Stem Cell Research and Regenerative Pharmacology and Toxicology

The research of our group is focused on the application of patient-specific induced pluripotent stem cells (iPS cells) for the establishment of in vitro disease models. We use these models to study the underlying molecular and pathophysiological mechanisms. Patient-specific iPS cells can be generated from somatic cells (e.g., skin cells, blood cells) by overexpressing specific transcription factors to “reprogram” cells to pluripotency. By this way, the cell gains two properties similar to embryonic stem cells. One is the self-renewal and the other is the differentiation potential (pluripotency). The self-renewal is a special form of unlimited proliferation to produce daughter cells that maintain the same properties of the parental stem cell. The differentiation potential allows the cells differentiating into specialized body cells (for example, heart cells, nerve cells, hepatocytes, and muscle cells). Patient-specific iPS-cell models can be applied in different research fields. In our case, we want to translate novel findings emerging from our basic biomedical research into the development of improved new therapeutics for fighting human diseases.

Fig. 1: Application of human iPS cells. Shown is the reprogramming of somatic cells into patient-specific iPS cells to study diseases in vitro. Reprogramming of human somatic cells is carried out by the over-expression of the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC). The generated iPS cells can differentiate into various cell types and allow us modeling diseases in vitro, screening drugs, and testing their toxicity.

I. Analysis of the development process in vitro

One important research topic is the analysis of complex developmental processes of early embryogenesis. Because in vitro differentiation of iPS cells can recapitulate the process of embryonic development, we are using this in vitro differentiation system to examine early developmental processes at the cellular and molecular level. We are particularly interested in the regulation of certain key molecules during early development of the heart and the pancreas. An example of the central regulators are phosphodiesterases (PDEs), which influence many processes in the body and play a central role in the regulation of cyclic nucleotide-mediated signaling pathways. We want to study the function of these potential key molecules in both healthy and diseased organs in order to better understand the causes of various diseases. Disease-causing molecules may also be used as the basis for the development of therapeutic targets, and are especially in focus for the development of new therapeutic strategies and selective drugs.

Fig. 2: In vivo and in vitro cardiogenesis. (A) Schematic illustration of a transverse section of early stage mammalian embryo showing counteracting signals which shape the cardiac precursor zone in vivo. (B) Sequential steps in cardiac differentiation in vitro from pluripotent stem cells into functional cardiomyocytes. (From Cyganek et al., 2013.)

II. iPS-cell-based disease modelling

Genome-wide association studies indicate that many human diseases can be caused by monogenic defects in genes encoding structural or regulatory proteins. One focus of our work is the application of iPS cells in the field of cardiovascular research. We have established various iPS cell lines from patients that suffer from genetically induced heart disease (eg, catecholaminergic polymorphic ventricular tachycardia, Brugada syndrome, long QT syndrome, Barth syndrome, Vici syndrome, and dilated cardiomyopathy). Because the iPS cells and the cells derived from the iPS cells have the identical genetic profile of each patient, we use the patient-specific iPS cells to investigate the disease phenotype in the cell culture dish. At present, we are trying to gain a deeper insight into the genetic, pathophysiological and signaling mechanisms involved in the development of the heart, heart failure and cardiac arrhythmias. To better understand and analyze the diversity of the particular disease better, we are using the CRISPR / Cas9 technology, an innovative approach that allows us to edit the mutated genes involved in the patient-specific iPS cells that thereby “correct” them.  Restoring the normal phenotype by correcting the target gene confirms that the mutation in the patient is the cause of the disease. Once we observe this, we can investigate how the malfunction of individual genes affects the development of the particular disease.

Fig. 3: Modelling Barth syndrome using patient-specific iPS-cardiomyocytes. (A) Irregularities in the contractile apparatus (sarcomere) in cardiomyocytes derived from patient-specific iPS cells. Staining are for cTNT, MLC (myosin light chain) 2a and alpha-actinin at day 60 post cardiac differentiation. (B)  Consumption rate of oxygen by iPS cell-derived cardiomyocytes from a patient suffering from Barth syndrome (TAZ10) is altered in comparison to control at basal conditions and after the administration of the indicated compounds. (From Dudek et al., 2015.)

III. Validation of therapeutic targets

Over the past two decades, many different animal models have been generated for studying human diseases to understand the onset, development and progression of the disease. However, animal models alone are often inadequate representations of human diseases due to genetic and physiological differences. Investigation and validation of disease mechanisms and targets in a human system remain limited. Our iPS cell-based model systems enable us to perform biochemical analysis and elucidate signaling pathways and key molecules involved in human physiological conditions. One aspect of our research is to investigate the role of cyclic nucleotides in cardiac disease. Cyclic nucleotides are known to have critical roles in the development of cardiac hypertrophy or heart failure. Recently, the altered expression, localization and activity of phosphodiesterases (PDEs) have been associated to the changes in cyclic nucleotide signaling in cardiovascular diseases. Accordingly, the pharmacological modification of PDEs is of particular interest in terms of the development of new treatments and prevention strategies. Our current research focuses on the establishment of a hypertrophic cell model in vitro by using cardiomyocytes differentiated from iPS cells. Functional effects of pharmacologically modified PDEs are studied by using this cardiac hypertrophic model. By this way, we can gain important insights into the validation of therapeutic targets (eg. PDEs) and effects of the substances that are relevant for the development of drugs.

IV. Establishment of in vitro drug testing 

About 30% of the drugs that enter clinical trials are abandoned because of a lack of efficacy. One major reason for this is that preclinical trials of drug development are based on animal models. For example, a number of drugs that showed therapeutic effects in rodent models of amyotrophic lateral sclerosis turned out to be ineffective in human patients, emphasizing the necessity of disease models using human cells. The human iPS cell-based cell culture and in vitro generated organ-like tissues (organoids) are a promising approach to fill the gap between animal models and clinical trials. We are currently focusing on the establishment of a directed differentiation of iPS cells into somatic cells, including cardiomyocytes, neurons and hepatocytes. In addition, a number of human tissues (organoids), which consist of structured three-dimensional cell clusters, are prepared (for example, cardiovascular, cerebral, liver organoids and etc.) from patient-specific iPS cells. We will test the effect that experimental substances (for example, anti-arrhythmic drugs) - with specific versus multi-targeted effectiveness - have on the properties of cardiomyocytes, particularly, the extent that they lead to improved function. Thus, patient-specific iPS cell-based models are particularly valuable and allow us investigating all pathophysiologic relevance when the disease affects multiple organs.

Fig. 4: Engineered heart tissue (EHT) generated from human iPS cell-derived cardiomyocytes. (A) Sarcomere structure in cardiomyocytes in EHTs shown by staining with actin (green) and alpha-actinin (red). Scale bars, 50 µm. (B) Isoproterenol-stimulation of an EHT. (From Streckfuss-Bömeke et al., 2013.)

V. Establishment of in vitro analyses for drug toxicity tests 

In addition to ineffectiveness of drugs in the human body, toxicity is another key issue of the potential drugs. Even though a minimum of two in vivo animal models (at least one rodent and one non-rodent) are used to estimate the bioavailability of a new compound during the drug development, a further 30% of the compounds that were tested in clinical trials were not approved due to safety concerns, such as cardiotoxicity and hepatotoxicity. This reduced approval suggests that the animal models are not ideal for estimating human bioavailability. Some forms of toxicity can be estimated by using a cell-based assay. Cardiomyocytes and hepatocytes derived from human iPS cells represent a unique and predictive model to investigate the potential cardiotoxic and hepatotoxic effects of new pharmacological agents during the early development of drugs. For this purpose, we establish effective assays for drug toxicity tests by using the somatic cells differentiated from iPS cells.