More Physiologically-Relevant Cellular Assays for Cardio and Neurotoxicity Testing- An Interview with Susanne Bremer-Hoffmann

Pharma IQ: How are you developing more physiologically-relevant cellular assays for cardio and neurotoxicity testing?

 Susanne Bremer-Hoffmann: In the last 3 years we have evaluated the potential of stem cells for cellular assays for cardio- and neurotoxicity testing in the framework of FP7 projects such as SCR&Tox and DETECTIVE in order to obtain standardized human cells in sufficient amounts to allow investigations of the mechanisms of toxicity. In the area of neurotoxicity, we have used stem cell derived neuronal cell cultures for the detection of physiological pathway perturbations. We were in particularly interested in the CREB pathway that is known to be involved in a number of neurodegenerative diseases. This work required the establishment of quality controls of stem cell cultures in order to obtain repeatable results. This was part of the SCR&Tox project and performed in collaboration with two SMEs (“Avantea” in Cremona Italy and “Life and Brain” Bonn/Germany). The hands-on experience allowed us to refine test development criteria for so-called 'pathway assays'. Further work is necessary in order to get an understanding of the toxicological relevance of these test systems.

 For the elucidation of pathways relevant to delayed cardiotoxicity, which is an adverse effect often seen in patients who have been treated with anthracyclines, we are currently using hiPSC derived cardiomyocytes. This work is done in collaboration with the University of Cologne in Germany. This way, we are able to combine different technologies, such as transcriptomics and high content imaging, to understand the mechanism of toxicity.

 All our work with human embryonic stem cell lines and induced pluripotent stem cell lines has been done in the framework of the above mentioned FP7 projects and, therefore, it had been reviewed and approved by an ethical committee of the funding body (European Commission) as well as by a local ethical committee in the country where the research is performed.

Pharma IQ: What are the advantages of using induced pluripotent stem cells over embryonic stem cells?

 Bremer-Hoffmann: The major advantage of iPSC is that they don’t pose the same ethical concerns as embryonic stem cells. Furthermore, the use of iPSCs has great potential in the development of new pharmaceuticals, in particular dealing with the side effects of drugs related to the polymorphism of the human population but also as building blocks in testing strategies for animal free hazard assessments.  However, the iPSC technology is rather new and the phase of test development is currently ongoing with huge investments of the European Commission and the pharmaceutical sector. In particular, several EU Member States might not accept toxicological data derived from test methods using hESC, because of ethical concerns.  In this case, the mutual acceptance of hESC-derived toxicological data would not be attainable.

 Pharma IQ: How do these combine with in silico tools to help identify the mode of action of new cosmetics and pharmaceuticals that animal models cannot?

 Bremer-Hoffmann: Stem cell assays are meant to be used within Integrated Testing Strategies (ITS) in order to address complex endpoints such as repeated dose toxicity or reproductive toxicity. In silico methods are often an integral part of ITS, in which they are used to optimise experimental testing as well as the translation of in vitro dose-response relationships into in vivo effects data that can be used in safety assessments.

 Pharma IQ: Why is the European Commission involved in harmonising toxicological tests for in vitro screening?

 Bremer-Hoffmann: According to Directive 2010/63/EU on the protection of animals used for scientific purposes, a European Union Reference Laboratory for Alternatives to Animal Testing (EURL ECVAM) has been established and assigned to the European Commission's Joint Research Centre. The mandate of EURL ECVAM is to (1) promote the development and use of alternative testing methods that serve the aim of the so-called 3Rs, i.e. replacing, reducing or refining testing on animals; (2) coordinate at the European level the validation of alternative methods; (3) disseminate information on alternative test methods; (4) act as a focal point for information exchange on development, use and acceptance of methods and to promote dialogue between all relevant players in the field.

In vitro toxicity testing is an essential part for reducing and replacing animal tests. Therefore, the JRC continues to look for innovative ways to translate the results of scientific research into useful and valid tools that can significantly reduce society's reliance on animal testing while ensuring the highest level of protection for human health and the environment and maintaining industrial and economic competitiveness of the EU.

 Pharma IQ: What are some of the challenges involved in obtaining ‘pure’ populations of cells from iPSCs? How do these complicate in vitro screening?

 Bremer-Hoffmann: Some of the difficulties we are facing are related to the fact that mature cells do not divide anymore and are often overgrown by other cell types. In order to overcome this situation, the cells of interest could be isolated, for example by cell sorting or the introduction of resistant genes into the pluripotent cells. However, cell sorting is stressful and might also result in a significant loss of cells, whereas the genetic manipulation of pluripotent cells is a challenge itself and might alter the toxic response of the cells.

However, unlike regenerative cell therapies, the need for pure cell cultures in toxicology testing is not imperative and also depends on the question being addressed. On the one hand, even if an assay with one cell type is needed, the requirements to purity are not as high. On the other hand, considering the fact that many toxicological effects involve more than one cell type, a 'pure' cell culture is not always needed.

Another challenge is that the interaction of different cell types derived from stem cell cultures in the development of a toxicological response has not yet been systematically investigated.

 Pharma IQ: In a recent paper published in Cell Stem Cell, Sandra Engle at Pfizer discussed some of the challenges involved in using human stem cell-derived cells.  They include being expensive, difficult to work with, and providing less quantitative information and fewer opportunities for large-scale screening than conventional cell lines.   How do these limitations this affect high-throughput and high-content screening studies?

 Bremer-Hoffmann: Indeed, up-scaling and automation of stem cell cultures are known challenges and some of the ongoing research activities (among them the FP7 project SCR&Tox) are already addressing these difficulties in their work programme. High-throughput studies are of importance when assessing the toxicological relevance of a mechanism-based in vitro assay.  For example, if we want to understand how often a pathway, e.g. the CREB pathway, is modulated in a group of classified neurotoxicants, HTS applications are needed. This information is relevant for the development of testing strategies, in which pathways with a high prevalence should be tested first.

 Due to the high sensitivity of some high content technologies, such as transcriptomics, highly standardized physiologically relevant cell systems are needed to generate a hypothesis on the hazard of a substance. Perturbation of pathways, as detected at transcriptome level, guide further and more complex investigations at higher biological levels where the toxicological effects are manifested. Another FP7 project “DETECTIVE” is developing this concept further.

 In this context it is worthwhile to invest in upscaling technologies as well as in the miniaturization of test system or single-cell analysis to allowing the above mentioned applications which are of high relevance for mechanism based safety assessments, to be tackled.

 Pharma IQ: Can you tell us more about your involvement with ScreenTox?

 Bremer-Hoffmann: SCR&Tox is part of the SEURAT-1 cluster and focuses on the development of physiologically relevant cell systems for toxicological testing. According to the mandate of EURL ECVAM to support test development, the JRC is leading the work package on test development in order to guide test developers towards standardized assays so that the test will reach the requirements for regulatory toxicity testing. Furthermore, we have participated in the development of a pathway based toxicity test for neurotoxicity in order to gain hands on experience of the quality control needs of stem cell based test systems.

 Pharma IQ: How are these shaping European legislation on the use of stem cells?  Is any new legislation being discussed concerning the use of human stem cell-derived cells for toxicology testing?

 Bremer-Hoffmann: The development of new legislation is the primary responsibility of policy DGs of the Commission, and the JRC provides scientific and technical support to this process on request. We are not aware of any legislative proposals where the above-described work will make a direct impact. However, as with all alternative methods to animal testing, efforts on development, standardisation and evaluation are supportive of Directive 2010/63/EU and may eventually impact on guidance revisions for regulatory testing.