@article{ReichertSchmalzlPrageretal.2013, author = {Reichert, Johannes and Schmalzl, Jonas and Prager, Patrick and Gilbert, Fabian and Quent, Verena M. C. and Steinert, Andre F. and Rudert, Maximilian and N{\"o}th, Ulrich}, title = {Synergistic effect of Indian hedgehog and bone morphogenetic protein-2 gene transfer to increase the osteogenic potential of human mesenchymal stem cells}, series = {Stem Cell Research \& Therapy}, journal = {Stem Cell Research \& Therapy}, doi = {10.1186/scrt316}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-97010}, year = {2013}, abstract = {Introduction To stimulate healing of large bone defects research has concentrated on the application of mesenchymal stem cells (MSCs). Methods In the present study, we induced the overexpression of the growth factors bone morphogenetic protein 2 (BMP-2) and/or Indian hedgehog (IHH) in human MSCs by adenoviral transduction to increase their osteogenic potential. GFP and nontransduced MSCs served as controls. The influence of the respective genetic modification on cell metabolic activity, proliferation, alkaline phosphatase (ALP) activity, mineralization in cell culture, and osteogenic marker gene expression was investigated. Results Transduction had no negative influence on cell metabolic activity or proliferation. ALP activity showed a typical rise-and-fall pattern with a maximal activity at day 14 and 21 after osteogenic induction. Enzyme activity was significantly higher in groups cultured with osteogenic media. The overexpression of BMP-2 and especially IHH + BMP-2 resulted in a significantly higher mineralization after 28 days. This was in line with obtained quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analyses, which showed a significant increase in osteopontin and osteocalcin expression for osteogenically induced BMP-2 and IHH + BMP-2 transduced cells when compared with the other groups. Moreover, an increase in runx2 expression was observed in all osteogenic groups toward day 21. It was again more pronounced for BMP-2 and IHH + BMP-2 transduced cells cultured in osteogenic media. Conclusions In summary, viral transduction did not negatively influence cell metabolic activity and proliferation. The overexpression of BMP-2 in combination with or without IHH resulted in an increased deposition of mineralized extracellular matrix, and expression of osteogenic marker genes. Viral transduction therefore represents a promising means to increase the osteogenic potential of MSCs and the combination of different transgenes may result in synergistic effects.}, language = {en} } @article{SteinertWeissenbergerKunzetal.2012, author = {Steinert, Andre F. and Weissenberger, Manuel and Kunz, Manuela and Gilbert, Fabian and Ghivizzani, Steven C. and Goebel, Sascha and Jakob, Franz and N{\"o}th, Ulrich and Rudert, Maximilian}, title = {Indian hedgehog gene transfer is a chondrogenic inducer of human mesenchymal stem cells}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-75425}, year = {2012}, abstract = {Introduction: To date, no single most-appropriate factor or delivery method has been identified for the purpose of mesenchymal stem cell (MSC)-based treatment of cartilage injury. Therefore, in this study we tested whether gene delivery of the growth factor Indian hedgehog (IHH) was able to induce chondrogenesis in human primary MSCs, and whether it was possible by such an approach to modulate the appearance of chondrogenic hypertrophy in pellet cultures in vitro. Methods: First-generation adenoviral vectors encoding the cDNA of the human IHH gene were created by cre-lox recombination and used alone or in combination with adenoviral vectors, bone morphogenetic protein-2 (Ad.BMP- 2), or transforming growth factor beta-1 (Ad.TGF-b1) to transduce human bone-marrow derived MSCs at 5 × 102 infectious particles/cell. Thereafter, 3 × 105 cells were seeded into aggregates and cultured for 3 weeks in serumfree medium, with untransduced or marker gene transduced cultures as controls. Transgene expressions were determined by ELISA, and aggregates were analysed histologically, immunohistochemically, biochemically and by RT-PCR for chondrogenesis and hypertrophy. Results: IHH, TGF-b1 and BMP-2 genes were equipotent inducers of chondrogenesis in primary MSCs, as evidenced by strong staining for proteoglycans, collagen type II, increased levels of glycosaminoglycan synthesis, and expression of mRNAs associated with chondrogenesis. IHH-modified aggregates, alone or in combination, also showed a tendency to progress towards hypertrophy, as judged by the expression of alkaline phosphatase and stainings for collagen type X and Annexin 5. Conclusion: As this study provides evidence for chondrogenic induction of MSC aggregates in vitro via IHH gene delivery, this technology may be efficiently employed for generating cartilaginous repair tissues in vivo.}, subject = {Medizin}, language = {en} }