iCARE - Induced pluripotent stem cells for clinically applicable heart repair
iCARE – a joint research project which is funded by the BMBF for a period of three years – officially started on April 1, 2017. In order to apply for the federal funding program “Innovative Stem Cell Technologies in Personalized Medicine”, a team was assembled headed by Prof. Ulrich Martin. This team hypothesized that the therapeutic application of iPSC-derived cardiomyocytes (CMs) is safe, and results in their structural integration and functional improvement in failing hearts. The aim of the joint project is to prepare the first worldwide clinical application of iPSC-based heart repair.
iCARE is one of the very few consortia worldwide that combines all necessary expertise and cutting edge technologies required for the clinical translation and exploitation of an iPSC-based therapy. The following departments, centers, and institutes are part of the joint project, which has been awarded a grant of more than 3 million Euro: The Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG) and the Institute of Cellular Therapeutics (ICT) of Hannover Medical School, the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), the German Primate Center GmbH, the Centre for Ethics and Law in the Life Sciences (CELLS) of the Leibniz University Hannover (LUH), Miltenyi Biotec as well as corlife.
The world’s aging population suffers from an increasing incidence of coronary heart disease. Indeed, the World Health Organization reported that in 2016 ischemic heart disease is still the major cause of mortality worldwide, accounting for about 9.4 million cases of deaths p.a.. Despite different therapeutic approaches, in most cases heart transplantation (HTx) remains the only option for affected patients. Left ventricular assist devices offer an alternative to HTx but in most cases serve as only a temporary solution. The number of heart transplantations is limited by the number of available donor organs and thus many patients remain without adequate treatment
Numerous stem cell (SC)-based approaches have been proposed for heart repair, but a multitude of experimental and clinical studies applying various adult stem cells have not achieved functional improvement, or showed only minor effects (Jiang, M., Expert opinion on biological therapy, 2010. 10: 667). More recently, however, patient-derived human induced pluripotent stem cells (hiPSCs), which, for the first time, provide a potential source of patient-derived cardiomyocytes (CMs) for heart repair, have become available. In order to generate these cells, easily accessible cell sources, e.g. blood cells, are reprogrammed into pluripotent stem cells in the laboratory. Intense research has driven forward dramatic progress in virtually all areas of iPSC technology relevant to the proposed project. This includes highly efficient reprogramming into iPS cells (Haase, A., Cell Stem Cell, 2009. 5: 434.; Lachmann, N., American journal of respiratory and critical care medicine, 2014. 189: 167), efficient genetic engineering of iPSCs (Merkert, S., Stem cell reports, 2014. 2: 107) as well as the development of technologies for the expansion and differentiation of iPSCs, e.g. CMs, at a clinically required large scale (Zweigerdt, R., Nat Protoc, 2011. 6: 689; Olmer, R., Tissue Eng Part C, 2012. 18: 772; Kempf, H., Stem cell reports, 2014. 6:1132). Intense research on safety issues concerning iPSC-based cell transplants, the most critical aspect for clinical applications, is ongoing. Importantly, the extremely low incidence of CM-derived tumours in human hearts suggests a low risk factor for a malignant transformation of terminally differentiated iPSC-derived CMs, and potential implant-related arrhythmias could be controlled through technical pacemaker devices.
A promising study (published in 2014) demonstrated, for the first time, the formation and functional coupling of large contractile human stem cell-derived heart muscle islands in a non-human primate (pig-tailed macaque, Macaca nemestrina) model of MI (Chong, J.J., Nature, 2014. 510: 273). In this case, embryonic stem cells were used instead of iPS cells. Within the iCARE consortium, in close collaboration with basic researchers, clinicians, veterinarians and experts from the German Primate Center, we will transplant human iPSC-based CMs into non-human primates (crab eating macaque or cynomolgus monkey; Macaca fascicularis) in order to examine their influence on the damaged heart. Cynomolgus monkeys belong to the family of Old Word monkeys and are frequently used in pharmaceutical industry, e.g. for safety and efficacy studies for certain drug approval.
Preclinical assessment of our cell therapy concept is crucial to cover both ethical and legal aspects. Based on the current state of research, these tests cannot be performed in small animals like mice or rats. Though these animals are well suited for many other animal studies, their hearts vary immensely from the human heart, especially concerning the beating rate. Compared to the human heart, the beating frequency is about 5-8 times higher in the rodent model. Thus, a functional integration of human CMs into the heart of mice or rats, including the required electrical and mechanical integration, seems impossible (Mummery, C.L., Sci Transl Med 2, 2010. 27: 29ps17; Garbern J.C., Cold Spring Harb Perspect Med 3, 2013. 4:a014019).
Heart size as well as physiology (including the beating frequency) of large animals like pigs more closely resemble the human heart (Garbern J.C., Cold Spring Harb Perspect Med 3, 2013. 4:a014019) and thus seem more suitable for the preclinical assessment of cell therapy concepts of heart diseases. Up to now, unfortunately, high quality iPSCs (and differentiated CMs) from pig or sheep have not been successfully generated, rendering it impossible to examine allogenic cardiomyocytes in this model. Our published results as well as data from other research groups additionally demonstrate the fact that survival of transplanted human cells in pigs is limited by an immunological barrier (Templin, C., Circulation 126, 2012. 4: 430; Kawamura, M., Circulation 127, 2012. 11 Suppl 1: S29). In contrast, recently published data (Chong, J.J., Nature, 2014. 510: 273) show that survival and functional integration of human CMs in NHPs is possible and might provide decisive information not only about the functionality of the desired therapy but also might demonstrate potential risks in a human-related animal model.
In conclusion, considering the current state of worldwide research, the use of NHPs is – from a scientific as well as from a clinical perspective - utterly necessary and in terms of urgently required novel therapeutic approaches, highly reasonable. From an ethical point of view, the stress for the animals caused by the experiments has to be weighed against the important clinical/practical insights gained for the treatment of patients with ischemic and, in many cases, lethal heart diseases. In view of the above points, we consider these animal experiments ethically justifiable. The principles of the 3R (Replacement, Reduction, Refinement) are of high priority and we will consider them in every experiment performed.
Here you can find further information regarding the importance of animal experiments with monkeys (German only):
- Generation and characterization of genetically enriched hiPSC derived CMs and transplantation in a preclinical non-human primate model.
- Development of GMP compliant processes for the reprogramming and genetic modification of iPSCs as well as scale up of hiPSC-CM production for clinical use.
- Enrichment of CM subtypes.
- Development of an in vitro potency assay.
- Discussion of the regulatory requirements for intramyocardial transplantation of autologous hiPSC-CMs in patients suffering from right-sided heart failure as a long-term consequence of a Mustard operation and submission of a first draft of an application protocol.
- Updating the ‘Freedom to Operate (FtO)’ analysis and the business and exploitation plan.
- Create a legal, ethical and commercial framework within which translation is facilitated.
The consortium is coordinated by Prof. Dr. Ulrich Martin (Leibniz Research Laboratories for Biotechnology and Artificial Organs, LEBAO) and Prof. Dr. Axel Haverich, both Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG). Further leaders of different subprojects at MHH are PD Dr. Ina Gruh and Dr. Robert Zweigerdt (LEBAO) as well as PD Dr. Serghei Cebotari and Prof. Dr. Samir Sarikouch (HTTG) and Prof. Dr. Ulrike Köhl (ICT). The external partners Prof. Dr. Braun from the Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Prof. Dr. Susann Boretius and Prof. Dr. Franz-Josef Kaup from the German Primate Center (DPZ), Prof. Dr. Nils Hoppe from the Centre for Ethics and Law in the Life Sciences (CELLS) of the Leibniz University Hannover (LUH), Dr. Sebastian Knöbel and Dr. Dominik Eckardt from Miltenyi Biotec as well as Dr. Michael Harder from corlife will also contribute significantly to the joint project.
The non-human primate experiments are performed at the German Primate Center (DPZ) in Göttingen in close collaboration with their scientists, veterinarians and animal caretakers and under strict obedience of legal requirements and animal welfare. Anesthetic procedures for primates established at the DPZ are applied by an anesthesiologist. Additionally, explanation and preparation of the samples for subsequent methods like electron microscopy, immunohistology of frozen and paraffin-embedded sections and molecular detection methods are well-established and are performed by experts in these fields. Clinically and experimentally involved staff from the Department of Cardiothoracic, Transplantation and Vascular Surgery (HTTG) of Hannover Medical School has long-standing experience in transplantation of organs, like lungs or heart, as well as the implantation of bioartificial tissue, like blood vessels or heart valves. Trans- and implantations of tissues are performed routinely in large animal models.
- Teilprojekt 1 (MHH: Cebotari, Martin; DPZ, ITEM): hiPS-Zell-basierte Therapie des Myokardinfarktes im nicht-humanen Primatenmodell
- Teilprojekt 2 (MHH: Köhl, Martin; Miltenyi): GMP-konforme Reprogrammierung und Genmodifikation von iPS-Zellen
- Teilprojekt 3 (MHH: Köhl, Zweigerdt; Miltenyi): Klinisches Upscaling der Zellproduktion
- Teilprojekt 4 (MHH: Zweigerdt; Miltenyi): Anreicherung von aus pluripotenten Stammzellen abgeleiteten Kardiomyozyten-Subtypen
- Teilprojekt 5 (MHH: Gruh): Entwicklung eines Potency Assays für iPS-Zell-basierte Kardiomyozyten
- Teilprojekt 6 (MHH: Haverich, Sarikouch): Entwicklung eines Protokolls zur klinischen Anwendung autologer humaner iPS-Zell-basierter Kardiomyozyten
- Teilprojekt 7 (corlife, CELLS): Entwicklung von Marketingstrategien unter Berücksichtigung ethischer und rechtlicher Fragen
- Teilprojekt C (MHH: Martin, Haverich): Koordination des iCARE-Verbundes
Subproject 1 (MHH: Cebotari, Martin; DPZ, ITEM): Transplantation of genetically enriched human iPSC (hiPSC) derived cardiomyocytes (CMs) in a preclinical model of heart repair (non-human primate)
Subproject 2 (MHH: Köhl, Martin; Miltenyi): GMP compliant scale up of iPSC-generation and genetic engineering
Subproject 3 (MHH: Köhl, Zweigerdt; Miltenyi): Clinical scale up of iPSC-CM and MSC production
Subproject 4 (MHH: Zweigerdt; Miltenyi): Development of techniques for enrichment of hPSC-derived cardiomyocyte subtypes
Subproject 5 (MHH: Gruh): Development of a potency assay for iPSCs for clinically applicable heart repair
Subproject 6 (MHH: Haverich, Sarikouch): Development of a treatment protocol for intramyocardial iPSC-CM transplantation
Subproject 7 (corlife, CELLS): Development of Marketing Strategies for iCARE from an ethical and legal perspective
Subproject C (MHH: Martin, Haverich): Coordination of the iCARE consortium
Weber N, Kowalski K, Holler T, Radocaj A, Fischer M, Thiemann S, et al. Advanced Single-Cell Mapping Reveals that in hESC Cardiomyocytes Contraction Kinetics and Action Potential Are Independent of Myosin Isoform. Stem Cell Reports. 2020(* Authors contributed equaly.).
Harder M. Herausforderungen innovativer Gewebemedizin aus unternehmerischer Sicht. In: Gerke S., Taupitz J., Wiesemann C., Kopetzki C., Zimmermann H. (eds) Die klinische Anwendung von humanen induzierten pluripotenten Stammzellen. Veröffentlichungen des Instituts für Deutsches, Europäisches und Internationales Medizinrecht, Gesundheitsrecht und Bioethik der Universitäten Heidelberg und Mannheim, vol 48. Springer, 2020.
Machado G. Induzierte pluripotente Stammzellen. Allgemeine arzneimittel- und gesundheitsrechtliche Fragen der klinischen Anwendung. In: Medizinrecht. 2020; 38 (4), S. 263–271.
Haase A, Glienke W, Engels L, Gohring G, Esser R, Arseniev L, et al. GMP-compatible manufacturing of three iPS cell lines from human peripheral blood. Stem cell research. 2019;35:101394.
Isu G, Morbiducci U, De Nisco G, Kropp C, Marsano A, Deriu MA, et al. Modeling methodology for defining a priori the hydrodynamics of a dynamic suspension bioreactor. Application to human induced pluripotent stem cell culture. J Biomech. 2019.
Halloin C, Coffee M, Manstein F, Zweigerdt R. Production of Cardiomyocytes from Human Pluripotent Stem Cells by Bioreactor Technologies. Methods in molecular biology (Clifton, NJ). 2019;1994:55-70.
Manstein F, Halloin C, Zweigerdt R. Human Pluripotent Stem Cell Expansion in Stirred Tank Bioreactors. Methods in molecular biology (Clifton, NJ). 2019;1994:79-91.
de la Roche J, Angsutararux P, Kempf H, Janan M, Bolesani E, Thiemann S, et al. Comparing human iPSC-cardiomyocytes versus HEK293T cells unveils disease-causing effects of Brugada mutation A735V of NaV1.5 sodium channels. Scientific reports. 2019;9(1):11173.
Halloin C, Schwanke K, Lobel W, Franke A, Szepes M, Biswanath S, et al. Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture. Stem Cell Reports. 2019;13(2):366-79.
Olmer R, Engels L, Usman A, Menke S, Malik MNH, Pessler F, et al. Differentiation of Human Pluripotent Stem Cells into Functional Endothelial Cells in Scalable Suspension Culture. Stem Cell Reports. 2018;10(5):1657-72.
Ackermann M, Kempf H, Hetzel M, Hesse C, Hashtchin AR, Brinkert K, et al. Bioreactor-based mass production of human iPSC-derived macrophages enables immunotherapies against bacterial airway infections. Nature communications. 2018; 9(1):5088.
Christoffersson J, Meier F, Kempf H, Schwanke K, Coffee M, Beilmann M, et al. A Cardiac Cell Outgrowth Assay for Evaluating Drug Compounds Using a Cardiac Spheroid-on-a-Chip Device. Bioengineering (Basel, Switzerland). 2018;5(2).
Koch L, Deiwick A, Franke A, Schwanke K, Haverich A, Zweigerdt R, et al. Laser bioprinting of human induced pluripotent stem cells-the effect of printing and biomaterials on cell survival, pluripotency, and differentiation. Biofabrication. 2018;10(3):035005.
Wolling H, Konze SA, Hofer A, Erdmann J, Pich A, Zweigerdt R, et al. Quantitative Secretomics Reveals Extrinsic Signals Involved in Human Pluripotent Stem Cell Cardiomyogenesis. Proteomics. 2018;18(14):e1800102.