Exploring novel molecular tools and strategies to analyze pluripotency induction in somatic cells and to derive factor-free iPS cells
Zusammenfassung der Projektergebnisse
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.
Projektbezogene Publikationen (Auswahl)
- Generation of transducible transcription factors Oct4 and Sox2. Biol. Chem. 389 (2008) 851-861
Bosnali, M & F. Edenhofer
(Siehe online unter https://doi.org/10.1515/BC.2008.106) - Engineering cell-permeant proteins. J. Visual. Exp. (2009) Dec 28;(34). pii: 1627
Münst, B., C. Patsch & F. Edenhofer
(Siehe online unter https://doi.org/10.3791/1627) - Exploring refined conditions for reprogramming cells by recombinant Oct4 protein Int. J. Dev. Biol. 54 (2010) 1713-21
Thier, M, B. Münst & F. Edenhofer
(Siehe online unter https://doi.org/10.1387/ijdb.103193mt) - Sox2 Is Essential for Formation of Trophectoderm in the Preimplantation Embryo, PLoS One (2010) Nov 12;5(11):e13952
M. Keramari, J. Razavi, K. A. Ingman, C. M. Ward, C. Patsch, F. Edenhofer & S. J. Kimber
(Siehe online unter https://doi.org/10.1371/journal.pone.0013952) - (2012). Induced neural stem cells. EP 12001981.5
Edenhofer F, Thier M, Wörsdörfer P, Lakes YB, Brüstle O
- Cellular reprogramming employing recombinant Sox2 protein, Stem Cells Intern. (2012);2012:549846. Epub 2012 May 29
Thier, M., B. Münst, S. Mielke & F. Edenhofer
(Siehe online unter https://doi.org/10.1155/2012/549846) - Direct conversion of fibroblasts into tripotent neural stem cells, Cell Stem Cell (2012) 10(4):473-9
Thier, M , P. Wörsdörfer, Y. B. Lakes, R. Gorris, S. Herms, T. Opitz, D. Seiferling, T. Quandel, P. Hoffmann, M. M. Nöthen, O. Brüstle & F. Edenhofer
(Siehe online unter https://doi.org/10.1016/j.stem.2012.03.003) - Non-Genetic Modulation of Notch Activity by Artificial Delivery of Notch Intracellular Domain into Neural Stem Cells, Stem Cell Rev. Rep. (2012) Jan 31
Haupt, S., L. Borghese, O. Brüstle & F. Edenhofer
(Siehe online unter https://doi.org/10.1007/s12015-011-9335-6) - Exploring the Neural Reprogrammome using Bioinformatics Approaches. J Stem Cell Res Ther (2013) 3:146
Sekaran T, Thummer R, F. Edenhofer
(Siehe online unter https://doi.org/10.4172/2157-7633.1000146) - Cell-permeant recombinant Nanog protein promotes pluripotency by inhibiting endodermal specification. Stem Cell Res (2014) Mar 5;12(3):680-689
Peitz M., B. Münst, R.P. Thummer, M. Helfen & F. Edenhofer
(Siehe online unter https://doi.org/10.1016/j.scr.2014.02.006) - Characterization of a novel cell penetrating peptide derived from human Oct4. Cell Regeneration (2014) 3:2
Harreither, Eva, H A Rydberg, H L Åmand, V Jadhav, L Fliedl, C Benda, M A Esteban, D Pei, N Borth, R Grillari-Voglauer, O Hommerding, F Edenhofer, B Nordén and J Grillari
(Siehe online unter https://doi.org/10.1186/2045-9769-3-2) - Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human iPS cells. Stem Cell Res Ther (2014) Apr 8;5(2):47
Kadari, A, Lu, M, Ming, Li, Sekaran, T., Thummer, R., Guyette, N., Chu, V. & F. Edenhofer
(Siehe online unter https://doi.org/10.1186/scrt435)
