, at 20
mL/min for 3 hours at 25°C. Scaffolds were then systematically processed by excising the reseeded
region from the matrix. Reseeded regions were cut into defined tissue blocks from base to apex (for
samples with 0.5 cm thickness), and then tissue blocks were placed in cassettes and submerged in 5%
formalin for 24 hours.
Tissue samples for both reseeded fibers and reseeded hearts were paraffin-embedded, sectioned, and
stained following standard protocols. Briefly, 5 µm sections were deparaffinized, and treated for 30
minutes in citrate buffer at 95°C for antigen retrieval. Primary antibodies were allowed to attach
overnight at 4 °C. Primary antibody dilutions were made with blocking buffer and were as follows: 1:100
anti-sarcomeric α-Actinin (cat #: A8711, Sigma-Aldrich, St. Louis, MO), 1:100 anti-myosin heavy chain
(cat #: SC-20641, Santa Cruz Biotechnology, Dallas, TX), and 1:100 anti-troponin T (cat #: ab8295,
Abcam Inc., Cambridge, MA). Secondary antibodies were applied for 1 hour at room temperature (25°C).
Secondary antibody dilutions were made with blocking buffer, as follows: 1:400 anti-mouse 488, 1:400
anti-mouse 594, 1:400 anti-rabbit 594 (all from Invitrogen, Eugene, OR). TUNEL assay was performed
using the DeadEndTM Fluorometric TUNEL System per the manufacture’s protocol (Promega
Corporation, Madison, WI). Tissues slides were mounted with Dapi Fluoromount-G (Southern Biotech,
Birmingham, AL). Masson’s Trichrome was performed using reagents and methods based on kit
manufacturer’s recommendations (American MasterTech, Lodi, CA). Images were recorded using a
Nikon Eclipse TE200 microscope (Nikon, Melville, NY).
In a systematic histological analysis of whole-heart experiments, reseeded regions were cut into
defined tissue blocks as described above. Tissue sections from each tissue block were then immuno-
stained for myosin heavy chain (MHC) for immunofluorescence detection, as described in the previous
paragraph. By measuring the areas of MHC-positive signal in several tissue sections throughout the
reseeded regions (from base to apex), we applied linear interpolation along with respect to the vertical
dimension to estimate a volumetric recellularization.
Vascular Histology. Samples of the left anterior descending (LAD) coronary artery were cut to 5 µm
cross-sections, which were then deparaffinized through xylene and alcohol washes. Pentachrome staining
was performed using the Russel-Movat Pentachrome Stain Kit Procedure, as per manufacturer’s
instructions (American MasterTech, Lodi, CA).
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Scanning Electron Microscopy of LAD. A cross sectional biopsy of a cadaveric and decellularized left
anterior descending arteries were fixed in half-strength Karnovsky's fixative after harvest, and stored for
24 hrs at 4°C. They were then rinsed in PBS, dehydrated through graded ethanols, critical point dried
(Autosamdri 795 Supercritical Point Dryer; Tousimis, Rockville, MD), mounted on specimen holders,
and sputter coated with Chromium in an ion beam coater (model 681; Gatan, Pleasanton, CA). Images
were acquired via a field emission scanning electron microscope (JSM-7401F; JEOL Ltd., Peabody, MA).
Coronary Angiography. All hearts were mounted with a cannula in the ascending aorta. Antegrade flow
was created via pumping normal saline through the cannula at a constant pressure into the aortic
root. Coronary angiography was then performed by selectively engaging each of the coronary artery with
the 5F Judkins catheters. Coronary cine was obtained using a GE OEC 9600 c-arm with injections of
Visipaque (1mL contains 652 mg of iodixanol which is 320 mg organically bound iodine) contrast. Cines
and still frame images were then captured onto USB via MediCapture.
Microfil® Preparation, µCT, and Analysis of Coronary Microvasculature. Microfil® Preparation:

Tissue samples were prepared by first accessing and cannulating the left and right main coronary arteries
in the whole-heart sample (either a cadaveric human heart or an intact acellular human cardiac scaffold).
Hearts were perfused with sterile PBS at a constant infusion pressure of 60 mm Hg for 15 minutes to
recruit vasculature and to visualize constant flow from the coronary sinus. Once the matrix was
rehydrated and vascular conduits were recruited, radiopaque Microfil® silicon-based injection compound
(MV-122, Flowtech, Carver, MA) was perfused into the matrix at a constant pressure of 60 mm Hg.
Microfil® perfusion was stopped when injection compound was observed flowing from the coronary
sinus. At this time, perfusion tubing was clamped off and the Microfil ®-infused heart was allowed to sit
for 90 minutes at room temperature for the compound to cure. Hearts were then placed in 70% ethanol,
and allowed to cure further at 4°C for 24 hours. µComputed Tomography (µCT): Once fully cured, full-
thickness samples were excised identifying a region of the LAD mid-way between the apex and base of
the heart, and then cutting a tissue cube with a 1x1 cm2 surface area that spanned from the epicardium to
the endocardium. Tissue cubes were stored in 70% ethanol until being scanned. µCT imaging was
performed on a piece of human heart using a high-resolution desktop imaging system (µCT40, Scanco
Medical AG, Bruttisellen, Switzerland). Transverse slices were acquired with 6 µm3 isotropic voxel size,
70 kVp peak x-ray tube potential, 114 uA intensity, 300 ms integration time, and were subjected to
Gaussian filtration. The resulting images were segmented based on voxel intensity in order to select the
vascular regions and 3D reconstructions were generated. Post-µCT Analysis of µVasculature: Two-
dimensional µCT images were analyzed for distribution and diameter. Image stacks from full-thickness
samples were first assessed to determine the starting epicardial slice and the ending endocardial slice, and
then the number of images was divided into equal thirds to determine epicardial, mid-myocardial, and
endocardial sub-regions. At least 3 representative images in each sub-region were analyzed, separated by
600-1200 µm, so as to assess the vascular landscape through the sub-regional thickness. Vessel densities
were determined for each 2D image and normalized to the analyzed tissue area, and images within sub-
regions were averaged. Using ImageJ to measure the minor axis of vessel cross-sections, each 2D image
was analyzed and binned for vessel diameters in each sub-region. µOptical Coherence Tomography
(µOCT) of Perfused µVasculature: To visualize perfusion through microvasculature in decellularized
human hearts, large full-thickness sections of myocardium were first excised along the left anterior
descending coronary artery. Epicardial conductance arteries were then cannulated, perfused with sterile
phosphate buffered saline (PBS), and major peripheral leaks were shunted with microvascular clips.
A 50 mL mixture of red fluorescent microbeads (2.06 µm diameter; cat #: FH-2040-2, Spherotech, Lake
Forest, IL) was then prepared to a dilution of 1:100. Diluted microbeads were then gravity-perfused
through the full-thickness piece of decellularized myocardium at 30 mm Hg. During gravity-perfusion of
microbeads, micro-optical coherence tomography (µOCT) was used to acquire image stacks ~200 µm
below the surface of the cut mid- and endocardium, at a frame rate of 40 fps for 30 seconds (µOCT
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method described below). Image analysis was completed using ImageJ software, equipped with a
Running Z Projector plugin, set to a running average size of 20, for maximum intensity projection.
Cell Culture, Cardiac Differentiation of iPS Cells, and Cell Characterization. iPS Culture: Human
cardiomyocytes were generated from the BJ-RiPS-1.1 cell line obtained from the Harvard Stem Cell
Institute, sourced from human BJ postnatal fibroblasts transfected with mRNAs encoding Klf4, c-Myc,
Oct4, Sox2, and Lin288. Passage 18-30 undifferentiated BJ-RiPS were cultured on growth factor reduced
MatrigelTM (diluted 1/100; cat. # CB 40230, Thermo Fisher Scientific Inc, Waltham, MA), in mTeSRTM1
media (cat. # 05870, Stem Cell Technologies Inc, Vancouver, BC, Canada). Cardiac Differentiation:
Cardiac differentiation of BJ-RiPS cells was performed using an established protocol based on a temporal
modulation of Wnt signaling with small molecules (Stemolecule™ CHIR99021, cat. # 04-0004-02;
Stemolecule™ Wnt Inhibitor IWP-4, cat. # 04-0036; Stemgent Inc, Cambridge, MA) 9. Cardiomyocyte
generation was confirmed by observation of functional contractility of beating cells in vitro.
Cardiomyocytes were maintained in RPMI 1640 medium (cat. # 11875-119, Life Technologies, Grand
Island, NY) supplemented with B-27 (cat. # 17504-044, Life Technologies), with media changes every 2
days. Human iPS-derived cardiomyocytes were cultured for at least 20 days after the onset of
differentiation before being harvested for recellularization experiments. Cell Characterization – RT-
PCR: Over the time-course of differentiation (days 0, 3, 5, 7, 14, 30, 60, 120, and 180), differentiating
iPS cells were scrapped off of culture plates, pelleted down by centrifugation (300 g, 5 min), and stored in
RNAlater (cat#: AM7020; Life Technologies, Grand Island, NY) at 4°C until analyzed. RNA was
extracted using an RNeasy® Plus Mini Kit (cat#: 74134; Qiagen, Valencia, CA), and quantified using a
NanoDrop 2000 Spectrophotometer (Thermo Scientific, Waltham, MA). cDNA was prepared using a
SuperScript® III First-Strand Synthesis SuperMix Kit for qRT-PCR (cat #: 11752-050; Life Technologies,
Grand Island, NY). Quantitative RT-PCR was performed using TaqMan® Gene Expression Master Mix
(cat. #: 4369016; Applied Biosystems, Foster City, CA), along with the following TaqMan® Probes (all
from Applied Biosystems): Oct4 (cat #: HS00999632_g1), Sox2 (cat #:HS04407947_m1), Nanog (cat #:
HS04260366_g1), MIXL1 (cat #: HS00430824_g1), T (cat #: HS00610080_m1), ISL-1 (cat #:
HS0015126_m1), TBX2 (cat #: HS00911929_m1), GATA4 (cat #: HS00171403_m1), Nkx2.5 (cat #:
HS00231763_m1), MEF2C (cat #: HS00231149_m1), TBX5 (cat #: HS00361155_m1), MYH6 (cat #:
HS01101425_m1), MYH7 (cat #: HS0110632_m1), MYL2 (cat #: HS00166405_m1), TNNT2 (cat#:
Hs00943911_m1), and 18S (cat #: HS99999901_s1). Samples were run on a StepOnePlus Real-Time
PCR System (Applied Biosystems), and analyzed using the delta-delta CT method with respect to
sample’s internal 18S signal and fold-differences to undifferentiated iPS cells. Cell Characterization –
Immunocytochemistry: BJ RiPS-derived cardiomyocytes between days 30 and 60 post-differentiation
were washed once with PBS, and then fixed with 4% paraformaldehyde for 15 minutes at 4°C. Cells were
then washed three times with PBS, and then permeabilized with 1.5% Triton-X solution for 10 minutes.
Cells were then washed three times with PBS, and then blocked with 1.5% donkey serum for 30 minutes.
Following blocking, primary antibodies were allowed to attach overnight at 4 °C. Primary antibody
dilutions were made with blocking buffer and were as follows: 1:100 anti-sarcomeric α-Actinin (cat #:
A8711, Sigma-Aldrich, St. Louis, MO), 1:100 anti-myosin heavy chain (cat #: SC-20641, Santa Cruz
Biotechnology, Dallas, TX), and 1:100 anti-troponin T (cat #: ab8295, Abcam Inc., Cambridge, MA).
Secondary antibodies were applied for 1 hour at room temperature (25°C). Secondary antibody dilutions
were made with blocking buffer, as follows: 1:400 anti-mouse 488, 1:400 anti-mouse 594, 1:400 anti-
rabbit 594 (all from Invitrogen, Eugene, OR). Cell nuclei were stained with Hoechst dye (cat. #: H3570,
Thermo Fisher Scientific, Inc., Waltham, MA), washed three times with PBS, and imaged in PBS. For
some cell preparations, cytoskeletal actin structures were counterstained with Phalloidin stain (cat. #:
O7466, Thermo Fisher Scientific, Inc.) for 20 minutes between the blocking step and application of
primary antibody. Cell Characterization – Flow Cytometry: iPS-derived cardiomyocytes between days
30 and 60 post-differentiation were washed twice in phosphate buffer saline (PBS) and dissociated into
single cells by treatment with 0.05% trypsin/EDTA. For the detection of cardiac sarcomeric α-actinin and
cardiac troponin T independently, the cells were fixed with 4% paraformaldehyde at room temperature for
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20 min, followed by 2 PBS washes. Cells were then permeabilized with 0.1% Triton X-100 in PBS on ice
for 20 min, washed twice with PBS, and centrifuged at 300 g for 5 min. Cells were blocked with 1% BSA
in PBS for 30 min, and then incubated with the primary antibody at room temperature for 1 h. After
washing in PBS, the cells were incubated with the secondary antibody at room temperature for 1 h. After
washing in PBS, the cells were suspended in 400 μl PBS and assayed by flow cytometry (FACS Calibur;
BD Biosciences, Franklin Lakes, NJ). Mouse IgG anti-α-sarcomeric-actinin (cat #: A7811, 1:100; Sigma,
St. Louis, MO) and mouse IgG cardiac troponin T (cat #: ab8295 – [1C11], 1:100; Abcam, Cambridge,
MA) served as primary antibodies. The secondary antibody was goat anti-mouse 488 (cat#: A-11001,
1:400; Invitrogen, Eugene, OR). Cells stained with primary isotype control antibody (normal mouse IgG1;
cat. #: sc-3877, Santa Cruz Biotechnology, Dallas, TX) and secondary fluorescence-conjugated antibody
served as controls. At least 50,000 cells per sample were acquired. Cardiac differentiation was quantified
by the percentage of α-actinin-positive cells subtracted by negative control after gating out the dead cells.
All of the data analyses were performed using the CellQuest software (Becton Dickinson).
Preparation and Recellularization of Human Acellular Myocardial Slices & Cardiac Fibers.
Myocardial Slices: To create acellular myocardial slices, full-thickness pieces of cardiac matrix were
excised from the left ventricle of acellular human hearts (~2x2x2 cm3), embedded in Tissue-Tek Optimal
Cutting Temperature (O.C.T.) Compound (cat#: 4583; Sakura Finetek, Torrance, CA), and cryo-sectioned
for 200-µm slices on a Leica CM3050 S cryostat (Leica Biosystems, Buffalo Grove, IL). Slices were
carefully transferred to 6-well tissue culture plates and allowed to adhere at room temperature. OCT
Compound was removed by a 5 min treatment with DI water, as the compound is water-soluble (per
manufacturer protocol). Slices were sterilized with treatment of 100% ethanol, followed by successive
PBS washing steps to rehydrate the matrix. Acellular slices were then primed with cardiomyocyte
maintenance media (RPMI 1640, with B-27) for 12 hours. Each primed myocardial slice was seeded with
1.5x106 human iPS-derived cardiomyocytes (unsorted), which were allowed to adhere for 24 hours at
37°C. Cardiomyocyte maintenance media was changed every 2 days following the initial seeding period,
spontaneously contracting tissue was observed 4-7 days after seeding, and functional tests were
performed after 28 days in culture. Cardiac Fibers: Cardiac fiber bundles (15 mm length, 2.5 mm
diameter) were cut from the left ventricular free wall of decellularized cardiac scaffolds, using sterile
surgical tools and aseptic technique in a biological hood. Acellular fibers were then transferred to
individual wells of a 12-well tissue culture plates, and then primed with cardiomyocyte maintenance
media (RPMI 1640, with B-27; 2 mL per well) for 12 hours. Each primed cardiac fiber was then injected
with 1.5x106 cardiomyocytes in 100 µL. Cell suspension that fell out or dripped off of the construct was
collected after injection, and seeded with the cardiac fiber in a rotating bioreactor tube for an additional
12 hours (similar to methods published previously).10 After the addition 12 hours of rotational bioreactor
seeding, recellularized fibers were removed from the bioreactor tube with sterile forceps, and placed into
6-well tissue culture plates onto custom 22-gauge stainless steel posts (1.5 mm apart). Cardiomyocyte
maintenance media was changed every 2 days following the initial seeding period, spontaneously
contracting tissue was observed 7-10 days after seeding, and functional tests were performed after 30 days
in culture.
Mechanical Function of Human Cardiac Slices & Fibers. Strain Analysis: Strain of contracting
myocardial slices and cardiac fibers was evaluated on successive image-stacks by using a high-spatial
resolution sub-pixel algorithm called high density mapping11, based on a method proposed by Foroosh
and Zerubia12. This technique is partially based on the capabilities of conventional particle tracking or
digital correlation techniques, displacements can be determined at hundreds of locations to provide a
whole field measurement, with a demonstrated accuracy of 0.09 pixels and precision of 0.04 pixels11, 13.
For myocardial slices, strain was assessed on both the cells (imaged using phase contrast, at the slice
surface) and underlying ECM (imaged using autofluorescence in the GFP channel, 20 µm below the slice
surface). Image stacks were acquired using a Nikon Eclipse TE200 microscope (Nikon, Melville, NY),
with a 1280 x 1024 pixel resolution, at a frame rate of 20 fps for 20 seconds. For cardiac fibers, strain was
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assessed on deforming matrix 100-200 µm below the fiber surface, using a method called micro-optical
coherence tomography (µOCT) to create image stacks at a frame rate of 40 fps for 30 seconds (µOCT
method described below). For HDM analysis, all images were converted to 8-bit grayscale for analysis.
Multiple contractions per sample were analyzed and averaged to determine area strain and frequency;
Unpaced slices (n=6), paced slices (n=4), unpaced fibers (n=4). Force analysis: Contractile force was
measured on submerged recellularized myocardial slices using an Aurora Scientific 403A Force
Transducer system. Since myocardial slices were fixed to tissue culture plastic, force was measured by
attaching a cantilever to the transducer, lowering the cantilever onto contractile cardiac tissue, and
detecting uniaxial deflections (n=9). Cardiac fibers reseeded with iPS-derived cardiomyocytes were
mounted lengthwise in our custom force measurement setup (Supplemental Fig.VI). Each fiber was
placed in a warmed (37°C) media bath and one end was attached to a fixed point via a 6-0 silk suture. The
other end was attached to a force transducer (403A Force Transducer system; Aurora Scientific) also via a
6-0 silk suture, with a preload of 50 mg (50 µN; n=4). In force measurements for both myocardial slices
and cardiac fibers, the force transducer was calibrated with 10-, 50-, and 100-mg weights, and data was
recorded at scale factor of 50 mg/volt. Signals were outputted to a PowerLab 16/35 digital acquisition
device (cat. # PL3516, AD Instruments Inc., Colorado Springs, CO) with a sampling rate of 1 kHz, and
then analyzed with LabChart software (cat. # MLU60/8, AD Instruments Inc.). Electrical Stimulation
During Mechanical Testing: During stimulation for strain and force measurements, we studied the
effects of electrical pacing. For myocardial slices, electrical pacing was conducted with a C-Pace EP
system (30 V, 5 ms; IonOptix LLC, Milton, MA) with increasing frequencies: 1, 1.5, 2, 2.5, or 3 Hz. For
cardiac fibers, electrical pacing was conducted with a Grass S88 Stimulator (30 V, 5 ms; Grass
Technologies, Warwick, RI) with increasing frequencies: 1.5, 2, and 2.5 Hz.
Micro-Optical Coherence Tomography (μOCT). The use of μOCT in this paper was based on methods
previously described.14 Briefly, axial (z) ranging for OCT was achieved by intereferometric measurement
of the optical delay of light returned from the sample. μOCT as implemented in this manuscript is based
on Spectral-domain OCT (SD-OCT).15 The application of μOCT in this setup employed very broad
bandwidth light source (800±150 nm laser-generated supercontinuum) and a common path reference arm
to achieve 1-μm depth or axial (z) resolution. To achieve high transverse resolutions, a high numerical-
aperture objective lens (numerical aperture = 0.12) was used to focus the beam onto the sample. We
further engineered the focus of the probe beam with an annular apodizer, which reduced the focal spot
size from 2.4 to 2.0 μm. Apodization and chromatic dispersion extended the focal depth to ~250 μm,
enabling cross-sectional imaging at high resolutions. The system operates with user-configurable line and
frame rates and customizable scan geometry; settings were set to 40 frames per second, 512 A-lines per
frame in a linear scan, and 0.5 mm by 0.5 mm (X by Z) for a cross-sectional image.
Cardiac Fiber Voltage Mapping. Recellularized cardiac fibers were treated with 20 mmol/L blebbistatin
for 30 min to arrest motion, and then loaded with Di-8-ANEPPS (5 mmol/L) for 20 min at 37°C
immediately prior to imaging. The cardiac fiber preparations were placed in a custom pacing and imaging
chamber, and voltage mapping of cardiac electric activity was performed with this chamber and a charge-
coupled device camera (CardioCCD-SMQ, RedShirt Imaging, Decatur, Ga) mounted on a Nikon TE2000
inverted microscope. The hearts were field paced at 60 bpm (Grass S48K Stimulator, West Warwick, RI).
Samples were illuminated with a 120-W metal-halide Exfo X-Cite 120 lamp with a 480/40 nm excitation
filter, and the fluorescence image was filtered through a 535/50 nm emission filter. Data were acquired at
a frame rate of 125 Hz. NeuroPlex software (RedShirtImaging) was used to view image sequences and
output darkfield-corrected mean fluorescence from regions of interest. Optical recordings were inverted
and low-pass filtered at 100 Hz. Fluorophore photobleaching was subtracted using a custom program, and
probe dynamics were fit using Clampfit software (Axon Instruments). Measurements of APDs were taken
as the average of 3 successive beats for each sample (n=4). APD was calculated at 80% repolarization
(APD80).
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Human Heart Bioreactor. Whole-heart acellular scaffolds were cultured in the human heart bioreactor
(Figs. 4A,E) which provides the organ with perfusion of media and mechanical stimulation. Perfusion
was achieved by driving media from the organ chamber into the main coronary arteries. In parallel with
the perfusion loop was a hollow-fiber oxygenator (Fiber oxygenator D150, cat. #73-3757, Harvard
Apparatus, Holliston, MA) fed with carbogen gas (95% oxygen, 5% carbon dioxide) to maintain media
buffering and oxygen content. The organ chamber also contained a small duct for collection of effluent
from the coronary sinus (CS, not shown in Fig. 4A). Mechanical stimulation was delivered to the left
ventricular wall via cyclic inflation and deflation of a fluid-filled balloon inserted into the LV via the
mitral valve (Fig. 4B). Both perfusion pressure and LV pressure were monitored for the duration of
culture (Fig. 4F). LV balloon inflation was controlled by oscillating the LV balloon pressure to deliver
strain to the left ventricular wall (Figs. 4B,C,F). The entire setup was then placed within an incubator at
37°C to maintain physiologic temperature.
Preparation and Recellularization of Human Acellular Whole-Heart Scaffolds in the Human Heart
Bioreactor. All preparations for whole-heart acellular scaffolds were performed using aseptic technique,
sterilized tools, sterilized tubing, sterilized bioreactor parts, and sterile-filtered media. Whole-heart
acellular scaffolds were prepared by dissecting down the aortic root to access the left main coronary
artery (LCA), and then accessing the coronary sinus (CS) through the right atrium. Both the LCA and CS
were cannulated. A latex balloon was inserted into the LV via the mitral valve, and prefilled to fill the
ventricular cavity. The acellular scaffold was then mounted in the organ chamber of the bioreactor. The
LCA cannula was attached to a perfusion line, the CS cannula was attached to a collection duct, and the
LV balloon cannula attached to an external fluid reservoir behind the ventricle pump. Whole-heart
acellular scaffolds were primed overnight with 1 L of cardiomyocyte maintenance media for 12 hours, at
a rate of 60 mL/min, at 37°C. After priming, organ chambers were brought into a clean biological safety
cabinet with laminar flow, whole-heart scaffolds were detached from the perfusion line, removed from the
organ chamber, and placed in a sterile surgical pan. Primed scaffolds were seeded with ~500x106 human
iPS-derived cardiomyocytes (unsorted) by intra-myocardial injection, into the epicardial surface of the
left ventricular free wall, between the LAD and left circumflex coronary artery (LCX; Fig. 4D).
Recellularized whole-heart scaffolds were then reattached to the perfusion line, the organ chamber was
sealed and returned back to the 37°C incubator, and cells attached under static culture for 3-4 hours. After
the static period, perfusion culture was started at 20 mL/min for the first 12 hours, and then increased to
60 mL/min on day 2. Media volume was increased to 1.5 L at the beginning of static culture, and changed
every 48 hours. Functional tests were performed after 14 days in culture. Six hearts were recellularized
and cultured in this fashion, which are listed Supplemental Table I.
Functional analysis of Human Acellular Whole-Heart Scaffolds. Metabolic Analysis: Pre-perfusion

media samples were collected from freshly prepared RPMI/B-27, prior to adding it to the bioreactor for
coronary circulation. Post-perfusion media samples were collected via the coronary sinus duct tubing,
every 48 hours (just before the next media exchange). Media samples were analyzed using an i-STAT®1
Analyzer (cat. # 04P75-01, Abbot Laboratories Inc., Abbot Park, IL) with CG8+ and CG4+ i-STAT
cartridges (cat. #s 03P88-25 and 03P85-25, respectively, Abbot Laboratories Inc.). Epicardial Force
Measurement: As defined intramyocardial regions were difficult to isolate without compromising whole-
heart scaffolds, contractile force was measured on recellularized whole-hearts using a method similar to
that used on recellularized myocardial slices. Briefly, a cantilever was attached to an Aurora Scientific
403A Force Transducer system, which was then lowered onto contractile cardiac tissue to detect uniaxial
deflections. LV Pressure Development: Left ventricular pressure traces were obtained using a calibrated
pressure transducer (cat. #: SPR-671; Millar, Inc., Houston, TX) that was guided through a pilot port and
inserted into the lumen of the LV balloon. The LV balloon was then prefilled to an isovolumetric pressure
of 20 mmHg, and pressure fluctuations were recorded within the closed volume. The pressure transducer
signals were recorded using a Transducer Amplifier Module (cat. # D-79232, Hugo Sachs Elektronik /
Harvard Apparatus, Holliston, MA), using a 150 Hz high-pass filter and a 0.1 Hz low-pass filter. Pressure
9
CIRCRES/2015/306874D/R2
signals were outputted to a PowerLab 16/35 digital acquisition device (cat #: PL3516; AD Instruments
Inc., Colorado Springs, CO), and then analyzed with LabChart software (cat #: MLU60/8; AD
Instruments Inc.). Electrical Pacing, ECG, and Electrical Activation Mapping: Electrical pacing was
conducted with a Grass S88 Stimulator (10-80 V, 5 ms, at 0.8 Hz; Grass Technologies, Warwick, RI) and
implanted temporary cardiac pacing wires (cat. # TPW50, Ethicon, Somerville, NJ) located at the border
of the recellularized volume. Electrocardiograms were recorded using an ECG Amplifier System and
micro-electrodes (cat. # D-79232, Hugo Sachs Elektronik / Harvard Apparatus, Holliston, MA), using a
150 Hz high-pass filter and a 0.1 Hz low-pass filter. ECG signals were outputted to a PowerLab 16/35
digital acquisition device (cat #: PL3516; AD Instruments Inc., Colorado Springs, CO), and then analyzed
with LabChart software (cat #: MLU60/8; AD Instruments Inc.). For electrical activation mapping, a
custom plaque electrode was constructed from an 8x8 PGA socket with gold/nickel plating (cat. #: 522-
13-064-08-000001; Mill-Max Manufacturing Corp., Oyster Bay, NY) to create an 8x8 pin array with 2.54
mm spacing between pins. Each socket in the 8x8 pin array was coupled to an input on a Prucka
Engineering CardioLab Amplifier (GE Healthcare, Wilmington, MA). The 8th row of pins in each column
were designated as reference channels, and electrical signals were recorded with a CardioLab 4000 EP
Recording System (GE Healthcare, Wilmington, MA), with a sampling rate of 1 kHz, and using a 150 Hz
high-pass filter and a 0.1 Hz low-pass filter.
Force and Pressure Trace Analysis. Force generation traces acquired from recellularized myocardial
slices, fibers, and recellularized whole-hearts were processed using custom Python scripts using SciPy16-18
to extract pulse amplitudes, periods, time to X% relaxation, and dominant frequency. A set of
approximately 8-12 pulses were analyzed per biological sample per condition and extracted parameters
were averaged within a set to obtain representative values. Amplitudes were calculated as the absolute
value between baseline and pulse peak. Periods were calculated as the time distance between consecutive
pulse peaks. Time to X% relaxation was calculated as the time distance between pulse peak and the signal
amplitude reducing by X% of peak amplitude. Dominant frequency was measured by first calculating a
fast-Fourier transform of the pulse set and then choosing the frequency corresponding to the highest peak
in the frequency domain. Left ventricular pressure traces from recellularized whole-hearts were also
processed using custom Python scripts to extract pulse peak pressure, maximum & minimum dP/dt, and
tau. Pulse peak pressure was calculated as the absolute value between baseline and pulse peak. Maximum
& minimum dP/dt was calculated as the maximum & minimum values of the derivative of the pressure
trace with respect to time within one pulse period. Tau was calculated by fitting an exponential decay
equation to the pressure trace between minimum dP/dt and the start of the next pressure pulse.19
Transmission Electron Microscopy. Tissue was fixed in Karnovsky fixative of 2.5% glutaraldehyde plus
2% paraformaldehyde and stored overnight at 4°C. Tissue was washed of fixative in 0.1 M sodium
cacodylate buffer (pH 7.2) then postfixed in 2% OsO4 in sodium cacodylate buffer, dehydrated in a
graded alcohol series, and embedded in Epon t812 (Tousimis, Rockville, MD). Ultrathin sections were cut
on a Reichert-Jung Ultracut E microtome (Vienna, Austria), collected on uncoated 100-mesh copper
grids, stained with uranyl acetate and lead citrate, and examined on a Philips CM-10 transmission electron
microscope (Eindhoven, The Netherlands).
Statistical Analysis. Results for coronary flow, vascular resistance, and biochemical analysis are
presented as mean ± standard deviation (SD). Results for mechanical testing of myocardium are
presented as mean ± standard deviation (SD). Comparisons for coronary flow and vascular resistance we
performed using a 2-way analysis of variance (ANOVA) for solution type and cause of death, followed
by the Tukey post hoc comparison test. Comparisons for biochemical analysis and biaxial testing on
myocardium were performed using a student’s t-test. In all tests, differences were considered statistically
significant at a value of p < 0.05. Data for area strain and force-curve metrics of myocardial slices are
represented as mean ± standard deviation, and comparisons were performed using a 2-way ANOVA with
10
CIRCRES/2015/306874D/R2
a Tukey post hoc test for unstimulated versus stimulated conditions. All plots (except for Fig. 3J) were
generated using the Python 2D plotting library matplotlib20.
For each study, preliminary data analysis or pilot experimentation was performed to determine the
magnitude of the effect between study groups and this data was used to perform a power analysis to
calculate sample size requirements. The sample size for animal studies was based on sample sizes used
for similar studies, previously described in the literature.21 As a pre-established criteria triaged by the
NEOB, donor hearts with a known medical history of coronary artery disease were excluded from our
study. Randomization was based on the availability of decellularization chambers and acquisition of
donor tissue. For example, if the decellularization chambers were occupied, the donor tissue was allocated
into the control group. No randomization methods were employed for animal studies. Based on the
processing technique utilized to prepare the samples, blinding was not incorporated into the analysis of
the experiments, nor was it incorporated in animal studies.
11
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Supplemental Table I. Donor heart data
Cause of
Heart Decell, Sex Age Height Weight BMI
Death Experimental Allocation
ID Control M, F DCD, DBD yrs. cm kg kg/m^2
1 D NA *** *** *** *** *** Establishment of the perfusion decellularization process; Matrix histology
Establishment of the perfusion decellularization process; Matrix histology; Biochemical analysis;
2 D M B 36 177.80 86.18 27.20
Sterility testing
3 D M B 72 177.80 105.69 33.40 Establishment of the perfusion decellularization process; Matrix histology
4 C NA *** *** *** *** *** Matrix histology
Refinement of perfusion decellularization; Flow/Resistance analysis; Matrix histology; Sterility
5 D M B 41 170.18 98.88 34.22
testing
Refinement of perfusion decellularization; Flow/Resistance analysis; Matrix histology; Sterility
6 D F C 52 160.02 76.20 29.80
testing
7 C NA *** *** *** *** *** Biochemical analysis; DNA content; Matrix histology; Biaxial mechanical testing
8 D M C 57 185.42 115.21 33.46 Flow/Resistance analysis; Biochemical analysis; DNA content; Proteomics
9 D M B 52 177.80 90.72 28.31 Flow/Resistance analysis; Matrix histology; Sterility testing
10 C M B 61 177.80 118.39 37.45 Biochemical analysis; DNA content; Biaxial mechanical testing
11 D F C 47 162.56 69.85 26.49 Flow/Resistance analysis; Biochemical analysis; DNA content; Proteomics; Sterility testing
Flow/Resistance analysis; Biochemical analysis; DNA content; Proteomics; Biaxial mechanical
12 D M B 57 170.18 83.91 28.97 testing; Sterility testing
13 D NA *** *** *** *** *** Flow/Resistance analysis; Sterility Testing
14 C M B 44 167.64 67.13 23.84 Matrix histology; Matrix mechanics
15 C M C 58 177.80 78.93 24.90 Vascular histology; Matrix mechanics
16 D F C 48 162.56 70.76 26.83 Flow/Resistance analysis; Biochemical analysis; DNA content; Proteomics
17 C M C 47 167.64 99.79 35.43 Flow/Resistance analysis; Biochemical analysis; Matrix mechanics
18 C M B 67 165.10 64.86 23.88 Biaxial mechanical testing; Biochemical analysis; Matrix histology
19 D F B 64 157.48 132.90 53.63 Flow/Resistance analysis; Biochemical analysis; DNA content; Proteomics
20 D M B 73 172.72 59.87 20.11 Biaxial mechanical testing
21 C M B 59 187.96 120.66 34.10 Biochemical analysis; DNA content
22 D M C 56 177.80 89.36 28.22 Biochemical analysis; Biaxial mechanical testing
23 D M B 53 182.88 99.79 29.90 Biochemical analysis; Biaxial mechanical testing
24 C M B 51 165.10 67.13 24.39 Vascular histology; Matrix mechanics
25 C M B 18 175.26 73.03 23.78 Biaxial mechanical testing
26 C F B 64 157.48 59.42 24.10 Biaxial mechanical testing
27 D F B 73 162.56 67.13 25.46 Flow/Resistance analysis; Matrix mechanics
28 D F B 59 162.56 113.85 43.14 Biaxial mechanical testing
29 D F B 78 165.10 63.96 23.48 Vascular analysis by angio
30 D F B 56 152.40 54.88 23.81 Vascular analysis by angio
31 D F B 64 160.02 83.01 32.37 Sterility testing
32 C M B 50 175.26 91.17 29.74 Vascular analysis by angio; Matrix mechanics
33 C M B 56 165.10 69.85 25.61 Vascular analysis by angio; Matrix mechanics
34 C F C 65 170.18 57.15 19.68 Micro-vascular analysis with contrast dye; Matrix mechanics
35 C F C 45 172.72 77.11 25.78 Micro-vascular analysis with contrast dye
Sterility testing; Recellularization (436.4 million cells); Perfusion culture; Force-generating
36 D M C 50 185.42 84.82 24.67
contraction testing; Histology
37 C F B 72 165.10 89.81 33.46 Micro-vascular analysis with micro-beads; Matrix mechanics
38 C F B 76 162.56 83.46 31.52 Micro-vascular analysis with micro-beads; Matrix mechanics
39 C F B 72 177.80 74.84 23.72 Micro-vascular analysis with iron particles
40 C M C 52 177.80 71.21 22.46 Micro-vascular analysis with Microfil®
41 C M C 61 172.72 88.45 29.71 Micro-vascular analysis with Microfil®
42 D F B 47 160.02 75.30 29.33 Biaxial mechanical testing
43 C M C 24 185.42 142.43 41.83 Vascular analysis by angio
45 D F B 56 157.48 49.90 20.16 Matrix slice recellularization
46 D M C 26 180.34 69.85 21.52 Matrix slice recellularization
47 D F C 66 182.88 104.33 31.22 Matrix slice recellularization
Sterility testing; Recellularization (467.2 million cells); Perfusion culture; Force-generating
48 D M C 21 185.42 63.50 18.47
contraction testing; Ventricular pressure development testing; Histology
49 C F C 58 160.02 68.95 26.88 Biaxial mechanical testing
Sterility testing; Recellularization (520.1 million cells); Biomimetic culture; Ventricular pressure
50 D M B 26 177.80 74.84 23.72
development testing; Histology; Reseeded volume assessment (27.97%)
51 C F C 25 180.34 107.05 33.02 RNA/Histology controls
Sterility testing; Recellularization (513.7 million cells); Biomimetic culture; Metabolic analysis;
52 D M C 38 185.42 119.75 34.77
Force-generating contraction testing; Histology
53 C F C 67 167.64 72.12 25.82 RNA/Histology controls
54 C M C 55 177.80 91.17 28.85 RNA/Control cell isolation
55 D F B 33 175.26 74.84 24.37 Matrix slice recellularization
56 C F B 66 162.56 66.22 24.98 RNA/Control cell isolation
57 D M B 58 193.04 84.82 22.56 Sterility testing; Matrix fiber recellularization
58 D F C 62 165.10 64.86 23.88 Sterility testing; Matrix fiber recellularization
59 D M B 49 180.34 80.29 24.72 Biaxial mechanical testing
61 D M B 57 172.72 77.11 25.73 Sterility testing; Matrix fiber recellularization
Sterility testing; Recellularization (485.6 million cells); Biomimetic culture; Ventricular pressure
62 D M C 55 165.10 67.13 24.61 development testing; Electrophysiology testing; Histology; Reseeded volume assessment
(32.53%)
63 C F C 58 172.72 76.20 25.48 Immunological response study
64 D F B 59 160.02 49.90 19.53 DNA content; Immunological response study
Sterility; Recellularization (562.3 million cells); Biomimetic culture; Ventricular pressure testing;
65 D F B 61 162.56 59.87 22.71
Electrophysiology testing; Histology; Reseeded volume assessment (50.17%)
66 D NA *** *** *** *** *** Sterility testing
67 D F B 51 172.72 60.78 20.35 Micro-vascular analysis with Microfil®
68 C F B 24 154.94 49.90 20.69 Micro-vascular analysis with Microfil®
69 C M C 45 185.42 118.84 34.61 Proteomics; Matrix Histology
70 D M C 51 177.80 94.80 30.05 Sterility testing; Matrix fiber recellularization
71 D M C 28 170.18 74.84 25.89 Proteomics; Matrix Histology
72 C M C 55 162.56 73.03 27.62 Proteomics; Matrix Histology
73 C F C 58 162.56 70.31 26.52 Proteomics; Matrix Histology
Decell: 40 M: 36 DCD: 29 52.43 170.96 81.54 27.76 DCD - donation after cardiac death, DBD - donation after brain death, NA - Not Available;
Control: 33 F: 32 DBD: 39 ± 14.29 ± 9.53 ± 20.53 ± 6.09 Highlighted hearts were recellularized.
Supplemental Table II. Decellularized Myocardium Peptide List
Decellularized Unique Proteins Spectral Counts Normalized Relative Abundance

Peptide Unique Confidence
Accession count peptides score Average St.Dev. Average St.Dev.
FBN1_HUMAN 114 109 9904.63 140.0 42.9 1134459622.73 113563050.71
CO4A2_HUMAN 19 19 2460.61 30.5 16.7 392909648.31 204818539.63
PGBM_HUMAN 75 75 7029.81 77.2 32.4 173234761.32 79209570.13
ALBU_HUMAN 39 39 3214.59 45.0 14.0 93995455.22 22667320.51
CO4A1_HUMAN 13 11 1403.38 13.0 8.0 88725437.86 68364377.64
LAMC1_HUMAN 44 44 3922.51 45.5 19.8 77874517.83 34795554.75
CO1A2_HUMAN 6 6 739.61 9.3 2.1 57691467.21 38304993.53
CO6A3_HUMAN 53 53 3620.71 46.7 2.8 57040103.92 10328972.57
LAMB2_HUMAN 45 44 3265.47 39.5 15.1 40323169.36 7013367.30
CO1A1_HUMAN 11 11 669.65 7.3 3.7 37615581.18 22165708.19
LAMA2_HUMAN 58 58 3845.32 40.7 26.9 26730050.04 13352495.51
CILP1_HUMAN 11 11 768.48 10.2 2.7 23633730.01 4960837.89
SAMP_HUMAN 6 6 437.31 6.0 0.6 20298689.22 3104574.42
MYH7_HUMAN 57 56 3736.39 38.5 20.7 17121399.11 11082852.12
FINC_HUMAN 27 27 1620.67 18.3 2.7 13022664.19 7210508.05
LAMB1_HUMAN 35 34 2271.76 23.0 12.1 10916579.09 4939717.03
CO6A2_HUMAN 11 11 636.04 8.8 2.3 10251159.58 7288349.36
CO6A1_HUMAN 11 11 982.91 11.5 1.0 9752546.73 2716022.36
LAMA5_HUMAN 34 34 2053.05 23.8 11.1 8265030.58 2971613.06
AMBP_HUMAN 8 8 622.88 6.0 2.8 7469426.22 2723317.28
MFAP2_HUMAN 2 2 171.19 1.8 1.0 7071690.33 2885558.93
VTNC_HUMAN 9 9 569.26 8.5 5.6 6845741.61 4350939.33
MYG_HUMAN 8 8 629.84 7.7 4.5 5952768.86 3927977.02
FBLN2_HUMAN 9 9 676.01 8.8 5.3 5878464.78 2346330.34
FIBG_HUMAN 8 8 433.50 3.8 1.0 5423399.53 6957620.89
CO5A3_HUMAN 2 2 110.45 1.7 0.8 4965755.81 3166135.70
EMIL1_HUMAN 15 15 922.56 9.8 6.8 4836736.76 6679099.14
IGKC_HUMAN 5 5 642.50 6.5 4.5 4429234.91 2908278.66
MFAP5_HUMAN 4 4 326.96 4.2 0.8 4032517.47 819970.49
FBLN3_HUMAN 11 11 809.37 6.3 3.8 3847198.28 1370414.33
TGM2_HUMAN 4 4 222.29 2.8 1.2 3807822.04 4657576.70
FHL2_HUMAN 8 8 469.15 4.2 3.5 3519248.78 2589215.79
MYL3_HUMAN 6 6 382.08 4.7 4.2 3408789.25 2627481.39
TIMP3_HUMAN 5 5 278.14 4.8 3.9 3241670.07 2371628.18
TPM1_HUMAN 10 7 824.93 4.7 4.3 3157494.14 2444111.41
ACTA_HUMAN 11 4 679.29 2.7 1.2 3140768.20 2092546.71
EMIL3_HUMAN 3 3 136.44 2.2 0.4 3092769.12 1565337.97
TITIN_HUMAN 52 52 2607.61 19.3 25.1 2985047.58 3194076.38
IGHG1_HUMAN 8 4 556.69 3.8 2.3 2980569.66 218906.81
FBLN5_HUMAN 9 9 457.08 6.0 2.4 2965564.64 826387.14
FABPH_HUMAN 7 7 405.70 3.8 2.3 2870557.06 1999161.38
HBB_HUMAN 5 5 413.58 5.3 1.2 2814557.57 2546396.38
CSPG2_HUMAN 9 9 753.48 8.2 5.2 2240995.81 237278.79
LDB3_HUMAN 9 9 620.10 4.7 4.6 2222835.35 1619079.00
LUM_HUMAN 8 8 494.19 7.3 3.5 1878572.90 689315.57
MLRV_HUMAN 7 7 372.94 4.3 2.9 1878335.55 1338712.33
ACTG_HUMAN 11 4 771.65 1.7 0.5 1874289.91 2178446.07
BGH3_HUMAN 5 5 291.70 4.2 2.3 1806924.49 711985.03
TRFE_HUMAN 11 11 585.45 5.2 3.9 1778840.14 799749.52
HBA_HUMAN 3 3 150.24 2.0 1.5 1737273.80 1153257.67
FHL1_HUMAN 4 4 205.06 1.7 1.0 1735646.86 853480.58
DCD_HUMAN 3 3 195.98 2.0 0.6 1701191.96 920201.13
THSD4_HUMAN 8 8 380.23 5.5 1.4 1645757.40 1066703.86
PGS2_HUMAN 3 3 155.93 2.3 1.2 1513594.38 615280.62
KCRM_HUMAN 6 6 335.17 4.0 2.7 1467889.88 1062579.49
CO8A1_HUMAN 4 4 196.51 2.3 1.9 1421496.20 329543.12
NID1_HUMAN 12 10 717.88 7.2 1.9 1418056.35 361864.33
DEF1_HUMAN 2 2 81.15 0.8 1.3 1294704.05 745804.08
DESM_HUMAN 7 6 459.47 3.3 2.7 1255126.46 904383.57
TENX_HUMAN 8 8 408.70 3.8 2.1 1210271.29 1466079.79
ACTN2_HUMAN 14 10 779.50 4.5 5.1 1096251.57 946458.86
LDHB_HUMAN 4 3 209.40 1.8 1.6 1081138.29 1255579.63
DESP_HUMAN 15 15 714.06 6.0 4.9 1058153.91 882337.06
EMIL2_HUMAN 3 3 186.79 1.7 1.4 1044015.56 451779.63
FBN2_HUMAN 6 2 450.28 0.7 0.8 1032494.64 1137230.53
G3P_HUMAN 5 5 305.60 3.2 2.3 1020150.86 917179.35
ATPA_HUMAN 6 6 352.09 4.0 2.8 1018732.74 442714.28
FLNA_HUMAN 4 4 173.66 2.8 2.2 939750.54 426080.81
NID2_HUMAN 7 6 372.99 2.5 1.4 918812.78 180029.44
FBLN1_HUMAN 4 4 183.06 1.7 1.2 839379.81 198465.44
NPNT_HUMAN 4 4 180.65 2.2 2.0 826376.08 621585.44
ADT1_HUMAN 5 5 228.14 2.7 0.5 809191.41 537089.84
EF1A2_HUMAN 3 3 179.40 1.8 1.2 807001.41 912786.32
FIBA_HUMAN 3 3 140.43 1.8 1.0 738941.21 897967.47
DAND5_HUMAN 2 2 172.45 3.2 2.5 724917.12 563120.56
CSRP3_HUMAN 2 2 115.68 1.2 1.5 711830.81 467164.75
LTBP1_HUMAN 4 4 224.42 2.5 0.8 661821.29 165524.43
HSPB6_HUMAN 2 2 75.40 0.7 1.0 649178.01 958503.40
PDLI5_HUMAN 6 6 310.82 3.2 3.0 646156.22 653402.22
TSP4_HUMAN 4 4 180.37 1.8 0.8 636212.58 163572.94
ATPB_HUMAN 5 5 270.71 2.3 1.2 593105.51 410581.22
MFAP4_HUMAN 2 2 116.29 1.2 1.2 573380.74 674810.14
TNNT2_HUMAN 4 4 206.07 2.2 2.3 572443.67 521655.11
PTGDS_HUMAN 3 3 260.42 2.5 2.0 564175.58 276841.71
CO3_HUMAN 9 9 456.03 5.5 2.4 545527.66 232431.62
POSTN_HUMAN 8 8 448.02 3.7 0.8 541336.40 502617.84
MFGM_HUMAN 5 5 263.00 2.3 2.9 537076.82 572638.67
FIBB_HUMAN 3 3 166.05 1.3 0.5 519143.16 611350.01
ODO2_HUMAN 2 2 81.54 1.8 1.2 479863.43 385724.17
KCRS_HUMAN 5 5 361.19 2.3 2.2 468363.55 357113.89
HSP7C_HUMAN 3 3 182.53 1.8 1.2 445748.14 444074.62
IGHA1_HUMAN 4 4 278.55 2.3 2.2 437976.70 268764.42
MDHC_HUMAN 3 3 153.71 1.8 1.2 435107.50 397476.37
SRBS2_HUMAN 4 4 196.92 2.2 2.6 414832.84 542214.03
MDHM_HUMAN 4 4 214.16 2.2 1.7 413716.38 340224.14
ITIH1_HUMAN 2 2 99.67 0.7 1.0 413424.58 589136.52
COLA1_HUMAN 2 2 104.18 1.0 0.9 411518.08 199301.96
TINAL_HUMAN 4 4 250.49 1.8 1.6 411319.86 321478.88
TNNC1_HUMAN 4 4 175.82 2.2 2.4 395744.49 374252.75
CO4A3_HUMAN 3 2 203.90 1.0 0.9 395648.84 284166.58
ACON_HUMAN 5 5 284.16 2.0 1.8 383863.74 311518.42
AACT_HUMAN 4 4 200.76 2.2 0.8 381401.79 264040.48
CO9_HUMAN 3 3 157.36 1.7 1.4 373139.96 320761.93
COCA1_HUMAN 3 3 193.11 2.2 1.7 353756.26 185065.66
CBPA3_HUMAN 5 5 268.59 2.3 2.3 338067.52 347095.66
ELN_HUMAN 2 2 104.14 0.8 1.3 332289.62 490245.22
LYSC_HUMAN 4 4 238.30 2.5 1.4 329591.23 157009.98
KPYM_HUMAN 2 2 104.42 0.7 0.8 320242.60 220362.81
CRYAB_HUMAN 3 3 197.68 1.7 1.9 310610.00 284958.83
COIA1_HUMAN 2 2 141.37 0.5 0.5 300927.19 201407.22
CO4A4_HUMAN 2 2 102.68 1.7 0.5 297698.18 35710.17
RS27A_HUMAN 2 2 103.40 1.3 0.8 273093.52 98845.03
S10A8_HUMAN 3 3 143.19 2.0 0.6 265138.83 117495.15
AATC_HUMAN 3 3 148.15 1.5 1.4 261136.29 285900.55
C1QB_HUMAN 2 2 92.09 1.3 1.0 233300.22 178967.62
CISY_HUMAN 3 3 176.97 1.3 1.0 233286.33 195335.88
H4_HUMAN 3 3 181.55 1.5 0.5 228775.76 190323.46
CMA1_HUMAN 4 4 184.42 2.2 1.8 226940.40 219996.45
DERM_HUMAN 2 2 252.78 1.8 1.6 225281.55 185242.98
PGS1_HUMAN 3 3 159.07 1.7 1.6 224981.76 152205.79
TPIS_HUMAN 4 4 252.77 1.8 1.5 220638.19 164620.11
ATL4_HUMAN 2 2 97.27 1.0 0.9 208976.52 217956.95
SAP_HUMAN 2 2 72.85 1.2 0.4 205667.59 222385.95
ALDOA_HUMAN 2 2 123.39 1.0 0.0 197886.13 175366.40
VDAC1_HUMAN 4 4 254.41 1.8 1.3 192493.62 145396.41
1433T_HUMAN 2 2 81.84 1.2 0.8 189351.93 136041.32
COFA1_HUMAN 4 4 216.71 2.5 1.2 178476.71 43909.49
HEMO_HUMAN 4 4 164.10 2.0 1.3 176310.56 24733.16
S10A9_HUMAN 2 2 122.02 1.3 1.0 175597.79 108151.23
MYPC3_HUMAN 3 3 175.15 1.5 0.5 173998.03 121955.34
FLNC_HUMAN 5 5 234.17 1.5 2.0 153428.88 90055.86
MYOZ2_HUMAN 2 2 86.08 0.5 0.8 133080.55 116144.11
COSA1_HUMAN 4 4 187.23 2.0 1.9 130769.13 100544.07
AOC3_HUMAN 4 4 159.31 2.0 1.8 130039.32 78375.13
HSPB1_HUMAN 2 2 88.84 1.3 0.5 125040.56 121791.32
PLAK_HUMAN 5 4 377.29 1.5 2.3 123049.02 170244.38
CO4B_HUMAN 2 2 81.14 0.7 0.5 121139.91 115390.67
MYH10_HUMAN 3 2 133.17 0.8 0.4 116013.70 58682.89
PRELP_HUMAN 3 3 167.98 1.3 1.4 114585.11 76245.71
WNT2B_HUMAN 2 2 148.31 1.7 1.4 114450.60 104794.67
PRDX6_HUMAN 3 3 149.14 2.2 1.3 113436.74 102548.39
SDF1_HUMAN 2 2 102.01 1.3 1.0 108005.83 70025.60
S10A7_HUMAN 2 2 83.43 1.2 0.8 101343.43 70942.79
VINC_HUMAN 3 3 135.40 1.0 1.5 95236.46 124814.05
VDAC2_HUMAN 2 2 98.73 0.8 1.0 90389.36 63634.94
CTNB1_HUMAN 4 3 194.24 0.8 1.3 88372.90 128259.64
IDHP_HUMAN 2 2 87.62 0.7 1.0 81168.96 63768.29
ANXA2_HUMAN 2 2 100.73 1.0 0.6 77119.46 15924.58
ASPN_HUMAN 3 3 151.43 1.3 1.0 75426.50 46353.33
CO4A5_HUMAN 4 2 389.90 1.0 0.0 67806.73 23632.94
ECM1_HUMAN 2 2 91.94 0.7 0.5 62456.43 73007.52
ADH1B_HUMAN 3 3 128.16 0.8 1.3 60925.34 61095.13
HPLN1_HUMAN 2 2 163.77 1.2 1.0 59810.09 51965.44
PPIA_HUMAN 2 2 82.67 0.5 0.5 54416.10 43693.12
ETFB_HUMAN 2 2 93.67 0.5 0.8 52111.80 52699.77
A2MG_HUMAN 2 2 100.14 1.2 1.0 49957.26 14469.71
VIME_HUMAN 3 2 150.86 0.8 0.4 46230.72 37880.84
PLEC_HUMAN 5 5 234.49 1.7 2.6 45892.70 69309.68
COEA1_HUMAN 2 2 107.17 0.8 1.0 44703.39 12671.62
MYOM1_HUMAN 2 2 102.93 0.7 1.0 39608.93 20661.81
MYOM2_HUMAN 2 2 102.59 0.5 0.8 18298.29 27462.88
ACADV_HUMAN 2 2 94.48 0.7 1.0 14303.97 22430.82
Supplemental Table III. Cadaveric Myocardium Peptide List
Cadaveric Unique Proteins Spectral Counts Normalized Relative Abundance

Peptide Unique Confidence
Accession count peptides score Average St.Dev. Average St.Dev.
MYH7_HUMAN 207 61 22334 94.2 10.6 1854575662.79 628397285.59
MYL3_HUMAN 14 12 1447.81 19.7 3.2 1231120573.43 306612860.04
TITIN_HUMAN 892 886 63390.64 787.2 75.5 1153003445.10 420215261.86
MYG_HUMAN 15 15 2097.59 30.8 3.8 911754840.71 357883463.84
MLRV_HUMAN 23 22 1888.1 34.0 2.8 816283949.65 167636791.27
ALBU_HUMAN 40 40 3358.48 49.5 6.0 600583340.49 398831601.05
ACTN2_HUMAN 52 33 4733.75 43.3 2.6 529661082.52 116470483.59
HBA_HUMAN 7 7 533.82 9.2 4.7 525622887.36 463577092.84
KCRM_HUMAN 17 16 1329.27 16.7 2.6 445722331.08 115906289.75
FABPH_HUMAN 12 11 1059.39 15.3 2.5 407336289.62 143603773.66
TPM1_HUMAN 31 13 3208.73 22.8 2.4 357578925.61 95768016.59
DESM_HUMAN 37 30 2823.84 29.7 4.8 296016112.63 95657251.17
TNNC1_HUMAN 9 9 640.35 8.2 2.2 290778199.92 32058834.15
TNNT2_HUMAN 16 16 1404.97 19.7 4.2 267183886.78 69024475.72
ATPB_HUMAN 25 24 2263.85 29.8 4.0 264567874.74 86391064.78
KCRS_HUMAN 18 17 1606.19 18.3 3.9 252761362.05 59267865.72
MYPC3_HUMAN 56 55 4471.75 56.3 6.5 238963210.81 64545652.30
ACON_HUMAN 32 32 3077.83 37.0 2.8 238604118.92 96694854.61
ATPA_HUMAN 21 21 1639.34 23.0 2.4 238474035.74 91332222.17
MDHM_HUMAN 15 15 1416.41 18.8 1.6 228192925.22 54459026.13
HBB_HUMAN 12 6 1192.38 8.8 2.8 218933580.06 212200777.07
G3P_HUMAN 14 14 1359.43 20.5 2.5 202699396.66 50038286.22
TNNI3_HUMAN 13 11 822.38 11.3 1.6 182841865.43 34371794.76
MDHC_HUMAN 9 9 926.83 12.0 1.7 169243364.65 54488729.24
ALDOA_HUMAN 19 18 1429.76 17.7 2.9 115832106.71 35481954.60
CRYAB_HUMAN 10 10 868.25 12.8 2.5 112415463.70 19526271.57
CISY_HUMAN 11 11 747.08 11.5 1.9 110430482.19 34864746.82
IDHP_HUMAN 23 20 1714.67 21.7 2.1 107479796.67 58588930.16
CYC_HUMAN 9 9 679.14 11.8 1.6 106803087.30 30292759.05
LDHB_HUMAN 16 14 1184.44 13.2 3.1 104571577.82 43809906.27
ECHA_HUMAN 27 27 1812.57 25.7 2.3 94557599.42 15327145.50
MYOM2_HUMAN 55 55 4197.01 51.7 2.0 91209727.71 28291217.56
QCR2_HUMAN 10 10 1059.83 13.3 1.0 77065570.44 29954136.46
ECHB_HUMAN 23 23 1681.64 22.3 3.1 75930072.61 14154919.82
FLNC_HUMAN 66 59 5125.22 51.7 8.9 74964682.03 26011636.61
ACADV_HUMAN 27 27 2172.69 27.7 3.3 74453483.72 14150846.46
TPIS_HUMAN 12 12 1148.07 14.0 1.5 69778596.34 22314727.79
NNTM_HUMAN 26 26 1850.75 26.7 1.5 68572600.73 27119366.53
KPYM_HUMAN 15 15 1380.61 16.8 2.3 67203754.73 17437252.34
THIL_HUMAN 11 11 1037.42 12.3 3.8 64856093.06 13901850.63
AATC_HUMAN 15 15 998.75 14.3 1.9 64847121.18 22672319.97
MYOM1_HUMAN 51 51 4114.2 49.2 2.1 63683350.67 18212481.55
FHL2_HUMAN 20 20 1606.26 23.7 3.1 62265570.33 21456031.10
DECR_HUMAN 9 9 592.85 7.5 2.1 61652815.16 28558475.54
ETFB_HUMAN 13 13 1085.2 14.0 2.1 61013137.93 19138343.82
AATM_HUMAN 17 17 1110.85 16.8 2.1 60896255.80 16958683.73
COX5B_HUMAN 6 6 626.32 6.5 2.1 60885659.81 16946596.83
QCR1_HUMAN 12 11 989.14 11.3 2.5 57109883.04 22271251.60
SDHA_HUMAN 17 17 1501.37 17.3 1.9 56214836.13 19111502.41
COX41_HUMAN 10 10 682.73 9.8 1.9 54994746.63 10581661.57
DLDH_HUMAN 11 11 963.6 11.3 2.0 54677044.13 24160875.56
COX5A_HUMAN 8 8 629.76 8.7 1.2 50428742.23 9702224.71
PGK1_HUMAN 20 20 1352.9 19.7 2.2 49053907.55 13445781.91
ACADM_HUMAN 13 13 903.7 12.7 1.8 47878012.38 5701309.02
MPCP_HUMAN 9 9 419.43 7.5 1.2 47865120.84 22022743.86
SPTN1_HUMAN 69 69 4703.14 60.5 8.6 47299085.60 9859465.39
AT2A2_HUMAN 24 24 1978.1 21.0 3.7 46831382.30 20214695.82
ATPO_HUMAN 7 7 484.84 7.2 2.2 45587923.25 23734875.29
CH60_HUMAN 24 24 1485.33 20.7 3.1 42509009.34 3101949.18
LDB3_HUMAN 20 20 1416.63 18.2 1.2 40907964.17 4966998.24
ETFA_HUMAN 11 11 914.29 10.7 2.5 40560630.26 12410439.20
HSPB1_HUMAN 9 9 910.87 14.0 2.5 40206974.38 11418200.71
VINC_HUMAN 29 29 2222.95 25.5 2.2 40105929.47 7649098.60
VDAC1_HUMAN 11 9 838.42 8.8 1.8 39466585.00 10418463.01
COX2_HUMAN 3 3 132.51 2.5 0.8 36291374.54 18515738.32
ODPA_HUMAN 14 14 919 12.2 1.3 35820086.28 9788911.58
GRP75_HUMAN 19 19 1713.55 19.7 2.4 34616780.20 3735102.86
PEBP1_HUMAN 10 10 870.69 11.3 2.2 34323049.61 9993846.09
VDAC2_HUMAN 10 9 911.74 9.7 1.6 34165315.33 7486498.89
GRP78_HUMAN 21 20 1836.22 19.7 3.8 33732834.86 11290804.35
ADT1_HUMAN 15 5 1023.71 6.7 1.4 33460347.74 9993113.65
ODO1_HUMAN 29 28 2135.69 25.5 4.4 32946781.01 12901644.34
COX6C_HUMAN 7 7 447.66 7.8 2.8 31656691.17 11412031.20
NDUS1_HUMAN 21 21 1351.57 18.7 3.4 31562490.68 9004970.17
ATP5H_HUMAN 7 7 650.67 8.7 1.2 31222089.74 7473206.66
ALDH2_HUMAN 18 16 1165.14 14.5 1.8 29743750.86 10380757.43
H4_HUMAN 8 8 654.35 9.3 2.3 29518050.67 11666105.25
ODO2_HUMAN 8 8 512.79 5.8 1.0 29335170.74 1162981.80
CATD_HUMAN 9 9 617.9 9.5 1.9 29229702.49 5649646.14
THIM_HUMAN 13 13 771.66 11.2 2.6 28226866.89 13807959.74
HCDH_HUMAN 9 9 649.93 8.3 1.8 27719095.79 6414690.76
PARK7_HUMAN 9 9 617.56 8.0 1.4 27428360.24 5423314.31
HPT_HUMAN 16 16 1180.67 11.7 10.5 27239148.96 32112478.02
ECH1_HUMAN 10 10 636.94 8.5 1.0 27053753.04 2237630.11
PPIA_HUMAN 8 8 593.68 8.3 2.0 26905540.17 7225156.12
ODPB_HUMAN 10 10 963.34 10.7 2.6 26904838.48 11986182.11
VIME_HUMAN 25 18 1687.36 14.3 1.6 26450525.34 4868836.77
ATPG_HUMAN 8 8 529.42 8.2 1.8 26390728.75 11907819.42
NDUV1_HUMAN 15 15 1095.31 15.2 2.6 25925576.65 5970994.55
MIC60_HUMAN 22 21 1593.21 18.2 2.1 25885797.82 6729671.07
ATPD_HUMAN 3 3 239.08 3.2 0.8 25782130.47 9738204.41
PGAM2_HUMAN 10 5 790.07 5.7 1.4 25468404.15 6319462.73
HSP7C_HUMAN 23 15 1869.21 16.5 4.2 25245199.76 9028937.59
LMNA_HUMAN 25 24 1596.78 17.5 2.5 25024989.82 1694821.60
TRFE_HUMAN 23 23 1469.2 17.8 2.0 24879236.14 17396000.61
FBN1_HUMAN 42 40 2764.31 30.8 7.8 24426214.64 9667372.55
CSRP3_HUMAN 8 8 658.67 6.7 1.5 23711392.71 11684385.65
PRDX1_HUMAN 12 11 701.96 10.0 2.4 23193325.57 6319800.64
FUMH_HUMAN 12 12 783.95 9.8 2.0 22854087.71 12509345.83
ATP5J_HUMAN 5 5 699.86 6.0 2.9 22592046.44 22583868.61
ATP5L_HUMAN 5 5 520.76 7.0 0.6 22146742.98 6311263.06
PRDX5_HUMAN 8 8 753.68 9.7 3.7 21903910.48 12811441.83
PRDX2_HUMAN 7 6 459.36 6.8 1.3 21869152.87 7213635.87
ETFD_HUMAN 12 12 836 9.8 1.9 21751268.99 6957765.20
ALDOC_HUMAN 13 12 943.81 11.2 2.4 21654048.50 5703606.12
CO6A3_HUMAN 40 39 2150.7 30.2 2.8 21559243.25 6803778.73
HSDL2_HUMAN 16 16 1050.09 12.3 2.5 21375691.35 8220109.61
ACBP_HUMAN 9 9 744.01 9.5 1.6 21223290.27 4958680.04
MYOM3_HUMAN 33 33 2406.95 29.0 5.0 20780416.12 8699829.78
SUCA_HUMAN 5 5 380.07 4.8 1.2 20517843.61 10974676.63
ECHM_HUMAN 10 10 815.04 10.2 2.1 20212443.95 7302453.61
PRDX3_HUMAN 9 9 632.29 8.3 2.0 19441342.41 3567211.70
SDHB_HUMAN 9 9 871.57 10.0 1.3 19348395.34 3768484.62
AIFM1_HUMAN 16 16 964.48 11.8 2.3 19114296.64 4723406.32
ACTN1_HUMAN 23 6 2066.09 7.7 2.5 18915597.41 9439025.88
ODP2_HUMAN 12 11 761.99 10.3 1.5 18905076.01 3614193.80
AT5F1_HUMAN 9 9 577.03 8.3 1.6 18656052.79 6499375.91
SPTB2_HUMAN 50 45 3189.71 35.7 2.6 18411233.76 2691792.23
KCRB_HUMAN 10 9 821.67 10.3 1.6 18184264.80 6211327.13
HS71A_HUMAN 16 10 1114.55 9.8 0.8 18107743.24 3031470.71
ATP5I_HUMAN 4 4 202.97 4.2 1.5 18000962.16 4882968.36
ANXA6_HUMAN 17 17 1265.38 15.7 2.1 17636805.13 3728770.56
SRCH_HUMAN 9 9 1304.45 14.8 1.9 17241545.76 5979042.30
LAMC1_HUMAN 28 28 1840.48 21.5 3.5 17201029.07 5469476.06
KAD1_HUMAN 6 6 408.75 6.3 1.2 16960731.65 9672275.69
CH10_HUMAN 6 6 368.96 5.5 1.0 16841863.77 2944806.79
DMD_HUMAN 40 39 2305.67 28.0 5.6 16736991.98 4873670.17
PGBM_HUMAN 44 44 2722.11 32.7 4.5 16584451.72 576444.06
G6PI_HUMAN 8 8 609.14 7.8 1.5 16458202.99 4349357.15
IGKC_HUMAN 3 3 409.35 4.2 1.7 16243977.87 4421834.80
ENOB_HUMAN 14 11 1079.29 11.7 3.6 16146251.36 3311467.82
SUCB1_HUMAN 9 9 513.99 8.0 1.5 16058038.66 7888000.33
PRDX6_HUMAN 9 9 549.84 8.5 3.1 15981688.10 5127377.87
SODM_HUMAN 6 6 478.29 6.2 0.8 15644558.36 2729489.64
FHL1_HUMAN 8 8 483.74 5.8 1.2 15573643.24 841566.94
H31T_HUMAN 3 3 117.53 2.5 0.5 15442826.21 6200365.97
MYOZ2_HUMAN 7 7 563.3 6.8 2.5 15375897.10 5096248.68
UCRI_HUMAN 5 5 229.1 3.2 1.0 15267246.21 5410139.08
CY1_HUMAN 3 3 376.43 5.0 1.5 15213497.93 5154703.58
GELS_HUMAN 12 11 966.09 12.7 1.8 15158891.90 1508886.19
SCOT1_HUMAN 7 7 535.68 6.7 1.2 15130263.24 10096422.40
IGHG1_HUMAN 7 4 486.6 4.0 0.9 14850730.27 2216914.15
PDLI5_HUMAN 12 12 776.85 11.3 2.0 14777409.84 487655.52
LEG1_HUMAN 3 3 263.97 3.0 0.9 14760709.35 3491779.67
SRCA_HUMAN 13 13 1130.6 14.0 4.4 14701934.00 5142748.84
ACSL1_HUMAN 17 16 958.53 13.7 1.6 14683909.20 3120871.70
CASQ2_HUMAN 8 7 543.15 7.5 1.0 14646121.63 3085792.60
PHB_HUMAN 8 8 431.87 6.5 0.8 14605026.57 3402302.48
NDUS2_HUMAN 9 9 465.43 8.3 1.2 14395940.87 4153095.49
SRBS2_HUMAN 12 11 629.89 7.7 2.2 14329005.95 4438072.12
H2AZ_HUMAN 3 3 125.52 2.2 1.0 14243750.20 5432318.02
PFKAM_HUMAN 15 12 1171.21 11.8 2.1 14241924.00 1473006.90
MYL4_HUMAN 11 8 945.75 8.2 4.3 13794721.80 13665481.00
NEBL_HUMAN 19 18 1110 15.0 2.5 13792852.48 3647200.49
PLEC_HUMAN 54 53 2930.55 39.7 6.0 13589272.99 4984922.48
USMG5_HUMAN 4 4 424.85 5.5 1.2 13242426.37 4118975.11
QCR7_HUMAN 6 6 336.65 5.2 1.2 13003641.63 3059145.69
HSPB6_HUMAN 5 5 385.08 6.3 1.2 12973944.73 2983427.66
LAMA2_HUMAN 39 39 2453.58 28.7 4.3 12873899.56 3103045.69
XIRP1_HUMAN 31 30 1690.71 21.2 7.0 12700350.88 7075656.44
PDIA1_HUMAN 16 16 1058.29 15.0 1.8 12375618.11 1466144.13
CMC1_HUMAN 18 18 936.41 12.5 1.5 12306125.95 2808107.18
NIPS2_HUMAN 5 4 357.57 4.8 1.9 12196057.30 3655593.44
TGM2_HUMAN 10 10 767.32 9.3 1.9 12173535.97 2427431.74
ACTC_HUMAN 26 4 2691.57 7.5 2.1 12118176.69 7545032.98
3HIDH_HUMAN 7 7 837.86 8.8 0.8 12062623.48 2467276.95
LDHA_HUMAN 9 7 613.93 6.7 2.0 12062261.52 4521927.34
COQ9_HUMAN 2 2 324.19 2.2 0.8 12058852.32 2381317.13
A1AT_HUMAN 12 10 697.8 8.5 0.8 11948656.79 7208264.17
ANXA2_HUMAN 13 13 804.95 13.7 2.3 11896744.63 2853462.87
NDUV2_HUMAN 9 9 695.53 8.3 0.8 11786741.16 3061252.60
NDUA4_HUMAN 3 3 145.27 2.5 0.8 11779921.26 7019019.10
CX6B1_HUMAN 4 4 306.42 4.2 1.3 11558910.83 7766095.88
KAD3_HUMAN 7 7 540.03 8.8 2.0 11551375.70 3467937.12
MYH6_HUMAN 155 17 15602.32 11.0 9.0 11410618.16 10913929.67
CO6A1_HUMAN 13 12 1049.48 12.5 1.9 10961783.11 4439084.48
NDUBA_HUMAN 7 7 376.46 6.3 1.4 10929724.58 3670774.10
ECI1_HUMAN 5 5 464.85 6.2 1.5 10910697.52 2506518.43
NDUB9_HUMAN 6 6 399.62 4.7 1.6 10793511.21 2493148.38
MIC19_HUMAN 6 6 515.75 6.2 1.2 10768828.53 2373786.20
TERA_HUMAN 17 17 1020.43 12.5 1.2 10505732.98 2830207.66
1433E_HUMAN 14 11 1026.4 11.2 2.3 10319061.33 2549299.30
FIBB_HUMAN 11 11 738.89 8.7 4.3 10140592.84 7225437.36
PDIA3_HUMAN 14 14 773.99 12.0 3.5 9932780.70 1248563.92
NDUA9_HUMAN 8 8 394.38 6.5 1.4 9847586.30 3642668.93
NDUS6_HUMAN 3 3 270.95 2.8 0.8 9843046.68 2768383.53
NDUA7_HUMAN 7 7 430.4 5.7 2.3 9792591.59 2687104.28
NDUBB_HUMAN 2 2 89.67 1.7 0.5 9671053.47 3853648.97
HS90A_HUMAN 20 10 1290.41 8.2 0.8 9654315.69 1213270.18
DHE3_HUMAN 13 13 791.38 10.5 1.9 9588313.87 3007006.71
HS90B_HUMAN 21 12 1254.31 9.2 1.2 9558615.74 2058065.68
MMSA_HUMAN 15 15 879.71 12.3 4.8 9483813.16 6224701.33
ALDR_HUMAN 7 6 379.49 5.2 1.0 9453050.95 2938151.71
GPD1L_HUMAN 10 9 559.76 6.8 1.6 9360949.52 6223633.23
CALM_HUMAN 4 4 238.47 4.8 1.0 9357599.30 2742369.65
GSTO1_HUMAN 8 8 410.77 6.8 1.8 9333662.18 3356637.24
NDUS5_HUMAN 6 6 392.84 5.2 1.0 9223719.91 3813173.18
LAMB2_HUMAN 28 25 1828.42 20.2 3.5 9075782.96 3073210.17
CPT1B_HUMAN 12 12 704.43 9.5 2.4 9053996.40 3749905.47
CAV1_HUMAN 3 3 148.43 2.0 0.6 8999523.03 2980787.80
VDAC3_HUMAN 7 5 467.27 5.0 0.9 8931699.66 1281153.58
HXK1_HUMAN 17 16 928 12.3 3.0 8833926.91 2057261.84
NDUAD_HUMAN 6 6 381.55 4.8 1.7 8782975.12 3277090.67
PGAM1_HUMAN 10 5 786.45 5.5 1.0 8588684.15 2401216.65
NDUB7_HUMAN 5 5 316.69 5.8 0.4 8373699.42 209243.94
CO6A2_HUMAN 8 8 491.44 7.2 1.3 8263898.69 1544891.54
FABP5_HUMAN 8 8 586.41 6.0 0.9 8220891.01 3681897.80
FABP4_HUMAN 4 3 235.86 2.8 0.8 8093734.58 2033533.99
NB5R3_HUMAN 6 6 316 4.5 1.9 8021052.17 1811794.87
NDUAA_HUMAN 8 8 570.21 7.7 1.2 7806255.51 4266911.57
CATB_HUMAN 7 6 481.13 5.8 1.5 7702209.68 1986614.06
CALR_HUMAN 10 10 623.31 8.5 1.4 7664279.78 2409983.74
NDUA2_HUMAN 6 6 500.81 5.5 1.4 7608920.29 1753827.09
PPIF_HUMAN 4 4 178.05 2.3 1.0 7585180.18 1447794.01
ENOA_HUMAN 9 8 794.36 8.3 1.4 7558895.06 396503.83
PHB2_HUMAN 8 8 520.36 6.5 1.8 7490986.89 1820456.58
TPM2_HUMAN 19 4 1861.85 3.5 2.2 7283297.20 2162188.25
NDUA5_HUMAN 5 5 350.12 4.8 0.8 7223900.34 961810.16
S10A6_HUMAN 2 2 79.89 1.7 0.5 7210978.45 2493476.17
RINI_HUMAN 11 11 753.62 8.2 0.8 7202311.81 1373958.69
NDUB3_HUMAN 4 4 165.03 3.3 0.8 7036856.08 1470380.39
PGM1_HUMAN 8 8 446.92 6.2 1.2 7030431.62 1454277.90
CALX_HUMAN 9 9 685.66 9.0 1.4 6938344.94 1968235.80
CO3_HUMAN 23 22 1158.24 14.8 4.7 6867807.74 3695835.06
KAD2_HUMAN 7 7 401.23 4.7 1.0 6834917.13 1576802.27
IGHA1_HUMAN 5 5 335.69 3.8 1.6 6808506.12 3038932.55
CISD1_HUMAN 3 3 245.31 3.2 1.2 6800548.68 2228361.61
PYGB_HUMAN 18 13 964.61 10.7 1.6 6692930.73 837902.07
QCR8_HUMAN 4 4 245.36 4.2 1.2 6688888.01 3150028.37
ANXA5_HUMAN 6 6 288.66 4.8 1.6 6646748.61 2788568.06
PTRF_HUMAN 8 8 755.76 7.5 2.3 6644887.08 1585846.11
MYH9_HUMAN 25 22 1623.8 15.7 4.7 6602480.47 2075683.92
FLNA_HUMAN 31 27 1991.72 19.8 2.6 6597602.64 1106564.07
ACOT1_HUMAN 8 8 446.53 5.3 0.8 6576500.70 3861030.89
ASAH1_HUMAN 6 6 259.53 4.2 1.0 6557031.70 1521885.73
EF2_HUMAN 12 12 750.08 9.5 2.3 6521596.07 692811.80
DESP_HUMAN 28 28 1442.19 18.2 2.4 6463057.54 612810.71
IGHG2_HUMAN 6 2 314.99 1.7 0.5 6380822.26 2403450.64
ACTS_HUMAN 25 3 2329.18 3.7 1.2 6277224.07 3109689.74
SLMAP_HUMAN 11 11 594.15 8.2 2.1 6200956.25 1623490.04
AHNK_HUMAN 36 36 1967.23 18.5 12.7 6033743.64 3509527.64
AT1B1_HUMAN 5 5 368.65 5.0 1.7 6019239.86 909913.41
ACADS_HUMAN 8 8 606.79 6.8 1.5 5973280.53 2171606.68
HEMO_HUMAN 7 7 434.65 5.0 1.3 5951004.05 5448321.03
FIBG_HUMAN 9 9 673.47 6.7 2.4 5867230.17 3530690.55
AL4A1_HUMAN 8 8 499.85 6.3 1.4 5699815.68 2567611.94
RT36_HUMAN 5 5 556.37 7.5 2.3 5589813.19 2876121.74
CYTB_HUMAN 3 3 315.23 4.0 1.3 5530407.36 2958280.16
NDUS3_HUMAN 8 8 503.53 6.5 1.5 5497883.55 2290110.18
PGM5_HUMAN 8 8 520.93 6.2 1.7 5471351.74 1585077.08
1433Z_HUMAN 7 4 426.84 2.8 1.0 5465791.50 885491.80
ENPL_HUMAN 11 11 523.79 7.8 2.9 5423087.67 1811483.41
THIO_HUMAN 2 2 125.31 1.7 0.5 5265516.47 1848388.07
ADHX_HUMAN 6 6 405.7 6.2 1.5 5251161.15 1475543.35
PPLA_HUMAN 2 2 71.94 1.7 0.8 5198023.78 2217525.12
1433G_HUMAN 6 2 404.11 2.2 0.8 5185233.99 1308335.89
CAD13_HUMAN 7 7 612.39 7.0 2.0 5088895.08 2772116.19
GLYG_HUMAN 7 7 398.48 4.5 1.0 5073030.18 1105012.15
SUCB2_HUMAN 8 8 507.92 5.2 1.2 5039729.94 2565937.09
CRYM_HUMAN 7 7 368.42 5.0 0.9 5016270.45 1201551.56
CD36_HUMAN 6 6 478.01 5.2 0.8 4967776.49 1890435.84
NACAM_HUMAN 20 18 1105.15 11.2 6.6 4925323.85 4300355.24
K22E_HUMAN 5 3 270.86 0.8 1.3 4908309.44 729793.85
NDUB4_HUMAN 4 4 314.7 4.2 1.0 4861908.93 2841140.86
PROF1_HUMAN 4 4 255.85 4.2 1.3 4744948.50 609922.98
TPP1_HUMAN 3 3 251.74 2.8 0.8 4705501.60 694596.57
GLRX1_HUMAN 3 3 198.86 3.3 0.8 4698821.23 1804363.82
BASI_HUMAN 6 6 381.82 5.5 1.5 4611953.04 884440.05
ADH1B_HUMAN 7 7 336 4.8 1.5 4604245.82 2119067.91
IDH3A_HUMAN 5 5 329.99 4.7 1.0 4509066.42 1376216.76
UGPA_HUMAN 8 8 534.15 6.7 0.5 4470139.40 1202064.33
AOFA_HUMAN 11 7 640.73 7.0 1.5 4447283.79 537630.62
AOFB_HUMAN 9 6 572.46 5.0 0.9 4418626.19 759605.85
DPYL2_HUMAN 11 11 528.42 6.2 1.5 4418505.59 1429738.23
S10A8_HUMAN 4 4 283.62 3.7 1.8 4395707.93 3059666.96
GSTP1_HUMAN 2 2 82.98 1.7 0.5 4325913.31 933213.16
DHPR_HUMAN 6 6 517.22 5.3 1.4 4304415.34 773313.49
LUM_HUMAN 6 6 264.14 4.8 0.8 4287966.67 1329857.15
GLO2_HUMAN 6 6 426.51 5.5 1.2 4255997.40 762464.83
HSPB7_HUMAN 3 3 276.71 2.8 1.0 4246363.01 851454.94
CACP_HUMAN 8 8 381.36 4.8 1.2 4173879.66 841608.84
NDUAC_HUMAN 6 6 357.92 4.7 0.8 4163068.58 1480801.72
MYL6_HUMAN 6 5 359.27 3.8 1.2 4159834.59 922736.94
PDLI1_HUMAN 7 7 490.1 5.3 0.8 4149027.24 931970.07
A2MG_HUMAN 14 14 816.32 8.3 4.6 4127055.38 2521293.21
SMYD1_HUMAN 9 9 567.43 6.8 2.9 4006422.35 935967.82
TACO1_HUMAN 3 3 167.73 1.5 1.0 3906773.36 1069076.33
LAMB1_HUMAN 12 11 721.18 7.0 2.2 3891409.98 1227952.82
RAN_HUMAN 3 3 197.23 2.0 0.9 3881487.98 4158904.70
ITB1_HUMAN 10 10 541.4 7.0 2.0 3874806.50 1175351.25
AT1A3_HUMAN 17 6 1240.33 5.0 1.4 3828119.57 894582.94
HBD_HUMAN 10 4 925.42 2.7 1.9 3744322.84 3830494.60
APOA1_HUMAN 11 11 633.85 4.8 6.7 3686916.88 4476713.29
ES1_HUMAN 3 3 264.54 3.8 1.7 3682758.43 3011033.22
NDUB1_HUMAN 2 2 70.87 1.5 0.5 3677505.34 1715193.85
MOES_HUMAN 14 9 848.81 7.3 1.2 3633386.20 289977.99
ODPX_HUMAN 6 6 306.9 4.8 1.9 3591193.21 1188737.33
MCCB_HUMAN 10 10 778.28 7.3 3.6 3588437.20 1843459.41
QOR_HUMAN 6 6 437.39 5.3 1.2 3528145.24 1113438.68
PCCA_HUMAN 10 10 579.93 7.2 1.2 3506728.92 536780.23
PLAK_HUMAN 11 9 705.06 7.3 2.1 3501068.82 402344.47
ECI2_HUMAN 7 7 611.01 7.0 1.4 3430522.76 1114719.19
PLIN4_HUMAN 13 13 885.5 7.3 4.6 3389545.27 1976966.46
H2B1B_HUMAN 3 2 145.55 1.5 0.5 3382813.30 2183397.89
PGS2_HUMAN 3 3 152.96 2.0 0.6 3352924.88 973460.95
VTDB_HUMAN 5 5 294.05 3.2 1.3 3347726.13 1216955.40
TMOD1_HUMAN 7 7 481.56 6.5 2.1 3344025.11 1353100.89
GDIR1_HUMAN 3 3 185.03 2.3 0.8 3341530.33 410291.43
CN159_HUMAN 10 10 708.68 6.5 6.6 3285643.77 3824516.92
EFTU_HUMAN 8 7 419.24 5.2 1.5 3280605.28 1062973.90
THTR_HUMAN 4 4 297.93 3.3 0.5 3276421.93 1048691.33
ESTD_HUMAN 5 5 394.83 5.0 1.3 3267922.61 959464.81
ANX11_HUMAN 9 8 400.41 6.3 1.2 3252028.96 360357.58
AL9A1_HUMAN 7 7 427.32 5.0 1.1 3205292.65 97854.23
PCCB_HUMAN 10 10 629.96 7.7 1.2 3202452.37 875208.28
CPT2_HUMAN 10 9 530.1 6.5 1.2 3151786.20 410737.74
BDH_HUMAN 8 8 349.03 5.0 2.4 3125786.82 1724143.10
KAD4_HUMAN 4 4 245.13 4.7 0.5 3079244.59 957876.07
ACS2L_HUMAN 8 7 430.66 5.2 1.2 3077098.88 1611056.50
IVD_HUMAN 4 4 261.78 3.2 0.8 3065359.13 529624.88
SAMP_HUMAN 5 5 304.93 3.7 0.5 3059260.20 525013.96
NID2_HUMAN 11 11 801.19 9.0 1.4 3050724.11 226706.80
MCCA_HUMAN 6 6 324.25 3.8 1.6 3039027.09 1803284.71
QCR6_HUMAN 2 2 162.24 2.0 1.1 3035505.35 687697.25
CX7A2_HUMAN 3 3 191.54 2.2 1.2 3021825.68 1014773.61
GDE_HUMAN 12 11 654.09 7.7 2.0 3011970.53 1215071.14
NDKB_HUMAN 3 3 288.87 2.5 1.0 2956364.89 1121091.83
FKB1A_HUMAN 2 2 164.67 1.0 0.6 2948692.79 1272685.65
LEG3_HUMAN 6 6 378.33 4.7 1.9 2942813.21 774464.75
SSDH_HUMAN 6 6 336.36 3.8 0.8 2940556.00 1315243.88
NDUB8_HUMAN 6 6 304.03 4.5 1.9 2904925.00 767049.39
CADH2_HUMAN 5 5 394.83 4.7 1.0 2903976.31 1032439.70
MPC2_HUMAN 4 4 234.72 4.0 1.3 2898922.43 1280102.11
MAP4_HUMAN 13 13 662.76 7.5 4.2 2888552.42 1797816.37
NDUS8_HUMAN 3 3 125.88 2.5 0.8 2884109.58 989266.03
AACT_HUMAN 6 6 281.78 2.8 2.1 2877309.70 1381185.16
PKP2_HUMAN 9 9 513.47 6.2 2.3 2872489.03 503263.03
CD59_HUMAN 3 3 204.22 2.3 1.0 2856537.00 1660997.38
ACOC_HUMAN 9 9 450.84 6.0 3.1 2809749.85 774230.40
ANKR2_HUMAN 9 9 613.7 7.0 2.2 2796871.34 1953039.97
MIF_HUMAN 2 2 100.94 1.7 0.5 2777878.38 1362915.48
AFG32_HUMAN 12 11 486.6 7.0 2.0 2775634.27 1083068.00
SBP1_HUMAN 9 9 478.81 5.8 0.8 2767676.80 338749.20
M2OM_HUMAN 8 8 435.07 5.3 0.8 2763019.42 877231.34
RYR2_HUMAN 17 16 926.64 10.3 2.3 2761667.85 780609.73
NDUA6_HUMAN 4 4 209.14 2.8 0.8 2740138.43 958141.39
SDPR_HUMAN 5 5 369.93 3.8 1.2 2720364.21 260047.54
NID1_HUMAN 10 10 544.57 7.0 2.0 2682362.05 366997.42
IMPA1_HUMAN 4 4 271.41 3.5 1.4 2664806.96 398586.42
DCXR_HUMAN 3 3 225.47 3.2 1.2 2620535.88 815663.24
ACO13_HUMAN 2 2 126.42 1.7 0.5 2617277.85 1346086.03
WDR1_HUMAN 7 7 461.54 6.3 0.8 2543690.78 354139.50
CLH1_HUMAN 10 10 568.24 8.0 2.2 2518300.81 292720.35
PDIA6_HUMAN 6 6 377.32 4.5 1.0 2492711.70 655932.46
PSA6_HUMAN 6 6 424.22 4.7 1.0 2452903.44 832484.27
BAG3_HUMAN 8 8 430.32 5.0 2.2 2444920.97 1029177.36
GYS1_HUMAN 7 7 327.33 4.5 2.1 2439493.01 629780.46
CO4A1_HUMAN 4 3 305.99 2.5 1.0 2417950.22 625184.10
SAM50_HUMAN 6 5 381.23 4.3 1.0 2416632.82 801195.66
DCMC_HUMAN 8 8 571.43 6.3 2.1 2399541.82 769145.19
K1C10_HUMAN 9 9 650.36 5.5 2.4 2381749.35 2164058.96
CX7A1_HUMAN 2 2 131.16 1.7 0.5 2357059.47 402495.13
TAGL_HUMAN 4 4 192.5 3.0 0.9 2354564.19 700744.61
MTCH2_HUMAN 5 5 291.87 4.5 1.0 2348357.18 597547.47
AL7A1_HUMAN 8 8 530.21 5.3 1.2 2345468.52 468629.64
BCAT2_HUMAN 7 7 402.19 3.5 1.5 2276008.60 810485.24
IF5A1_HUMAN 4 4 267.82 5.0 1.3 2267340.87 701270.89
ANXA1_HUMAN 7 7 503.04 5.0 1.8 2242900.38 437055.75
MAOM_HUMAN 7 7 416.74 4.7 1.2 2212313.64 562777.64
OPA1_HUMAN 14 14 665.11 8.2 1.3 2200538.14 710197.85
NDUA8_HUMAN 4 4 198.7 3.2 0.8 2200306.36 533323.59
CO4A2_HUMAN 4 4 210.36 2.5 1.2 2167794.74 222766.30
TLN2_HUMAN 23 19 1286.95 12.8 2.2 2163494.97 431531.30
ARHL1_HUMAN 6 6 229.63 2.8 1.5 2148598.11 1380212.84
TLN1_HUMAN 18 14 1337.56 11.2 2.2 2135576.87 406184.62
CAH1_HUMAN 5 5 248.8 3.3 2.7 2130135.44 1399193.30
NDUS4_HUMAN 2 2 168.85 2.5 0.5 2111635.34 1320626.78
GSTK1_HUMAN 4 4 266.43 2.8 1.6 2092403.94 1224862.51
IDH3B_HUMAN 3 3 315.5 3.2 1.0 2083929.49 1292557.65
DOPD_HUMAN 4 4 214.49 3.0 0.6 2037836.48 1181327.02
LPPRC_HUMAN 17 15 803.18 9.7 1.9 2029835.35 774989.55
NDUAB_HUMAN 3 3 222.3 2.7 0.5 2012317.97 997516.42
AT1A1_HUMAN 18 9 1276.78 5.2 2.3 2002667.69 568164.55
2AAA_HUMAN 6 6 276.54 4.3 1.5 1985530.95 982472.45
DYHC1_HUMAN 18 18 870.26 11.8 0.8 1965874.49 325371.95
HCD2_HUMAN 5 5 330.8 3.3 2.0 1938856.20 871584.71
OBSCN_HUMAN 19 18 979.77 9.8 3.8 1936158.91 557721.27
VTNC_HUMAN 4 4 201.21 2.7 1.0 1934243.44 118009.28
PSA5_HUMAN 4 4 263.3 3.2 1.2 1930003.17 664098.24
CTNB1_HUMAN 6 4 372.24 2.2 1.2 1900265.43 364258.30
CFAB_HUMAN 6 6 330.37 2.7 1.9 1898422.80 824829.66
CTNA1_HUMAN 10 10 591.23 6.5 1.6 1883655.59 386922.45
H2B1L_HUMAN 3 2 148.24 1.7 0.5 1873007.17 784221.26
ABLM1_HUMAN 10 10 551.73 5.8 1.6 1866041.47 424914.44
ODB2_HUMAN 6 6 357.2 3.7 3.1 1858275.21 1325956.50
DYL2_HUMAN 3 3 253.93 3.3 0.5 1855693.56 284768.29
SAHH_HUMAN 8 8 332.45 5.8 1.6 1855012.51 631588.95
GSTM3_HUMAN 7 6 514.2 5.5 1.0 1829848.09 286847.38
SRBS1_HUMAN 15 13 709.55 8.7 1.6 1807556.65 138364.75
RS3_HUMAN 5 5 202.58 3.7 0.5 1804218.10 327171.00
PACN3_HUMAN 7 7 477.54 4.5 1.0 1782983.03 554671.78
TBB4B_HUMAN 14 2 1061.36 2.2 0.8 1771782.50 745823.63
FINC_HUMAN 5 5 225.43 2.7 1.4 1771214.53 146495.99
AUHM_HUMAN 6 6 318.2 4.3 0.5 1762507.71 857381.06
PYGM_HUMAN 11 6 515.2 4.3 0.5 1757176.36 336655.47
ATP5E_HUMAN 2 2 75.79 1.5 0.5 1747273.09 1211638.72
DAG1_HUMAN 5 5 247.74 3.5 1.2 1718455.46 499596.54
UBA1_HUMAN 7 7 531.62 5.8 1.2 1709924.96 448285.11
HEBP2_HUMAN 3 3 154.09 2.0 0.9 1697399.15 557855.11
VAT1_HUMAN 4 4 226.92 2.2 1.2 1682954.32 1357001.46
PIMT_HUMAN 2 2 227.48 2.8 0.4 1676043.59 513383.09
APOH_HUMAN 4 4 220.95 3.3 1.0 1670083.77 388514.95
F10A5_HUMAN 4 4 214.3 2.5 0.5 1660326.88 344047.34
ANXA3_HUMAN 6 6 353.13 4.8 1.2 1656549.43 419174.39
GPX3_HUMAN 3 3 196.21 2.5 1.0 1633513.45 817758.61
ODBA_HUMAN 5 5 226.46 3.3 2.3 1628574.28 852784.40
IPYR2_HUMAN 5 5 308.26 3.7 0.5 1621838.24 571140.88
GLU2B_HUMAN 7 7 393.28 4.7 1.4 1603032.83 229566.79
MIC27_HUMAN 4 4 213.96 3.0 1.4 1600997.41 730479.96
ATPK_HUMAN 3 3 178.55 2.3 1.4 1596730.27 840931.81
GRHPR_HUMAN 5 5 397.17 4.7 1.0 1593778.23 264251.34
ACTG_HUMAN 20 3 1519.04 1.3 1.0 1589881.10 190591.56
F162A_HUMAN 4 4 229.55 2.7 2.1 1583046.86 1035557.36
DYSF_HUMAN 11 11 692.17 7.2 1.5 1568487.30 402047.60
ACDSB_HUMAN 6 6 329.38 4.5 0.5 1563774.82 590364.96
CAPZB_HUMAN 3 3 165.81 2.8 1.2 1557305.07 426874.22
NCAM1_HUMAN 4 4 203.71 2.2 1.2 1543889.17 316925.18
SGCD_HUMAN 2 2 96.26 1.7 0.8 1537089.62 375023.86
MACD1_HUMAN 2 2 154.48 0.8 0.8 1531660.86 492237.86
PP1B_HUMAN 4 4 289.83 3.0 0.6 1530709.62 421113.52
CERU_HUMAN 7 7 458.33 4.5 2.3 1521838.87 1292235.09
TBA4A_HUMAN 10 2 607.89 1.5 0.5 1503198.32 325806.45
PSME1_HUMAN 5 5 296.57 4.2 1.2 1496027.89 683342.77
S10AA_HUMAN 2 2 76.68 1.5 0.5 1484168.92 299682.23
S10A9_HUMAN 2 2 219.41 2.0 1.7 1464645.26 995709.69
ITA7_HUMAN 5 5 237.55 2.5 1.0 1455495.78 168702.03
NPS3B_HUMAN 5 4 277.92 2.8 0.8 1430703.91 453711.32
K2C1_HUMAN 11 7 688.53 3.2 2.3 1424585.76 1083178.64
SSBP_HUMAN 2 2 127.67 1.7 0.5 1424391.00 593808.97
RLA2_HUMAN 4 4 238.94 2.8 1.0 1423055.31 319809.92
PCYOX_HUMAN 6 6 293.43 3.8 2.1 1421859.09 573323.22
1433T_HUMAN 7 3 446.45 3.0 1.1 1393617.55 659503.76
TAGL2_HUMAN 5 5 258.4 4.0 1.1 1375898.85 147653.60
L2HDH_HUMAN 5 5 242.76 3.7 1.8 1374223.01 293540.03
PPIB_HUMAN 4 4 212.37 3.3 0.8 1367226.34 357787.01
ICAL_HUMAN 8 8 440.12 3.5 3.1 1362787.48 1310303.57
PSA_HUMAN 10 10 563.16 7.0 1.5 1360776.93 330504.47
TCPQ_HUMAN 8 8 447.64 4.8 1.6 1358087.41 319864.33
EHD2_HUMAN 7 6 315.53 4.5 1.5 1344535.85 324161.27
GBLP_HUMAN 5 5 285.3 2.8 0.8 1326530.57 364016.22
AT1A2_HUMAN 15 5 1083.8 3.2 1.7 1316089.68 384631.79
PP2AA_HUMAN 5 4 307.17 3.8 1.3 1310879.94 346484.34
LETM1_HUMAN 5 5 292.1 3.8 0.8 1309395.26 525882.29
EHD4_HUMAN 9 8 510.93 4.3 1.6 1308066.36 345517.65
CAH2_HUMAN 3 3 192.96 2.5 1.6 1304851.93 928983.31
CATA_HUMAN 7 7 397.54 3.8 1.5 1280538.92 558362.81
TALDO_HUMAN 3 3 257.94 3.0 1.3 1276265.80 368135.92
MTX2_HUMAN 5 5 256.95 3.5 1.4 1259871.02 136086.53
RL10A_HUMAN 3 3 121.45 2.3 0.5 1258580.27 223088.92
PHP14_HUMAN 2 2 177.13 2.5 0.8 1245544.10 843414.57
FRDA_HUMAN 2 2 147.12 1.7 0.5 1241197.37 286393.94
NIBAN_HUMAN 6 6 358.04 3.2 1.6 1237907.69 576181.66
GSHR_HUMAN 3 3 154.41 2.0 0.9 1223061.73 336216.43
PGS1_HUMAN 4 4 289.26 2.5 1.4 1222801.53 399354.95
ML12A_HUMAN 4 2 251.88 1.7 0.5 1203590.69 345055.49
STIP1_HUMAN 5 5 249.78 2.7 0.5 1203535.27 448148.10
ACOT9_HUMAN 5 5 214.72 3.5 1.4 1201178.17 584683.29
LYPA1_HUMAN 3 3 116.17 1.7 1.0 1196419.61 100968.39
SGCA_HUMAN 4 4 244.83 2.8 1.3 1196025.17 414116.47
RLA0_HUMAN 4 4 321.02 2.8 0.8 1185752.67 287616.09
S10AD_HUMAN 2 2 143 1.8 1.2 1174326.75 387991.88
GDIA_HUMAN 6 5 406.98 4.0 1.1 1158429.96 358541.88
UK114_HUMAN 2 2 122.62 1.2 0.4 1146444.24 340019.71
CNDP2_HUMAN 5 5 259.41 3.2 0.8 1140190.54 217281.77
GSTM2_HUMAN 5 4 333.45 4.0 1.3 1138233.25 356087.39
NEXN_HUMAN 4 4 235.28 2.5 1.2 1136026.41 383621.68
TRXR2_HUMAN 5 4 509.58 3.8 1.0 1119518.25 328250.46
FRIH_HUMAN 6 5 324.58 4.0 0.9 1116293.99 123760.09
GANAB_HUMAN 4 4 223.93 3.2 0.8 1115111.60 122840.10
LMNB2_HUMAN 9 7 370.97 4.0 1.8 1113230.97 188997.80
IDH3G_HUMAN 3 3 145.82 2.0 0.9 1106473.64 324058.15
KAP2_HUMAN 5 4 364.55 2.5 1.4 1104444.97 437872.48
RHOA_HUMAN 3 3 142.56 2.0 1.1 1103645.59 252704.19
SPB6_HUMAN 4 4 237.15 3.2 1.0 1101195.27 317032.82
ACTN4_HUMAN 21 5 1622.87 3.3 1.2 1098979.77 221012.87
EF1G_HUMAN 4 4 212.02 2.3 0.8 1097115.87 193041.34
MECR_HUMAN 3 3 127.28 2.0 0.6 1095449.42 448552.89
RL12_HUMAN 3 3 190.33 2.2 0.8 1078094.57 248894.35
HIBCH_HUMAN 4 4 244.71 2.0 0.9 1077482.18 782015.71
NB5R1_HUMAN 3 3 145.67 1.8 0.8 1073430.86 246920.01
TM143_HUMAN 5 5 223.84 2.5 0.8 1068063.59 262961.83
MSRB2_HUMAN 5 5 210.24 3.7 1.0 1062188.63 309099.61
IDHC_HUMAN 7 6 446.5 5.3 1.8 1056458.00 274526.66
HINT2_HUMAN 2 2 117.23 1.5 0.8 1054515.45 486364.30
LACTB_HUMAN 4 4 183.23 3.5 0.8 1049968.19 475033.44
EZRI_HUMAN 9 4 551.11 2.8 1.0 1045178.41 486149.12
PSA1_HUMAN 5 5 270.96 4.2 1.0 1042645.02 359564.78
AMPL_HUMAN 5 4 274.98 3.0 0.9 1041835.12 62484.74
UB2V1_HUMAN 3 3 117.98 1.8 1.7 1031418.14 229629.67
PNPO_HUMAN 4 4 158.03 2.7 0.8 1027592.83 71374.45
PSB6_HUMAN 3 3 178.73 2.5 1.2 1025581.06 318452.15
ORN_HUMAN 3 3 161.95 2.3 0.5 1025061.54 178324.82
DHB4_HUMAN 4 4 175.94 2.3 1.0 1022154.59 310007.39
GDIB_HUMAN 4 3 229.64 2.2 0.8 1007673.29 315828.04
LG3BP_HUMAN 3 3 190.31 3.2 1.2 1005015.41 264373.38
NTF2_HUMAN 2 2 122.54 1.7 0.5 1003673.32 335822.75
CPNE3_HUMAN 5 5 223.79 3.5 0.8 999967.67 421704.99
SPTB1_HUMAN 13 9 531.59 5.0 1.3 982149.23 156718.08
RS4X_HUMAN 5 5 284.91 3.7 0.5 968387.93 173509.79
RL14_HUMAN 2 2 97.74 1.3 1.2 960667.66 539114.17
TELT_HUMAN 4 4 218.07 2.8 0.8 959286.05 122291.14
SAP_HUMAN 2 2 98.46 1.7 0.5 947281.00 80124.16
NEST_HUMAN 10 10 536.34 5.8 1.2 929790.62 208952.28
CAN2_HUMAN 5 5 252.13 3.2 0.4 925474.46 129708.32
TIM44_HUMAN 5 5 351.23 4.5 0.5 925382.25 213839.82
RS6_HUMAN 4 4 154.33 2.0 1.1 925236.79 231558.93
LONM_HUMAN 7 7 394.41 4.3 2.4 924236.66 366869.62
EF1D_HUMAN 7 4 388.6 3.3 0.5 923059.48 179335.90
RL5_HUMAN 3 3 231.85 2.7 1.2 909884.49 188880.13
SIR5_HUMAN 4 4 201.16 2.3 1.2 902183.12 544656.02
FETUA_HUMAN 2 2 160.02 1.7 0.5 868694.52 394681.04
RS8_HUMAN 5 5 305.73 2.8 1.0 858181.39 235998.33
CO9_HUMAN 2 2 81.88 0.7 1.0 855822.81 480698.72
SYWC_HUMAN 5 5 304.76 3.3 1.2 841975.84 169672.23
SYNEM_HUMAN 6 6 310.62 3.5 0.5 839779.80 198214.47
COF1_HUMAN 3 2 219.11 1.5 0.8 839708.81 356203.67
ACAD8_HUMAN 4 4 227.03 3.2 1.5 832395.84 203378.07
ROA2_HUMAN 3 3 187.05 2.2 0.8 819987.87 278757.65
RS3A_HUMAN 2 2 117.59 1.0 0.6 819351.00 173116.61
CRP_HUMAN 2 2 91.18 1.7 0.5 810281.47 523842.07
MOT1_HUMAN 2 2 133.49 1.7 1.4 801509.15 197942.68
SODC_HUMAN 2 2 166.98 1.8 1.2 800643.70 413375.95
RAB2A_HUMAN 4 4 225.51 3.0 0.6 800445.84 129628.03
HEMH_HUMAN 6 6 318.43 3.5 1.2 799777.55 165638.51
CPNS1_HUMAN 2 2 98.83 1.5 0.5 796403.58 219983.86
HOT_HUMAN 5 5 262.71 3.8 0.8 792490.99 190219.66
FUND2_HUMAN 2 2 136.94 0.7 0.8 792171.46 279304.14
NPS3A_HUMAN 4 3 223.66 2.7 0.8 790574.00 106836.89
RMD1_HUMAN 4 4 189.79 2.7 1.6 787973.16 360481.09
ISOC1_HUMAN 3 3 225.91 2.5 1.0 783636.40 269478.54
HMGB1_HUMAN 3 3 215.23 2.5 1.2 782372.58 238420.04
RL23A_HUMAN 2 2 112.16 1.2 0.8 773790.29 188738.14
TCPG_HUMAN 7 7 323.1 4.0 1.7 773474.34 185056.41
RS16_HUMAN 2 2 132.77 1.7 0.8 771758.52 208388.13
MUTA_HUMAN 4 4 222.81 3.0 2.5 769990.33 466756.21
SGCG_HUMAN 3 3 201.64 2.5 0.5 768166.00 262577.16
NAC1_HUMAN 4 4 195.86 2.8 1.5 766570.61 253473.32
RL7A_HUMAN 3 3 170.22 1.8 0.4 764840.10 250008.08
PSB4_HUMAN 3 3 135.13 1.8 0.8 760826.03 194936.13
MCEE_HUMAN 2 2 71.21 1.3 0.5 750043.23 166225.00
BCS1_HUMAN 4 4 172.99 3.2 0.8 749896.25 235210.89
C1QBP_HUMAN 2 2 157.22 1.8 0.8 743131.70 152482.97
FIBA_HUMAN 2 2 134.93 1.3 0.8 742653.63 612472.74
FERM2_HUMAN 2 2 135.93 1.3 0.8 739946.15 63100.27
EST2_HUMAN 4 4 216.34 4.0 1.4 736404.01 239936.55
COQ7_HUMAN 2 2 120.58 1.7 0.5 734454.64 181522.80
SYP2L_HUMAN 4 4 233.85 1.8 1.3 733077.05 396025.33
HNRDL_HUMAN 3 3 165.83 2.3 1.4 732162.44 235721.20
ELOC_HUMAN 2 2 138.76 1.7 0.5 730667.05 307555.37
UN45B_HUMAN 5 5 234.48 2.7 1.0 727191.27 278708.56
ERP29_HUMAN 4 4 229.52 3.3 0.5 720154.49 182563.88
S10AB_HUMAN 2 2 91.71 0.7 0.5 714116.02 115592.57
SPRE_HUMAN 3 3 175.69 2.3 0.5 712261.50 93972.96
ILK_HUMAN 3 3 120.94 2.3 1.2 711275.99 116699.33
GLOD4_HUMAN 5 5 240.74 3.2 1.2 710231.41 152953.05
RS20_HUMAN 2 2 69.85 1.2 0.4 709052.07 43920.43
PRAF3_HUMAN 2 2 82.63 1.2 0.8 696064.78 145768.42
DUS3_HUMAN 3 3 215.81 1.7 0.8 694960.72 532051.88
CATZ_HUMAN 3 2 139.63 1.0 0.6 693060.87 227181.60
ODBB_HUMAN 2 2 121.89 1.7 0.5 689596.40 309009.55
HSP74_HUMAN 8 7 492.36 4.2 2.0 687114.96 279945.75
SGCB_HUMAN 3 3 188.99 2.0 0.6 683275.98 94730.87
TMM65_HUMAN 2 2 122.39 2.5 1.2 682888.38 149558.65
NQO2_HUMAN 2 2 126.43 1.5 0.5 680437.01 407316.77
NNRE_HUMAN 2 2 120.12 1.8 1.3 679922.98 439909.90
FIS1_HUMAN 2 2 97.01 1.7 0.5 679279.64 331516.71
CLCB_HUMAN 2 2 81.66 1.2 0.8 672135.59 133265.30
A2GL_HUMAN 4 3 179.32 2.2 1.8 667701.72 310166.75
CDC37_HUMAN 3 3 144.2 2.0 1.3 665616.13 131870.35
RS25_HUMAN 2 2 121.74 1.5 0.5 663920.51 203601.74
CALU_HUMAN 2 2 165.23 1.8 0.8 662218.04 178768.95
BCAM_HUMAN 4 4 227.29 2.5 0.8 658420.88 154816.08
RAB7A_HUMAN 3 3 189.34 2.0 0.9 657550.53 134504.93
TIM13_HUMAN 2 2 135.68 1.7 0.5 656085.76 196506.09
KAP0_HUMAN 4 4 239.87 3.7 1.4 655860.70 111271.10
CLIC4_HUMAN 2 2 178.53 1.8 1.3 654213.52 465791.75
MYPT2_HUMAN 4 4 339.31 2.8 1.0 651511.96 187705.16
TXNL1_HUMAN 3 3 142.44 2.3 0.8 647650.30 143615.58
K1C9_HUMAN 6 6 429.12 2.8 2.1 647485.53 542466.24
MCAT_HUMAN 3 3 124.43 2.0 0.9 632903.79 196422.33
RS9_HUMAN 3 3 97.5 2.3 0.5 632577.45 37644.99
RL11_HUMAN 2 2 97.45 1.7 1.0 630663.28 108087.30
PSB1_HUMAN 2 2 86.81 1.2 0.4 629389.41 198729.04
JPH2_HUMAN 5 5 242.39 3.5 1.5 628570.80 271013.39
ABEC2_HUMAN 2 2 77.81 1.5 0.5 627064.72 91592.22
TCPB_HUMAN 3 3 197.88 1.8 0.8 626548.45 353989.33
NFS1_HUMAN 5 5 321.27 3.7 2.0 623384.78 237156.54
TCPA_HUMAN 6 5 259.46 3.7 1.0 611324.33 175181.86
PCBP1_HUMAN 3 2 183.33 1.5 0.5 608876.79 233179.22
68MP_HUMAN 2 2 67.13 1.5 0.5 608454.90 302313.31
LIS1_HUMAN 3 3 215.33 2.3 0.8 604594.08 162198.03
NFU1_HUMAN 3 2 244.7 2.5 0.5 600281.14 230931.19
TRAP1_HUMAN 7 6 435.52 3.0 1.3 598603.43 329493.41
RL15_HUMAN 2 2 107.87 1.0 0.6 598319.86 108781.61
ALAT1_HUMAN 4 4 224.67 2.5 1.0 592720.77 198254.70
SAA1_HUMAN 2 2 132.23 1.8 1.2 591906.24 341123.40
ECHD3_HUMAN 3 3 159.7 1.8 1.0 590968.12 202685.89
HSPB2_HUMAN 2 2 126.21 1.5 0.5 582672.81 237339.98
PSB5_HUMAN 3 3 182.84 1.8 0.8 580187.90 213708.71
SFXN1_HUMAN 3 3 178.27 2.5 0.5 577973.23 156065.09
SUOX_HUMAN 3 3 123.66 2.3 0.8 577649.59 110922.48
RTCB_HUMAN 4 4 174.97 2.3 0.5 576705.90 130301.70
RAC1_HUMAN 2 2 90.93 1.7 0.5 571299.40 87664.32
LGUL_HUMAN 3 3 139.36 2.5 0.5 566323.60 201460.29
HNRPK_HUMAN 3 3 189.25 1.5 1.0 561585.39 231968.03
CAN1_HUMAN 4 4 196.49 2.7 1.0 558201.58 121722.72
HEBP1_HUMAN 3 3 182.58 2.5 0.8 556498.58 102357.94
IGHG3_HUMAN 7 2 393.65 1.7 2.6 548028.89 661712.61
HNRH1_HUMAN 4 4 160.2 2.7 0.8 547837.87 95092.19
LMAN1_HUMAN 4 4 196.43 2.7 1.8 541573.24 327051.88
USO1_HUMAN 4 4 253.37 2.0 1.3 539109.97 159262.58
COFA1_HUMAN 3 3 142.28 2.2 0.8 538570.42 179001.39
CTNA3_HUMAN 6 6 369.57 3.0 2.0 538135.40 197285.80
DDB1_HUMAN 6 6 287.62 4.3 1.0 537709.22 238008.02
COQ5_HUMAN 3 3 136.88 1.8 1.0 537652.75 185396.74
SNAA_HUMAN 5 5 275.88 3.5 1.0 535891.34 95953.16
CLUS_HUMAN 3 3 180.67 1.0 1.3 533187.71 324584.20
ADT2_HUMAN 10 2 481.34 0.7 0.5 532606.54 168396.80
CKAP4_HUMAN 5 4 322.25 3.5 0.8 530789.10 130335.70
TPM4_HUMAN 11 2 761.81 0.8 0.8 523085.47 144860.55
NDUA3_HUMAN 2 2 75.68 1.0 0.0 519601.56 303098.22
NP1L4_HUMAN 3 3 163.62 2.3 0.5 519413.30 184070.74
PDIA4_HUMAN 6 6 318.6 4.3 2.3 518427.19 209357.26
NUCL_HUMAN 2 2 88.82 1.0 0.6 512574.52 224958.09
PNPT1_HUMAN 6 6 307.48 3.3 1.8 511788.30 293473.24
TXND5_HUMAN 4 4 207.78 2.7 0.5 509129.68 125547.70
XRCC6_HUMAN 3 3 171.25 1.5 0.5 507938.39 98249.92
A1BG_HUMAN 3 3 128.12 1.2 1.2 505661.02 372968.75
IMB1_HUMAN 2 2 93.97 1.7 0.5 504726.01 156031.27
SPRY4_HUMAN 2 2 91.25 1.2 0.4 497048.48 345483.13
CBR1_HUMAN 4 4 245.42 3.2 0.8 492307.95 110511.45
PHS2_HUMAN 2 2 91.01 1.2 0.4 491815.70 84282.05
NCEH1_HUMAN 3 3 134.55 2.0 1.4 490276.30 215502.43
RD23B_HUMAN 2 2 71.54 0.5 0.5 489898.52 165100.79
PDC6I_HUMAN 4 4 196.74 2.7 1.0 487635.79 139371.64
TINAL_HUMAN 3 3 172.86 2.2 1.2 483287.22 175106.90
AAMDC_HUMAN 2 2 132.04 1.2 0.8 481873.56 261926.15
THRB_HUMAN 3 3 135.5 1.0 1.5 480516.93 469216.71
PDLI3_HUMAN 3 3 212.43 2.8 0.8 476512.76 151979.87
PSA4_HUMAN 5 5 275.29 3.5 1.2 474311.64 167692.23
HEM2_HUMAN 3 3 141.33 1.8 1.3 471902.60 212127.56
LAMA5_HUMAN 9 9 519.82 3.7 2.9 471458.07 196051.62
PCBP2_HUMAN 4 3 227.61 1.8 1.0 468538.94 176325.50
CYB5_HUMAN 2 2 104.07 1.7 0.5 465145.25 103384.02
MTCH1_HUMAN 5 3 253.98 2.5 0.8 463517.29 129963.91
NIPS1_HUMAN 4 3 166.61 2.0 1.3 461998.07 109937.26
MPPB_HUMAN 5 4 204.55 2.8 0.8 459741.10 134302.39
NRAP_HUMAN 5 5 256.83 2.7 1.9 458808.95 103105.15
MYO1C_HUMAN 5 5 225.88 2.3 1.2 458025.91 182918.12
PPAC_HUMAN 2 2 127.85 1.0 0.6 455890.49 100321.88
CA2D1_HUMAN 6 6 263.48 3.3 1.4 455394.30 146811.97
1433F_HUMAN 6 3 409.2 2.2 1.0 454155.31 82545.65
RL8_HUMAN 2 2 107.58 1.3 0.8 448862.31 208843.95
OLA1_HUMAN 2 2 75.81 1.2 0.8 446215.45 154392.94
PA2G4_HUMAN 3 3 124.51 1.7 1.0 444137.94 95391.08
PFKAP_HUMAN 8 5 369.15 3.5 2.4 443326.77 83543.52
SNTA1_HUMAN 2 2 100.79 1.5 0.5 442361.71 145370.04
UBC12_HUMAN 3 3 128.01 1.7 0.8 438820.13 95989.24
PRS7_HUMAN 4 4 246.08 2.2 0.8 438666.13 138367.57
BST2_HUMAN 2 2 101.43 1.2 1.0 438099.00 249122.87
HYOU1_HUMAN 2 2 127.01 1.7 0.5 437921.88 139388.78
CCD58_HUMAN 3 3 144.61 1.5 0.5 437368.15 127002.13
IPYR_HUMAN 3 3 216.88 2.2 0.8 437353.90 68066.19
SYNP2_HUMAN 4 4 223.68 2.0 0.6 436367.38 251758.51
CO4B_HUMAN 6 6 236.61 2.5 2.1 436238.95 409276.46
NAMPT_HUMAN 3 3 171.65 2.2 1.2 434295.67 145249.05
ARK72_HUMAN 3 3 152.51 1.8 0.8 428779.34 19118.13
SCPDL_HUMAN 3 2 152.47 1.7 0.5 426482.65 249661.22
NIT2_HUMAN 6 6 347.58 4.0 1.8 421584.92 106274.76
TFAM_HUMAN 3 3 145.62 2.5 0.8 418627.93 143903.89
RL18_HUMAN 3 3 202.33 2.0 0.6 417606.21 125686.05
PGES2_HUMAN 2 2 134.01 1.7 0.5 417045.29 57103.98
RAB1B_HUMAN 4 2 192.44 1.2 0.4 416245.63 76183.99
H10_HUMAN 2 2 97.06 1.3 0.5 402019.68 166647.97
RM12_HUMAN 2 2 109.49 1.5 0.5 400792.99 159367.90
MAOX_HUMAN 2 2 66.27 1.3 0.8 400672.66 167341.26
MIC26_HUMAN 2 2 109.87 1.5 0.5 393660.07 248808.14
LKHA4_HUMAN 4 4 280.3 2.5 1.6 391505.13 188936.01
PABP4_HUMAN 3 3 158.58 1.8 0.8 391490.60 136397.69
TCTP_HUMAN 2 2 82.77 1.0 0.6 390362.27 140735.62
DNJA3_HUMAN 2 2 122.83 1.7 0.5 390108.18 91282.89
RL6_HUMAN 2 2 104.27 1.7 0.5 388406.75 48113.65
MGST3_HUMAN 2 2 113.36 1.3 1.0 385812.38 158560.16
B2MG_HUMAN 2 2 73.86 1.3 0.5 380747.47 155739.29
AL1A1_HUMAN 6 3 254.58 1.5 0.5 378740.93 18761.88
TCPD_HUMAN 2 2 77.52 1.0 0.6 372069.64 111234.88
DTNB_HUMAN 2 2 102.33 1.7 0.5 370538.01 150939.52
AK1A1_HUMAN 5 4 238.39 3.3 1.2 368987.12 117879.75
PSMD2_HUMAN 3 3 184.36 2.5 1.0 364138.52 70657.62
FA9_HUMAN 4 4 210.4 1.5 1.6 362390.51 398571.48
DDX1_HUMAN 4 4 204.01 2.2 1.8 361896.59 115117.30
SNX3_HUMAN 2 2 76.79 1.3 0.5 361889.24 307628.05
CALD1_HUMAN 4 4 245.8 2.0 1.7 358335.81 167206.34
RL3L_HUMAN 3 3 175.11 2.2 0.8 358169.52 79518.53
OST48_HUMAN 2 2 94.43 1.5 0.5 356986.87 128140.71
C1TC_HUMAN 4 4 203.52 2.7 1.2 354574.27 176131.95
CO8A_HUMAN 2 2 80.97 0.8 1.0 352534.34 350980.41
PDPR_HUMAN 2 2 82.61 1.2 1.0 345951.08 104875.08
EMAL2_HUMAN 2 2 126.27 1.2 1.0 342265.24 150099.80
COA6_HUMAN 2 2 106.5 1.7 1.0 339906.26 248128.10
GLRX5_HUMAN 3 3 156.32 1.5 1.0 337103.60 176403.61
KCY_HUMAN 4 4 205.07 2.2 1.2 335626.33 198354.06
PTGDS_HUMAN 2 2 167.52 1.7 0.5 334617.78 128203.07
GRPE1_HUMAN 3 3 158.37 1.7 0.8 334261.70 176100.56
RPN1_HUMAN 3 3 145.83 2.5 0.5 331741.69 145694.46
SYIM_HUMAN 4 3 250.76 1.5 0.5 331515.54 130295.86
HNRPQ_HUMAN 2 2 89.88 1.3 0.5 330541.47 98953.28
PRELP_HUMAN 3 3 114.11 1.5 0.5 322557.37 173190.04
GLGB_HUMAN 4 4 218.49 3.0 0.9 322462.69 116036.23
RL13_HUMAN 3 3 124.14 1.2 1.0 322016.26 139217.24
PREP_HUMAN 2 2 98.68 1.2 0.8 321934.00 174561.56
RAP1B_HUMAN 2 2 85.09 1.0 0.6 318384.85 104888.48
ENOG_HUMAN 4 2 234.2 1.5 0.8 316721.42 80575.62
F136A_HUMAN 2 2 88.23 0.5 0.8 315804.36 50544.30
ARP3_HUMAN 2 2 95.47 1.7 0.5 314535.71 107619.14
CAP2_HUMAN 3 3 144.38 1.8 0.8 311969.80 98495.46
TATD1_HUMAN 3 3 157.55 1.3 0.8 311517.97 57095.80
RET4_HUMAN 3 3 124.41 1.5 1.2 309897.79 356567.01
PALLD_HUMAN 3 2 185.06 1.3 0.8 308282.08 75516.83
MFN2_HUMAN 4 4 196.37 2.7 0.8 306992.41 32736.70
ITIH2_HUMAN 3 3 217.59 1.3 1.0 306863.45 300368.35
GSHB_HUMAN 3 3 142.33 1.7 1.0 305492.21 136326.72
6PGD_HUMAN 2 2 115.68 1.2 1.0 305384.44 107979.09
CSN4_HUMAN 3 3 159.17 0.8 1.2 302566.54 114360.31
KTN1_HUMAN 4 4 248.95 3.2 1.0 300930.92 100180.38
DCTN1_HUMAN 3 3 209.61 1.2 1.0 298604.09 34766.32
ANT3_HUMAN 2 2 110.07 1.3 0.5 297029.65 240586.50
LYPL1_HUMAN 2 2 75.65 1.2 1.0 296302.53 150791.82
VPS35_HUMAN 3 3 160.36 1.5 1.0 296042.47 88454.42
RS19_HUMAN 2 2 126.85 1.7 0.5 288317.31 71922.26
EST1_HUMAN 2 2 89.56 1.3 0.5 283085.01 94268.93
TCPE_HUMAN 3 3 121.15 1.3 1.2 276948.47 136546.34
PGRC2_HUMAN 2 2 138.25 1.5 0.8 275978.97 101112.37
SIAS_HUMAN 3 3 130.56 1.0 1.1 274629.21 103053.56
SODE_HUMAN 3 3 164.93 2.2 1.0 273634.04 19317.47
GPX1_HUMAN 2 2 109.34 1.7 0.5 269505.97 94161.90
MFAP5_HUMAN 2 2 104.95 1.5 0.8 268758.89 194633.12
MYH14_HUMAN 7 5 419.34 1.3 1.5 268176.72 37995.99
MARC2_HUMAN 3 3 160.49 2.3 0.8 267506.13 51774.32
SFPQ_HUMAN 3 3 129.01 1.2 0.8 266140.33 82981.62
CLIC1_HUMAN 2 2 98.68 1.5 0.5 263190.89 107701.93
ROA1_HUMAN 2 2 91.5 1.2 0.4 260002.63 25987.24
RL4_HUMAN 2 2 99.12 1.5 0.5 257719.29 83100.78
BGH3_HUMAN 2 2 88.96 1.7 0.5 257674.90 78448.19
MPPA_HUMAN 2 2 82.69 1.3 0.8 254332.87 44323.46
IC1_HUMAN 2 2 131.5 1.2 1.5 252480.90 286092.36
TMLH_HUMAN 3 3 130.33 2.2 0.8 252292.81 67485.28
AT2B4_HUMAN 2 2 143.61 1.2 0.4 250222.88 77121.45
FLNB_HUMAN 8 4 407.73 1.7 0.8 247851.56 48197.12
ANXA4_HUMAN 2 2 113.8 1.7 0.8 247666.72 16776.11
TIM9_HUMAN 2 2 108.24 1.0 0.9 245486.53 202715.54
PTGR1_HUMAN 2 2 156.54 1.3 1.0 242988.53 165956.20
ILEU_HUMAN 2 2 105.35 0.5 0.5 242487.78 128291.22
MARCS_HUMAN 3 3 185.15 1.0 1.3 241248.95 215477.33
CK054_HUMAN 2 2 94.86 1.2 1.0 239601.27 80285.87
HNRPM_HUMAN 4 4 201.69 2.7 1.0 239054.40 22316.17
PSMD6_HUMAN 2 2 116.9 1.5 0.8 238771.07 116446.48
RS13_HUMAN 2 2 93.45 1.7 0.8 238197.20 58053.86
ACAD9_HUMAN 2 2 76.52 1.2 0.8 235832.22 79070.25
RL7_HUMAN 2 2 78.72 1.2 0.4 233787.81 42190.91
RAB18_HUMAN 2 2 95.42 1.5 0.5 233678.48 92657.15
NSF1C_HUMAN 3 3 231.13 2.2 1.0 232104.27 108227.50
COQ6_HUMAN 3 3 127.71 1.8 1.0 230392.69 85327.41
MVP_HUMAN 2 2 111.39 1.3 0.5 229339.02 52166.58
PRPS1_HUMAN 3 3 159.23 2.2 0.8 225778.03 50339.97
DNJB6_HUMAN 4 4 268.32 3.7 0.8 223394.60 88027.89
GPDA_HUMAN 2 2 123.72 1.3 0.5 222955.93 49357.07
TENS1_HUMAN 3 3 205.42 1.5 0.5 220555.05 83285.87
MUC18_HUMAN 2 2 74.36 1.5 0.8 218602.34 60202.61
FBLN5_HUMAN 3 3 179.23 1.7 0.8 218338.87 125834.22
SQRD_HUMAN 4 4 167.84 2.0 1.7 218087.11 100569.88
KCD12_HUMAN 3 3 145.28 2.2 0.8 216247.87 48671.27
VAPB_HUMAN 4 2 230.03 1.0 0.6 209970.58 34848.87
PDXK_HUMAN 2 2 78.06 0.8 0.8 208335.19 123528.90
NDUF4_HUMAN 2 2 86.21 1.7 0.5 200975.58 90693.12
PFD2_HUMAN 2 2 93.01 1.5 0.8 199724.94 83258.40
MAON_HUMAN 2 2 83.14 0.5 0.5 197965.83 33032.58
STRAP_HUMAN 3 3 132.98 2.0 0.0 197784.89 34008.57
ITIH1_HUMAN 2 2 93.82 1.3 0.5 196630.86 134086.98
SGTA_HUMAN 2 2 115.76 1.2 0.8 195059.24 104370.42
HNRPU_HUMAN 2 2 96.05 1.5 0.5 193076.34 19371.39
PLSL_HUMAN 3 2 168.35 1.0 0.9 191894.52 154398.20
SYK_HUMAN 2 2 86.39 0.8 0.4 191528.62 49197.82
ZADH2_HUMAN 2 2 115.56 1.2 0.8 190260.37 80696.50
C163A_HUMAN 3 3 164.8 1.3 1.0 189113.48 77087.84
AOC3_HUMAN 2 2 72.89 1.3 0.8 187685.89 68348.15
UFD1_HUMAN 3 3 167.56 1.5 1.2 186556.14 92811.45
RAB5B_HUMAN 2 2 79.63 0.7 0.8 184933.42 31937.90
RL30_HUMAN 2 2 121.06 1.7 0.8 183995.52 92183.00
DNM1L_HUMAN 3 3 138.32 1.5 0.8 183264.30 94710.90
RHG01_HUMAN 2 2 106.6 1.0 0.6 182145.55 27191.97
UBP14_HUMAN 2 2 124.75 1.3 1.0 180700.94 103222.46
LBP_HUMAN 2 2 84.86 1.5 0.5 179666.28 87061.12
DSG2_HUMAN 2 2 113.93 1.3 0.5 178859.31 113095.23
XPP1_HUMAN 3 2 158.44 1.0 0.6 177926.97 67895.23
CC141_HUMAN 2 2 65.1 1.2 0.8 173274.83 67729.43
STOM_HUMAN 2 2 78.23 0.8 0.4 170578.01 39268.70
ETHE1_HUMAN 2 2 112.26 1.2 0.4 170402.31 130775.26
ATPF1_HUMAN 2 2 108.3 1.7 0.5 168175.58 27776.58
RT23_HUMAN 2 2 86.79 1.3 0.8 167187.52 53654.04
LMOD2_HUMAN 2 2 102.54 1.2 0.8 165829.48 61616.06
RS10_HUMAN 2 2 93.01 0.7 1.0 165593.60 89522.16
TCPH_HUMAN 2 2 122.19 1.7 0.5 164954.89 68771.30
PDK4_HUMAN 2 2 90.33 0.8 1.0 163635.79 206464.90
ARF1_HUMAN 4 2 173.06 1.3 0.8 159691.90 37753.92
SYEP_HUMAN 4 4 197.86 1.8 1.3 159456.89 92695.88
MLEC_HUMAN 2 2 107.18 1.5 1.2 159325.90 58152.40
SMD3_HUMAN 2 2 107.91 1.2 0.4 158991.24 51112.83
STML2_HUMAN 3 3 167.46 2.3 0.5 157746.35 42548.99
RS12_HUMAN 2 2 88.02 1.2 0.8 156826.21 40314.30
PDCD5_HUMAN 2 2 105.14 0.8 0.4 155287.46 98226.18
MYPN_HUMAN 3 2 142.13 1.7 0.5 154569.15 52748.27
CBX3_HUMAN 2 2 86.21 0.8 0.4 154379.00 66766.31
COX20_HUMAN 2 2 94.6 1.2 0.4 151526.82 118605.77
DLRB1_HUMAN 2 2 132.16 1.0 0.6 151524.97 42251.85
LMO7_HUMAN 3 3 101.71 1.3 0.8 149502.43 61972.67
IQGA1_HUMAN 2 2 107.33 1.5 0.5 148958.43 37712.81
PPT1_HUMAN 2 2 163.19 1.7 0.5 147587.36 81126.22
PSMD9_HUMAN 3 3 123.41 1.5 0.8 144682.65 26752.49
TOM70_HUMAN 2 2 94.06 1.2 1.0 143591.45 102565.83
XRCC5_HUMAN 2 2 87.75 1.7 0.8 143093.76 20626.16
CFAH_HUMAN 2 2 73.25 1.2 0.8 142208.93 57382.27
BAG2_HUMAN 2 2 169.06 1.3 1.0 140837.50 37152.58
DNJB4_HUMAN 2 2 108.97 1.3 1.0 140176.95 40360.53
TSP4_HUMAN 2 2 73.71 0.7 0.8 138214.43 74948.68
AMPB_HUMAN 2 2 92.6 1.0 0.9 137983.42 19375.31
MYLK3_HUMAN 2 2 146.86 1.3 0.5 137963.88 141655.94
PPCE_HUMAN 2 2 100.09 0.7 0.5 137617.28 46226.02
SCRB2_HUMAN 3 2 162.79 1.7 0.5 131661.55 24568.97
TNNI1_HUMAN 2 2 75.56 0.7 0.8 131377.52 148050.24
CAH3_HUMAN 2 2 99.68 1.5 1.2 130168.34 124061.80
NLRX1_HUMAN 3 3 139.71 1.2 1.2 128042.88 105051.72
APOB_HUMAN 3 3 136.44 0.7 1.2 124989.00 183739.01
TFR1_HUMAN 2 2 115.01 1.0 0.9 124829.05 53433.16
CHCH7_HUMAN 2 2 89.68 1.7 1.0 124683.72 19412.00
CREL1_HUMAN 2 2 92.26 0.7 1.0 122724.23 35249.22
SEPT7_HUMAN 3 2 116.89 0.7 0.8 121801.07 48643.57
PLMN_HUMAN 2 2 83.26 1.0 0.9 120979.17 128449.95
CMBL_HUMAN 2 2 92.21 1.7 0.5 119769.99 50856.43
BAP29_HUMAN 2 2 84.87 1.7 0.5 119407.23 45562.70
HDHD2_HUMAN 2 2 91.6 1.7 0.5 117198.38 20588.87
MAP1B_HUMAN 3 3 130.38 2.2 0.8 113854.32 28076.10
PLIN3_HUMAN 2 2 163.99 0.7 0.8 113234.33 85795.40
PDK1_HUMAN 2 2 108.54 1.2 0.4 110804.01 47113.01
EMIL2_HUMAN 2 2 121.83 1.2 0.8 108932.95 42025.97
CISD2_HUMAN 2 2 129.27 1.2 1.0 106348.55 24621.62
GNPI1_HUMAN 2 2 114.63 1.2 1.0 106253.01 40741.21
UFC1_HUMAN 2 2 75.01 1.2 0.8 105268.30 37449.82
MTX1_HUMAN 2 2 110.13 1.0 0.6 104235.39 74283.75
PLCX3_HUMAN 2 2 78.08 0.8 0.8 102784.49 61209.04
SH3L1_HUMAN 2 2 76.34 1.0 0.6 101226.29 20130.38
PSA7_HUMAN 2 2 121.77 0.8 0.8 100399.72 46387.76
PDCD6_HUMAN 2 2 103.54 0.8 0.4 99922.67 31944.43
PCY2_HUMAN 2 2 74.36 0.3 0.8 97606.82 76780.28
1A69_HUMAN 6 2 335.19 1.0 0.9 96480.77 101807.00
SLIRP_HUMAN 2 2 107.67 0.7 0.5 95204.70 69616.20
HV303_HUMAN 2 2 90.52 1.2 0.8 94740.99 50814.72
ACY1_HUMAN 2 2 71.4 1.0 0.6 92511.42 28292.19
SYNC_HUMAN 2 2 129.52 1.5 0.5 91050.15 31412.47
CSN7A_HUMAN 3 3 183.26 1.3 0.5 90749.94 37371.13
IPO5_HUMAN 2 2 168.14 1.5 0.5 89617.82 39053.47
ARF4_HUMAN 3 2 140.8 1.2 0.8 88849.71 28657.33
TNPO1_HUMAN 2 2 126.27 1.3 0.5 88632.39 46357.19
BZW2_HUMAN 2 2 81.74 1.0 0.9 88333.30 90680.60
RDH13_HUMAN 2 2 69.74 0.5 0.5 88311.47 72191.32
NNRD_HUMAN 2 2 96.77 1.0 0.9 87798.50 14957.58
D2HDH_HUMAN 2 2 94.42 0.8 0.8 83391.45 24024.27
SYNPO_HUMAN 2 2 120.82 1.0 0.6 82268.58 16218.68
MIPEP_HUMAN 2 2 98.15 1.3 1.0 80261.31 55861.95
AL1B1_HUMAN 3 2 125.06 0.8 1.0 78695.64 73629.71
FRIL_HUMAN 2 2 103.09 1.3 1.2 77591.97 32298.41
NAR3_HUMAN 2 2 75.82 1.0 0.9 75424.29 34832.43
SURF1_HUMAN 2 2 76.29 1.3 0.5 74939.36 50223.05
APOD_HUMAN 2 2 133.58 0.8 1.0 74749.55 51242.53
NLTP_HUMAN 2 2 86.08 0.7 0.8 73245.53 60280.92
U2AF2_HUMAN 2 2 84.43 1.5 0.5 72798.14 29525.73
PEDF_HUMAN 2 2 109.62 0.8 1.0 72695.62 57906.42
MAT2B_HUMAN 2 2 80.19 0.7 0.5 71785.95 16434.16
LAMA4_HUMAN 2 2 108.4 1.2 0.8 70414.37 25931.24
ACSF2_HUMAN 2 2 105.13 0.5 0.8 70218.52 49176.79
MLIP_HUMAN 2 2 116.74 0.8 1.0 68075.08 100403.81
STBD1_HUMAN 2 2 115.84 1.3 0.5 67317.75 37869.56
ARC1A_HUMAN 2 2 94.66 1.0 0.6 65396.56 42114.76
CSN6_HUMAN 2 2 74.61 1.2 0.4 62952.92 29997.81
ITIH4_HUMAN 2 2 76.95 1.0 0.9 62122.85 62535.49
PTCD3_HUMAN 2 2 103.08 0.7 1.0 59442.85 41778.92
EP15R_HUMAN 2 2 128.9 1.5 0.5 57798.42 28756.68
TMM43_HUMAN 2 2 74.71 1.7 0.5 56835.45 15613.19
SND1_HUMAN 2 2 66.56 0.5 0.5 55356.29 23471.55
ANK3_HUMAN 3 2 143.33 0.8 0.4 49450.56 13153.34
MYH11_HUMAN 6 2 381.95 1.0 0.6 45154.73 12066.81
TPP2_HUMAN 2 2 87.55 0.8 0.8 43974.06 7734.63
SPG7_HUMAN 2 2 99.4 1.5 0.8 40716.20 16727.90
MIRO1_HUMAN 2 2 87.89 1.2 0.8 39217.60 11696.36
TENX_HUMAN 2 2 80.68 1.2 1.0 36404.00 18999.57
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VIDEO FILE LEGENDS
Supplemental Video I
µbead perfusion through the conductance artery and microvasculature within the mid-myocardium of a
decellularized human heart, acquired using µ-optical coherence tomography (µOCT).
Supplemental Video II
Spontaneously contracting human iPS-derived cardiomyocytes.
Supplemental Video III

Spontaneously contracting cardiac fiber reseeded with human iPS-derived cardiomyocytes.
Supplemental Video IV
Micro-optical coherence tomography of a spontaneously contracting cardiac fiber reseeded with iPS-
derived cardiomyocytes.
Supplemental Video V
Strain delivery to the LV wall of a decellularized porcine heart under mechanical stimulation in our
bioreactor.
Supplemental Video VI
Recellularized human heart mounted in our human heart bioreactor under perfusion and mechanical
stimulation.
Supplemental Video VII

Visible contractions of a reseeded human heart.
Jacques P Guyette, Jonathan Charest, Robert W Mills, Bernhard Jank, Pilipp T Moser, Sarah E
Gilpin, Joshua R Gershlak, Tatsuya Okamoto, Gabriel Gonzalez, David J Milan, Glenn R Gaudette
and Harald C Ott
Circ Res. published online October 26, 2015;

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Bioengineering Human Myocardium on Native Extracellular Matrix

Running title: Human Myocardium on Native Extracellular Matrix

Address correspondence to:

Nonstandard Abbreviations and Acronyms:

ECM – extracellular matrix MFI – mean fluorescence intensity

Perfusion decellularization of human cadaveric hearts.

Immunogenic profile of decellularized human cardiac matrix.

Decellularized coronary vasculature.

to atherosclerotic plaques (Supplemental Fig. VII). To visualize perfusion of microvasculature within

To evaluate the preservation of microvasculature, we perfused cadaveric human hearts and

In vitro cardiac differentiation of BJ RiPS.

Recellularization of myocardial slices and fibers.

After 14 days in culture, we performed electromechanical functional analysis on the regenerated

31. Xi J, Khalil M, Shishechian N, Hannes T, Pfannkuche K, Liang H, Fatima A, Haustein M, Suhr F,

65. Masumoto H, Matsuo T, Yamamizu K, Uosaki H, Narazaki G, Katayama S, Marui A, Shimizu T,

82. Hulsmann J, Aubin H, Kranz A, Godehardt E, Munakata H, Kamiya H, Barth M, Lichtenberg A,

Figure 1: Biological and mechanical characterization of decellularized human myocardium.

Figure 2: Immunological characterization of decellularized myocardium.

Figure 3: Characterization of vasculature in decellularized human myocardium.

Figure 4: Generation of human iPS-derived cardiac myocytes.

Figure 5: Generation of functional tissue constructs based on decellularized human myocardium.

Figure 6: Human heart bioreactor.

TABLE 1. Metabolic measurements during whole-heart culture.

NOVELTY AND SIGNIFICANCE

What New Information Does This Article Contribute?

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Bioengineering Human Myocardium on Native Extracellular Matrix

Histology and Immunocytochemistry. For immunohistochemistry, tissue samples were fixed in 5%

Microfil® Preparation, µCT, and Analysis of Coronary Microvasculature. Microfil® Preparation:

Functional analysis of Human Acellular Whole-Heart Scaffolds. Metabolic Analysis: Pre-perfusion

Suppl ement alFigur eI I

Decellularized Unique Proteins Spectral Counts Normalized Relative Abundance

Cadaveric Unique Proteins Spectral Counts Normalized Relative Abundance

Supplemental Video III

Supplemental Video VII

Circ Res. published online October 26, 2015;

Data Supplement (unedited) at:

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