Aiming at the personalized treatment of cardiac and pulmonary diseases, we place a particular focus on the reprogramming of iPS cells, the establishment of iPS-derived patient- or disease-specific cell lines, the detailed examination of genetic abnormalities of iPS cells, as well as the targeted genetic modification of iPS cells.
Reprogramming somatic cells into iPS cells
In 2006, the induction of pluripotent stem cells was first demonstrated by K. Takahashi und S. Yamanaka by the addition of four defined factors through retroviral transduction using adult mouse fibroblasts (Cell, 2006). Soon after, other groups also reported on the generation of induced pluripotent stem cells using human somatic cells. In the meantime, numerous patient-specific iPS-cell lines have been produced in order to further investigate various genetic diseases. Reprogramming somatic cells into pluripotent stem cells has thus succeeded in becoming a major breakthrough in stem cell research. In the research group of Prof. Martin, reprogramming somatic cells derived from different species is now established as a standard method, but with continuous improvements.
As a first step, reprogramming somatic cells into iPS cells requires the introduction of a defined combination of different cellular factors, usually with the help of so-called viral vectors which integrate into the genome of the target cells. The introduction of reprogramming factors into differentiated cells triggers a cascade of genetic events including alteration in DNA methylation status, chromatin remodeling, and gene expression. So far, the mechanisms leading to the generation of iPS cells are not yet fully understood.
One goal of the RG Martin is the development of clinically applicable protocols for the efficient generation of iPS cells. For this, a better understanding of the underlying molecular mechanisms of reprogramming is required. To fully realize the potential of in vitro reprogrammed cells, we need to understand the epigenetic and molecular determinants that convert a differentiated cell into a pluripotent stem cell. Detailed investigations in the RG Martin are performed using cells derived from mice, humans (blood cells, endothelial cells, epithelial cells, etc.), and various large animals, enabling a direct comparison of the different species and underlying reprogramming mechanisms.
Somatic cells are reprogrammed into induced pluripotent stem cells after isolation from the patient. These cells can be genetically modified, e.g., in order to correct for disease-specific mutations, to generate reporter cell lines or to introduce selection markers. After differentiation into the desired cell type, cells can be used for the investigation of the underlying molecular mechanisms of different diseases in order to further develop existing drugs or to discover novel substances for the treatment of various diseases.
Potential risks of iPS cells and iPS cell-derived cells
Transplantation of undifferentiated iPS cells in mice results in the formation of teratomas, as observed in ES cells. Thanks to the advanced purification of differentiated cells, this risk can be minimized. Additionally, the use of viral vectors for the reprogramming of somatic cells into iPS cells and for the genetic correction of iPS cells harbors the risk of activating neighboring proto-oncogenes. As an alternative, transgene-free reprogramming methods are used, e.g., synthetic mRNAs or non-integrating RNA viruses such as Sendai virus. Nowadays, designer-nucleases (ZFN, TALEN; see below), which allow targeted integration into the genome, are used in order to minimize the risk of insertional mutagenesis during the genetic correction of iPS cells. Thus, it is feasible to integrate the genes into safe harbor loci without any physiological consequences.
Recently, it has been shown that iPS cells may exhibit an increased amount of genetic abnormalities. In the RG Martin, we thus focus on the comparison of adult vs. young cell sources. In the future, juvenile cell sources may be used for cellular therapies in order to reduce the risk for mutations.
Genetic modifications of pluripotent stem cells
Many cardiovascular diseases, e.g., affecting the heart or the respiratory system, are based on genetic defects or mutations. In principle, regenerative therapies based on the isolation of patient specific cells, their reprogramming into iPS cells and subsequent genetic correction of the disease are conceivable. So far, genetic modifications of stem cells have mostly been performed employing randomly integrating viral vectors or plasmids. This bears the risk of activating neighboring alleles including proto-oncogenes via insertional mutagenesis. Alternative methods for gene modification with reduced risks need to be established for the future clinical application of patient-specific iPS cells.
Novel technologies for targeted genome editing are based on artificial enzymes, so-called designer nucleases, comprising zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the CRISPR/Cas9 system. These enzymes can be designed to introduce a DNA double/single strand break in a specific genomic locus. Together with cellular repair mechanisms, this allows the targeted modification of the genome. In comparison to conventional gene targeting and integrated vectors, ZFN- or TALEN-based homologues recombination is both safer and significantly more efficient. Together with the ground-breaking discovery of iPS cells, these designer nucleases now for the first time offer the opportunity to develop patient-specific cellular therapies for numerous genetic diseases.
Genetic modifications of pluripotent stem cells, e.g., for patient-specific therapies.
Within the RG Martin, a TALEN-platform was established allowing the routine production of various target-specific TALENs. These TALENs will be used, inter alia, for the generation of reporter cell lines for the differentiation of iPS cells into cardiomyocytes and respiratory cells (REBIRTH Unit 1.2).
Potential applications of genetically modified stem cells
In cooperation with T. Cathomen at the University Medical Center Freiburg, the RG Martin is working on ZNF- and TALEN-based genetic correction of patient-specific cells for the treatment of cystic fibrosis (CF). CF is a rare and fatal hereditary disease of the airways which affects between 1:2000 to 1:3000 newborns in Europe and Northern America. The disease is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
Since no effective treatment targeting CF is available to date, the long-term goal of this project funded by the Mukoviszidose Institut gGmbH is the development of an innovative cell replacement therapy. In a first step, patient-specific iPS cells are generated, characterized and genetically corrected, followed by the differentiation of pluripotent cells into progenitor / airway cells. Genetically corrected autologous iPS cells and their derivatives constitute an innovative strategy that can be used to replace damaged airway cells and deliver normal CFTR to the lung.
Screening for novel drugs represents an additional approach for the treatment of lung diseases like CF. TALENs are a valuable tool to efficiently introduce reporter constructs into disease-specific iPS cells in order to establish transgenic reporter cell lines used in such high-throughput screening approaches. Changing fluorescence intensities within the established cell lines thus allow the identification of potential drugs, e.g., in case of CF by measuring the activity of the CFTR channel.
Using the ZFN/TALEN method, gene constructs can be inserted into the cells which express reporter or selection genes under the control of cell type-specific promoters. These can be highly beneficial for the optimization of stem cell culture conditions, the analysis of cardiac and pulmonary differentiation potential, and the purification of stem cell-derived cardiomyocytes and lung epithelial cells.