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Exploring novel molecular tools and strategies to analyze pluripotency induction in somatic cells and to derive factor-free iPS cells

Subject Area Cell Biology
Term from 2008 to 2015
Project identifier Deutsche Forschungsgemeinschaft (DFG) - Project number 66434078
 
Final Report Year 2014

Final Report Abstract

The reports by Yamanaka and colleagues that adult somatic cells can be reprogrammed into induced pluripotent (iPS) stem cells by a combination of the factors Oct4, Sox2, Klf4 and c-Myc paved the way for a new field of biomedical research holding immense promise for a wide range of developmental biological studies and therapeutic applications. iPS cells could provide a virtually unlimited cellular source for analyzing human diseases and screening potential drugs in the cell culture dish. In order to realize the promising potential of iPS cells several hurdles need to be overcome. Moreover, the molecular mechanism underlying cellular reprogramming needs comprehensive investigations. This research project was aimed at getting further insights into the reprogramming process, modifying it and identifying novel reprogramming pathways. First, crucial safety issues were addressed in order to make iPS cells clinically useful. The viral transduction method initially used for iPS derivation carries the risk of oncogenic transformation. Additionally, some of the reprogramming genes play a role in tumor formation. Within this project we developed several strategies to avoid this complication. One approach was aimed at engineering reprogramming-competent proteins that are able to induce conversion without genetic manipulation. Through the application of protein transduction technology we developed recombinant versions of the cell-permeant reprogramming factors Oct4 and Sox2 representing an alternative to viral transduction. We demonstrated that those reprogramming factors efficiently translocate into cells and exhibit biological function. Cell-permeant biologically active versions of Klf4 and c-Myc could not be generated due to biochemical instability of the recombinant proteins. However, recombinant Oct4 and Sox2 were able to replace viral factors during iPS-type reprogramming of fibroblast cells, demonstrating proof-of-principle for protein-based reprogramming. In a second approach we developed a protocol to derive iPS cells by viral transduction of a polycistronic construct that can be subsequently removed by site specific DNA recombination. After viral transduction of human fibroblasts we derived stably reprogrammed iPS cells carrying a reprogramming transgene flanked by loxP recombinase recognition sites. Upon application of the cell-permeant DNA recombinase Cre we showed that the transgene can be deleted with an exceptionally high efficiency and demonstrated that ‘cleaned’ iPS cells resemble human ES cells more than iPS cells still carrying reprogramming transgenes. By this, we developed a rapid and robust strategy to derive patient-specific transgene-free iPS cells. A thorough understanding of the mechanism underlying cellular reprogramming does not only enable the development of more robust and effective protocols of iPS derivation but also the targeted modification of the reprogramming outcome. By constitutively inducing Sox2, Klf4, and c-Myc while strictly limiting Oct4 activity to the initial phase of reprogramming through Oct4 protein transduction we generated neuro-spherelike colonies. Those colonies could be isolated and expanded for more than 50 passages while their proliferation does not depend on sustained expression of the reprogramming factors. These induced neural stem cells (iNSCs) uniformly display morphological and molecular features of NSCs, such as the expression of Nestin, Pax6, and Olig2, and have a genome-wide transcriptional profile similar to that of brain-derived NSCs. Moreover, iNSCs can differentiate into neurons, astrocytes, and oligodendrocytes. These results demonstrating for the first time that functional somatic stem cells can be generated from somatic cells by factor-driven induction received widespread recognition also through public media. In conclusion, the results of this DFG-funded research project provided enhanced protocols for the derivation of safe iPS cells and was instrumental to explore and characterize new pathways of cellular reprogramming yielding novel reprogrammed cell populations that will be of high interest for future research and biomedical applications such as cell replacement therapy and disease modeling.

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