Marina Reinsch

Universität Hamburg

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Thomas Eschenhagen

German Centre for Cardiovascular Research

Florian Weinberger

University Medical Center Hamburg-Eppendorf

Arne Hansen

German Centre for Cardiovascular Research

Aya Shibamiya

University Medical Center Hamburg-Eppendorf

Bärbel Ulmer

Universität Hamburg

Birgit Klampe

University Medical Center Hamburg-Eppendorf

Thomas G. Schulze

Universitätsmedizin Göttingen

Sigrid Fuchs

Universität Hamburg

Richard J. Jabbour

University Hospital Southampton NHS Foundation Trust

Birgit Geertz

University Medical Center Hamburg-Eppendorf

Cooperative Institutions

German Centre for Cardiovascular Research

University Medical Center Hamburg-Eppendorf

Universität Hamburg

Imperial College London

Hammersmith Hospital

Goethe University Frankfurt

Technical University of Munich

Cardiovascular Research Center

Klinikum rechts der Isar

Ludwig-Maximilians-Universität München

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Cell Banking of hiPSCs: A Practical Guide to Cryopreservation and Quality Control in Basic Research

Current Protocols in Stem Cell Biology (2020)

Aya Shibamiya E. Schulze Dana Krauß Christa Augustin Marina Reinsch

Abstract The reproducibility of stem cell research relies on the constant availability of quality‐controlled cells. As the quality of human induced pluripotent stem cells (hiPSCs) can deteriorate in the course of a few passages, cell banking is key to achieve consistent results and low batch‐to‐batch variation. Here, we provide a cost‐efficient route to generate master and working cell banks for basic research projects. In addition, we describe minimal protocols for quality assurance including tests for sterility, viability, pluripotency, and genetic integrity. © 2020 The Authors. Basic Protocol 1 : Expansion of hiPSCs Basic Protocol 2 : Cell banking of hiPSCs Support Protocol 1 : Pluripotency assessment by flow cytometry Support Protocol 2 : Thawing control: Viability and sterility Support Protocol 3 : Potency, viral clearance, and pluripotency: Spontaneous differentiation and qRT‐PCR Support Protocol 4 : Identity: Short tandem repeat analysis

Viability assay

10.1002/cpsc.127

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Citations (15)

BS27 Development and preclinical testing of a large heart muscle patch

Richard J. Jabbour Thomas J. Owen Marina Reinsch Pragati Pandey Cesare M. Terracciano

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 × 2cm × 1.5mm) able to contain up to 50 million human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) (figure 1A/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 (figure 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 ± 1.7days relative to day 0 [n=5] and KU80 (human specific marker) staining confirmed that this was of human origin. CD31 (figure 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 associated with any significant changes in arrhythmogenicity.

Conclusion

We successfully upscaled hiPSC-CM derived EHT to a clinically relevant size and were able to demonstrate 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.

Conflict of interest

none

10.1136/heartjnl-2019-bcs.190

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

European Heart Journal (2019)

Richard J. Jabbour Thomas J. Owen Marina Reinsch Pragati Pandey Brian Wang

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.

10.1093/eurheartj/ehz746.0345

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

Richard J. Jabbour Thomas J. Owen Pragati Pandey Marina Reinsch Godfrey L. Smith

Introduction

Engineered heart tissue (EHT) strategies, by combining cells within a hydrogel matrix may overcome the limitations of intracoronary/myocardial cell delivery routes. EHTs regenerate heart muscle in small animal models but data regarding clinically relevant engineered heart tissue (EHT) patches large enough for first-in-human studies are lacking.

Methods

An upscaled EHT patch (approx. 3 cm × 2 cm × 1.5 mm) consisting of 15–20 million human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) embedded in a fibrin based hydrogel was developed. A rabbit myocardial infarction model was then developed to test for feasibility and efficacy of EHT grafting.

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). Late EHTs contained hiPSC-CMs which were more aligned; showed better contraction kinetics, and faster calcium transients. We then tested the EHT patch in-vivo using a rabbit model. Patches were applied to infarcted hearts (n=14 [n=7 EHT vs n=7 sham]). Sham operations used non-cellular fibrin patches. Blinded echocardiographic analysis revealed a significant improvement in function in infarcted hearts that underwent EHT patch grafting (n=7; absolute difference of 10.04 ± 3.1% over sham group; fractional area change, P<0.01). In-vivo telemetry recordings (n=5 MI/sham vs n=7 MI/EHT) indicated that no clinically relevant arrhythmia was seen in the MI/EHT group and arrhythmia provocation protocols (ex vivo n=5 MI/sham vs n=6 MI/EHT) confirmed that the patch was not pro-arrhythmic (arrhythmia inducibility score 5.6 ± 1.0 [MI/patch] vs 5.0 ± 0.6 [MI/sham]; p=ns).

Conclusion

An upscaled clinically relevant EHT patch was developed and improved function in infarcted hearts without causing arrhythmia. Therefore EHT may have specific advantages over the direct intramyocardial injection of cells.

Ex vivo

10.1136/heartjnl-2020-bcs.137

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Comparison of Human Induced Pluripotent Stem Cells Cultured at Different Oxygen Concentrations by Proteomic Analysis Suggests Normoxia-induced Cellular Senescence

Free Radical Biology and Medicine (2022)

Janice Raabe Ilka Wittig Aya Shibamiya Sandra D. Laufer Birgit Klampe

Senescence

Cellular senescence

10.1016/j.freeradbiomed.2021.12.047

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