In vivo grafting of large engineered heart tissue patches for cardiac repair

Engineered Heart Tissue (EHT) from neonatal rat cardiomyocytes has been used successfully as in vitro model and in cardiac repair. Here, we hypothesized that human embryonic stem cells (hESC) can be used to generate EHT with properties of native myocardium. Methods: hESC (hES3-ENVY) were differentiated in embryoid bodies, enzymatically dispersed, and subjected to EHT-generation in circular casting molds (1.5x10 6 cells, 0.4 mg collagen, 10% Matrigel/EHT; inner/outer diameter - 2/4 mm). Contractile function was assessed 10 days after casting under isometric conditions (37°C, 1.5 Hz, Tyrode’s solution). Action potentials (AP) were recorded in spontaneously contracting EHTs with intracellular electrodes (37°C, Tyrode’s solution). Calcium gradients were assessed by confocal laser scanning microscopy (CLSM) after rhod-2 loading. EHT-morphology was examined by CLSM and electron microscopy (EM). Results: hESC-EHTs contracted synchronously and spontaneously at 1.1±0.1 Hz (n=3). Increasing concentrations of extracellular calcium (0.2–2.4 mM) enhanced force of contraction from 53±8 to 199±22 μN (n=8, p<0.05; EC 50 : 0.8±0.04 mM). Isoprenaline (1 μM) at 0.4 mM calcium increased twitch tension from 61±7 to 108±15 μN (n=8, p<0.05) and shortened relaxation time from 111±6 to 87±4 ms (n=3, p<0.05). Cardiomyocytes within EHTs formed a functional syncytium composed of predominantly oriented muscle strands with a high degree of sarcomere differentiation (CLSM, EM). Cell-cell contacts through adherens junctions were identified by EM. Synchronous calcium gradient spread in spontaneously contracting EHTs indicated electrical coupling of individual cells within the multicellular constructs. AP recordings identified pacemaker cells (spontaneous diastolic depolarization) and cells with a flat phase 4 of the AP (working myocardium-like cells). Pharmacological studies demonstrated the presence and functional relevance of I Na (10–30 μM flecainide), I Ca (1 μM nisoldipine), and I Kr (1–5 μM E4031). Conclusion: Human force-generating EHT with functional and morphological properties of native myocardium can be generated. Ultimately, hESC-EHTs may constitute a model system for substance screening and could further be utilized in cardiac repair.

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Abstract 16949: Failing Fontan: Genesis of a Subpulmonary Neo-ventricle From Engineered Heart Tissue

Circulation ◽

10.1161/circ.132.suppl_3.16949 ◽

2015 ◽

Vol 132 (suppl_3) ◽

Author(s):

Daniel Biermann ◽

Alexandra Eder ◽

Hatim Seoudy ◽

Florian Arndt ◽

Tillmann Schuler ◽

...

Keyword(s):

Blood Flow ◽

Pulmonary Blood Flow ◽

Connexin 43 ◽

Wistar Rats ◽

Vena Cava ◽

Heart Tissue ◽

Long Term Outcome ◽

Engineered Heart Tissue ◽

The Right

Introduction: Fontan palliation is the treatment of choice for patients with morphological or functional univentricular hearts. The unphysiologic and non-pulsatile pulmonary blood flow results in multiorgan complications and a poor long-term outcome. We evaluated graft survival and histomorphology of a pulsatile Fontan conduit generated from Engineered Heart Tissue (EHT) after implantation in a rat model. Hypothesis: We hypothesized that EHT matures and remains contractile in the setting of venous (Fontan-like) preload. Methods: EHT was generated from ventricular cardiomyocytes of neonatal Wistar rats, fibrinogen, thrombin and DMEM. After culture for 14 days constructs were implanted around the right superior vena cava of Wistar rats (n=12, 300-350 g). Immunosupression was achieved by daily subcutaneous injection of Cyclosporin A (25 mg/kg BW) and Methylprednisolone (2 mg/kg BW). MRI (Bruker) was used to assess condensation of EHTs in vivo. Animals were euthanized after 7, 14, 28 and 56 days postoperatively for histomorphological analysis. Transmission electron microscopy was used to evaluate sarcomeric integrity of cardiomyocytes within the construct. Results: In culture, EHTs started beating around day 8 and remained contractile in vivo throughout the experiment (d7=3/3, d14=2/3, d28=3/3, d56=2/3). All animals survived circumferential implantation of EHTs around the right SVC via a right thoracotomy. MRI (d14, n=3) revealed no constriction or stenosis of the SVC by the constructs. Hematoxylin and Eosin staining showed densely packed bundles of cardiomyocytes within the EHT conduit and intense vascularisation. Immunolabeling of actinin and connexin 43 indicated adequate maturation of cardiomyocytes after grafting around the right SVC in rats. Conclusions: EHTs placed around the superior caval vein of Wistar rats survive and contract for a considerable time after implantation. Histomorphology revealed a matured phenotype of grafted cardiomyocytes and an adequate vascularisation. The functional benefit of a contractile neo-ventricle to propel pulmonary blood flow in Fontan patients remains to be evaluated.

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P5385Development and preclinical testing of upscaled engineered heart tissue for use in translational studies

European Heart Journal ◽

10.1093/eurheartj/ehz746.0345 ◽

2019 ◽

Vol 40 (Supplement_1) ◽

Author(s):

R Jabbour ◽

T Owen ◽

M Reinsch ◽

P Pandey ◽

B Wang ◽

...

Keyword(s):

Myocardial Infarction ◽

Tissue Engineering ◽

Stem Cell ◽

Ex Vivo ◽

Rabbit Model ◽

Heart Tissue ◽

Border Zone ◽

Preclinical Testing ◽

Engineered Heart Tissue

Abstract Introduction The lack of efficacy of stem cell therapy for the treatment of heart failure may be related to the poor retention rates offered by existing delivery methods (intra-coronary/ intramyocardial). Tissue engineering strategies improve cell retention in small animal models but data regarding engineered heart tissue (EHT) patches large enough for human studies are lacking. Purpose To upscale EHT to a clinically relevant size and mature the patch in-vitro. Once matured to undergo preclinical testing in a rabbit model of myocardial infarction. Methods We developed an upscaled EHT patch (3cm x 2cm x 1.5mm) able to contain up to 50 million human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM; Fig A/B). Myocardial infarction model was performed by permanent ligation. Results The patches began to beat spontaneously within 3 days of fabrication and after 28 days of dynamic culture (Late EHTs) showed the development of several mature characteristics when compared to early patches (<14 days from fabrication). For example, late EHTs contained hiPSC-CMs which were more aligned (hiPSC-CM accumulative angle change: early 2702±778 degrees [n=4] vs late 922±186 [n=5], p=0.042); showed better contraction kinetics (early peak contraction amplitude 87.9±5.8a.u. versus late 952±304a.u.; p<0.001) and faster calcium transients (time to peak: early 200.8±8.8ms [n=5] vs late 147.7±10.2ms [n=6], p=0.004; time to 75% decay: early 274±9.7ms vs late 219.9±2.7ms, p=0.0003). We then tested the EHT patch in-vivo using a rabbit model (Fig C). Patches were applied to normal (n=5) or infarcted hearts (n=8). Sham operations used non-cellular fibrin patches (n=5). The mean fraction of troponin positive cells in the graft was 27.8±10.3% at 25.2±1.7 days relative to day 0 [n=5] and KU80 (human specific marker) staining confirmed that this was of human origin. CD31 (Fig D) and KU80 staining revealed that the grafts were well vascularized and that the vasculature was not human in origin (therefore were originating from the host). Ex-vivo optical mapping revealed evidence of electrical coupling between the graft and host at 2 weeks and preliminary experiments indicated that the patch improved left ventricular function when grafted onto infarcted hearts. Telemetry recordings in vivo and arrhythmia provocation protocols (ex vivo) indicated that the patch was not proarrhythmic. Figure 1. A/B) EHT Images; C) 20x troponin T (brown) of rabbit myocardium/EHT (2 weeks after grafting), blue counterstain = haematoxylin, red lines = EHT borders; D) 63x CD31 staining (brown) rabbit/EHT border zone (2 weeks after grafting), blue stain = haematoxylin, red lines = graft/host border zones. Conclusion We successfully upscaled hiPSC-CM derived EHT to a clinically relevant size and demonstrated feasibility and integration using a rabbit model of myocardial infarction. Tissue engineering strategies may be the preferred modality of cell delivery for future cardiac regenerative medicine studies.

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Fibrin-based engineered heart tissue for cardiac repair: Initial results after transplantation

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0030-1269168 ◽

2011 ◽

Vol 59 (S 01) ◽

Cited By ~ 1

Author(s):

L Conradi ◽

A Vogelsang ◽

A Hansen ◽

A Eder ◽

M Hirt ◽

...

Keyword(s):

Heart Tissue ◽

Cardiac Repair ◽

Engineered Heart Tissue ◽

Initial Results

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Human iPS-cell-derived engineered heart tissue for cardiac repair

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0034-1367125 ◽

2014 ◽

Vol 62 (S 01) ◽

Cited By ~ 1

Author(s):

S. Pecha ◽

F. Weinberger ◽

K. Breckwoldt ◽

B. Geertz ◽

J. Starbatty ◽

...

Keyword(s):

Heart Tissue ◽

Cardiac Repair ◽

Ips Cell ◽

Engineered Heart Tissue

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Cardiac repair in guinea pigs with human engineered heart tissue from induced pluripotent stem cells

Science Translational Medicine ◽

10.1126/scitranslmed.aaf8781 ◽

2016 ◽

Vol 8 (363) ◽

pp. 363ra148-363ra148 ◽

Cited By ~ 106

Author(s):

F. Weinberger ◽

K. Breckwoldt ◽

S. Pecha ◽

A. Kelly ◽

B. Geertz ◽

...

Keyword(s):

Stem Cells ◽

Induced Pluripotent Stem Cells ◽

Pluripotent Stem Cells ◽

Guinea Pigs ◽

Heart Tissue ◽

Cardiac Repair ◽

Engineered Heart Tissue ◽

Induced Pluripotent

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Integration of a new engineered heart tissue into rat hearts in vivo

The Thoracic and Cardiovascular Surgeon ◽

10.1055/s-0029-1246961 ◽

2010 ◽

Vol 58 (S 01) ◽

Author(s):

F Schlegel ◽

S Leontjev ◽

C Spath ◽

M Nichtitz ◽

R Schmiedel ◽

...

Keyword(s):

Heart Tissue ◽

Engineered Heart Tissue ◽

Rat Hearts

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Transcriptome analysis of non human primate-induced pluripotent stem cell-derived cardiomyocytes in 2D monolayer culture vs. 3D engineered heart tissue

Cardiovascular Research ◽

10.1093/cvr/cvaa281 ◽

2020 ◽

Author(s):

Huaxiao Yang ◽

Ningyi Shao ◽

Alexandra Holmström ◽

Xin Zhao ◽

Tony Chour ◽

...

Keyword(s):

Stem Cell ◽

Monolayer Culture ◽

Induced Pluripotent Stem Cell ◽

Heart Tissue ◽

Paracrine Signalling ◽

Engineered Heart Tissue ◽

Direct Cell ◽

Induced Pluripotent ◽

Transcriptome Level

Abstract Aims Stem cell therapy has shown promise for treating myocardial infarction via re-muscularization and paracrine signalling in both small and large animals. Non-human primates (NHPs), such as rhesus macaques (Macaca mulatta), are primarily utilized in preclinical trials due to their similarity to humans, both genetically and physiologically. Currently, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) are delivered into the infarcted myocardium by either direct cell injection or an engineered tissue patch. Although both approaches have advantages in terms of sample preparation, cell–host interaction, and engraftment, how the iPSC-CMs respond to ischaemic conditions in the infarcted heart under these two different delivery approaches remains unclear. Here, we aim to gain a better understanding of the effects of hypoxia on iPSC-CMs at the transcriptome level. Methods and results NHP iPSC-CMs in both monolayer culture (2D) and engineered heart tissue (EHT) (3D) format were exposed to hypoxic conditions to serve as surrogates of direct cell injection and tissue implantation in vivo, respectively. Outcomes were compared at the transcriptome level. We found the 3D EHT model was more sensitive to ischaemic conditions and similar to the native in vivo myocardium in terms of cell–extracellular matrix/cell–cell interactions, energy metabolism, and paracrine signalling. Conclusion By exposing NHP iPSC-CMs to different culture conditions, transcriptome profiling improves our understanding of the mechanism of ischaemic injury.

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