Integrative Physiology

Bioengineering Human Myocardium on

Native Extracellular Matrix
Jacques P. Guyette, Jonathan M. Charest, Robert W. Mills, Bernhard J. Jank,
Philipp T. Moser, Sarah E. Gilpin, Joshua R. Gershlak, Tatsuya Okamoto, Gabriel Gonzalez,
David J. Milan, Glenn R. Gaudette, Harald C. Ott

Rationale: More than 25 million individuals have heart failure worldwide, with 4000 patients currently awaiting
heart transplantation in the United States. Donor organ shortage and allograft rejection remain major limitations
with only 2500 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
induced pluripotent stem cellderived cardiomyocytes.
Methods and Results: To provide a clinically relevant tissue scaffold, we translated perfusion-decellularization to
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

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 cardiomyocytes derived from nontransgenic human induced pluripotent stem cells and generated
tissues of increasing 3-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 induced pluripotent stem cellderived cardiomyocytes.
Under biomimetic culture, the seeded constructs developed force-generating human myocardial tissue and 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 induced pluripotent stem cellderived cardiomyocytes and enable the
bioengineering of functional human myocardial-like tissue of multiple complexities.(Circ Res. 2016;118:56-72.
DOI: 10.1161/CIRCRESAHA.115.306874.)
Key Words: cardiomyocytes extracellular matrix induced pluripotent stem cells regeneration

M ore than 5 million people in the United States live with

heart failure (HF), with >800000 new cases diagnosed
each year.1 Patients have poor quality of life and require fre-
quent interventions, leading to >1 million hospital discharges
and a cost of >$39 billion per year.2 Although artificial me-
chanical 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 1500 heart transplant candidates will endure
extended waitlist periods while facing 5-year mortality rates
of 50%.2,3 Although heart transplant recipients benefit from
significantly improved short-term survival rates, long-term
survival remains limited because of chronic rejection and
adverse effects from life-long immunosuppression.4 The en-
gineering of bioartificial myocardium, and ultimately of bio-
artificial hearts, from patient-derived cells could theoretically
solve both of these problems. Although many hurdles remain,
the creation of bioartificial functional human myocardium
from adult-derived, genetically nonaltered cells is the first ver-
tical step toward this goal and can provide new pathways for
repair and replacement of myocardium in patients with HF.
In This Issue, see p 2
Editorial, see p 12

Original received May 20, 2015; revision received October 6, 2015; accepted October 26, 2015. In September 2015, the average time from submission
to first decision for all original research papers submitted to Circulation Research was 12.75 days.
From the Center for Regenerative Medicine (J.P.G., J.M.C., B.J.J., P.T.M., S.E.G., T.O., G.G., H.C.O.), Cardiovascular Research Center (R.W.M.,
D.J.M.), Division of Cardiology (D.J.M.), and Division of Thoracic Surgery, Department of Surgery (H.C.O.), Massachusetts General Hospital, Boston,
MA; Harvard Medical School, Boston, MA (J.P.G., B.J.J., P.T.M., S.E.G., G.G., H.C.O.); Department of Biomedical Engineering, Worcester Polytechnic
Institute, Worcester, MA (J.R.G., G.R.G.); and Harvard Stem Cell Institute, Cambridge, MA (H.C.O.).
The online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.
115.306874/-/DC1.
Correspondence to Harald C. Ott, MD, Division of Thoracic Surgery, Department of Surgery, Harvard Medical School, Massachusetts General Hospital,
55 Fruit St, Blake 15 Boston, MA 02114. E-mail hott@partners.org
2015 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.115.306874

56
 Guyette et al Human Myocardium on Native Extracellular Matrix 57
Nonstandard Abbreviations and Acronyms
CT microcomputed tomography
BJ RiPS BJ fibroblast RNA-induced pluripotent stem (cells)
DBD donation after brain death
DCD donation after cardiac death
ECM extracellular matrix
HF heart failure
HLA human leukocyte antigen
iPSC induced pluripotent stem cell
MFI mean fluorescence intensity
MHC myosin heavy chain
The onset of HF is preceded by myocardial necrosis,
fibrosis, and extracellular matrix (ECM) remodeling, re-
sulting in progressive loss of architecture and contractile
function.1,2,5 Partial reverse remodeling and myocardial
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
augmentation have been accomplished by direct delivery
of contractile cardiomyocytes and passive biomaterials.69
Following similar principles, different scaffold materi-
als and cells have been combined to generate tissue-engi-
neered myocardium. In both approaches, contractile graft
thickness is limited because of poor diffusion and neo-
vascularization that fails to meet the demand of cardiac
myocyte metabolism.1012 Graft architecture and electro-
mechanical integration are constrained by nonphysiologi-
cal scaffold properties and the altered properties of scarred
myocardium. Successful clinical application of myocardial
regeneration will depend on the ability to establish physi-
ological tissue architecture and immediate graft perfusion
on implantation. Native human heart matrix provides the
physiological micro- and macroarchitecture, ECM com-
position, and the perfusable hierarchical vascular bed,
serving as a foundational scaffold to engineer bioartificial
myocardium.13,14
By translating our previous work to human scale,15
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 im-
munologic profile that induces a constructive remodeling
response. By comparing brain death and donation after
cardiac death (DCD) donors, we validate this new regen-
erative platform from >5500 currently unused donor hearts
per year.3 By deriving cardiomyocytes from nontransgen-
ic adult-derived induced pluripotent stem cells (iPSC) at
clinical scale, we provide the necessary building blocks
for clinically relevant myocardial regeneration. By repop-
ulating 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 myocar-
dial function and serve as a patient-specific development
platform.
Methods
All Methods and associated references are included in the online Data
Supplement.
Results
Perfusion-Decellularization of Human Cadaveric
Hearts
In a first set of experiments, we scaled perfusion-decellu-
larization to donated human hearts that were recovered by
the New England Organ Bank and considered unusable for
clinical transplantation. Between 2012 and 2015, 73 human
nontransplantable hearts were recovered for research after
specific consent was obtained. Donor records (n=73; 5 patient
records unavailable) indicate a distribution of 36 men ver-
sus 32 women, 29 DCD versus 39 donation after brain death
(DBD), an average age of 52.4314.29 years, an average
height of 170.969.53 cm, an average weight of 81.5420.53
kg, and an average body mass index of 27.766.09 kg/m2.
Heart identification numbers, corresponding donor data, and
associated experimental tests are outlined in Online Table I.
We applied aseptic pressure-controlled antegrade coronary
flow to 40 hearts in a custom organ chamber (60 mmHg,
average cold ischemia time of 455184 minutes; Online
Figure I). Hearts were perfusion-decellularized with 1% SDS
(168 hours), deionized H2O (24 hours), 1% Triton-X100 (24
hours), and PBS washes (168 hours) following a standard-
ized protocol (Figure1A).16 Decellularized hearts contained
346.1727.92 ng/mg wet tissue of residual double-stranded
DNA, which could be removed by treatment with endonu-
clease (Figure1B). Subsequent perfusion of decellularized
whole hearts with 25 U/mL of endonuclease at room tem-
perature for 24 hours demonstrated a 99.05% reduction in
double-stranded DNA (3.273.12 ng/mg wet tissue; P<0.05),
with thresholds meeting established criteria for decellular-
ized biomaterials.17 Biochemical analysis of perfusion-decel-
lularized hearts from both DCD and DBD donors indicated
a high retention of insoluble collagen, moderate decrease of
sulfated glycosaminoglycans, and lower concentrations of -
elastin and soluble collagen (Figure1C; Online Figure II).
Importantly, we did not detect measureable amounts of re-
sidual SDS in decellularized hearts (<0.02% wt/vol 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 decellular-
ized hearts (Figure1D). Matrisome proteins were identi-
fied by querying our cadaveric and decellularized lists of
unique proteins with the UniProt database for the subcel-
lular location search terms ECM and basement membrane,
exclusively and combined, as defined cellular component
terms by the Gene Ontology Consortium.19,20 Acellular
heart scaffolds showed a marked retention of 36 matrisome
proteins following the perfusion-decellularization process
(Figure1D). Normalized relative abundances of uniquely
identified matrisomal proteins in both cadaveric and decel-
lularized human myocardium were further categorized into
ECM-protein families (Figure1E). The 4 largest ECM-
protein families are conserved across cadaveric and decel-
lularized 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 -1. Whole lists of unique
 58Circulation ResearchJanuary 8, 2016
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

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 postperfusion with
nuclease (n=3 hearts). Normalized to tissue wet weight. Error bars, SD. *P<0.05 compared with preperfusion with nuclease. C, Biochemical
analysis for insoluble collagen (hydroxyproline), soluble collagen, -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 (Continued)
 Guyette et al Human Myocardium on Native Extracellular Matrix 59

Figure 1 Continued. per heart per component, and the average of those 3 was used as the representative value for the component
in that heart. Normalized to tissue wet weight. Error bars, SD. *P<0.05 compared with 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 a part of the extracellular matrix or basement
membrane. Other (gray) represents nonmatrisome 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 m, except
MHC, 100 m. G, Biaxial mechanical testing of cadaveric (C; n=8 hearts) and decellularized (D; n=10 hearts) human myocardium.
Error bars, SD. H, Anisotropy observed in biaxial mechanical testing of cadaveric (C, n=8 hearts) and decellularized (D, n=10) human
myocardium. Error bars, SD. B/A indicates base to apex axis; and S/FW, septum to free-wall axis.

proteins for both cadaveric and decellularized myocardium
are provided (Online Tables II and III). Histological evalu-
ation confirmed the retention of collagens (I, III, and IV),
laminin, and fibronectin within acellular human heart scaf-
folds (Figure1F; Online Figure III). Cardiac matrix fiber
composition and architecture were maintained, showing
vacant spacing between fibers with a loss of nuclei and
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
Analyst were used to quantify macrophage phenotype across
images (Online Figure V).21,22 From all cells counted in each
high-power field (number of fields/group: 281; number of
cells/field: 1152.5294.2), cadaveric human tissue contained
70.491.72% CD68+ cells, whereas decellularized human
matrix contained 79.633.22% and decellularized porcine
matrix contained 66.446.17% CD68+ cells (Figure 2B).

the cardiac myocyte marker myosin heavy chain (MHC).
Insoluble adipose tissue matrix and lipid molecules re-
main on the epicardial surface after decellularization
(Figure1A), but further histological analysis confirmed
the absence of cells and preservation of matrix structure
(Online Figure IV). In evaluation of how the decellular-
ization process affects the material properties of the car-
diac matrix scaffold, equibiaxial mechanical testing of
transmural samples displayed similar moduli between ca-
daveric and decellularized samples, along both the base/
apex axis (376.3191.7 versus 370.8150.9 kPa) and the
septum/free-wall axis (581.4325.5 versus 451.4257.8
kPa; Figure1G). Anisotropic elastic behavior was mea-
sured by an anisotropy ratio comparing the difference and
the sum of the 2 orthogonal moduli within a tissue sample.
A value of zero indicates perfect biaxial isotropy, whereas
a value approaching 1 indicates a high degree of anisot-
ropy. Cadaveric and decellularized samples exhibited mea-
surable anisotropic ratios (0.3140.196 and 0.1670.158;
Figure1H) 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 toward the potential use of our human
cardiac scaffolds in clinical applications, we assessed the im-
munogenic profile of perfusion-decellularized myocardium
after subcutaneous implantation in SpragueDawley rats for
2 weeks. Comparisons were made versus cadaveric human
myocardium, as well as decellularized porcine myocardium
that was perfusion-decellularized in the same manner as do-
nated human hearts described above.16 Histological evalua-
tion of explanted material by immunofluorescence showed
a considerable number of CD68+ mononuclear cells in all 3
groups. There appeared to be a more discernable M1 mac-
rophage proinflammatory response by cadaveric human and
decellularized porcine myocardium, whereas decellular-
ized human myocardium presented with a more definitive
M2-macrophage immunomodulatory response (Figure 2A).
A CellProfiler image processing pipeline and CellProfiler
Costaining with CD80 demonstrated the presence of M1 mac-
rophage phenotype in cadaveric human tissue (63.139.79%),
decellularized human matrix (52.117.05%), and decellular-
ized porcine matrix (67.733.01%) from all cells counted per
field. Costaining for CD163 on adjacent sections revealed
that cadaveric tissue implants elicited only 38.307.46%
M2+ cells, whereas decellularized human matrix implants
elicited 69.548.44% and decellularized porcine matrix elic-
ited 53.526.80% M2+ immunomodulatory cells (P<0.01).
A CD163/M2+ macrophage response has previously been
shown as an indication of reconstructive remodeling de-
scribed in decellularized matrices.23 During tissue explan-
tation 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 white blood cell components (Online
Figure VI). Sera were also tested against human leukocyte
antigen (HLA) single antigen beads, where experimental
groups were normalized to the sham group, and antibody re-
activity to beads with specific HLA alleles was determined
by mean fluorescence intensity using HLA Fusion software
(version 3.0; One Lambda). Implanted decellularized human
matrix showed reactivity to only 2 HLA beads, yielding val-
ues of <100 mean fluorescence intensity that are well below
the positive cutoff value of 500 mean fluorescence intensity
(Figure 2C). Implanted cadaveric myocardium showed reac-
tivity to >50 HLA beads, yielding values that ranged between
5000 and 7900 mean fluorescence intensity, and showing
high reactivity against patient-specific HLA types attributed
to the implanted cadaveric myocardium (Figure 2C). The sin-
gle antigen bead assay confirmed our qualitative assessment
(by immunofluorescence) that the decellularization process
removes HLA from the cardiac matrix as cells are removed
(Figure 2D).
Decellularized Coronary Vasculature
In assessment of acellular coronary vasculature, DCD hearts
had slightly lower coronary flow (Figure3A) and higher vas-
cular resistance (Figure3B) than DBD hearts during detergent
perfusion but achieved similar flow and resistance by the end
PBS washes. This initial difference did not reach statistical
 60Circulation ResearchJanuary 8, 2016
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

Figure 2. Immunologic 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 2 wk implantation in a
rat model. Scale bars, 100 m. 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 (35 fields averaged
per explant) after 2-wk implantation in a rat model. Error bars, SD. *P<0.05 between groups using an ANOVA with Tukey post hoc test.
C,Human leukocyte antigen (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). *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 m.

significance. Histological evaluation by pentachrome stain
showed decellularized vasculature retained structural blood
vessel features, with preservation of the intima, media, ad-
ventitia, and elastic laminas (Figure3C). Scanning electron
microscopy further confirmed that the decellularization pro-
cess removed all endothelial cells, without evidence of base-
ment membrane disruption (Figure3D and 3E). Cardiac
computed tomographic scans revealed conservation of the
conductance arteries of the coronary tree, showing structure
and patent lumens stemming from the left and right main cor-
onaries (Figure3F). Angiography of acellular hearts from pa-
tients presenting with coronary artery disease confirmed the
preservation of perfusable coronary vessels, but revealed that
the decellularization process does not resolve luminal nar-
rowing related to atherosclerotic plaques (Online Figure VII).
To visualize perfusion of microvasculature within decellu-
larized hearts, we cannulated and directly perfused epicar-
dial conductance arteries with resuspended 0.2-m-diameter
fluorescent microspheres and tracked microsphere movement
through midmyocardial and endocardial regions using micro-
optical coherence tomography. Using a Z-projection tool to
generate a running average from the acquired video sequenc-
es, we confirmed bead-flow through distinct pathways or mi-
crovascular channels (Online Movie I).
 Guyette et al Human Myocardium on Native Extracellular Matrix 61
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

Figure 3. Characterization of vasculature in decellularized human myocardium. A, Mean flow rates of each solution under constant
pressure perfusion for nondonation after cardiac death (DCD; n=3 hearts) and DCD (n=4) populations. Error bars, SEM. B., Mean
vascular resistance calculated under constant pressure perfusion for non-DCD (n=3 hearts) and DCD (n=4) populations. Error bars, SEM.
C, Pentachrome staining of decellularized left anterior descending coronary artery (LAD) coronary artery. Adventitia (a), tunica media
(m), tunica intima (i), exterior elastic lamina (ext), and interior elastic lamina (int). Scale bar, 500 m. 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 m (left) and 20 m (right). E, Scanning electron micrograph
of decellularized LAD coronary artery. Dotted white square indicates region of higher magnification. Scale bar, 500 m (left) and 20
m (right). F, Cardiac computed tomography of a decellularized human heart. Left, long-axis view; right, posterior view. G, Three-
dimensional reconstruction of microvasculature in a left ventricular segment of decellularized human heart. Scale bar, 1 mm. H, Vessel
density in epicardium (epi), midmyocardium (mid), and endocardium (endo) of cadaveric (C, n=3) and decellularized (D, n=7) heart
samples. I, Distribution of vessel sizes in the epi-, mid-, and endocardial of cadaveric human heart samples (n=3). J, Distribution of vessel
sizes in the epi-, mid-, and endocardial of decellularized human heart samples (n=7). AM indicates acute marginal coronary artery; LCA,
left coronary artery; LCX, left circumflex coronary artery; OM, obtuse marginal coronary artery; and RCA, right coronary artery.
 62Circulation ResearchJanuary 8, 2016

To evaluate the preservation of microvasculature, we densities between cadaveric myocardium (25.930.40

perfused cadaveric human hearts and decellularized human
hearts with radio-opaque silicone and performed a system-
atic comparison of full-thickness left ventricular samples
by microcomputed tomography (CT). Reconstructions
of high-resolution CT 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 m (Figure3G). Analyzing
2-dimensional (2D) CT 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 the decellularized hearts (Figure3H).
In the subepicardial region, no statistical difference in
vascular density was found between cadaveric myocar-
dium (15.943.03 vessels/mm2) and decellularized matrix
(13.441.38). Likewise, both midmyocardial and subendo-
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
and 20.643.52, respectively) and decellularized matrix
(22.442.04 and 18.303.6, respectively). Further analysis
of the CT images was done to measure the minor axis of
vessel cross sections as an indication of vessel diameter,
which confirmed similar vessel diameter distribution be-
tween the cadaveric and the decellularized samples in all 3
myocardial subregions (Figure3I and 3J).
In Vitro Cardiac Differentiation of BJ Fibroblast
RNA-Induced Pluripotent Stem (Cells)
To examine the translational value of our approach, we
sought to generate a clinically relevant number of human
cardiomyocytes from a nontransgenic cell source. Therefore,
we obtained and expanded human BJ fibroblast RNA-iPSCs
(BJ fibroblast RNA-induced pluripotent stem cells [BJ RiPS],
clone 1.1).24 To induce cardiac differentiation, we applied
temporal modulation of canonical Wnt/-catenin signaling

cardial regions showed no statistical difference in vascular to BJ RiPS cultured in monolayer.25 We then mapped gene

Figure 4. Generation of human induced pluripotent stem cell (iPS)derived cardiomyocytes. A, Timeline of human induced
pluripotent stem cell differentiation into late-stage cardiomyocytes (days 0, 3, 5, 7, 14, 30, 60, 120, and 180) with corresponding gene
activation as measured by polymerase chain reaction (n=3). Error bars, SD. Flow chart relates culture time with media, supplement, and
cell type. B, Immunofluorescence staining on differentiated cells isolated between days 30 to 60 for cardiac markers -actinin, troponin
T, and myosin heavy chain (MHC). Scale bars, 50 m. C, Flow cytometry analysis of a representative culture of human iPSderived
cardiomyocytes positive for -actinin (left) and troponin T (right), isolated between days 30 to 60 post differentiation.
 Guyette et al Human Myocardium on Native Extracellular Matrix 63
expression profiles of 15 markers by reverse transcriptase-
polymerase chain reaction 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 dif-
ferentiation and maintenance, and to define checkpoints for
quality control required at a clinically relevant scale (Figure
4A). Before 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, whereas expression of
genes typically found in mesendoderm and mesodermal tis-
sues was 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, whereas 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
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
(MYH6, MYH7, MYL2, and TNNT2) as large areas of dif-
ferentiated cardiomyocytes began spontaneously contracting
by day 14 (Online Movie II). Early- and late-stage cardiac
markers tended to increase by day 30, and expression persist-
ed through days 60, 120, and 180 postdifferentiation. 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 expres-
sion, BJ RiPSderived cardiomyocytes isolated between days
30 and 60 post differentiation showed positive cardiac protein
expression and structure for sarcomeric -actinin, cardiac
troponin T, and MHC by immunofluorescence (Figure 4B).
Cardiac protein expression in day 30 to 60 cardiomyocytes
was further confirmed by flow cytometry, yielding abundant
populations positive for sarcomeric -actinin and cardiac tro-
ponin T (Figure 4C), achieving differentiation efficiencies of
>85%. As day 30 to 60 cardiomyocytes seemed to be the most
robust for cardiac gene and protein expression, cells within
this time frame were enzymatically dispersed and isolated for
seeding experiments.
Recellularization of Myocardial Slices and Fibers
To test whether perfusion-decellularized human cardiac
matrix can support engraftment of cardiomyocytes and en-
able myocardial tissue formation, we reseeded matrix sec-
tions (200 m) with human iPSderived cardiomyocytes
in nonperfused 2D culture (Figure 5A). Acellular human
heart matrix (Figure 5Ai) supported the attachment, viabil-
ity, and function of human cardiomyocytes (Figure 5Aii) by
evidence of spontaneously contracting slices within 4 to 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 cul-
ture. To characterize the mechanical function, we first used
high spatial-resolution imaging (high-density mapping)26 to
determine the area deformation of small defined regions of
interest (Online Figure VIII). By independently analyzing
matrix-seeded cardiomyocytes, as well as the underlying
cardiac ECM within the same region (20 m below the slice
surface), we confirmed stable cellmatrix interaction and
quantified the degree to which cell contractility translated
to matrix deformation. In spontaneously contracting slices,
cardiomyocytes achieved 0.590.26% area strain at a natural
frequency of 1.360.32 Hz, which translated to 0.470.17%
area strain of the ECM with an average natural frequency
of 1.380.43 Hz (Figure 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 (Figure 5B). Area strain of myocardial matrix
could be significantly increased to 1.550.55% when paced
at 1 Hz and decreased to 1.020.31% when paced at 2 Hz.
The technique to measure in vitro area strain is concurrent
with the in vivo regional cardiac functional metric of systol-
ic area contraction.27 Regenerated human myocardial slices
achieved strain below normal adult myocardium (20%);
however, the integration of human cardiomyocytes with hu-
man 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 physiological 3D model, we reseeded
nonperfused cardiac fibers (15-mm length, 2.5-mm diameter)
cut from the left ventricular free wall of decellularized cardiac
scaffolds (Figure 5C). Reseeded cardiac fibers supported the
attachment, viability, and function of human cardiomyocytes
by evidence of spontaneously contracting tissue within 7 to
10 days, which could be maintained for 60 days in culture
(Online Movie III). Using microoptical coherence tomogra-
phy that allows for imaging contractility 250 m within car-
diac fibers with high spatial and temporal resolution (Figure
5C; Online Movie IV),28 we then applied high-density map-
ping and postprocessing for strain analysis. Areas 100 to 200
m within spontaneously contracting cardiac fibers achieved
8.320.74% strain (Figure 5D) at a frequency of 1.490.06
Hz (Figure 5E).
We measured force generation on myocardial slices by
measuring deflections of contractile nodes using a custom
setup for adhered slices (Online Figure IXA). Force could
be measured in spontaneously contracting cardiac tis-
sues and was modulated by electrical pacing (Figure 5F).
Spontaneously beating areas within unstimulated slices gen-
erated 14.933.85 N of contractile force, which was de-
creased to 10.302.81 N when electrically stimulated at 2
Hz. Force generation in human myocardial slices was orders
of magnitude higher than single human iPSderived cardio-
myocytes,29,30 achieving 33.8% of native murine embryonic
heart tissue (53.015.5 N),31 suggesting multicellular con-
tractile tissue formation.
Using a more traditional setup to measure contractile force
in muscle tissue (Online Figure IXB), spontaneously contract-
ing cardiac fibers generated 102.776.3 N of force, whereas
electrical pacing at 2 Hz could increase force generation to
124.194.7 N (Figure 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% re-
laxation, and time to 90% relaxation under different pacing
conditions (Figure 5H; Online Figure X). Cardiac fibers gen-
erated contractile force at an order of magnitude higher than
myocardial slices, whereas decreasing trends for period, time
 64Circulation ResearchJanuary 8, 2016
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

Figure 5. Generation of functional tissue constructs based on decellularized human myocardium. A, Cardiac matrix slices:
decellularized human cardiac matrix slice (200 m) in a culture well (i), seeded with induced pluripotent stem cell (iPS)derived
cardiomyocytes (ii) for 120-d culture. Ruler ticks are 1 mm. B, Area strain of matrix-seeded iPS-derived cardiomyocytes 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, SD. Black and blue asterisks (*) indicate P<0.05 compared with unstimulated cells and matrix, respectively. C, Left, Macroimage
of a reseeded cardiac fiber. Scale bar, 5 mm. Right, Representative microoptical coherence tomography still-frame, depicting a
longitudinal cross section within a reseeded cardiac fiber. Red shading indicates example high-density mapping (HDM) region of interest.
Scale bar, 100 m. D, HDM computed area strain under unpaced conditions (n=4 fibers). Error bar, SD. E, HDM-captured frequencies
under unpaced conditions (n=4 fibers). Error bar, SD. 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). 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,
and 3 for unpaced, 1, 2, and 3 Hz, respectively) and fibers (red triangles; n=4, 3, 4, and 2 for unpaced, 1.5, 2, and 2.5 Hz, respectively)
under paced and unpaced (U) conditions. Error bars, SD. I, Conductivity map (left) and example action potential (right) from a reseeded
cardiac fiber. J, Immunofluorescence staining on reseeded cardiac fibers for cardiac markers -actinin, troponin T, and myosin heavy
chain (MHC). Scale bars, 25 m. K, Transmission electron micrographs of recellularized cardiac fibers. Dashed black box indicates region
of higher magnification. Left, Red arrows indicate Z bands, right, Z, I, A, H, and mt indicate Z bands, I bands, A bands, H bands, and
mitochondria, respectively. Scale bars, 2 m and 500 nm for left and right, respectively.

to 50% relaxation, and time to 90% relaxation were similar
for both constructs (Figure 5H). Captured frequencies of both
slices and fibers effectively correlated with pacing frequen-
cies (Online Figure XI). Loading fiber-seeded cardiomyocytes
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 ve-
locity of 3256 cm/s (Figure 5I). Action potentials at mul-
tiple points within conduction areas were assessed based on
relative fluorescence intensity signal, with an average action
 Guyette et al Human Myocardium on Native Extracellular Matrix 65

potential duration at 80% of 405116 ms. Immunofluorescent
staining of reseeded fibers for cardiac markers sarcomeric -
actinin, cardiac troponin T, and MHC showed large areas of
cardiomyocytes with a striated phenotype, and a high degree
of alignment (Figure 5J). Further analysis by transmission
electron microscopy confirmed cardiac myocyte elongation
and myocardial tissue formation, demonstrated by aligned
myofibrillogenesis (Figure 5K, left) and definitive sarcomeric
structures (right).
Whole-Heart Experiments
Whole-heart recellularization experiments were performed
using a custom human heart bioreactor capable of provid-
ing coronary perfusion and LV wall mechanical stimulation
(Figure6A). Mechanical stimulation was achieved by os-
cillating the pressure inside a balloon placed within the LV
to determined targets, thus imparting strain to the LV wall
(Figure6B). Validation of strain delivery with oscillating bal-
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
descending and the left circumflex coronary arteries (Figure
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 (Figure 6E).6 LV bal-
loons were implanted through the mitral valve post cell de-
livery to prevent balloon puncture, and then prefilled 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 (Figure 6F; Online Movie VI).
Metabolic function was monitored during culture by sam-
pling perfusate from the coronary sinus to compare against
baseline media (Table). 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 sug-
gested consumption (baseline, 186.331.53 mg/dL; culture,

loon pressure was performed using high-density mapping on
the presumed reseeding area of the LV wall of a decellularized
porcine heart under mechanical stimulation in our bioreactor
(Figure6C; Online Movie V).
We reseeded whole acellular human hearts with 500
million human BJ RiPSderived cardiomyocytes by making
5 intramyocardial injections into the basal anterior and mid
anterior segments of the left ventricle (using 500 L/injec-
tion, for total volume of 2.5 mL), between the left anterior
155.756.70 mg/dL; P<0.01). Conversely, significant lac-
tate production was detected (baseline, 0 mmol/L; culture,
2.440.19 mmol/L; P<0.01).
After 14 days of culture, we performed electromechanical
functional analysis on the regenerated myocardium, using a
bench-top perfusion setup. Electrical stimulation was deliv-
ered through cardiac leads placed within the anterolateral wall
of the left ventricle (0.8 Hz, 1080 V, 5 ms). Stimulated myo-
cardium generated visible contractions (Online Movie VII),

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. , strain; P, pressure; Q, volumetric flow; MV, mitral valve. C, Representative left
ventricular (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 high-density mapping 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 LV balloon during mechanical stimulation. F, Example pressure (top) and flow rate (bottom)
traces in the coronary arteries (red) and LV (blue) during mechanical stimulation in the human heart bioreactor.
 66Circulation ResearchJanuary 8, 2016

Table. Metabolic Measurements During Whole-Heart Culture

Measurement Baseline 48-h Media Changes P Value
Units Mean SD Mean SD
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

with recordable repolarizations (Figure 7A). Using a force stimulation at 0.8 Hz could generate regional contractions
transducer to measure force based on deflections of contract- measuring 350 N (Figure 7B). By introducing a pressure
ing regions on the epicardial surface, continuous electrical transducer into the LV balloon and prefilling the balloon to
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

Figure 7. Repopulation of decellularized human myocardium in whole hearts with human induced pluripotent stem cell (iPSC)
derived cardiomyocytes. A, ECG 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 (PLV) development in a recellularized human
acellular cardiac scaffold under electrical stimulation at 0.8 Hz including corresponding meanSD peak pressure (P), max dP/dt, min dP/
dt, and (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. F, Cell coverage from base to apex of recellularized human cardiac scaffolds. Error
bars are SD of measurements taken from 2 adjacent tissue sections. G, Fluorescent TUNEL of recellularized human cardiac scaffolds.
Scale bar, 200 m. H, Trichrome staining of recellularized human cardiac scaffolds. Scale bar, 200 m. I, Immunofluorescent staining
on recellularized human myocardium for cardiac markers -actinin, troponin T, and myosin heavy chain (MHC). Scale bars, 50 m. LAD
indicates left anterior descending coronary artery; LCX, left circumflex artery; and TUNEL, terminal deoxynucleotidyl transferase dUTP
nick end labeling.
 Guyette et al Human Myocardium on Native Extracellular Matrix 67
an isovolumetric pressure of 20 mmHg, stimulated myocar-
dial contractions generated developed pressures of 2.40.1
mmHg, with a dP/dt max of 139.75.7 mmHg/s, a dP/dt incomplete heparinization of DCD donors did not have any
min of 41.63.5 mmHg/s, and a calculated of 47.310.6 detectable detrimental effect on the decellularization process
ms (Figure 7C). Using an 88-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 (Figure 7D).
Histological analysis revealed dense regions of engrafted
iPS-derived cardiomyocytes in the depth of the left ventricular
wall, indicated by large areas of MHC-positive cells in succes-
sive sections throughout recellularized volumes (Figure 7E).
By linear interpolation of the areas of MHC-positive signal
in tissue sections across reseeded regions, we detected a tis-
sue repopulation of 50% within the target region of 5 cm3
(Figure 7F; Online Table I). Coronary perfusion during whole-
heart culture provided sufficient nutrient and oxygen supply
to maintain >90% viability after 14 days in culture (Figure
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
7G). Trichrome staining of recellularized areas confirmed en-
graftment, with reseeded cells integrating with cardiac matrix
(Figure 7H). Recellularized regions of whole-heart scaffolds
contained large areas of cardiomyocytes that expressed sar-
comeric -actinin, cardiac troponin T, and MHC by immu-
nofluorescence (Figure 7I). Reseeded cardiomyocytes within
biomimetically stimulated hearts appeared to demonstrate a
range of maturity, where some cardiomyocytes showed round-
ed immature sarcomere formation, whereas other cardiomyo-
cytes displayed elongated and striated phenotypes.
Discussion
Loss of viable myocardium leads to architectural and func-
tional decline and ultimately results in the development of HF.
Bioengineered myocardium generated from patient-derived
cells could provide an alternative to allotransplantation and
mechanical support. Recent reports on the native ECM plat-
form and robust protocols for directed differentiation of hu-
man pluripotent stem cells provide key building blocks that
could be combined toward that goal. Herein, we describe re-
generation of functional human myocardial tissue constructs
based on decellularized human cardiac ECM and human iPS
derived cardiomyocytes, 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
immunologic response; the directed differentiation of adult
derived, nontransgenic iPSC to generate cardiomyocytes at a
clinical scale; the engraftment of human iPS-derived cardio-
myocytes on native ECM for the development of functionally
contractile tissue; and the up-scaling to biomimetic organ cul-
ture to generate functional myocardial tissue in the context of
a human heart.
Perfusion-decellularization has been applied to hearts
from several species.15,3234 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 pub-
lished detergent-based perfusion-decellularization protocol
to generate scaffolds of reproducible characteristics.15,35,36 In
our studies, the increased warm ischemia time and potentially
or the resulting scaffold when compared with hearts from
DBD donors. This finding is of importance, given that 1600
DCD donor hearts are currently available per year in the
United States,3,37 and this number is expected to increase.3840
Although human donors represent a more heterogeneous pool
than 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
(eg, shape and pulmonary venous anatomy) may prove to be
relevant in regenerated myocardial grafts.41,42 From a regula-
tory perspective, perfusion-decellularized human heart matrix
could be a readily available and safe first step on a pathway
toward clinical translation.
As it has been reported in other human organs,43 decellu-
larization of hearts with structural disease and atherosclerotic
lesions yielded scaffolds with corresponding matrix defects.
In line with previous works,15,35,44 we found preservation of
a hierarchical network of macro- and microvascular chan-
nels after decellularization, providing the foundation for the
formation of metabolically active myocardium. Our analysis
revealed preserved vascular density and binned diameter dis-
tribution in decellularized myocardium when compared with
cadaveric controls and data reported in human hearts and ani-
mal models.45,46 Using high-resolution CT imaging (6 m),
this finding extended to compare lower-boundary density and
distribution of small-diameter vessels (<80 m) in decellular-
ized and cadaveric hearts. Although there was no statistical
difference found in vessel density across regions, decellular-
ized hearts tended to demonstrate slightly lower densities, vis-
ibly noticeable in the CT of mid- and endocardial regions.
This represents a technical limitation, as the loss of endothe-
lium and tissue-turgor makes the recruitment of deeper vas-
cular conduits more challenging. For culture of recellularized
hearts, perfusion pressure must be increased toward the higher
end of physiological range to ensure media flow to reseeded
regions. Active and passive mechanical properties of native
myocardium are a result of mutually orthogonal fiber direc-
tions and play a key role in translating mechanical function
from cells, to tissue, and to organ.47 In human hearts, perfu-
sion-decellularization did not affect the anisotropic behavior
of myocardium on passive mechanical testing, indicating the
preservation of the native fiber framework within the myocar-
dial matrix.48 Artificial scaffolds designed to reproduce some
of the anisotropic behavior of myocardial tissue have been
repopulated with cardiomyocytes to create engineered myo-
cardial tissue grafts with anisotropic properties.49,50 However,
reproduction of the multidimensional complexity of native
cardiac fiber architecture in a synthetic whole-organ scaffold
has not been accomplished to date. On the basis of our re-
sults, we concluded that perfusion-decellularization of whole
human hearts captures a structural blueprint that could guide
 68Circulation ResearchJanuary 8, 2016
the formation of electrically and mechanically orthotropic
myocardium.
In the present study, perfusion-decellularization of hu-
man hearts resulted in acellular scaffolds consisting of major
ECM components, such as collagens, elastin, and glycos-
aminoglycans, which is in line with similar data published
in other organ systems and species,15,35,36 and decellularized
human heart sections.51 Quantitative analyses of ECM com-
ponents were similar in DCD and DBD hearts, with preser-
vation of insoluble collagen and glycosaminoglycans, and
reduction of soluble collagen and elastin. This is consistent
with a loss of mainly immature collagen, whereas more
mature, crosslinked, high-strength collagen is preserved re-
gardless of normalization to wet weight (Figure 1C) or dry
weight (Online Figure II).52 Histological and immunohisto-
chemical analysis showed absence of nuclei and cytoplasmic
components such as MHC. Further compositional analysis
using mass spectrometrybased label-free proteomics re-
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
vealed an 89.14% reduction of total protein content to 105
proteins and confirmed preservation of 36 matrisome pro-
teins, including important structural proteins such as fibril-
lin-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 trans-
plantation has yet to be determined. The potential clinical
value of any organ matrix depends on complete removal of
cell surface xeno- or alloantigens, 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 transplantation.17 We further
confirmed the absence of HLAs by immunostaining of decel-
lularized hearts, and the absence of antibody induction in an
immunocompetent animal model in vivo. Although we did
not observe the generation of panel reactive antibodies, de-
cellularized human heart matrixes promoted a CD163 heavy
macrophage response, consistent with reconstructive remod-
eling described in noncrosslinked, decellularized matrix
implants.23,5355 Notably, decellularized porcine myocardium
showed a similar immunogenic profile as decellularized hu-
man myocardium by histology although we did not examine
the humoral response to porcine matrix. These early results
hold promise from a device perspective because in vivo re-
modeling and graft homeostasis will depend on the absence
of chronic inflammation and the promotion of a reconstruc-
tive response.56
Since iPSCs were first described, the generation of human
iPSderived cardiomyocytes holds immediate promise for
cardiac applications.57,58 In cardiac regeneration, techniques
have been developed for the delivery of human iPSderived
cardiomyocytes to improve cardiac function postinfarct,7,59
provide cardioprotection,60 and enhance homing and survival
in infarcted myocardium.61 These approaches build on the
extensive body of literature on ESC (embryonic stem cell)-
derived cardiomyocytes, which have been recently reported
to mediate myogenesis in a primate model of cardiac repair.6
In parallel, the field of myocardial tissue engineering has pro-
vided a solid foundation for protocols to repopulate various
scaffold materials with human iPSderived cardiomyocytes
and create functional tissue.7,33,6265 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-through-
put screening and as a foundational basis for creating human
myocardial grafts of clinically relevant scale.
With potential translation in mind, we chose to derive
cardiomyocytes from a nongenetically altered pluripotent
cell source that could be generated from patient-derived
somatic cells via repeated administration of synthetic mes-
senger RNAs.24,66 This method is characterized by lack of
vector integration, high efficiency, and low rate of chromo-
somal abnormalities and has been validated by several human
reprogramming laboratories.67 To generate cardiomyocytes
at a clinically relevant scale, we adapted a directed differen-
tiation protocol based on temporal modulation of canonical
Wnt/-catenin signaling to BJ RiPS cultured in monolayer.25
This method yielded 5108 cardiomyocytes 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 de-
velopment.68 Therefore, with Wnt/-catenin signaling,25 we
identified definitive stages for the development of precardi-
ac mesoderm (day 3), cardiac mesoderm (day 5), heart field
specific progenitors (day 7), and iPS-derived cardiomyocytes
(days 14, 30, 60, 120, and 180). Although embryoid body
methods have been applied to this cell line previously,69 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 cardiomyocytes with a high
efficiency.
Using 3D models of increasing complexity, we were then
able to document the formation of mechanically and electri-
cally active myocardial tissue. In slice experiments, we first
confirmed biocompatibility, cell-mediated matrix deforma-
tion, and force generation. The formation of contractile tis-
sue on myocardial slices is evidence of re-establishment of
cellcell adhesions, as well as cellmatrix coupling, which
are essential features of the myocardial microenvironment.70
Despite being a stationary model, the observed development
of strain and force generation in cardiac slices further confirms
that native ECM provides a physiological microenvironment
known to be beneficial to pluripotent cellderived cardiomyo-
cytes and myofibers.13,71 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 station-
ary slices, but still nonperfused, the resulting tissue showed
enhanced cell engraftment and orientation, increased strain
and force generation, electrical conductivity, and improved
fiber microarchitecture. This is in line with the observation
that physiological substrate stiffness improves contractility of
neonatal and iPSC-derived cardiomyocytes.72,73 The observed
improvement in dynamic electrical and mechanical tissue
function in fibers may further be related to improved rigidity
matching and resulting cardiomyocytestissue synchroniza-
tion within the native cardiac ECM.74
 Guyette et al Human Myocardium on Native Extracellular Matrix 69
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.6,62,75,76 Therefore, we attempted tissue assembly
in whole human heart matrices similar to previous reports in
rat and mouse hearts.15,33 Depending on its size, 1 billion
cardiomyocytes are lost because of a myocardial infarction,77
whereas several billion cardiomyocytes would be required to
completely repopulate an acellular human heart. To examine
the clinical feasibility of myocardial regeneration, we at-
tempted to deliver 500 million cardiomyocytes to a highly
vascularized 5 cm3 volume of the left ventricle, between
the left anterior descending and the left circumflex coronary
arteries. Efficient cell delivery with minimal matrix damage
could be achieved via five injections to multiple depths with-
in the ventricular wall over a square epicardial region (Figure
6E).
To enable tissue development, we designed and built a bio-
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
mimetic bioreactor system to accommodate whole-heart scaf-
folds, largely based on reported isolated working heart models,
and the well-documented role of mechanical stimulation in
the formation of myocardial tissue constructs.7881 Media per-
fusion via the native coronary vascular channels provided suf-
ficient nutrient and oxygen supply to enable the formation of
myocardial tissue within the context of the whole-heart scaf-
fold. After a culture period of 14 days, repopulated myocar-
dial segments were metabolically active, provided contractile
function, and responded to electrical stimulation. Myocardial
tissue was immature, showing a range of rounded to elon-
gated sarcomeric phenotypes, which corresponds to reported
morphological features of developing myocardium82,83 and
the in vivo fate of pluripotent cell derived cardiomyocytes.84
This end point 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 highlights the
translational value of the human acellular cardiac matrix 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 recellular-
ization of the myocardial matrix and valve structures are nec-
essary for generating sustainable ventricular pump function.
Achieving this necessitates improved recellularization tech-
niques to enhance cell coverage without damaging the matrix
(eg, unidirectional or bidirectional vascular techniques). In
addition, biomimetic culture conditions (ie, static or dynamic
stretch, electrical stimulation, and media components) must
be optimized to elicit the maturity of reseeded cardiomyo-
cytes and drive tissue development. Considerations should
potentially be made to donor matrix age and composition, for
the promotion of improved cellmatrix integration. A larger-
scale immunologic study is needed for a more complete un-
derstanding of hostresponse to cardiac matrices over time,
as well as to understand the modulatory or reconstructive ef-
fects 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 (ie, 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 analytic purposes. Undoubtedly, many hurdles to-
ward 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 sur-
geons 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
National Institutes of Health grants DK43351 and DK57521). We
thank Jeremy Song, MD, for his contributions to decellularization
process development. In addition, 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 high-density mapping functional
analysis. We thank Patricia Della Pelle, Gertraude Warren, Jed Barger,
and Susan Saidman, PhD, for their help with immunogenic profiling
and human leukocyte antigen testing. For vascular analysis, we thank
Brian Ghoshhajra, MD, from the Department of Radiology at MGH.
Furthermore, we thank Daniel Brooks, Mathew Scott, and Adriana
Martinez-Betancourt from the Center for Advanced Orthopedic
Studies at Beth Israel Deaconess Medical Center for their assistance
with microcomputed tomography of microvasculature. In addition,
we thank Megan Scanlan for her assistance with transmission electron
microscopy on cardiac fibers. Finally, we thank Kengyeh Chu, PhD,
and Joseph Gardecki, PhD, of the Wellman Center for Photomedicine
at MGH for their assistance with microoptical coherence tomogra-
phy on cardiac fibers and microvasculature.
Sources of Funding
The present study was supported by the US National Institutes of
Health Directors New Innovator DP2 OD008749-01, and by the
National Heart Lung and Blood Institute R21 HL108663-2 and R01
HL115282.
Disclosures
Dr Ott is founder and stockholder of IVIVA Medical Inc. This rela-
tionship did not affect the content or conclusions contained in this
article. The other authors report no conflicts.
References
1. Go AS, Mozaffarian D, Roger VL, et al; American Heart Association
Statistics Committee and Stroke Statistics Subcommittee. Heart dis-
ease and stroke statistics2014 update: a report from the American
Heart Association. Circulation. 2014;129:e28e292. doi: 10.1161/01.
cir.0000441139.02102.80.
2. Braunwald E. Heart failure. JACC Heart Fail. 2013;1:120. doi: 10.1016/j.
jchf.2012.10.002.
3. Organ Procurement and Transplantation Network (OPTN)National
Data Reports; U.S. Department of Health and Human Services, Health
Resources Services Administration (HRSA). 2014
 70Circulation ResearchJanuary 8, 2016
4. Tonsho M, Michel S, Ahmed Z, Alessandrini A, Madsen JC. Heart trans-
plantation: challenges facing the field. Cold Spring Harb Perspect Med.
2014;4:. doi: 10.1101/cshperspect.a015636.
5. Hill JA, Olson EN. Cardiac plasticity. N Engl J Med. 2008;358:1370
1380. doi: 10.1056/NEJMra072139.
6. Chong JJ, Yang X, Don CW, et al. Human embryonic-stem-cell-de-
rived cardiomyocytes regenerate non-human primate hearts. Nature.
2014;510:273277. doi: 10.1038/nature13233.
7. Kawamura M, Miyagawa S, Miki K, Saito A, Fukushima S, Higuchi
T, Kawamura T, Kuratani T, Daimon T, Shimizu T, Okano T, Sawa Y.
Feasibility, safety, and therapeutic efficacy of human induced pluripo-
tent stem cell-derived cardiomyocyte sheets in a porcine ischemic car-
diomyopathy model. Circulation. 2012;126:S29S37. doi: 10.1161/
CIRCULATIONAHA.111.084343.
8. Seif-Naraghi SB, Singelyn JM, Salvatore MA, et al. Safety and efficacy of
an injectable extracellular matrix hydrogel for treating myocardial infarc-
tion. Sci Transl Med. 2013;5:173ra25. doi: 10.1126/scitranslmed.3005503.
9. Leor J, Tuvia S, Guetta V, Manczur F, Castel D, Willenz U, Petnehzy
O, Landa N, Feinberg MS, Konen E, Goitein O, Tsur-Gang O, Shaul M,
Klapper L, Cohen S. Intracoronary injection of in situ forming alginate
hydrogel reverses left ventricular remodeling after myocardial infarc-
tion in Swine. J Am Coll Cardiol. 2009;54:10141023. doi: 10.1016/j.
jacc.2009.06.010.
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
10. Coulombe KL, Bajpai VK, Andreadis ST, Murry CE. Heart regeneration
with engineered myocardial tissue. Annu Rev Biomed Eng. 2014;16:128.
doi: 10.1146/annurev-bioeng-071812-152344.
11. Zimmermann WH, Melnychenko I, Eschenhagen T. Engineered heart tis-
sue for regeneration of diseased hearts. Biomaterials. 2004;25:16391647.
12. Radisic M, Deen W, Langer R, Vunjak-Novakovic G. Mathematical model
of oxygen distribution in engineered cardiac tissue with parallel chan-
nel array perfused with culture medium containing oxygen carriers. Am
J Physiol Heart Circ Physiol. 2005;288:H1278H1289. doi: 10.1152/
ajpheart.00787.2004.
13. Engler AJ, Carag-Krieger C, Johnson CP, Raab M, Tang HY, Speicher
DW, Sanger JW, Sanger JM, Discher DE. Embryonic cardiomyocytes beat
best on a matrix with heart-like elasticity: scar-like rigidity inhibits beat-
ing. J Cell Sci. 2008;121:37943802. doi: 10.1242/jcs.029678.
14. Buckberg G, Hoffman JI, Mahajan A, Saleh S, Coghlan C. Cardiac
mechanics revisited: the relationship of cardiac architecture to ven-
tricular function. Circulation. 2008;118:25712587. doi: 10.1161/
CIRCULATIONAHA.107.754424.
15. Ott HC, Matthiesen TS, Goh SK, Black LD, Kren SM, Netoff TI, Taylor
DA. Perfusion-decellularized matrix: using natures platform to engineer a
bioartificial heart. Nat Med. 2008;14:213221. doi: 10.1038/nm1684.
16. Guyette JP, Gilpin SE, Charest JM, Tapias LF, Ren X, Ott HC. Perfusion
decellularization of whole organs. Nat Protoc. 2014;9:14511468. doi:
10.1038/nprot.2014.097.
17. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole
organ decellularization processes. Biomaterials. 2011;32:32333243. doi:
10.1016/j.biomaterials.2011.01.057.
18. Rusconi F, Valton E, Nguyen R, Dufourc E. Quantification of sodium
dodecyl sulfate in microliter-volume biochemical samples by vis-
ible light spectroscopy. Anal Biochem. 2001;295:3137. doi: 10.1006/
abio.2001.5164.
19. Magrane M, Consortium U. UniProt Knowledgebase: a hub of integrat-
ed protein data. Database (Oxford). 2011;2011:bar009. doi: 10.1093/
database/bar009.
20. Ashburner M, Ball CA, Blake JA, et al. Gene ontology: tool for the unifica-
tion of biology. The Gene Ontology Consortium. Nat Genet. 2000;25:25
29. doi: 10.1038/75556.
21. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman
O, Guertin DA, Chang JH, Lindquist RA, Moffat J, Golland P, Sabatini
DM. CellProfiler: image analysis software for identifying and quan-
tifying cell phenotypes. Genome Biol. 2006;7:R100. doi: 10.1186/
gb-2006-7-10-r100.
22. Jones TR, Carpenter AE, Lamprecht MR, Moffat J, Silver SJ, Grenier JK,
Castoreno AB, Eggert US, Root DE, Golland P, Sabatini DM. Scoring
diverse cellular morphologies in image-based screens with iterative feed-
back and machine learning. Proc Natl Acad Sci U S A. 2009;106:1826
1831. doi: 10.1073/pnas.0808843106.
23. Badylak SF, Valentin JE, Ravindra AK, McCabe GP, Stewart-Akers AM.
Macrophage phenotype as a determinant of biologic scaffold remodeling.
Tissue Eng Part A. 2008;14:18351842. doi: 10.1089/ten.tea.2007.0264.
24. Warren L, Manos PD, Ahfeldt T, et al. Highly efficient reprogram-
ming to pluripotency and directed differentiation of human cells with
synthetic modified mRNA. Cell Stem Cell. 2010;7:618630. doi:
10.1016/j.stem.2010.08.012.
25. Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK,
Zhang J, Kamp TJ, Palecek SP. Robust cardiomyocyte differentiation
from human pluripotent stem cells via temporal modulation of canonical
Wnt signaling. Proc Natl Acad Sci U S A. 2012;109:E1848E1857. doi:
10.1073/pnas.1200250109.
26. Kelly DJ, Azeloglu EU, Kochupura PV, Sharma GS, Gaudette GR.
Accuracy and reproducibility of a subpixel extended phase correlation
method to determine micron level displacements in the heart. Med Eng
Phys. 2007;29:154162. doi: 10.1016/j.medengphy.2006.01.001.
27. Kochupura PV, Azeloglu EU, Kelly DJ, Doronin SV, Badylak SF,
Krukenkamp IB, Cohen IS, Gaudette GR. Tissue-engineered myo-
cardial patch derived from extracellular matrix provides regional me-
chanical function. Circulation. 2005;112:I144I149. doi: 10.1161/
CIRCULATIONAHA.104.524355.
28. Liu L, Gardecki JA, Nadkarni SK, Toussaint JD, Yagi Y, Bouma BE,
Tearney GJ. Imaging the subcellular structure of human coronary ath-
erosclerosis using micro-optical coherence tomography. Nat Med.
2011;17:10101014. doi: 10.1038/nm.2409.
29. Liu J, Sun N, Bruce MA, Wu JC, Butte MJ. Atomic force mechanobi-
ology of pluripotent stem cell-derived cardiomyocytes. PLoS One.
2012;7:e37559. doi: 10.1371/journal.pone.0037559.
30. Rodriguez ML, Murry CE, Sniadecki NJ. Assessment of induced pluripo-
tent stem cell-derived cardiac myocyte contractility using micropost ar-
rays, ASME 2013 Summer Bioengineering Conference; SBC2013-14640,
The American Society of Mechanical Engineers, pp. V01AT15A005,
2013.
31. Xi J, Khalil M, Shishechian N, et al. Comparison of contractile behavior
of native murine ventricular tissue and cardiomyocytes derived from em-
bryonic or induced pluripotent stem cells. FASEB J. 2010;24:27392751.
doi: 10.1096/fj.09-145177.
32. Wainwright JM, Czajka CA, Patel UB, Freytes DO, Tobita K, Gilbert TW,
Badylak SF. Preparation of cardiac extracellular matrix from an intact por-
cine heart. Tissue Eng Part C Methods. 2010;16:525532. doi: 10.1089/
ten.TEC.2009.0392.
33. Lu TY, Lin B, Kim J, Sullivan M, Tobita K, Salama G, Yang L.
Repopulation of decellularized mouse heart with human induced plu-
ripotent stem cell-derived cardiovascular progenitor cells. Nat Commun.
2013;4:2307. doi: 10.1038/ncomms3307.
34. Snchez PL, Fernndez-Santos ME, Costanza S, et al. Acellular human
heart matrix: A critical step toward whole heart grafts. Biomaterials.
2015;61:279289. doi: 10.1016/j.biomaterials.2015.04.056.
35. Song JJ, Guyette JP, Gilpin SE, Gonzalez G, Vacanti JP, Ott HC.
Regeneration and experimental orthotopic transplantation of a bioengi-
neered kidney. Nat Med. 2013;19:646651. doi: 10.1038/nm.3154.
36. Gilpin SE, Guyette JP, Gonzalez G, Ren X, Asara JM, Mathisen DJ,
Vacanti JP, Ott HC. Perfusion decellularization of human and porcine
lungs: bringing the matrix to clinical scale. J Heart Lung Transplant.
2014;33:298308. doi: 10.1016/j.healun.2013.10.030.
37. Osaki S, Anderson JE, Johnson MR, Edwards NM, Kohmoto T. The poten-
tial of cardiac allografts from donors after cardiac death at the University
of Wisconsin Organ Procurement Organization. Eur J Cardiothorac Surg.
2010;37:7479. doi: 10.1016/j.ejcts.2009.07.005.
38. Zych B, Popov AF, Amrani M, Bahrami T, Redmond KC, Krueger H,
Carby M, Simon AR. Lungs from donation after circulatory death donors:
an alternative source to brain-dead donors? Midterm results at a single in-
stitution. Eur J Cardiothorac Surg. 2012;42:542549. doi: 10.1093/ejcts/
ezs096.
39. Alvarez J, del Barrio R, Arias J, Vzquez S, Snchez A, Iglesias J, Barra
C, Ibarguren C. Non-heart-beating donors: estimated actual potential.
Transplant Proc. 2001;33:11011103.
40. Garca Sez D, Elbetanony A, Lezberg P, Hassanein A, Bowles CT, Popov
AF, Zych B, Sabashnikov A, Mohite P, Simon AR. Ex vivo heart perfusion
after cardiocirculatory death; a porcine model. J Surg Res. 2015;195:311
314. doi: 10.1016/j.jss.2014.12.039.
41. Crick SJ, Sheppard MN, Ho SY, Gebstein L, Anderson RH. Anatomy of
the pig heart: comparisons with normal human cardiac structure. J Anat.
1998;193(pt 1):105119.
42. Lelovas PP, Kostomitsopoulos NG, Xanthos TT. A comparative anatomic
and physiologic overview of the porcine heart. J Am Assoc Lab Anim Sci.
2014;53:432438.
43. Sokocevic D, Bonenfant NR, Wagner DE, Borg ZD, Lathrop MJ, Lam
YW, Deng B, Desarno MJ, Ashikaga T, Loi R, Hoffman AM, Weiss
DJ. The effect of age and emphysematous and fibrotic injury on the
 Guyette et al Human Myocardium on Native Extracellular Matrix 71
re-cellularization of de-cellularized lungs. Biomaterials. 2013;34:3256
3269. doi: 10.1016/j.biomaterials.2013.01.028.
44. Ott HC, Clippinger B, Conrad C, Schuetz C, Pomerantseva I, Ikonomou
L, Kotton D, Vacanti JP. Regeneration and orthotopic transplantation of a
bioartificial lung. Nat Med. 2010;16:927933. doi: 10.1038/nm.2193.
45. Rodriguez-Porcel M, Lerman A, Ritman EL, Wilson SH, Best PJ, Lerman
LO. Altered myocardial microvascular 3D architecture in experimental
hypercholesterolemia. Circulation. 2000;102:20282030.
46. Sangaralingham SJ, Ritman EL, McKie PM, Ichiki T, Lerman A, Scott CG,
Martin FL, Harders GE, Bellavia D, Burnett JC Jr. Cardiac micro-computed
tomography imaging of the aging coronary vasculature. Circ Cardiovasc
Imaging. 2012;5:518524. doi: 10.1161/CIRCIMAGING.112.973057.
47. Cansz FB, Dal H, Kaliske M. An orthotropic viscoelastic mate-
rial model for passive myocardium: theory and algorithmic treatment.
Comput Methods Biomech Biomed Engin. 2015;18:11601172. doi:
10.1080/10255842.2014.881475.
48. Holzapfel GA, Ogden RW. Constitutive modelling of passive myocardium:
a structurally based framework for material characterization. Philos Trans
A Math Phys Eng Sci. 2009;367:34453475. doi: 10.1098/rsta.2009.0091.
49. Engelmayr GC Jr, Cheng M, Bettinger CJ, Borenstein JT, Langer R, Freed
LE. Accordion-like honeycombs for tissue engineering of cardiac anisot-
ropy. Nat Mater. 2008;7:10031010. doi: 10.1038/nmat2316.
50. Costa KD, Lee EJ, Holmes JW. Creating alignment and anisotropy
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
in engineered heart tissue: role of boundary conditions in a model
three-dimensional culture system. Tissue Eng. 2003;9:567577. doi:
10.1089/107632703768247278.
51. Oberwallner B, Brodarac A, Choi YH, Saric T, Ani P, Morawietz L,
Stamm C. Preparation of cardiac extracellular matrix scaffolds by decellu-
larization of human myocardium. J Biomed Mater Res A. 2014;102:3263
3272. doi: 10.1002/jbma.35000.
52. Friess W. Collagenbiomaterial for drug delivery. Eur J Pharm Biopharm.
1998;45:113136.
53. Badylak SF, Gilbert TW. Immune response to biologic scaffold materials.
Semin Immunol. 2008;20:109116. doi: 10.1016/j.smim.2007.11.003.
54. Brown BN, Londono R, Tottey S, Zhang L, Kukla KA, Wolf MT, Daly
KA, Reing JE, Badylak SF. Macrophage phenotype as a predictor of con-
structive remodeling following the implantation of biologically derived
surgical mesh materials. Acta Biomater. 2012;8:978987. doi: 10.1016/j.
actbio.2011.11.031.
55. Brown BN, Ratner BD, Goodman SB, Amar S, Badylak SF. Macrophage
polarization: an opportunity for improved outcomes in biomaterials and
regenerative medicine. Biomaterials. 2012;33:37923802. doi: 10.1016/j.
biomaterials.2012.02.034.
56. Ariganello MB, Simionescu DT, Labow RS, Lee JM. Macrophage
differentiation and polarization on a decellularized pericardi-
al biomaterial. Biomaterials. 2011;32:439449. doi: 10.1016/j.
biomaterials.2010.09.004.
57. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K,
Yamanaka S. Induction of pluripotent stem cells from adult human fi-
broblasts by defined factors. Cell. 2007;131:861872. doi: 10.1016/j.
cell.2007.11.019.
58. Zhang J, Wilson GF, Soerens AG, Koonce CH, Yu J, Palecek SP, Thomson
JA, Kamp TJ. Functional cardiomyocytes derived from human induced
pluripotent stem cells. Circ Res. 2009;104:e30e41. doi: 10.1161/
CIRCRESAHA.108.192237.
59. Citro L, Naidu S, Hassan F, Kuppusamy ML, Kuppusamy P, Angelos MG,
Khan M. Comparison of human induced pluripotent stem-cell derived
cardiomyocytes with human mesenchymal stem cells following acute
myocardial infarction. PLoS One. 2014;9:e116281. doi: 10.1371/journal.
pone.0116281.
60. Carpenter L, Carr C, Yang CT, Stuckey DJ, Clarke K, Watt SM. Efficient
differentiation of human induced pluripotent stem cells generates cardiac
cells that provide protection following myocardial infarction in the rat.
Stem Cells Dev. 2012;21:977986. doi: 10.1089/scd.2011.0075.
61. Malecki M, Putzer E, Sabo C, Foorohar A, Quach C, Stampe C, Beauchaine
M, Malecki R, Tombokan X, Anderson M. Directed cardiomyogenesis of
autologous human induced pluripotent stem cells recruited to infarcted
myocardium with bioengineered antibodies. Mol Cell Ther. 2014;2. doi:
10.1186/2052-8426-2-13.
62. Kawamura M, Miyagawa S, Fukushima S, Saito A, Miki K, Ito E,
Sougawa N, Kawamura T, Daimon T, Shimizu T, Okano T, Toda K, Sawa
Y. Enhanced survival of transplanted human induced pluripotent stem cell-
derived cardiomyocytes by the combination of cell sheets with the pedicled
omental flap technique in a porcine heart. Circulation. 2013;128:S87S94.
doi: 10.1161/CIRCULATIONAHA.112.000366.
63. Masumoto H, Ikuno T, Takeda M, Fukushima H, Marui A, Katayama
S, Shimizu T, Ikeda T, Okano T, Sakata R, Yamashita JK. Human iPS
cell-engineered cardiac tissue sheets with cardiomyocytes and vascu-
lar cells for cardiac regeneration. Sci Rep. 2014;4:6716. doi: 10.1038/
srep06716.
64. Masumoto H, Matsuo T, Yamamizu K, Uosaki H, Narazaki G, Katayama
S, Marui A, Shimizu T, Ikeda T, Okano T, Sakata R, Yamashita JK.
Pluripotent stem cell-engineered cell sheets reassembled with defined
cardiovascular populations ameliorate reduction in infarct heart func-
tion through cardiomyocyte-mediated neovascularization. Stem Cells.
2012;30:11961205. doi: 10.1002/stem.1089.
65. Fujita J, Itabashi Y, Seki T, Tohyama S, Tamura Y, Sano M, Fukuda
K. Myocardial cell sheet therapy and cardiac function. Am J
Physiol Heart Circ Physiol. 2012;303:H1169H1182. doi: 10.1152/
ajpheart.00376.2012.
66. Soares FA, Sheldon M, Rao M, Mummery C, Vallier L. International co-
ordination of large-scale human induced pluripotent stem cell initiatives:
Wellcome Trust and ISSCR workshops white paper. Stem Cell Reports.
2014;3:931939. doi: 10.1016/j.stemcr.2014.11.006.
67. Schlaeger TM, Daheron L, Brickler TR, Entwisle S, Chan K, Cianci A,
DeVine A, Ettenger A, Fitzgerald K, Godfrey M, Gupta D, McPherson J,
Malwadkar P, Gupta M, Bell B, Doi A, Jung N, Li X, Lynes MS, Brookes
E, Cherry AB, Demirbas D, Tsankov AM, Zon LI, Rubin LL, Feinberg
AP, Meissner A, Cowan CA, Daley GQ. A comparison of non-integrating
reprogramming methods. Nat Biotechnol. 2015;33:5863
68. Mummery CL, Zhang J, Ng ES, Elliott DA, Elefanty AG, Kamp TJ.
Differentiation of human embryonic stem cells and induced plu-
ripotent stem cells to cardiomyocytes: a methods overview. Circ Res.
2012;111:344358. doi: 10.1161/CIRCRESAHA.110.227512.
69. Mehta A, Verma V, Nandihalli M, Ramachandra CJ, Sequiera GL, Sudibyo
Y, Chung Y, Sun W, Shim W. A systemic evaluation of cardiac differentia-
tion from mRNA reprogrammed human induced pluripotent stem cells.
PLoS One. 2014;9:e103485. doi: 10.1371/journal.pone.0103485.
70. McCain ML, Lee H, Aratyn-Schaus Y, Klber AG, Parker KK.
Cooperative coupling of cell-matrix and cell-cell adhesions in cardiac
muscle. Proc Natl Acad Sci U S A. 2012;109:98819886. doi: 10.1073/
pnas.1203007109.
71. Engler AJ, Griffin MA, Sen S, Bnnemann CG, Sweeney HL, Discher DE.
Myotubes differentiate optimally on substrates with tissue-like stiffness:
pathological implications for soft or stiff microenvironments. J Cell Biol.
2004;166:877887. doi: 10.1083/jcb.200405004.
72. Rodriguez AG, Han SJ, Regnier M, Sniadecki NJ. Substrate stiffness
increases twitch power of neonatal cardiomyocytes in correlation with
changes in myofibril structure and intracellular calcium. Biophys J.
2011;101:24552464. doi: 10.1016/j.bpj.2011.09.057.
73. Marsano A, Maidhof R, Wan LQ, Wang Y, Gao J, Tandon N, Vunjak-
Novakovic G. Scaffold stiffness affects the contractile function of
three-dimensional engineered cardiac constructs. Biotechnol Prog.
2010;26:13821390. doi: 10.1002/btpr.435.
74. Hrning M, Kidoaki S, Kawano T, Yoshikawa K. Rigidity match-
ing between cells and the extracellular matrix leads to the stabilization
of cardiac conduction. Biophys J. 2012;102:379387. doi: 10.1016/j.
bpj.2011.12.018.
75. Shiba Y, Fernandes S, Zhu WZ, et al. Human ES-cell-derived cardio-
myocytes electrically couple and suppress arrhythmias in injured hearts.
Nature. 2012;489:322325. doi: 10.1038/nature11317.
76. Zhang F, Song G, Li X, Gu W, Shen Y, Chen M, Yang B, Qian L, Cao
K. Transplantation of iPSc ameliorates neural remodeling and reduces
ventricular arrhythmias in a post-infarcted swine model. J Cell Biochem.
2014;115:531539. doi: 10.1002/jcb.24687.
77. Laflamme MA, Murry CE. Regenerating the heart. Nat Biotechnol.
2005;23:845856. doi: 10.1038/nbt1117.
78. Hill AJ, Laske TG, Coles JA Jr, Sigg DC, Skadsberg ND, Vincent
SA, Soule CL, Gallagher WJ, Iaizzo PA. In vitro studies of hu-
man hearts. Ann Thorac Surg. 2005;79:168177. doi: 10.1016/j.
athoracsur.2004.06.080.
79. Hlsmann J, Aubin H, Kranz A, Godehardt E, Munakata H, Kamiya H,
Barth M, Lichtenberg A, Akhyari P. A novel customizable modular bio-
reactor system for whole-heart cultivation under controlled 3D biome-
chanical stimulation. J Artif Organs. 2013;16:294304. doi: 10.1007/
s10047-013-0705-5.
80. Wang B, Wang G, To F, Butler JR, Claude A, McLaughlin RM, Williams
LN, de Jongh Curry AL, Liao J. Myocardial scaffold-based cardiac tissue
engineering: application of coordinated mechanical and electrical stimula-
tions. Langmuir. 2013;29:1110911117. doi: 10.1021/la401702w.
 72Circulation ResearchJanuary 8, 2016
81. Vunjak-Novakovic G, Tandon N, Godier A, Maidhof R, Marsano
83. Yang X, Pabon L, Murry CE. Engineering adolescence: maturation
A, Martens TP, Radisic M. Challenges in cardiac tissue engineer-
ing. Tissue Eng Part B Rev. 2010;16:169187. doi: 10.1089/ten.
TEB.2009.0352.
82. Du A, Sanger JM, Sanger JW. Cardiac myofibrillogenesis inside in-
tact embryonic hearts. Dev Biol. 2008;318:236246. doi: 10.1016/j.
ydbio.2008.03.011.
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 car-
diac tissue graft or a bioartificial heart, theoretically holds the promise
to improve contractile function to the injured heart.
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016
What New Information Does This Article Contribute?
Human hearts were decellularized using aseptic pressure-controlled
perfusion to create acellular cardiac scaffolds with preserved extra-
cellular matrix components, architecture, material properties, and
microvasculature.
Characterization of the acellular human scaffolds with thorough pro-
teomics, microvasculature, and immunogenicity analyses.
Human cardiac myocytes were derived from a nontransgenic induced
pluripotent stem cell line, to generate cardiac myocytes at a clinically
relevant scale and to create force-generating human myocardial-
like tissue slices, fibers, and ventricular segments on whole-heart
scaffolds.
In a first step toward the creation of bioartificial myocardium,
we sought to engineer myocardial tissue that combines acellular
of human pluripotent stem cell-derived cardiomyocytes. Circ Res.
2014;114:511523. doi: 10.1161/CIRCRESAHA.114.300558.
84. Lundy SD, Zhu WZ, Regnier M, Laflamme MA. Structural and func-
tional maturation of cardiomyocytes derived from human pluripo-
tent stem cells. Stem Cells Dev. 2013;22:19912002. doi: 10.1089/
scd.2012.0490.
human cardiac matrix and clinically relevant human induced
pluripotent stem cellderived 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 con-
tent 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 con-
tained a preserved microvasculature, with a vessel distribution
similar to cadaveric hearts. Using a directed cardiac differentia-
tion protocol on a nontransgenic induced pluripotent stem cell
line, we generated a clinically relevant number of human cardiac
myocytes. We then reseeded acellular human cardiac matrix with
human induced pluripotent stem cellderived cardiac myocytes
to create force-generating myocardial-like tissues of increasing
3-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
and to show that functional myocardial tissue could be built on a
whole-heart platform.
 Bioengineering Human Myocardium on Native Extracellular Matrix
Jacques P. Guyette, Jonathan M. Charest, Robert W. Mills, Bernhard J. Jank, Philipp T. Moser,
Sarah E. Gilpin, Joshua R. Gershlak, Tatsuya Okamoto, Gabriel Gonzalez, David J. Milan,
Downloaded from http://circres.ahajournals.org/ by guest on December 6, 2016

Glenn R. Gaudette and Harald C. Ott

Circ Res. 2016;118:56-72; originally published online October 26, 2015;

doi: 10.1161/CIRCRESAHA.115.306874
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright 2015 American Heart Association, Inc. All rights reserved.
Print ISSN: 0009-7330. Online ISSN: 1524-4571

The online version of this article, along with updated information and services, is located on the
World Wide Web at:
http://circres.ahajournals.org/content/118/1/56

Data Supplement (unedited) at:

http://circres.ahajournals.org/content/suppl/2015/10/26/CIRCRESAHA.115.306874.DC1.html

Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published
in Circulation Research can be obtained via RightsLink, a service of the Copyright Clearance Center, not the
Editorial Office. Once the online version of the published article for which permission is being requested is
located, click Request Permissions in the middle column of the Web page under Services. Further information
about this process is available in the Permissions and Rights Question and Answer document.

Reprints: Information about reprints can be found online at:

http://www.lww.com/reprints

Subscriptions: Information about subscribing to Circulation Research is online at:

http://circres.ahajournals.org//subscriptions/
 CIRCRES/2015/306874D/R2

SUPPLEMENTAL MATERIAL

Bioengineering Human Myocardium on Native Extracellular Matrix

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 4C.

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
manufacturers 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 L of a
solution containing 10 mM Tris (pH = 8.0), 5 mM EDTA, 0.1 M NaCl, 1% SDS, and 650 g/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

2
 CIRCRES/2015/306874D/R2

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 4C 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 m. 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 (25C). 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 4C 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 m. Sections were deparaffinized with xylene
and rehydration through a graded ethanol series, and then treated for 30 minutes in citrate buffer at 95C
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;

3
 CIRCRES/2015/306874D/R2

Abcam, Cambridge, MA). Secondary antibody was applied for 1 hour at room temperature (25C).
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 m sections were
deparaffinized, and treated for 30 minutes in citrate buffer at 95C 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 4C 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 25C. 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 m sections were deparaffinized, and treated for 30
minutes in citrate buffer at 95C 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 -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 (25C).
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 manufactures protocol (Promega
Corporation, Madison, WI). Tissues slides were mounted with Dapi Fluoromount-G (Southern Biotech,
Birmingham, AL). Massons Trichrome was performed using reagents and methods based on kit
manufacturers 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 m
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 manufacturers
instructions (American MasterTech, Lodi, CA).

4
 CIRCRES/2015/306874D/R2

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 4C. 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, CT, 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 4C for 24 hours. Computed Tomography (CT): 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. CT imaging was
performed on a piece of human heart using a high-resolution desktop imaging system (CT40, Scanco
Medical AG, Bruttisellen, Switzerland). Transverse slices were acquired with 6 m3 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-CT Analysis of Vasculature: Two-
dimensional CT 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 m, 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. Optical Coherence Tomography
(OCT) of Perfused Vasculature: 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 m 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 (OCT) was used to acquire image stacks ~200 m
below the surface of the cut mid- and endocardium, at a frame rate of 40 fps for 30 seconds (OCT

5
 CIRCRES/2015/306874D/R2

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 4C 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
samples 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 4C. 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 -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 (25C). 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 -actinin and
cardiac troponin T independently, the cells were fixed with 4% paraformaldehyde at room temperature for

6
 CIRCRES/2015/306874D/R2

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 l PBS and assayed by flow cytometry (FACS Calibur;
BD Biosciences, Franklin Lakes, NJ). Mouse IgG anti--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 -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-m 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
37C. 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 L. 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 m 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

7
 CIRCRES/2015/306874D/R2

assessed on deforming matrix 100-200 m below the fiber surface, using a method called micro-optical
coherence tomography (OCT) to create image stacks at a frame rate of 40 fps for 30 seconds (OCT
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 (37C) 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 N; 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 (OCT). The use of OCT 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. OCT as implemented in this manuscript is based
on Spectral-domain OCT (SD-OCT).15 The application of OCT in this setup employed very broad
bandwidth light source (800150 nm laser-generated supercontinuum) and a common path reference arm
to achieve 1-m 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 m. Apodization and chromatic dispersion extended the focal depth to ~250 m,
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 37C
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).

8
 CIRCRES/2015/306874D/R2

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
37C 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 37C. 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 37C 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-STAT1
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 4C. 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 students 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
Suppl
ement alFi
gureI
Phot
ographoftheperfusi
ondecell
ulari
zati
onsetup.From l
efttori
ght:Lapt
oprunning
pumpcontr
olsoft
ware,pumpcont r
oll
ers,chambercontai
ningahumanhear tundergo-
i
ngthedecell
ular
izat
i
onpr ocess,r
oll
erpump,50Lcar boyflui
dreservoi
r.
 **

** **

Suppl ement alFigur eI I

Biochemical analysi
sf orinsolublecollagen(hydr
oxyproli
ne)
,solublecoll
agen,-elas-
ti
n,andsul fatedglycosami nogl ycans( s-
GAGs)onnor malcadaver i
chumanmyocar -
dium (C,n=5hear t
s) ,decellular
izedhumancar diacmatri
xfrom heartsdonatedafter
braindeath( B,n=4) ,anddecel l
ulari
zedhumancar diacmat r
ixf
rom heartsdonated
aftercar
diacdeat h( D,n=4) . Threeti
ssuesampleswer eanalyzedperhear tpercom-
ponentandt heaver ageoft hoset hreewasusedast herepresentat
ivevalueforthe
componenti nthathear t.Normal i
zedt oti
ssuedryweight.Errorbarsarestandardde
vi
at i
on.Asterisks(*)indicatep<0. 05compar edtocadavericconditi
on.
Suppl
ement alFigureII
I
I
mmunohist
ochemi calstai
ning ofcadaveri
cmyocar
dium f
orcol
lagensI
,II
I,I
V,f
i
-
br
onect
in,l
aminin,andmyosi nheavychain(MHC)
.Scalebar
,200m.
Suppl
ement alFi
gureIV
Adi
poset
issuemat r
ixr
emai
ningaf
tert
hedecel
l
ular
izat
ionpr
ocess.Scal
ebar
,100m.
Suppl
ement alFi
gureV
Quant
if
icat
ionofcel
lmar
ker
s(CD68,CD80,CD163)usi
ngCel
l
Prof
il
erandCel
l
Prof
il
erAnal
yst
.

A.Grayscaleimagesofanucl earstain(
DAPI )ar
ereadint
oCellPr
ofi
l
er(www.cel
lprofi
ler
.or
g)andnucl
ei
obj
ectsarei dent
if
ied.Nucleiobjectsarepropagatedout
wardsbyaspeci f
ieddistance(20pixel
s)t
o
obt
aincellobject
s.Cytopl
asm objectsar
ethengener at
edbysubtr
act
ingnucl
eiobjectsfr
om cel
lobj
ect
s.
Nucl
ei,cel
l,andcyt opl
asm object
sshareaone- t
o-onerel
ati
onshi
p.

B.Correspondi
ngi magesoft hemarker(
s)ofint
erestareoverl
aidonthenuclei
,cell
,andcytoplasm ob-
j
ects.Vari
ousar ea,shape,int
ensi
t
y,andt ext
urefeatur
esareextract
edperobjectforeachobjectcl
ass
andstoredinaSQLi t
edatabase.Toextractt
hesef eat
ures,aCellPr
ofi
l
erpipel
inewasusedbasedon:
JonesTR,etal .ProcNat AcadSciUSA.2009Feb10;
l 106(6)
:1826-
31.Featuresareextr
actedforall
markersofint
erestwithi
nani mage(e.g.CD68onl y
,CD68wi thCD80,orCD68wi t
hCD163) .

C.Imaget humbnai
lsoftheidenti
fi
edandmeasuredobj ectsarethenpresentedtot
heuser /r
esearcher
whogener atesanexamplesetofposit
iveandnegati
vecel l
s(atr
ainingset)
.UsingCell
Pr of
il
erAnalyst
,
aclassi
fi
eristhentr
ainedwiththi
ssetandsubsequentlyusedt oscoreallofthecel
lsidenti
fiedi
nt he
i
nputimages.Theoutputist
henumberofcel l
sbelongi
ngt oeachclass(posi
ti
veornegative,redorblue
overl
ayrespecti
vel
yinexample)perimage.
 A

B C

F G

SupplementalFigureVI
Whit
ebloodcell(WBC)componentper
cent
ages(
n=3r
atspercondi
t
ion)
.Er
rorbar
sar
e
st
andarddeviat
i
on.fL,f
emtol
i
ter
s.

A.Sub-populati
onsofWBCsasaper cent
ageoftot
alWBCs.LYM,lymphocyt
es;MONO,
monocytes;GRAN,gr anul
ocyt
es
B.HCT,hemat ocrit
C.RBC,r edbloodcel l
s
D.MCV,meancel lvolumeofRBCs;RDWa,r edcel
lwidth
E.HGB,hemogl obinconcent
rat
ion;MCHC,meancel lhemogl
obi
nconcent
rat
ion
F.PLT,t
otalplat
eletcount
G.MPV,meanpl ateletvol
ume
Decel
l
ular
izedHHwi
t
houtcor
onar
yar
ter
ydi
sease

LCA RCA
LCX

LAD
OM
AM

Decel
l
ular
izedHHwi
thcor
onar
yar
ter
ydi
sease

Suppl
ement
alFi
gur
eVI
I

Top:Coronar
yangi ogr
aphyofl
eftandrightmaincoronaryart
eriesi
ndecell
ular
ized
humanhear t
s.Lef
tcoronaryar
ter
y(LCA) ,l
eftant
eri
ordescendingart
ery(LAD),lef
t
ci
rcumfl
exarter
y( LCX),obt
usemar gi
nalart
ery(OM) ,ri
ghtcoronar
yarter
y( RCA),
acutemargi
nalart
ery(AM).

Bott
om:Angiogr
aphyofanacell
ularhear
tfrom apati
entpresent
ingwithcoronar
y
ar
terydi
sease.Whit
e ar
rowsindi
cateluminalnar
rowi
ng r
elated t
o at
heroscl
erot
ic
pl
aques.
 Cardi
omyocyt
e
Defor
mati
on

A0 A1
Car
diomyocyt
es

10m
200m
A0 A1
Ac
ell
ularCar
diacMat
ri
x

CardiacMatr
ix
Deformat
ion

Suppl
ement
alFi
gur
eVI
II

Schemat
icofHDM anal
ysi
sforar
eastr
ainofmat
ri
x-seededhumancar
diomy-
ocyt
esandunder
lyi
ngcardi
acmatri
x.
 A

Medi
a For
ce
Tr
ansducer
Sl
i
ce

Cont
ainer

Medi
a For
ce
Fi
ber
Tr
ansducer

Cont
ainer

Suppl
ement
alFi
gur
eIX

A.Schemat i
cdepict
ingsetupf ormeasuringf
orcegenerat
ioninr eseededcar diac
sl
ices.Thesl
icei
sattachedtothecontai
nerwhi
lethefor
cetransducerisbroughtinto
contactwi
thcont
ract
i
lenodules.Greenarrowsi
ndicat
eaxisofnodulecontract
il
i
ty.

B.Schematicdepicti
ngsetupf
ormeasuri
ngforcegenerat
ioni nr
eseededcardi
ac
f
iber
s.Thelefthookisfi
xedwhi
let
herighthookisfr
ee-
movi ngandatt
achedtothe
f
orcetr
ansducer.Greenar
rowsi
ndi
cat
eaxisoffi
bercont
ract
il
ity
.
SupplementalFigureX
Schematicoff
orce-
curvemetri
csil
l
ustr
ati
ngampl
i
tude,t
imet
o50% r
elaxat
ion
(
TTR50),andti
met o90% r
elaxat
ion(TTR90)
.
 A

SupplementalFigureXI
Captur
edf r
equenciesofr
eseededcar
diacsl
i
ces(A)andr
eseededcar
diac
f
iber
s( B)underunpaced(
U)andpacedcondi
ti
ons.
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
44 C F B 29 170.18 99.79 34.52 Biaxial mechanical testing
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
60 C F B 51 154.94 62.60 26.12 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
REFERENCES

1. Guyette JP, Gilpin SE, Charest JM, Tapias LF, Ren X, Ott HC. Perfusion decellularization of whole
organs. Nature protocols. 2014;9:1451-1468
2. Crapo PM, Gilbert TW, Badylak SF. An overview of tissue and whole organ decellularization processes.
Biomaterials. 2011;32:3233-3243
3. Magrane M, Consortium U. Uniprot knowledgebase: A hub of integrated protein data. Database : the
journal of biological databases and curation. 2011;2011:bar009
4. Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS,
Eppig JT, Harris MA, Hill DP, Issel-Tarver L, Kasarskis A, Lewis S, Matese JC, Richardson JE,
Ringwald M, Rubin GM, Sherlock G. Gene ontology: Tool for the unification of biology. The gene
ontology consortium. Nature genetics. 2000;25:25-29
5. Rodella LF, Rezzani R, Buffoli B, Bonomini F, Tengattini S, Laffranchi L, Paganelli C, Sapelli PL,
Bianchi R. Role of mast cells in wound healing process after glass-fiber composite implant in rats.
Journal of cellular and molecular medicine. 2006;10:946-954
6. Rezzani R, Rodella L, Tartaglia GM, Paganelli C, Sapelli P, Bianchi R. Mast cells and the inflammatory
response to different implanted biomaterials. Archives of histology and cytology. 2004;67:211-217
7. Carpenter AE, Jones TR, Lamprecht MR, Clarke C, Kang IH, Friman O, Guertin DA, Chang JH,
Lindquist RA, Moffat J, Golland P, Sabatini DM. Cellprofiler: Image analysis software for identifying
and quantifying cell phenotypes. Genome biology. 2006;7:R100
8. Maherali N, Ahfeldt T, Rigamonti A, Utikal J, Cowan C, Hochedlinger K. A high-efficiency system for
the generation and study of human induced pluripotent stem cells. Cell stem cell. 2008;3:340-345
9. Lian X, Hsiao C, Wilson G, Zhu K, Hazeltine LB, Azarin SM, Raval KK, Zhang J, Kamp TJ, Palecek SP.
Robust cardiomyocyte differentiation from human pluripotent stem cells via temporal modulation of
canonical wnt signaling. Proc Natl Acad Sci U S A. 2012;109:E1848-1857
10. Guyette JP, Fakharzadeh M, Burford EJ, Tao ZW, Pins GD, Rolle MW, Gaudette GR. A novel suture-
based method for efficient transplantation of stem cells. J Biomed Mater Res A. 2013;101:809-818
11. Kelly DJ, Azeloglu EU, Kochupura PV, Sharma GS, Gaudette GR. Accuracy and reproducibility of a
subpixel extended phase correlation method to determine micron level displacements in the heart. Med
Eng Phys. 2007;29:154-162
12. Foroosh H, Zerubia J. Extension of phase correlation to subpixel registration. IEEE Transactions on
Image Processing. 2002;11:188-200
13. Gaudette GR, Todaro J, Krukenkamp IB, Chiang FP. Computer aided speckle interferometry: A
technique for measuring deformation of the surface of the heart. Ann Biomed Eng. 2001;29:775-780
14. Liu L, Gardecki JA, Nadkarni SK, Toussaint JD, Yagi Y, Bouma BE, Tearney GJ. Imaging the
subcellular structure of human coronary atherosclerosis using micro-optical coherence tomography. Nat
Med. 2011;17:1010-1014
15. Ha Usler G, Lindner MW. "Coherence radar" and "spectral radar"-new tools for dermatological diagnosis.
Journal of biomedical optics. 1998;3:21-31
16. Oliphant TE. Python for scientific computing. IEEE COMPUTER SOC. 2007;9:10-20
17. Aivazis KJMaM. Python for scientists and engineers. IEEE COMPUTER SOC. 2011;13:9-12
18. Du P, Kibbe WA, Lin SM. Improved peak detection in mass spectrum by incorporating continuous
wavelet transform-based pattern matching. Bioinformatics. 2006;22:2059-2065
19. De Mey S, Thomas JD, Greenberg NL, Vandervoort PM, Verdonck PR. Assessment of the time constant
of relaxation: Insights from simulations and hemodynamic measurements. Am J Physiol Heart Circ
Physiol. 2001;280:H2936-2943
20. Hunter JD. Matplotlib: A 2d graphics environment. IEEE COMPUTER SOC. 2007;9:90-95
21. Badylak SF, Valentin JE, Ravindra AK, McCabe GP, Stewart-Akers AM. Macrophage phenotype as a
determinant of biologic scaffold remodeling. Tissue Eng Part A. 2008;14:1835-1842
VIDEO FILE LEGENDS

Supplemental Video I
bead perfusion through the conductance artery and microvasculature within the mid-myocardium of a
decellularized human heart, acquired using -optical coherence tomography (OCT).

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.