Expressionsmodulation stammzell-assoziierter Gene mittels kurzer Polypeptide
Stem cells and especially embryonic stem cells are of great interest in basic research and regenerative medicine. Due to massive ethical concerns, the work with embryonic stem cells is strictly regulated. The therapeutic application of induced pluripotent stem cells (iPS) represents a promising approach. Therefore the discovery of reprogramming somatic cells to a pluripotent state by viral transduction of four transcription factors Oct3/4, Sox2, Klf4 and c-Myc has been of great value. However, for cell replacement therapy, techniques utilizing viral gene transfer can not be used due to their uncalculable risk of deleterious mutations. To overcome this issue, alternative strategies were investigated, including the use of promoter based reporter assays to identify small molecules. The aim of this study was to establish a promoter based screening tool, to discover instead of small molecules short polypeptides, which modulate the expression of stem cell related genes such as oct4. Basically a tri functional reporter construct LEP (Luciferase, EGFP, Puromycin) was designed and stable expressed in human embryonic kidney cells permitting a high-throughput screening with a peptide library. A simultaneous screening procedure via antibiotic or flow cytometry yielded for both selection strategies putative candidates. In detail the flow cytometry selection resulted in more positive hits in less time than the antibiotic selection. All obtained polypeptides were confirmed by Rescue RCR and additional sequence homology analysis revealed connections to cell cycle processes. Besides the identification of novel short peptide aptamers activating hoct4, the developed experimental platform can also be used to study other stem cell related genes such as nanog or sox2. Consequently, the results are of great benefit to understand and optimize the generation of iPS, which remains a desirable approach for cell therapy.