Listen to the Heart Beat

Collaboration will be vital to help advance the field of advanced medicine. TECHNOBEAT (Tools and Technologies for Breakthrough in Heart Therapies), funded by the European Commission’s Horizon2020, is one example of a collaborative project that is addressing unmet medical needs in cardiovascular disease. Here, we speak with Robert Zweigerdt, a principal investigator at Hannover Medical School and coordinator of TECHNOBEAT, and Katharina Kinast at Eppendorf, one of the partners in the project.

How did the TECHNOBEAT project came about?

Robert Zweigerdt: After James Thomson, in collaboration with Joseph Itskovitz-Eldor (who is one of the partners in TECHNOBEAT) and co-workers, published the derivation of human embryonic stem cells (hESC) in 1998, the field of stem cell research received a massive boost. The unlimited proliferation potential of human embryonic stem cells (hESC) and their ability to differentiate into, in principle, any somatic cell type in vitro, has opened a whole new universe of exciting possibilities in regenerative medicine, pharmacological research, human developmental biology and basic stem cell research.

The possibilities where further stimulated by the derivation of induced pluripotent stem cells from mice (2006) and humans (2007) by Shinya Yamanaka and his team, through a technology enabling the so called “reprograming of somatic cells” from adult patients into an ESC-like state. These findings laid the foundations for the routine use of stem cells in medicine because they allowed the scientific community to bypass the ethical issues associated with the use of human embryonic stem cells. Human pluripotent stem cells (hPSC; an umbrella term for hESC and hiPSC) can serve as a universal cell source for the derivation of unlimited amounts of functional somatic cells to help in a variety of conditions, such as loss of dopaminergic neurons in the brain leading to Parkinson’s disease, the autoimmune-triggered loss of beta cells in the pancreas underlying diabetes type 1, or age-related blindness demanding the replacement of retinal pigment epithelial cells to stop progression of – and ideally cure – macular degeneration.

Cardiovascular diseases, particularly ischemic cardiomyopathies, remain the major global causes of morbidity and mortality affecting millions of patients worldwide. The obstruction of coronary arteries, which normally supply the heart with oxygenated blood, triggers ischemia in areas downstream of the occluded vessel, a condition known as myocardial infarction. The condition often leads to the terminal loss of billions of heart muscle cells, which are not replaced by endogenous repair mechanisms and may result in reduced heart function and ultimately heart failure.

TECHNOBEAT aims to develop new treatment options for patients suffering from heart failure caused by the loss of heart muscle tissue following a heart attack. The consortium calls on the expertise of a network of leading European entities in the cardiovascular field, including Hannover Medical School – a medical center with a strong focus on translating cell-based regenerative medicine for several organs; Leiden University, which offers leading expertise in basic mechanism of cardiovascular differentiation of hPSCs; and Utrecht Medical Centre, which brings top expertise in experimental cardiology and development of pre-clinical animal models. The consortium also involves partners with leading know-how in clinical stem cell production: Paracelsus University in Salzburg provides expertise in the derivation and clinical application of adult stem cells (in particular, mesenchymal stem cells, an important cellular component for organ repair) and Kadimastem, located in Rohovot, Israel, is developing protocols for the clinically compliant manufacturing of hPSCs and their progenies, as well as expertise in handling the regulatory requirements of regenerative medicine. In addition, technical innovation in hardware development for stem cell bioprocessing, monitoring and analysis is essential to our project – as well as the whole cell therapy field. In the area of bioreactor development, Eppendorf provides their support, while OVIZIO provides innovative solutions in the monitoring of cells and more complex cell aggregates.

It also goes without saying that safety is of great importance in cell-based organ repair – and that specifically requires monitoring of the genomic integrity of mass-expanded and differentiated stem cells to avoid process-induced cell transformation and the potential development of tumors. Thus, our project partner at the University of Sheffield in the UK is applying its long-standing expertise in analyzing the genomic stability of hESC lines to the field of hiPSCs manufacturing (hiPSC are the exclusive pluripotent stem cell source applied in TECHNOBEAT in compliance with European regulations).

The consortium aims to establish novel tools and technologies required for the controlled, clinically- and regulatory compliant mass production of hiPSC and their functional progenies required for meaningful heart therapies. We also aim to establish efficient transplantation and functional integration of donor cells in pre-clinical animal models, and to demonstrate safety and therapeutic efficiency. In addition to these cell production and application goals, the project aims to advance cardiac tissue engineering and  maturation technologies, supporting the utility of (typically immature) hiPSC progenies such as cardiomyocytes in vitro – thereby providing improved tools for the use of hiPSC-cardiomyocytes in drug discovery and drug safety in pharmacological research.

Katharina Kinast: For some time, Eppendorf has been aiming to improve bioprocessing equipment for the cultivation of stem cells. As part of this engagement, we have a long-standing cooperation with Robert Zweigerdt’s group at Hannover Medical School.

Depending on the therapeutic application, stem cell-based therapies need ^~1 x 10⁵ to 1 x 10¹⁰ cells per treatment dose per patient. It is difficult to reproducibly produce such high cell numbers (i.e. those > 1 x 10⁸ required for most disease including heart repair) in conventional, two-dimensional culture systems, such as flasks or dishes, and stirred-tank bioreactor systems have clear advantages; they require less lab space than the corresponding number of cell culture flasks, can help with process scale-up, and allow for close monitoring and control.  Within the TECHNOBEAT project, we will develop new impeller and vessel designs to optimize hiPSC culture mixing and shear characteristics. Furthermore, in cooperation with the project partners from OVIZIO, we plan to integrate holographic microscopy to monitor cell aggregate formation in real time. We will facilitate communication of the device with our bioprocess control software, so that values measured by the microscopic device are communicated to the software in real-time. Thus, they can be used for automated bioprocess control, in addition to conventionally measured parameters such as pH, dissolved oxygen, and temperature.

How could cell therapies help with cardiovascular disease?

Zweigerdt: The heart is one of the least regenerative organs in the body. To replace the loss of cardiomyocytes post myocardial infarction, clinicians have – in the last two decades – transplanted   numerous somatic cell types to the heart, such as skeletal muscle-derived myoblasts, and bone marrow or peripheral blood-derived cells isolated by several technologies and markers. However, these can have drawbacks. Skeletal muscle-derived myoblasts can, for example, induce life threatening arrhythmias.

hiPSC have been demonstrated to efficiently differentiate into bona fide cardiomyocytes in vitro and could provide promising building blocks for heart repair. This may be achieved either by direct cell injection into the damaged heart, or by the transplantation of in vitro, pre-engineered, heart muscle tissue. Moreover, hiPSC can also be directed into the formation of endothelial cells lining the inner lumen of the heart and all vessels and capillaries of our organs.

Previous work conducted by TECHNOBEAT partners and others has revealed that the combination of cardiomyocytes with endothelial cells and the addition of interstitial cells, such as MSCs or fibroblasts, is favorable for cardiac tissue formation in vitro and the overall retention, survival and engraftment of cells transplanted to the heart.

What progress has the consortium made so far?

Zweigerdt: TECHNOBEAT investigator Christine Mummery (Leiden University) was one of the first to establish protocols for directed cardiomyocyte differentiation of hESC – and recently developed efficient protocols for hiPSC differentiation into endothelial cells, which will be further advanced and scaled-up in the project. Ulrich Martin (Hannover Medical School) has applied his extensive know-how on iPSC derivation and differentiation to be the first to demonstrate formation of functional cardiomyocytes from this cell source. Lead by Robert Zweigerdt, Hannover Medical School investigators have teamed-up with Eppendorf to translate conventional 2D tissue culture strategies for hiPSC cultivation towards industry-compliant stirred tank bioreactors in 3D suspension culture. Given this prior work, the project was successfully kick-started. Bioreactor-based cultivation processes for hiPSC expansion and differentiation have been scaled-up from 100-250 ml bioreactor scale, into advanced bioreactors in 500-1,000 ml process scale. Moreover, novel transgenic reporter hiPSC lines have been generated, which allow monitoring of iodine isotope-labelled hiPSC-cardiomyocytes by SPECT-CT imaging in the heart – even in large animal models such as pigs. Bioprocess-derived hiPSC-cardiomyocytes and hiPSC-endothelial cells are currently combined with clinical-grade MSCs provided by Dirk Stunk (Paracelsus University) to form novel multicellular µ-tissues. These µ-tissues, which are mass generated in a highly homogeneous manner by a novel platform technology, are currently tested for transplantation in a pig model of myocardial infarction by the team of Joost Sluijter at (Utrecht Medical Centre). In parallel, Kadimastem has defined clinically-compliant protocols for hiPSC production in close exchange with competent regulatory authorities – in particular the US FDA. These examples highlight the multi-dimensional swift progress of the work along the ambitious timelines defined in the project.

Kinast: We have developed a stirred-tank bioreactor for the cultivation of hiPSCs in a working volume of 100 ml, including a new impeller design to optimize culture mixing and shear characteristics. With a prototype we were already able to significantly improve the mixing of a hiPSCs aggregate culture compared to conventional impellers. These results are very encouraging! The challenge is now to adapt the design to larger bioreactors with a working volume of 1000 ml – and larger.

When working with stem cells, what specific needs should to be considered in terms of processing equipment?

Kinast: Stem cells are much trickier to cultivate than, say, CHO cells. There are several reasons for this. One critical point is culture mixing. hiPSCs are very shear sensitive, and they do not grow as a single-cell suspension, but must be cultivated as cell aggregates or on microcarriers. This imposes special requirements on the impeller design to ensure their homogenous distribution while keeping the shear stress low. Another important point is that in stem cell cultivation (unlike protein production), the cell is the product and hence has to be in an optimal state. In the course of expansion, stem cells have to maintain pluripotency and their potential to differentiate, and then must differentiate into the desired cell type. Various factors influence this, including shear forces, oxygen tension, and pH. To produce stem cells of constant quality, the tight, reproducible control of process parameters, as well as cell monitoring in real-time, is even more critical than in a protein production process.

In summary, the cultivation of stem cells demands special process conditions and a level of cell monitoring that might be not required for the production of a monoclonal antibody, and the bioreactors used must accommodate this.

What do you think are the main challenges that lie ahead for the cell therapy field?

Zweigerdt: This comment relates to the field of pluripotent stem cells only, but not the field of adult stem cells. Despite significant progress in the last decade, the reproducible mass production of specific, functional and ideally fully matured hPSC-progenies remains a challenge. In particular, the up-scaling and transition of established laboratory-scale strategies for the directed differentiation of hPSC into clinically/industry-compliant production process is not established for most hPSC progenies of therapeutic interest.

However, in disease areas including the treatment of diabetes, age-related blindness, spinal cord repair, and recently in ischemic heart diseases, hPSC-progenies have now entered the clinical arena – following almost 20 years of research after the first description of hESC in 1998. Therefore, this is a very exciting time! The next 5 to 10 years will reveal the feasibility of integrating hPSC progenies into patients’ diseased tissues and organs, such that they can indeed restore organs’ function. The topic of ensuring long-lasting, functional integration of transplanted cells into a diseased organ is, in my opinion, the most challenging next hurdle in the field of regenerative medicine. This challenge is also a key topic that we are addressing in TECHNOBEAT by developing appropriate technologies for the efficient retention and functional integration of transplanted hiPSC-progenies in the hearts of pre-clinical animal models.

How do you envision the future of heart therapies?

Zweigerdt: In terms of hiPSC-progenies, hiPSC-derived cardiomyocytes, endothelial cells and other functional cell types will play a significant role in developing novel treatment strategies beyond direct cell transplantation into diseased hearts. The use of hiPSC-based in vitro models to mimic the function and malfunction of heart cells and engineered heart tissue in a dish is currently providing a substantial addition of technologies to the drug development process in pharma companies. Such hiPSC-based in vitro models, which allow direct correlation of a patient’s clinical symptoms with cells’ function in a dish, have already enabled a much better understanding of mechanisms underlying specific diseases, including electrophysiological and metabolic disorders of the heart. Thus, the hiPSC technology will substantially improve the effectiveness, specificity and safety of drug discovery and thereby improve the conventional, pharmacological treatment of cardiovascular diseases in addition to the envisioned cell therapies.

Notably, the hiPSC-based cardiac µ-tissue that we are developing in TECHNOBEAT could serve as both injectable building blocks for cell transplantation into failing hearts, as well as comprehensive three dimensional (3D) in vitro models of heart tissue for drug discovery in pharma.

Anything else you’d like to add?

Zweigerdt: Trans-European funding by the EU-commission has been essential for this project! Due to its complexity and early stage of development, the field of hiPSC-based cell therapies currently lacks a common trans-European and global regulatory framework. The funding of projects such as TECHNOBEAT is fostering communication between stakeholders in the field: researchers, clinicians, companies and regulatory authorities, thereby providing the required input for establishing a regulatory framework, which is now evolving.