@article{KadariLuLietal.2014, author = {Kadari, Asifiqbal and Lu, Min and Li, Ming and Sekaran, Thileepan and Thummer, Rajkumar P. and Guyette, Naomi and Chu, Vi and Edenhofer, Frank}, title = {Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells}, series = {Stem Cell Research \& Therapy}, volume = {5}, journal = {Stem Cell Research \& Therapy}, number = {2}, issn = {1757-6512}, doi = {10.1186/scrt435}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120578}, pages = {47}, year = {2014}, abstract = {Integrating viruses represent robust tools for cellular reprogramming; however, the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here, we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies.}, language = {en} } @article{HarreitherRydbergAmandetal.2014, author = {Harreither, Eva and Rydberg, Hanna A. and {\AA}mand, Helene L. and Jadhav, Vaibhav and Fliedl, Lukas and Benda, Christina and Esteban, Miguel A. and Pei, Duanqing and Borth, Nicole and Grillari-Voglauer, Regina and Hommerding, Oliver and Edenhofer, Frank and Nord{\´e}n, Bengt and Grillari, Johanne}, title = {Characterization of a novel cell penetrating peptide derived from human Oct4}, series = {Cell Regeneration}, volume = {3}, journal = {Cell Regeneration}, number = {2}, issn = {2045-9769}, doi = {10.1186/2045-9769-3-2}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-120999}, year = {2014}, abstract = {BACKGROUND: Oct4 is a transcription factor that plays a major role for the preservation of the pluripotent state in embryonic stem cells as well as for efficient reprogramming of somatic cells to induced pluripotent stem cells (iPSC) or other progenitors. Protein-based reprogramming methods mainly rely on the addition of a fused cell penetrating peptide. This study describes that Oct4 inherently carries a protein transduction domain, which can translocate into human and mouse cells. RESULTS: A 16 amino acid peptide representing the third helix of the human Oct4 homeodomain, referred to as Oct4 protein transduction domain (Oct4-PTD), can internalize in mammalian cells upon conjugation to a fluorescence moiety thereby acting as a cell penetrating peptide (CPP). The cellular distribution of Oct4-PTD shows diffuse cytosolic and nuclear staining, whereas penetratin is strictly localized to a punctuate pattern in the cytoplasm. By using a Cre/loxP-based reporter system, we show that this peptide also drives translocation of a functionally active Oct4-PTD-Cre-fusion protein. We further provide evidence for translocation of full length Oct4 into human and mouse cell lines without the addition of any kind of cationic fusion tag. Finally, physico-chemical properties of the novel CPP are characterized, showing that in contrast to penetratin a helical structure of Oct4-PTD is only observed if the FITC label is present on the N-terminus of the peptide. CONCLUSIONS: Oct4 is a key transcription factor in stem cell research and cellular reprogramming. Since it has been shown that recombinant Oct4 fused to a cationic fusion tag can drive generation of iPSCs, our finding might contribute to further development of protein-based methods to generate iPSCs. Moreover, our data support the idea that transcription factors might be part of an alternative paracrine signalling pathway, where the proteins are transferred to neighbouring cells thereby actively changing the behaviour of the recipient cell.}, language = {en} } @article{PeitzMuenstThummeretal.2014, author = {Peitz, Michael and M{\"u}nst, Bernhard and Thummer, Rajkumar P. and Helfen, Martina and Edenhofer, Frank}, title = {Cell-permeant recombinant Nanog protein promotes pluripotency by inhibiting endodermal specification}, series = {Stem Cell Research}, volume = {12}, journal = {Stem Cell Research}, number = {3}, issn = {1876-7753}, doi = {10.1016/j.scr.2014.02.006}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-119740}, pages = {680-689}, year = {2014}, abstract = {A comprehensive understanding of the functional network of transcription factors establishing and maintaining pluripotency is key for the development of biomedical applications of stem cells. Nanog plays an important role in early development and is essential to induce natural pluripotency in embryonic stem cells (ESCs). Inducible gain-of-function systems allowing a precise control over time and dosage of Nanog activity would be highly desirable to study its vital role in the establishment and maintenance of pluripotency at molecular level. Here we engineered a recombinant cell permeable version of Nanog by fusing it with the cell penetrating peptide TAT. Nanog-TAT can be readily expressed in and purified from E. coli and binds to a consensus Nanog DNA sequence. At cellular level it enhances proliferation and self-renewal of ESCs in the absence of leukemia inhibitory factor (LIF). Nanog-TAT together with LIF acts synergistically as judged by enhanced clonogenicity and activation of an Oct4-promoter-driven GFP reporter gene. Furthermore Nanog-TAT, in the absence of LIF, promotes pluripotency by inhibiting endodermal specification in a Stat3-independent manner. Our results demonstrate that Nanog protein transduction is an attractive tool allowing control over dose and time of addition to the cells for studying the molecular control of pluripotency without genetic manipulation.}, language = {en} }