%0 journal article %@ 2380-6761 %A Moradian, H., Schwestka, M., Roch, T., Gossen, M. %D 2023 %J Bioengineering & Translational Medicine %P e10622 %R doi:10.1002/btm2.10622 %T Deconvolution of synthetic mRNA expression: nucleoside chemistry alters translatability %U https://doi.org/10.1002/btm2.10622 %X Recent technological advances in the production of in vitro transcribed messenger RNA (IVT-mRNA) facilitate its clinical use as well as its application in basic research. In this regard, numerous chemical modifications, which are not naturally observed in endogenous mRNA, have been implemented primarily to address the issue of immunogenicity and improve its biological performance. However, recent findings suggested pronounced differences between expression levels of IVT-mRNAs with different nucleoside modifications in transfected cells. Given the multistep process of IVT-mRNA delivery and subsequent intracellular expression, it is unclear which step is influenced by IVT-mRNA chemistry. Here, we deconvolute this process and show that the nucleoside modification does not interfere with complexation of carriers, their physicochemical properties, and extracellular stability, as exemplified by selected modifications. The immediate effect of mRNA chemistry on the efficiency of ribosomal protein synthesis as a contributor to differences in expression was quantified by in vitro cell-free translation. Our results demonstrate that for the nucleoside modifications tested, translatability was the decisive step in determining overall protein production. Also of special importance for future work on rational selection of tailored synthetic mRNA chemistries, our findings set a workflow to identify potentially limiting, modification-dependent steps in the complex delivery process. %0 journal article %@ 2162-2531 %A Radloff, K., Gutbier, B., Dunne, C.M., Moradian, H., Schwestka, M., Gossen, M., Ahrens, K., Kneller, L., Wang, Y., Moga, A., Gkionis, L., Keil, O., Fehring, V., Tondera, D., Giese, K., Santel, A., Kaufmann, J., Witzenrath, M. %D 2023 %J Molecular Therapy Nucleic Acids %P 102068 %R doi:10.1016/j.omtn.2023.102068 %T Cationic LNP-formulated mRNA expressing Tie2-agonist in the lung endothelium prevents pulmonary vascular leakage %U https://doi.org/10.1016/j.omtn.2023.102068 %X Dysfunction of endothelial cells (ECs) lining the inner surface of blood vessels are causative for a number of diseases. Hence, the ability to therapeutically modulate gene expression within ECs is of high therapeutic value in treating diseases such as those associated with lung edema. mRNAs formulated with lipid nanoparticles (LNPs) have emerged as a new drug modality to induce transient protein expression for modulating disease-relevant signal transduction pathways. In the study presented here, we tested the effect of a novel synthetic, nucleoside-modified mRNA encoding COMP-Ang1 (mRNA-76) formulated into a cationic LNP on attenuating inflammation-induced vascular leakage. After intravenous injection, the respective mRNA was found to be delivered almost exclusively to the ECs of the lung, while sparing other vascular beds and bypassing the liver. The mode of action of mRNA-76, such as its activation of the Tie2 signal transduction pathway, was tested by pharmacological studies in vitro and in vivo in respective mouse models. mRNA-76 was found to prevent lung vascular leakage/lung edema as well as neutrophil infiltration in a lipopolysaccharide-challenging model. %0 journal article %@ 1757-6512 %A Bachamanda Somesh, D., Klose, K., Maring, J.A., Kunkel, D., Jürchott, K., Protze, S.I., Klein, O., Nebrich, G., Becker, M., Krüger, U., Nazari-Shafti, T.Z., Falk, V., Kurtz, A., Gossen, M., Stamm, C. %D 2023 %J Stem Cell Research and Therapy %P 296 %R doi:10.1186/s13287-023-03519-w %T Cardiomyocyte precursors generated by direct reprogramming and molecular beacon selection attenuate ventricular remodeling after experimental myocardial infarction %U https://doi.org/10.1186/s13287-023-03519-w %X Background: Direct cardiac reprogramming is currently being investigated for the generation of cells with a true cardiomyocyte (CM) phenotype. Based on the original approach of cardiac transcription factor-induced reprogramming of fibroblasts into CM-like cells, various modifications of that strategy have been developed. However, they uniformly suffer from poor reprogramming efficacy and a lack of translational tools for target cell expansion and purification. Therefore, our group has developed a unique approach to generate proliferative cells with a pre-CM phenotype that can be expanded in vitro to yield substantial cell doses. Methods: Cardiac fibroblasts were reprogrammed toward CM fate using lentiviral transduction of cardiac transcriptions factors (GATA4, MEF2C, TBX5, and MYOCD). The resulting cellular phenotype was analyzed by RNA sequencing and immunocytology. Live target cells were purified based on intracellular CM marker expression using molecular beacon technology and fluorescence-activated cell sorting. CM commitment was assessed using 5-azacytidine-based differentiation assays and the therapeutic effect was evaluated in a mouse model of acute myocardial infarction using echocardiography and histology. The cellular secretome was analyzed using mass spectrometry. Results:We found that proliferative CM precursor-like cells were part of the phenotype spectrum arising during direct reprogramming of fibroblasts toward CMs. These induced CM precursors (iCMPs) expressed CPC- and CM-specific proteins and were selectable via hairpin-shaped oligonucleotide hybridization probes targeting Myh6/7-mRNA–expressing cells. After purification, iCMPs were capable of extensive expansion, with preserved phenotype when under ascorbic acid supplementation, and gave rise to CM-like cells with organized sarcomeres in differentiation assays. When transplanted into infarcted mouse hearts, iCMPs prevented CM loss, attenuated fibrotic scarring, and preserved ventricular function, which can in part be attributed to their substantial secretion of factors with documented beneficial effect on cardiac repair. Conclusions: Fibroblast reprogramming combined with molecular beacon-based cell selection yields an iCMP-like cell population with cardioprotective potential. Further studies are needed to elucidate mechanism-of-action and translational potential. %0 journal article %@ 1420-682X %A Batool, L., Hariharan, K., Xu, Y., Kaßmann, M., Tsvetkov, D., Gohlke, B.O., Kaden, S., Gossen, M., Nürnberg, B., Kurtz, A., Gollasch, M. %D 2023 %J Cellular and Molecular Life Sciences %N 9 %P 256 %R doi:10.1007/s00018-023-04901-w %T An inactivating human TRPC6 channel mutation without focal segmental glomerulosclerosis %U https://doi.org/10.1007/s00018-023-04901-w 9 %X Transient receptor potential cation channel-6 (TRPC6) gene mutations cause familial focal segmental glomerulosclerosis (FSGS), which is inherited as an autosomal dominant disease. In patients with TRPC6-related FSGS, all mutations map to the N- or C-terminal TRPC6 protein domains. Thus far, the majority of TRPC6 mutations are missense resulting in increased or decreased calcium influx; however, the fundamental molecular mechanisms causing cell injury and kidney pathology are unclear. We report a novel heterozygous TRPC6 mutation (V691Kfs*) in a large kindred with no signs of FSGS despite a largely truncated TRPC6 protein. We studied the molecular effects of V691Kfs* TRPC6 mutant using the tridimensional cryo-EM structure of the tetrameric TRPC6 protein. The results indicated that V691 is localized at the pore-forming transmembrane region affecting the ion conduction pathway, and predicted that V691Kfs* causes closure of the ion-conducting pathway leading to channel inactivation. We assessed the impact of V691Kfs* and two previously reported TRPC6 disease mutants (P112Q and G757D) on calcium influx in cells. Our data show that the V691Kfs* fully inactivated the TRCP6 channel-specific calcium influx consistent with a complete loss-of-function phenotype. Furthermore, the V691Kfs* truncation exerted a dominant negative effect on the full-length TRPC6 proteins. In conclusion, the V691Kfs* non-functional truncated TRPC6 is not sufficient to cause FSGS. Our data corroborate recently characterized TRPC6 loss-of-function and gain-of-function mutants suggesting that one defective TRPC6 gene copy is not sufficient to cause FSGS. We underscore the importance of increased rather than reduced calcium influx through TRPC6 for podocyte cell death. %0 journal article %@ 1944-8244 %A Listek, M., Hönow, A., Gossen, M., Hanack, K. %D 2023 %J ACS Applied Materials and Interfaces %N 37 %P 43219–43222 %R doi:10.1021/acsami.3c05317 %T Comment on “Monoclonal Antibody Discovery based on precise selection of single transgenic hybridomas with an On-Cell-Surface and Antigen-Specific Anchor” %U https://doi.org/10.1021/acsami.3c05317 37 %X In the original paper, Li and co-workers [ACS Appl. Mater. Interfaces 2022, 14, 17128–17141] described their approach to select specific hybridoma cells from a polyclonal hybridoma pool by using a cell surface anchor to catch the secreted antibody. The antigen-specific detection was performed with streptavidin-labeled antigen and a PE-labeled anti-F(ab′)2 antibody. The present comment offers a clearer description of the selection system originally published by Listek et al. in 2020 and provides further information about the importance of controls and recent adaptations made by our lab. %0 journal article %@ 2041-1723 %A Melo, U.S., Jatzlau, J., Prada-Medina, C.A., Flex, E., Hartmann, S., Ali, S., Schöpflin, R., Bernardini, L., Ciolfi, A., Moeinzadeh, M.H., Klever, M.K., Altay, A., Vallecillo-Garcia, P., Carpentieri, G., Delledonne, M., Ort, M.J., Schwestka, M., Battista Ferrero, G., Tartaglia, M., Brusco, A., Gossen, M., Strunk, D., Geißler, S., Mundlos, S., Stricker, S., Knaus, P., Giorgio, E., Spielmann, M. %D 2023 %J Nature Communications %P 2034 %R doi:10.1038/s41467-023-37585-8 %T Enhancer hijacking at the ARHGAP36 locus is associated with connective tissue to bone transformation %U https://doi.org/10.1038/s41467-023-37585-8 %X Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFβ and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFβ, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease. %0 journal article %@ 2190-3948 %A Stahn, L., Rasińska, J., Dehne, T., Schreyer, S., Hakus, A., Gossen, M., Steiner, B., Hemmati-Sadeghi, S. %D 2023 %J Drug Delivery and Translational Research %P 1745-1765 %R doi:10.1007/s13346-023-01289-9 %T Sleeping beauty transposon system for GDNF overexpression of entrapped stem cells in fibrin hydrogel in a rat model of Parkinson’s disease %U https://doi.org/10.1007/s13346-023-01289-9 %X There is currently no causal treatment available for Parkinson’s disease (PD). However, the use of glial cell line–derived neurotrophic factor (GDNF) to provide regenerative effects for neurons is promising. Such approaches require translational delivery systems that are functional in diseased tissue. To do so, we used a non-viral Sleeping Beauty (SB) transposon system to overexpress GDNF in adipose tissue–derived mesenchymal stromal cells (adMSCs). Entrapment of cells in fibrin hydrogel was used to boost potential neurorestorative effects. Functional GDNF-adMSCs were able to secrete 1066.8 ± 169.4 ng GDNF/120,000 cells in vitro. The GDNF-adMSCs were detectable for up to 1 month after transplantation in a mild 6-hydroxydopamine (6-OHDA) hemiparkinson male rat model. Entrapment of GDNF-adMSCs enabled GDNF secretion in surrounding tissue in a more concentrated manner, also tending to prolong GDNF secretion relatively. GDNF-adMSCs entrapped in hydrogel also led to positive immunomodulatory effects via an 83% reduction of regional IL-1β levels compared to the non-entrapped GDNF-adMSC group after 1 month. Furthermore, GDNF-adMSC-treated groups showed higher recovery of tyrosine hydroxylase (TH)-expressing cells, indicating a neuroprotective function, although this was not strong enough to show significant improvement in motor performance. Our findings establish a promising GDNF treatment system in a PD model. Entrapment of GDNF-adMSCs mediated positive immunomodulatory effects. Although the durability of the hydrogel needs to be extended to unlock its full potential for motor improvements, the neuroprotective effects of GDNF were evident and safe. Further motor behavioral tests and other disease models are necessary to evaluate this treatment option adequately. %0 journal article %@ 0142-9612 %A Drzeniek, N.M., Kahwaji, N., Schlickeiser, S., Reinke, P., Geißler, S., Volk, H.-D., Gossen, M. %D 2023 %J Biomaterials %P 121971 %R doi:10.1016/j.biomaterials.2022.121971 %T Immuno-engineered mRNA combined with cell adhesive niche for synergistic modulation of the MSC secretome %U https://doi.org/10.1016/j.biomaterials.2022.121971 %X Integrating minimally immunogenic mRNA technology with predesigned matrix-derived cues allows for the synergistic combination of multiple dimensions of cell manipulation and opens routes for biomaterial-based delivery of mRNA-engineered cell products. Such multimodal systems could present a more biologically relevant way to therapeutically address complex multifactorial processes such as tissue ischemia, angiogenesis, and regeneration. %0 journal article %@ 1474-9726 %A Brauer, E., Lange, T., Keller, D., Görlitz, S., Cho, S., Keye, J., Gossen, M., Petersen, A., Kornak, U. %D 2023 %J Aging Cell %N 3 %P e13744 %R doi:10.1111/acel.13744 %T Dissecting the influence of cellular senescence on cell mechanics and extracellular matrix formation in vitro %U https://doi.org/10.1111/acel.13744 3 %X Tissue formation and healing both require cell proliferation and migration, but also extracellular matrix production and tensioning. In addition to restricting proliferation of damaged cells, increasing evidence suggests that cellular senescence also has distinct modulatory effects during wound healing and fibrosis. Yet, a direct role of senescent cells during tissue formation beyond paracrine signaling remains unknown. We here report how individual modules of the senescence program differentially influence cell mechanics and ECM expression with relevance for tissue formation. We compared DNA damage-mediated and DNA damage-independent senescence which was achieved through over-expression of either p16Ink4a or p21Cip1 cyclin-dependent kinase inhibitors in primary human skin fibroblasts. Cellular senescence modulated focal adhesion size and composition. All senescent cells exhibited increased single cell forces which led to an increase in tissue stiffness and contraction in an in vitro 3D tissue formation model selectively for p16 and p21-overexpressing cells. The mechanical component was complemented by an altered expression profile of ECM-related genes including collagens, lysyl oxidases, and MMPs. We found that particularly the lack of collagen and lysyl oxidase expression in the case of DNA damage-mediated senescence foiled their intrinsic mechanical potential. These observations highlight the active mechanical role of cellular senescence during tissue formation as well as the need to synthesize a functional ECM network capable of transferring and storing cellular forces. %0 journal article %@ 1076-3279 %A Brauer, E., Lange, T., Keller, D., Görlitz, S., Cho, S., Keye, J., Gossen, M., Petersen, A., Kornak, U. %D 2023 %J Tissue Engineering A %N 13-14 %R doi:10.1089/ten.tea.2023.29043.abstracts %T Towards the role of cellular senescence on cell mechanics and ECM formation %U https://doi.org/10.1089/ten.tea.2023.29043.abstracts 13-14 %X %0 journal article %@ 1932-6254 %A Rasińska, J., Klein, C., Stahn, L., Maidhof, F., Pfeffer, A., Schreyer, S., Gossen, M., Kurtz, A., Steiner, B., Hemmati-Sadeghi, S. %D 2022 %J Journal of Tissue Engineering and Regenerative Medicine %N 6 %P 515-529 %R doi:10.1002/term.3296 %T Transposon-mediated glial cell line-derived neurotrophic factor overexpression in human adipose tissue-derived mesenchymal stromal cells: A potential approach for neuroregenerative medicine? %U https://doi.org/10.1002/term.3296 6 %X Glial cell line-derived neurotrophic factor (GDNF) has neuroprotective effects and may be a promising candidate for regenerative strategies focusing on neurodegenerative diseases. As GDNF cannot cross the blood–brain barrier to potentially regenerate damaged brain areas, continuous in situ delivery with host cells is desired. Here, a non-viral Sleeping Beauty transposon was used to achieve continuous in vitro overexpression of GDNF in immune-privileged human adipose tissue-derived mesenchymal stromal cells (GDNF-tASCs). In addition, in vivo survival, tolerance, and effectiveness of transfected cells were tested in a very mild 6-hydroxydopamine (6-OHDA)-induced dopamine depletion rat model by means of intrastriatal injection on a sample basis up to 6 months after treatment. GDNF-tASCs showed vast in vitro gene overexpression up to 13 weeks post-transfection. In vivo, GDNF was detectable 4 days following transplantation, but no longer after 1 month, although adipose tissue-derived mesenchymal stromal cells (ASCs) could be visualized histologically even after 6 months. Despite successful long-term in vitro GDNF overexpression and its in vivo detection shortly after cell transplantation, the 6-OHDA model was too mild to enable sufficient evaluation of in vivo disease improvement. Still, in vivo immunocompatibility could be further examined. ASCs initially induced a pronounced microglial accumulation at transplantation site, particularly prominent in GDNF-tASCs. However, 6-OHDA-induced pro-inflammatory immune response was attenuated by ASCs, although delayed in the GDNF-tASCs group. To further test the therapeutic potential of the generated GDNF-overexpressing cells in a disease-related context, a follow-up study using a more appropriate 6-OHDA model is needed. %0 journal article %@ 2162-2531 %A Moradian, H., Roch, T., Anthofer, L., Lendlein, A., Gossen, M. %D 2022 %J Molecular Therapy Nucleic Acids %P 854-869 %R doi:10.1016/j.omtn.2022.01.004 %T Chemical modification of uridine modulates mRNA-mediated proinflammatory and antiviral response in primary human macrophages %U https://doi.org/10.1016/j.omtn.2022.01.004 %X In vitro transcribed (IVT)-mRNA has been accepted as a promising therapeutic modality. Advances in facile and rapid production technologies make IVT-mRNA an appealing alternative to protein- or virus-based medicines. Robust expression levels, lack of genotoxicity and their manageable immunogenicity benefit its clinical applicability. We postulated that innate immune responses of therapeutically relevant human cells can be tailored or abrogated by combinations of 5’-end and internal IVT-mRNA modifications. Using primary human macrophages as targets, our data show the particular importance of uridine modifications for IVT-mRNA performance. Among five nucleotide modification schemes tested, 5-methoxy-uridine outperformed other modifications up to 4-fold increased transgene expression, triggering moderate proinflammatory and non-detectable antiviral responses. Macrophage responses against IVT-mRNAs exhibiting high immunogenicity (e.g., pseudouridine) could be minimized upon HPLC purification. Conversely, 5’-end modifications, had only modest effects on mRNA expression and immune responses. Our results revealed how the uptake of chemically modified IVT-mRNA impacts human macrophages, responding with distinct patterns of innate immune responses concomitant with increased transient transgene expression. We anticipate our findings are instrumental to predictively address specific cell responses required for wide range of therapeutic applications from eliciting controlled immunogenicity in mRNA vaccines to, e.g., completely abrogating cell activation in protein replacement therapies. %0 journal article %@ 2159-6859 %A Moradian, H., Gossen, M., Lendlein, A. %D 2022 %J MRS Communications %P 145-153 %R doi:10.1557/s43579-021-00128-7 %T Co-delivery of genes can be confounded by bicistronic vector design %U https://doi.org/10.1557/s43579-021-00128-7 %X Maximizing the efficiency of nanocarrier-mediated co-delivery of genes for co-expression in the same cell is critical for many applications. Strategies to maximize co-delivery of nucleic acids (NA) focused largely on carrier systems, with little attention towards payload composition itself. Here, we investigated the effects of different payload designs: co-delivery of two individual “monocistronic” NAs versus a single bicistronic NA comprising two genes separated by a 2A self-cleavage site. Unexpectedly, co-delivery via the monocistronic design resulted in a higher percentage of co-expressing cells, while predictive co-expression via the bicistronic design remained elusive. Our results will aid the application-dependent selection of the optimal methodology for co-delivery of genes. %0 journal article %@ 1386-0291 %A Lau, S., Gossen, M., Lendlein, A., Jung, F. %D 2022 %J Clinical Hemorheology and Microcirculation %N 3 %P 191-203 %R doi:10.3233/CH-211294 %T Differential sensitivity of assays for determining vein endothelial cell senescence %U https://doi.org/10.3233/CH-211294 3 %X In vivo endothelialization of polymer-based cardiovascular implant materials is a promising strategy to reduce the risk of platelet adherence and the subsequent thrombus formation and implant failure. However, endothelial cells from elderly patients are likely to exhibit a senescent phenotype that may counteract endothelialization. The senescence status of cells should therefore be investigated prior to implantation of devices designed to be integrated in the blood vessel wall. Here, human umbilical vein endothelial cells (HUVEC) were cultivated up to passage (P) 4, 10 and 26/27 to determine the population doubling time and the senescence status by four different methods. Determination of the senescence-associated β-galactosidase activity (SA-β-Gal) was carried out by colorimetric staining and microscopy (i), as well as by photometric quantification (ii), and the expression of senescence-associated nuclear proteins p16 and p21 as well as the proliferation marker Ki67 was assessed by immunostaining (iii), and by flow cytometry (iv). The population doubling time of P27-cells was remarkably greater (103±65 h) compared to P4-cells (24±3 h) and P10-cell (37±15 h). Among the four different methods tested, the photometric SA-β-Gal activity assay and the flow cytometric determination of p16 and Ki67 were most effective in discriminating P27-cells from P4- and P10-cells. These methods combined with functional endothelial cell analyses might aid predictions on the performance of implant endothelialization in vivo. %0 conference poster %@ %A Ullah, I., Nie, Y., Gossen, M., Kurtz, A., Ma, N., Lendlein, A. %D 2022 %J 40. Jahrestagung der Deutschen Gesellschaft für Klinische Mikrozirkulation und Hämorheologie e.V. %T A simple approach towards the efficient generation of 3D blood vessels organoids %U %X %0 journal article %@ 2329-0501 %A Peter, L., Wendering, D.J., Schlickeiser, S., Hoffmann, H., Noster, R., Wagner, D.L., Zarrinrad, G., Münch, S., Picht, S., Schulenberg, S., Moradian, H., Mashreghi, M.-F., Klein, O., Gossen, M., Roch, T., Babel, N., Reinke, P., Volk, H.-D., Amini, L., Schmueck-Henneresse, M. %D 2022 %J Molecular Therapy. Methods & Clinical Development %P 52-73 %R doi:10.1016/j.omtm.2022.02.012 %T Tacrolimus-resistant SARS-CoV-2-specific T cell products to prevent and treat severe COVID-19 in immunosuppressed patients %U https://doi.org/10.1016/j.omtm.2022.02.012 %X Solid organ transplant (SOT) recipients receive therapeutic immunosuppression that compromises their immune response to infections and vaccines. For this reason, SOT patients have a high risk of developing severe coronavirus disease 2019 (COVID-19) and an increased risk of death from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. Moreover, the efficiency of immunotherapies and vaccines is reduced due to the constant immunosuppression in this patient group. Here, we propose adoptive transfer of SARS-CoV-2-specific T cells made resistant to a common immunosuppressant, tacrolimus, for optimized performance in the immunosuppressed patient. Using a ribonucleoprotein approach of CRISPR-Cas9 technology, we have generated tacrolimus-resistant SARS-CoV-2-specific T cell products from convalescent donors and demonstrate their specificity and function through characterizations at the single-cell level, including flow cytometry, single-cell RNA (scRNA) Cellular Indexing of Transcriptomes and Epitopes (CITE), and T cell receptor (TCR) sequencing analyses. Based on the promising results, we aim for clinical validation of this approach in transplant recipients. Additionally, we propose a combinatory approach with tacrolimus, to prevent an overshooting immune response manifested as bystander T cell activation in the setting of severe COVID-19 immunopathology, and tacrolimus-resistant SARS-CoV-2-specific T cell products, allowing for efficient clearance of viral infection. Our strategy has the potential to prevent severe COVID-19 courses in SOT or autoimmunity settings and to prevent immunopathology while providing viral clearance in severe non-transplant COVID-19 cases. %0 conference lecture %@ %A Maring, J., Becker, M., Tung, W., Gossen, M., Stamm, C., Ma, N., Lendlein, A. %D 2022 %J 40. Jahrestagung der Deutschen Gesellschaft für Klinische Mikrozirkulation und Hämorheologie e.V. %T Immune response to electrospun PEEU fiber meshes %U %X %0 journal article %@ 2161-5063 %A Duran, A., Schwestka, M., Nazari-Shafti, T., Neuber, S., Stamm, C., Gossen, M. %D 2022 %J ACS Synthetic Biology %N 8 %P 2623-2635 %R doi:10.1021/acssynbio.2c00036 %T Limiting Transactivator Amounts Contribute to Transgene Mosaicism in Tet-On All-in-One Systems %U https://doi.org/10.1021/acssynbio.2c00036 8 %X MicroRNAs play an essential role in cell homeostasis and have been proposed as therapeutic agents. One strategy to deliver microRNAs is to genetically engineer target cells to express microRNAs of interest. However, to control dosage and timing, as well as to limit potential side-effects, microRNAs’ expression should ideally be under exogenous, inducible control. Conditional expression of miRNA-based short hairpin RNAs (shRNAmirs) via gene regulatory circuits such as the Tet-system is therefore a promising strategy to control shRNAmirs’ expression in research and therapy. Single vector approaches like Tet-On all-in-one designs are more compatible with potential clinical applications by providing the Tet-On system components in a single round of genetic engineering. However, all-in-one systems often come at the expense of heterogeneous and unstable expression. In this study, we aimed to understand the causes that lead to such erratic transgene expression. By using a reporter cell, we found that the degree of heterogeneity mostly correlated with reverse tetracycline transactivator (rtTA) expression levels. Moreover, the targeted integration of a potent rtTA expression cassette into a genomic safe harbor locus functionally rescued previously silenced rtTA-responsive transcription units. Overall, our results suggest that ensuring homogenous and stable rtTA expression is essential for the robust and reliable performance of future Tet-On all-in-one designs. %0 conference lecture %@ %A Lau, S., Gossen, M., Lendlein, A., Jung, F. %D 2022 %J 40. Jahrestagung der Deutschen Gesellschaft für Klinische Mikrozirkulation und Hämorheologie (DGKMH) %T Differential sensitivity of assays for determining vein endothelial cell senescence %U %X %0 conference lecture %@ %A Moradian, H., Lendlein, A., Gossen, M. %D 2021 %J Virtual MRS Spring Meeting 2021 %T Effect of nucleotide chemistry on expression of in vitro transcribed mRNA %U %X %0 journal article %@ 1422-0067 %A Lau, S., Gossen, M., Lendlein, A., Jung, F. %D 2021 %J International Journal of Molecular Sciences %N 2 %P 978 %R doi:10.3390/ijms22020978 %T Venous and Arterial Endothelial Cells from Human Umbilical Cords: Potential Cell Sources for Cardiovascular Research %U https://doi.org/10.3390/ijms22020978 2 %X Although cardiovascular devices are mostly implanted in arteries or to replace arteries, in vitro studies on implant endothelialization are commonly performed with human umbilical cord-derived venous endothelial cells (HUVEC). In light of considerable differences, both morphologically and functionally, between arterial and venous endothelial cells, we here compare HUVEC and human umbilical cord-derived arterial endothelial cells (HUAEC) regarding their equivalence as an endothelial cell in vitro model for cardiovascular research. No differences were found in either for the tested parameters. The metabolic activity and lactate dehydrogenase, an indicator for the membrane integrity, slightly decreased over seven days of cultivation upon normalization to the cell number. The amount of secreted nitrite and nitrate, as well as prostacyclin per cell, also decreased slightly over time. Thromboxane B2 was secreted in constant amounts per cell at all time points. The Von Willebrand factor remained mainly intracellularly up to seven days of cultivation. In contrast, collagen and laminin were secreted into the extracellular space with increasing cell density. Based on these results one might argue that both cell types are equally suited for cardiovascular research. However, future studies should investigate further cell functionalities, and whether arterial endothelial cells from implantation-relevant areas, such as coronary arteries in the heart, are superior to umbilical cord-derived endothelial cells. %0 conference lecture %@ %A Lau, S., Liu, Y., Maier, A., Braune, S., Gossen, M., Lendlein, A. %D 2021 %J Virtual MRS Spring Meeting %T In vitro assessment of polymer thrombogenicity: the effect of endothelial culture conditions on platelet responses %U %X %0 journal article %@ 0884-0431 %A Rössler, U., Hennig, A., Stelzer, N., Bose, S., Kopp, J., Søe, K., Cyganek, L., Zifarelli, G., Ali, S., von der Hagen, M., Strässler, E., Hahn, G., Pusch, M., Stauber, T., Izsvák, Z., Gossen, M., Stachelscheid, H., Kornak, U. %D 2021 %J Journal of Bone and Mineral Research %N 8 %P 1621-1635 %R doi:10.1002/jbmr.4322 %T Efficient generation of osteoclasts from human induced pluripotent stem cells and functional investigations of lethal CLCN7-related osteopetrosis %U https://doi.org/10.1002/jbmr.4322 8 %X Human induced pluripotent stem cells (hiPSCs) hold great potential for modeling human diseases and the development of innovative therapeutic approaches. Here, we report on a novel, simplified differentiation method for forming functional osteoclasts from hiPSCs. The three-step protocol starts with embryoid body formation, followed by hematopoietic specification, and finally osteoclast differentiation. We observed continuous production of monocyte-like cells over a period of up to 9 weeks, generating sufficient material for several osteoclast differentiations. The analysis of stage-specific gene and surface marker expression proved mesodermal priming, the presence of monocyte-like cells, and of terminally differentiated multinucleated osteoclasts, able to form resorption pits and trenches on bone and dentine in vitro. In comparison to peripheral blood mononuclear cell (PBMC)-derived osteoclasts hiPSC-derived osteoclasts were larger and contained a higher number of nuclei. Detailed functional studies on the resorption behavior of hiPSC-osteoclasts indicated a trend towards forming more trenches than pits and an increase in pseudoresorption. We used hiPSCs from an autosomal recessive osteopetrosis (ARO) patient (BIHi002-A, ARO hiPSCs) with compound heterozygous missense mutations p.(G292E) and p.(R403Q) in CLCN7, coding for the Cl−/H+-exchanger ClC-7, for functional investigations. The patient's leading clinical feature was a brain malformation due to defective neuronal migration. Mutant ClC-7 displayed residual expression and retained lysosomal co-localization with OSTM1, the gene coding for the osteopetrosis-associated transmembrane protein 1, but only ClC-7 harboring the mutation p.(R403Q) gave strongly reduced ion currents. An increased autophagic flux in spite of unchanged lysosomal pH was evident in undifferentiated ARO hiPSCs. ARO hiPSC-derived osteoclasts showed an increased size compared to hiPSCs of healthy donors. They were not able to resorb bone, underlining a loss-of-function effect of the mutations. In summary, we developed a highly reproducible, straightforward hiPSC-osteoclast differentiation protocol. We demonstrated that osteoclasts differentiated from ARO hiPSCs can be used as a disease model for ARO and potentially also other osteoclast-related diseases. © 2021 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR). %0 conference lecture %@ %A Moradian, H., Lendlein, A., Gossen, M. %D 2021 %J Virtual MRS Spring Meeting 2021 %T Nucleic acid co-delivery: How to modulate protein co-expression by formulation of payload %U %X %0 journal article %@ 1422-0067 %A Lau, S., Gossen, M., Lendlein, A. %D 2021 %J International Journal of Molecular Sciences %N 23 %P 13120 %R doi:10.3390/ijms222313120 %T Designing Cardiovascular Implants Taking in View the Endothelial Basement Membrane %U https://doi.org/10.3390/ijms222313120 23 %X Insufficient endothelialization of cardiovascular grafts is a major hurdle in vascular surgery and regenerative medicine, bearing a risk for early graft thrombosis. Neither of the numerous strategies pursued to solve these problems were conclusive. Endothelialization is regulated by the endothelial basement membrane (EBM), a highly specialized part of the vascular extracellular matrix. Thus, a detailed understanding of the structure–function interrelations of the EBM components is fundamental for designing biomimetic materials aiming to mimic EBM functions. In this review, a detailed description of the structure and functions of the EBM are provided, including the luminal and abluminal interactions with adjacent cell types, such as vascular smooth muscle cells. Moreover, in vivo as well as in vitro strategies to build or renew EBM are summarized and critically discussed. The spectrum of methods includes vessel decellularization and implant biofunctionalization strategies as well as tissue engineering-based approaches and bioprinting. Finally, the limitations of these methods are highlighted, and future directions are suggested to help improve future design strategies for EBM-inspired materials in the cardiovascular field. %0 journal article %@ 1422-0067 %A Lau, S., Maier, A., Braune, S., Gossen, M., Lendlein, A. %D 2021 %J International Journal of Molecular Sciences %N 13 %P 7006 %R doi:10.3390/ijms22137006 %T Effect of Endothelial Culture Medium Composition on Platelet Responses to Polymeric Biomaterials %U https://doi.org/10.3390/ijms22137006 13 %X Near-physiological in vitro thrombogenicity test systems for the evaluation of blood-contacting endothelialized biomaterials requires co-cultivation with platelets (PLT). However, the addition of PLT has led to unphysiological endothelial cell (EC) detachment in such in vitro systems. A possible cause for this phenomenon may be PLT activation triggered by the applied endothelial cell medium, which typically consists of basal medium (BM) and nine different supplements. To verify this hypothesis, the influence of BM and its supplements was systematically analyzed regarding PLT responses. For this, human platelet rich plasma (PRP) was mixed with BM, BM containing one of nine supplements, or with BM containing all supplements together. PLT adherence analysis was carried out in six-channel slides with plasma-treated cyclic olefin copolymer (COC) and poly(tetrafluoro ethylene) (PTFE, as a positive control) substrates as part of the six-channel slides in the absence of EC and under static conditions. PLT activation and aggregation were analyzed using light transmission aggregometry and flow cytometry (CD62P). Medium supplements had no effect on PLT activation and aggregation. In contrast, supplements differentially affected PLT adherence, however, in a polymer- and donor-dependent manner. Thus, the use of standard endothelial growth medium (BM + all supplements) maintains functionality of PLT under EC compatible conditions without masking the differences of PLT adherence on different polymeric substrates. These findings are important prerequisites for the establishment of a near-physiological in vitro thrombogenicity test system assessing polymer-based cardiovascular implant materials in contact with EC and PLT. %0 journal article %@ 2159-6859 %A Lau, S., Liu, Y., Maier, A., Braune, S., Gossen, M., Neffe, A., Lendlein, A. %D 2021 %J MRS Communications %N 5 %P 559-567 %R doi:10.1557/s43579-021-00072-6 %T Establishment of an in vitro thrombogenicity test system with cyclic olefin copolymer substrate for endothelial layer formation %U https://doi.org/10.1557/s43579-021-00072-6 5 %X In vitro thrombogenicity test systems require co-cultivation of endothelial cells and platelets under blood flow-like conditions. Here, a commercially available perfusion system is explored using plasma-treated cyclic olefin copolymer (COC) as a substrate for the endothelial cell layer. COC was characterized prior to endothelialization and co-cultivation with platelets under static or flow conditions. COC exhibits a low roughness and a moderate hydrophilicity. Flow promoted endothelial cell growth and prevented platelet adherence. These findings show the suitability of COC as substrate and the importance of blood flow-like conditions for the assessment of the thrombogenic risk of drugs or cardiovascular implant materials. %0 journal article %@ 2045-2322 %A Taieb, H., Garske, D., Contzen, J., Gossen, M., Bertinetti, L., Robinson, T., Cipitria, A. %D 2021 %J Scientific Reports %N 1 %P 13455 %R doi:10.1038/s41598-021-92054-w %T Osmotic pressure modulates single cell cycle dynamics inducing reversible growth arrest and reactivation of human metastatic cells %U https://doi.org/10.1038/s41598-021-92054-w 1 %X Biophysical cues such as osmotic pressure modulate proliferation and growth arrest of bacteria, yeast cells and seeds. In tissues, osmotic regulation takes place through blood and lymphatic capillaries and, at a single cell level, water and osmoregulation play a critical role. However, the effect of osmotic pressure on single cell cycle dynamics remains poorly understood. Here, we investigate the effect of osmotic pressure on single cell cycle dynamics, nuclear growth, proliferation, migration and protein expression, by quantitative time-lapse imaging of single cells genetically modified with fluorescent ubiquitination-based cell cycle indicator 2 (FUCCI2). Single cell data reveals that under hyperosmotic stress, distinct cell subpopulations emerge with impaired nuclear growth, delayed or growth arrested cell cycle and reduced migration. This state is reversible for mild hyperosmotic stress, where cells return to regular cell cycle dynamics, proliferation and migration. Thus, osmotic pressure can modulate the reversible growth arrest and reactivation of human metastatic cells. %0 conference lecture %@ %A Gossen, M. %D 2021 %J 2021 Virtual MRS Spring Meeting %T RNA Delivery %U %X %0 journal article %@ 1758-5082 %A Drzeniek, N., Mazzocchi, A., Schlickeiser, S., Forsythe, S., Moll, G., Geißler, S., Reinke, P., Gossen, M., Gorantla, V., Volk, H., Soker, S. %D 2021 %J Biofabrication %P 045002 %R doi:10.1088/1758-5090/ac0a32 %T Bio-instructive hydrogel expands the paracrine potency of mesenchymal stem cells %U https://doi.org/10.1088/1758-5090/ac0a32 %X The therapeutic efficacy of clinically applied mesenchymal stromal cells (MSCs) is limited due to their injection into harsh in vivo environments, resulting in the significant loss of their secretory function upon transplantation. A potential strategy for preserving their full therapeutic potential is encapsulation of MSCs in a specialized protective microenvironment, for example hydrogels. However, commonly used injectable hydrogels for cell delivery fail to provide the bio-instructive cues needed to sustain and stimulate cellular therapeutic functions. Here we introduce a customizable collagen I-hyaluronic acid (COL-HA)-based hydrogel platform for the encapsulation of MSCs. Cells encapsulated within COL-HA showed a significant expansion of their secretory profile compared to MSCs cultured in standard (2D) cell culture dishes or encapsulated in other hydrogels. Functionalization of the COL-HA backbone with thiol-modified glycoproteins such as laminin led to further changes in the paracrine profile of MSCs. In depth profiling of more than 250 proteins revealed an expanded secretion profile of proangiogenic, neuroprotective and immunomodulatory paracrine factors in COL-HA-encapsulated MSCs with a predicted augmented pro-angiogenic potential. This was confirmed by increased capillary network formation of endothelial cells stimulated by conditioned media from COL-HA-encapsulated MSCs. Our findings suggest that encapsulation of therapeutic cells in a protective COL-HA hydrogel layer provides the necessary bio-instructive cues to maintain and direct their therapeutic potential. Our customizable hydrogel combines bioactivity and clinically applicable properties such as injectability, on-demand polymerization and tissue-specific elasticity, all features that will support and improve the ability to successfully deliver functional MSCs into patients. %0 conference lecture %@ %A Gossen, M., Lau, S., Moradian, H., Behl, M., Lendlein, A. %D 2021 %J 2nd Joint Meeting of The European Society for Clinical Hemorheology and Microcirculation; The International Society for Clinical Hemorheology; and The International Society of Biorheology (ESCHM-ISCHM-ISB) %T Nanoparticle-mediated delivery of nucleic acids in primary human endothelial cells %U %X %0 journal article %@ 1479-5876 %A Liu, Z., Klose, K., Neuber, S., Jiang, M., Gossen, M., Stamm, C. %D 2020 %J Journal of Translational Medicine %P 437 %R doi:10.1186/s12967-020-02605-4 %T Comparative analysis of adeno-associated virus serotypes for gene transfer in organotypic heart slices %U https://doi.org/10.1186/s12967-020-02605-4 %X We have established a readily available mouse organotypic heart slice culture model and provided evidence that AAV6 may be a promising gene therapy vector for heart failure and other cardiac diseases. %0 journal article %@ 0946-2716 %A Moradian, H., Lendlein, A., Gossen, M. %D 2020 %J Journal of Molecular Medicine %P 1767-1779 %R doi:10.1007/s00109-020-01956-1 %T Strategies for simultaneous and successive delivery of RNA %U https://doi.org/10.1007/s00109-020-01956-1 %X Advanced non-viral gene delivery experiments often require co-delivery of multiple nucleic acids. Therefore, the availability of reliable and robust co-transfection methods and defined selection criteria for their use in, e.g., expression of multimeric proteins or mixed RNA/DNA delivery is of utmost importance. Here, we investigated different co- and successive transfection approaches, with particular focus on in vitro transcribed messenger RNA (IVT-mRNA). Expression levels and patterns of two fluorescent protein reporters were determined, using different IVT-mRNA doses, carriers, and cell types. Quantitative parameters determining the efficiency of co-delivery were analyzed for IVT-mRNAs premixed before nanocarrier formation (integrated co-transfection) and when simultaneously transfecting cells with separately formed nanocarriers (parallel co-transfection), which resulted in a much higher level of expression heterogeneity for the two reporters. Successive delivery of mRNA revealed a lower transfection efficiency in the second transfection round. All these differences proved to be more pronounced for low mRNA doses. Concurrent delivery of siRNA with mRNA also indicated the highest co-transfection efficiency for integrated method. However, the maximum efficacy was shown for successive delivery, due to the kinetically different peak output for the two discretely operating entities. Our findings provide guidance for selection of the co-delivery method best suited to accommodate experimental requirements, highlighting in particular the nucleic acid dose-response dependence on co-delivery on the single-cell level. %0 journal article %@ 2045-2322 %A Listek, M., Hönow, A., Gossen, M., Hanack, K. %D 2020 %J Scientific Reports %P 1664 %R doi:10.1038/s41598-020-58571-w %T A novel selection strategy for antibody producing hybridoma cells based on a new transgenic fusion cell line %U https://doi.org/10.1038/s41598-020-58571-w %X The use of monoclonal antibodies is ubiquitous in science and biomedicine but the generation and validation process of antibodies is nevertheless complicated and time-consuming. To address these issues we developed a novel selective technology based on an artificial cell surface construct by which secreted antibodies were connected to the corresponding hybridoma cell when they possess the desired antigen-specificity. Further the system enables the selection of desired isotypes and the screening for potential cross-reactivities in the same context. For the design of the construct we combined the transmembrane domain of the EGF-receptor with a hemagglutinin epitope and a biotin acceptor peptide and performed a transposon-mediated transfection of myeloma cell lines. The stably transfected myeloma cell line was used for the generation of hybridoma cells and an antigen- and isotype-specific screening method was established. The system has been validated for globular protein antigens as well as for haptens and enables a fast and early stage selection and validation of monoclonal antibodies in one step. %0 journal article %@ 2045-2322 %A Moradian, H., Roch, T., Lendlein, A., Gossen, M. %D 2020 %J Scientific Reports %P 4181 %R doi:10.1038/s41598-020-60506-4 %T mRNA Transfection-Induced Activation of Primary Human Monocytes and Macrophages: Dependence on Carrier System and Nucleotide Modification %U https://doi.org/10.1038/s41598-020-60506-4 %X Monocytes and macrophages are key players in maintaining immune homeostasis. Identifying strategies to manipulate their functions via gene delivery is thus of great interest for immunological research and biomedical applications. We set out to establish conditions for mRNA transfection in hard-to-transfect primary human monocytes and monocyte-derived macrophages due to the great potential of gene expression from in vitro transcribed mRNA for modulating cell phenotypes. mRNA doses, nucleotide modifications, and different carriers were systematically explored in order to optimize high mRNA transfer rates while minimizing cell stress and immune activation. We selected three commercially available mRNA transfection reagents including liposome and polymer-based formulations, covering different application spectra. Our results demonstrate that liposomal reagents can particularly combine high gene transfer rates with only moderate immune cell activation. For the latter, use of specific nucleotide modifications proved essential. In addition to improving efficacy of gene transfer, our findings address discrete aspects of innate immune activation using cytokine and surface marker expression, as well as cell viability as key readouts to judge overall transfection efficiency. The impact of this study goes beyond optimizing transfection conditions for immune cells, by providing a framework for assessing new gene carrier systems for monocyte and macrophage, tailored to specific applications. %0 journal article %@ 0892-6638 %A Klose, K., Gossen, M., Stamm, C. %D 2019 %J The FASEB Journal %N 1 %P 49-70 %R doi:10.1096/fj.201800712R %T Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies %U https://doi.org/10.1096/fj.201800712R 1 %X To date, no viable therapeutic options exist for the effective and sustained reversal of cardiac failure, other than heart transplantation and mechanical circulatory assist devices. Therefore, divergent strategies aiming at the de novo formation of contractile tissue, as a prerequisite for the restoration of cardiac pump function, are currently being pursued. Clinical trials involving the transplantation of somatic progenitor cells failed. The search for alternative cell-based strategies to combat the consequences of ischemic injury has sparked widespread interest in the genetic and pharmacologic reprogramming of fibroblasts into cardiomyocytes, harnessing the abundant in vivo pool of cardiac fibroblasts. Here, we provide a comprehensive overview of in vitro and in vivo cardiac reprogramming studies identified in an extensive literature search. We systematically review and evaluate feasibility, efficiency, and reproducibility of the different technologies currently being explored. Finally, we discuss potential safety issues deduced from preclinical studies and identify obstacles that must be overcome before clinical translation.—Klose, K., Gossen, M., Stamm, C. Turning fibroblasts into cardiomyocytes: technological review of cardiac transdifferentiation strategies. %0 journal article %@ 1873-5061 %A Henning, A.F., Roessler, U., Boiti, F., Hagen, M.von der, Gossen, M., Kornak, U., Stachelscheid, H. %D 2019 %J Stem Cell Research %P 101367 %R doi:10.1016/j.scr.2018.101367 %T Generation of a human induced pluripotent stem cell line (BIHi002-A) from a patient with CLCN7-related infantile malignant autosomal recessive osteopetrosis %U https://doi.org/10.1016/j.scr.2018.101367 %X Autosomal recessive osteopetrosis (ARO) is a genetic bone disease that can be caused by mutations in the CLCN7 gene preventing osteoclast-mediated bone resorption. We generated a human induced pluripotent stem cell (hiPSC) line, BIHi002-A, from peripheral blood mononuclear cells of an ARO patient carrying the CLCN7 mutations c.875G>A and c.1208G>A using Sendai viral vectors. The pluripotent identity of the BIHi002-A line was confirmed by their expression of typical markers for undifferentiated hiPSCs, their capacity to differentiate into cells of the three germ layers and by PluriTest analysis. The BIHi002-A line provides a tool for disease modelling and therapy development. %0 conference poster %@ %A Moradian, H., Lendlein, A., Gossen, M. %D 2019 %J Polydays 2019 - Polymer Science and Engineering in View of Digitalization %T Co-transfection Strategies for In Vitro Transcribed mRNA %U %X %0 conference poster %@ %A Moradian, H., Lendlein, A., Gossen, M. %D 2019 %J Advanced Functional Polymers for Medicine (AFPM) 2019 %T Co-transfection Strategies for In Vitro Transcribed mRNA %U %X %0 journal article %@ 1946-6242 %A Baron, U., Werner, J., Schildknecht, K., Schulze, J.J., Mulu, A., Liebert, U.-G., Sack, U., Speckmann, C., Gossen, M., Wong, R.J., Stevenson, D.K., Babel, N., Schuermann, D., Baldinger, T., Bacchetta, R., Gruetzkau, A., Borte, S., Olek, S. %D 2018 %J Science Translational Medicine %N 452 %P 3508 %R doi:10.1126/scitranslmed.aan3508 %T Epigenetic immune cell counting in human blood samples for immunodiagnostics %U https://doi.org/10.1126/scitranslmed.aan3508 452 %X Immune cell profiles provide valuable diagnostic information for hematologic and immunologic diseases. Although it is the most widely applied analytical approach, flow cytometry is limited to liquid blood. Moreover, either analysis must be performed with fresh samples or cell integrity needs to be guaranteed during storage and transport. We developed epigenetic real-time quantitative polymerase chain reaction (qPCR) assays for analysis of human leukocyte subpopulations. After method establishment, whole blood from 25 healthy donors and 97 HIV+ patients as well as dried spots from 250 healthy newborns and 24 newborns with primary immunodeficiencies were analyzed. Concordance between flow cytometric and epigenetic data for neutrophils and B, natural killer, CD3+ T, CD8+ T, CD4+ T, and FOXP3+ regulatory T cells was evaluated, demonstrating substantial equivalence between epigenetic qPCR analysis and flow cytometry. Epigenetic qPCR achieves both relative and absolute quantifications. Applied to dried blood spots, epigenetic immune cell quantification was shown to identify newborns suffering from various primary immunodeficiencies. Using epigenetic qPCR not only provides a precise means for immune cell counting in fresh-frozen blood but also extends applicability to dried blood spots. This method could expand the ability for screening immune defects and facilitates diagnostics of unobservantly collected samples, for example, in underdeveloped areas, where logistics are major barriers to screening. %0 journal article %@ 2050-750X %A Li, Z., Wang, W., Xu, X., Kratz, K., Zou, J., Lysyakova, L., Heuchel, M., Kurtz, A., Gossen, M., Ma, N., Lendlein, A. %D 2017 %J Journal of Materials Chemistry B %N 35 %P 7415-7425 %R doi:10.1039/C7TB01232B %T Integrin β1 activation by micro-scale curvature promotes pro-angiogenic secretion of human mesenchymal stem cells %U https://doi.org/10.1039/C7TB01232B 35 %X Fine tuning of the substrate properties to modulate the function of mesenchymal stem cells (MSCs) has emerged as an attractive strategy to optimize their therapeutic potential. In the context of the mechanotransduction process, the conformational change of integrin (integrin activation) plays a critical role in perceiving and transmitting various signals. In this study, polymeric cell culture inserts with defined bottom roughness were fabricated as a model system for cell cultivation. We showed that the conformational change of integrin and its downstream signaling cascade of human adipose-derived mesenchymal stem cells (hADSCs) could be modulated by the curvature of the cell–material interface. The curvature of the substrate surface with a roughness in the size range of a single cell could strongly increase the high-affinity β1 integrin level of hADSCs without alteration of the total β1 integrin level. Further, the integrin downstream FAK/ERK and Rho/ROCK pathways were activated and resulted in upregulated VEGF secretion of hADSCs. A conditioned medium on such a surface exhibited a strong pro-angiogenic effect, with an increased formation of the tubular structure, a higher migration velocity of endothelial cells and an enhanced blood vessel density in an ex vivo hen's egg test-chorioallantoic membrane (HET-CAM). These results highlighted the clinical potential to manipulate the topographic features of the cell culture substrate, whereby to regulate integrin affinity states and further control MSC functions. %0 conference object %@ 1043-0342 %A Roessler, U., Hennig, A.F., Stachelscheid, H., Gossen, M., Izvak, Z., Kornak, U. %D 2017 %J Human Gene Therapy %N 12 %P A 50-A51 %R doi:10.1089/hum.2017.29055.abstracts %T ESGCT XXV Anniversary Congress in Collaboration with the German Society for Gene Therapy - Osteoclasts differentiated from iPS cells as a test system for gene therapeutic approaches for CLCN7-related autosomal recessive osteopetrosis %U https://doi.org/10.1089/hum.2017.29055.abstracts 12 %X No abstract %0 conference poster %@ %A Roessler, U., Hennig, A.F., Stachelscheid, H., Gossen, M., Izvak, Z., Kornak, U. %D 2017 %J ESGCT XXV Anniversary Congress in Collaboration with the German Society for Gene Therapy %R doi:10.1089/hum.2017.29055.abstracts %T Osteoclasts differentiated from iPS cells as a test system for gene therapeutic approaches for CLCN7-related autosomal recessive osteopetrosis %U https://doi.org/10.1089/hum.2017.29055.abstracts %X %0 conference object %@ 1043-0342 %A Hennig, A.F., Roessler, U., Corrado, A., Werner, D.L., Stachelscheid, H., Gossen, M., Kornak, U. %D 2017 %J Human Gene Therapy %N 12 %P A 60 %R doi:10.1089/hum.2017.29055.abstracts %T ESGCT XXV Anniversary Congress in Collaboration with the German Society for Gene Therapy - Optimisation of CRISPR/Cas9-mediated knock-in of large inserts into the AAVS1 safe harbor locus %U https://doi.org/10.1089/hum.2017.29055.abstracts 12 %X No abstract %0 conference poster %@ %A Hennig, A.F., Roessler, U., Corrado, A., Werner, D.L., Stachelscheid, H., Gossen, M., Kornak, U. %D 2017 %J ESGCT XXV Anniversary Congress in Collaboration with the German Society for Gene Therapy %R doi:10.1089/hum.2017.29055.abstracts %T Optimisation of CRISPR/Cas9-mediated knock-in of large inserts into the AAVS1 safe harbor locus %U https://doi.org/10.1089/hum.2017.29055.abstracts %X %0 journal article %@ 1525-7797 %A Wang, W., Naolou, T., Ma, N., Deng, Z., Xu, X., Mansfeld, U., Wischke, C., Gossen, M., Neffe, A.T., Lendlein, A. %D 2017 %J Biomacromolecules %N 11 %P 3819-3833 %R doi:10.1021/acs.biomac.7b01034 %T Polydepsipeptide Block-Stabilized Polyplexes for Efficient Transfection of Primary Human Cells %U https://doi.org/10.1021/acs.biomac.7b01034 11 %X The rational design of a polyplex gene carrier aims to balance maximal effectiveness of nucleic acid transfection into cells with minimal adverse effects. Depsipeptide blocks with an Mn ∼ 5 kDa exhibiting strong physical interactions were conjugated with PEI moieties (2.5 or 10 kDa) to di- and triblock copolymers. Upon nanoparticle formation and complexation with DNA, the resulting polyplexes (sizes typically 60–150 nm) showed remarkable stability compared to PEI-only or lipoplex and facilitated efficient gene delivery. Intracellular trafficking was visualized by observing fluorescence-labeled pDNA and highlighted the effective cytoplasmic uptake of polyplexes and release of DNA to the perinuclear space. Specifically, a triblock copolymer with a middle depsipeptide block and two 10 kDa PEI swallowtail structures mediated the highest levels of transgenic VEGF secretion in mesenchymal stem cells with low cytotoxicity. These nanocarriers form the basis for a delivery platform technology, especially for gene transfer to primary human cells. %0 journal article %@ 2045-2322 %A Phan, Q.V., Contzen, J., Seemann, P., Gossen, M. %D 2017 %J Scientific Reports %P 17771 %R doi:10.1038/s41598-017-17651-0 %T Site-specific chromosomal gene insertion: Flp recombinase versus Cas9 nuclease %U https://doi.org/10.1038/s41598-017-17651-0 %X Site-specific recombination systems like those based on the Flp recombinase proved themselves as efficient tools for cell line engineering. The recent emergence of designer nucleases, especially RNA guided endonucleases like Cas9, has considerably broadened the available toolbox for applications like targeted transgene insertions. Here we established a recombinase-mediated cassette exchange (RMCE) protocol for the fast and effective, drug-free isolation of recombinant cells. Distinct fluorescent protein patterns identified the recombination status of individual cells. In derivatives of a CHO master cell line the expression of the introduced transgene of interest could be dramatically increased almost 20-fold by subsequent deletion of the fluorescent protein gene that provided the initial isolation principle. The same master cell line was employed in a comparative analysis using CRISPR/Cas9 for transgene integration in identical loci. Even though the overall targeting efficacy was comparable, multi-loci targeting was considerably more effective for Cas9-mediated transgene insertion when compared to RMCE. While Cas9 is inherently more flexible, our results also alert to the risk of aberrant recombination events around the cut site. Together, this study points at the individual strengths in performance of both systems and provides guidance for their appropriate use. %0 journal article %@ 1386-0291 %A Li, Z., Xu, X., Wang, W., Kratz, K., Sun, X., Zou, J., Deng, Z., Jung, F., Gossen, M., Ma, N., Lendlein, A. %D 2017 %J Clinical Hemorheology and Microcirculation %N 3-4 %P 267-278 %R doi:10.3233/CH-179208 %T Modulation of the mesenchymal stem cell migration capacity via preconditioning with topographic microstructure %U https://doi.org/10.3233/CH-179208 3-4 %X Controlling mesenchymal stem cells (MSCs) behavior is necessary to fully exploit their therapeutic potential. Various approaches are employed to effectively influence the migration capacity of MSCs. Here, topographic microstructures with different microscale roughness were created on polystyrene (PS) culture vessel surfaces as a feasible physical preconditioning strategy to modulate MSC migration. By analyzing trajectories of cells migrating after reseeding, we demonstrated that the mobilization velocity of human adipose derived mesenchymal stem cells (hADSCs) could be promoted by and persisted after brief preconditioning with the appropriate microtopography. Moreover, the elevated activation levels of focal adhesion kinase (FAK) and mitogen-activated protein kinase (MAPK) in hADSCs were also observed during and after the preconditioning process. These findings underline the potential enhancement of in vivo therapeutic efficacy in regenerative medicine via transplantation of topographic microstructure preconditioned stem cells. %0 conference lecture %@ %A Li, Z., Xu, X., Wang, W., Kratz, K., Sun, X., Zou, J., Deng, Z., Jung, F., Gossen, M., Ma, N., Lendlein, A. %D 2017 %J 36th Conference of the German Society for Clinical Microcirculation and Hemorheology %T Modulation of the mesenchymal stem cell migration capacity via preconditioning with topographic microstructure %U %X %0 journal article %@ 0898-6568 %A Hildebrand, L., Stange, K., Deichsel, A., Gossen, M., Seemann, P. %D 2017 %J Cellular Signalling %P 23-30 %R doi:10.1016/j.cellsig.2016.10.001 %T The Fibrodysplasia Ossificans Progressiva (FOP) mutation p.R206H in ACVR1 confers an altered ligand response %U https://doi.org/10.1016/j.cellsig.2016.10.001 %X In this study we compared the signalling responses of ACVR1WT and ACVR1R206H to different ligands. ACVR1WT, but not ACVR1R206H inhibited BMP signalling of BMP2 or BMP4 in a ligand binding domain independent manner. Likewise, the basal BMP signalling activity of the receptor BMPR1A or BMPR1B was inhibited by ACVR1WT, but enhanced by ACVR1R206H. In comparison, BMP6 or BMP7 activated ACVR1WT and caused a hyper-activation of ACVR1R206H. These effects were dependent on an intact ligand binding domain. Finally, the neofunction of Activin A in FOP was tested and found to depend on the ligand binding domain for activating ACVR1R206H. We conclude that the FOP mutation ACVR1R206H is more sensitive to a number of natural ligands. The mutant receptor apparently lost some essential inhibitory interactions with its ligands and co-receptors, thereby conferring an enhanced ligand-dependent signalling and stimulating ectopic bone formation as observed in the patients. %0 conference lecture %@ %A Li, Z., Xu, X., Wang, W., Kratz, K., Sun, X., Zou, J., Deng, Z., Jung, F., Gossen, M., Ma, N., Lendlein, A. %D 2017 %J 36. Jahrestagung der Deutschen Gesellschaft für Klinische Mikrozirkulation und Hämorheologie (DGKMH) %T Preconditioning with topographic microstructure of materials promotes the migration capacity of adipose derived mesenchymal stem cells %U %X transplantation of topographic microstructure preconditioned stem cells. %0 journal article %@ 1386-0291 %A Li, Z., Wang, W., Kratz, K., Kuechler, J., Xu, X., Zou, J., Deng, Z., Sun, X., Gossen, M., Ma, N., Lendlein, A. %D 2016 %J Clinical Hemorheology and Microcirculation %N 3 %P 355-366 %R doi:10.3233/CH-168121 %T Influence of surface roughness on neural differentiation of human induced pluripotent stem cells %U https://doi.org/10.3233/CH-168121 3 %X Induced pluripotent stem cells (iPSCs) own the capacity to develop into all cell types of the adult body, presenting high potential in regenerative medicine. Regulating and controlling the differentiation of iPSCs using the surface topographic cues of biomaterials is a promising and safe approach to enhance their therapeutic efficacy. In this study, we tested the effects of surface roughness on differentiation of human iPSCs into neural progenitor cells and dopaminergic neuron cells using polystyrene with different roughness (R0: flat surface; R1: rough surface, Rq ∼ 6 μm; R2: rough surface, Rq ∼ 38 μm). Neural differentiation of human iPSCs could be influenced by surface roughness. Up-regulated neuronal markers were found in cells on rough surface, as examined by real-time PCR and immunostaining. Particularly, the R1 surface significantly improved the neuronal marker expression, as compared to R0 and R2 surface. This study demonstrates the significance of surface roughness, depending on the roughness level, in promoting differentiation of human iPSCs towards the neuronal lineage. Our study suggests the potential applications of surface roughness in iPSCs based treatment of neural disorder diseases, and highlights the importance of design and development of biomaterials with effective surface structures to regulate stem cells. %0 conference lecture %@ %A Li, Z., Wang, W., Kratz, K., Kuechler, J., Xu, X., Zou, J., Deng, Z., Sun, X., Gossen, M., Ma, N., Lendlein, A. %D 2016 %J 35th Conference of the German Society for Clinical Microcirculation and Hemorheology %T Influence of surface roughness on neural differentiation of human induced pluripotent stem cells %U %X %0 conference poster %@ %A Li, Z., Wang, W., Kratz, K., Küchler, J., Xu, X., Zou, J., Deng, Z., Sun, X., Gossen, M., Ma, N., Lendlein, A. %D 2016 %J 35. Jahrestagung der Deutschen Gesellschaft für klinische Mikrozirkulation und Hämorheologie (DGKMH) %T Neural differentiation of human induced pluripotent stem cells on the structured surface %U %X lnduced pluripotent stem cells (iPSCs) own the capacity to develop into all cell types of the adult body, presenting high potential in regenerative medicine. Regulating and controlling the differentiation of iPSCs via the surface topographic cues of biomaterials is a promising and safe approach to enhance their therapeutic efficacy. In this study, we tested the effects of microscale roughness on differentiation of human iPSCs into neural progenitor cells and dopaminergic neuron cells using polystyrene surface with different roughness levels. Neural differentiation of human iPSCs could be strongly influenced by microscale roughness. Upregulated neuronal markers were detected in iPSCs cells seeded on rougher surface, as examined by real-time PCR and immunost aini ng. Particularly, the intermedium rough surface significantly improved the neuronal marker expression as compared other surfaces. This study demonstrates that a surface with an appropriated microscale roughness level can promote the differentiation of human iPSCs towards the neuronal lineage. Our study suggests the potential applications of controllable iPSCs cell differentiation via surface structure, and highlights the strategy of design and development of structured surface on regulating stem cell development. %0 journal article %@ 1873-5061 %A Hildebrand, L., Rossbach, B., Kuehnen, P., Gossen, M., Kurtz, A., Reinke, P., Seemann, P., Stachelscheid, H. %D 2016 %J Stem Cell Research %N 1 %P 54-58 %R doi:10.1016/j.scr.2015.11.017 %T Generation of integration free induced pluripotent stem cells from fibrodysplasia ossificans progressiva (FOP) patients from urine samples %U https://doi.org/10.1016/j.scr.2015.11.017 1 %X Here, we describe the derivation and characterization of two hiPSC lines from two FOP patients, both carrying the mutation R206H. Cells were isolated from urine and reprogrammed using integration free Sendai virus vectors under defined conditions. %0 journal article %@ 0168-3659 %A Wang, W., Balk, M., Deng, Z., Wischke, C., Gossen, M., Behl, M., Ma, N., Lendlein, A. %D 2016 %J Journal of Controlled Release %P 71-79 %R doi:10.1016/j.jconrel.2016.08.004 %T Engineering biodegradable micelles of polyethylenimine-based amphiphilic block copolymers for efficient DNA and siRNA delivery %U https://doi.org/10.1016/j.jconrel.2016.08.004 %X Polycationic micelles have shown advantageous properties as nucleic acid delivery vectors both in vitro and in vivo. In contrast to polycationic micelles reported so far, we designed particles integrating a sufficient nucleic acid condensation capability by polycationic polyethylenimine (PEI) segments as well as only a mild cytotoxic behavior. The micelles composed of a hydrophobic oligoester core with glycolide units resulting in fast degradation after cellular internalization in combination with PEG moieties acting as shielding agents. By grafting branched 25 kDa polyethylenimine (PEI25) and poly(ethylene glycol) (PEG) on poly[(ε-caprolactone)-co-glycolide] (CG), amphiphilic PEI-CG-PEI and PEG-CG block copolymers were used to form a series of micelles via self-assembly of PEI-CG-PEI or co-assembly of both copolymers for DNA and siRNA delivery. This modular system enabled a systematic investigation of different parameters and their synergetic effects as different functions were introduced. The polyplex formation and serum stability, cytotoxicity, and transfection activity could be tailored by changing the CG chain length in PEI-based copolymer, incorporating PEG-CG, and varying the N/P ratio. All micelle-based polyplex compositions showed high DNA transfection activity according to reporter gene-expression and an exceptionally high knockdown in siRNA delivery experiments. Remarkably, the GFP expression of > 99% cells was successfully knocked down by micelle-mediated siRNA interference, resulting in a decrease of two orders of magnitude in fluorescence intensity. Incorporation of PEG-CG in the micelles reduced the PEI-related cytotoxicity, and markedly enhanced the serum stability of both DNA and siRNA polyplexes. Compared with homo-PEI25, these micelles showed several advantages including the lower toxicity, higher siRNA transfection efficiency and higher polyplex stability in the presence of serum. This study therefore provides an effective approach to tune the structure, property and function of polycationic micelles for efficient DNA and siRNA delivery, which could contribute to the design and development of novel non-viral transfection vectors with superb functionality. %0 journal article %@ 1465-3249 %A Schwerk, A., Altschueler, J., Roch, M., Gossen, M., Winter, C., Berg, J., Kurtz, A., Steiner, B. %D 2015 %J Cytotherapy %N 2 %P 199-214 %R doi:10.1016/j.jcyt.2014.09.005 %T Human adipose-derived mesenchymal stromal cells increase endogenous neurogenesis in the rat subventricular zone acutely after 6-hydroxydopamine lesioning %U https://doi.org/10.1016/j.jcyt.2014.09.005 2 %X The acute neurogenic effects and neurotrophic factor expression of MSC could help to restore the SVZ-OB axis in PD. %0 journal article %@ 1420-682X %A Hildebrand, L., Seemann, P., Kurtz, A., Hecht, J., Contzen, J., Gossen, M., Stachelscheid, H. %D 2015 %J Cellular and Molecular Life Sciences %N 23 %P 4671-4680 %R doi:10.1007/s00018-015-1957-4 %T Selective cell targeting and lineage tracing of human induced pluripotent stem cells using recombinant avian retroviruses %U https://doi.org/10.1007/s00018-015-1957-4 23 %X Human induced pluripotent stem cells (hiPSC) differentiate into multiple cell types. Selective cell targeting is often needed for analyzing gene function by overexpressing proteins in a distinct population of hiPSC-derived cell types and for monitoring cell fate in response to stimuli. However, to date, this has not been possible, as commonly used viruses enter the hiPSC via ubiquitously expressed receptors. Here, we report for the first time the application of a heterologous avian receptor, the tumor virus receptor A (TVA), to selectively transduce TVA+ cells in a mixed cell population. Expression of the TVA surface receptor via genetic engineering renders cells susceptible for infection by avian leucosis virus (ALV). We generated hiPSC lines with this stably integrated, ectopic TVA receptor gene that expressed the receptor while retaining pluripotency. The undifferentiated hiPSCTVA+ as well as their differentiating progeny could be infected by recombinant ALV (so-called RCAS virus) with high efficiency. Due to incomplete receptor blocking, even sequential infection of differentiating or undifferentiated TVA+ cells was possible. In conclusion, the TVA/RCAS system provides an efficient and gentle gene transfer system for hiPSC and extends our possibilities for selective cell targeting and lineage tracing studies. %0 journal article %@ 1746-0751 %A Schwerk, A., Altschueler, J., Roch, M., Gossen, M., Winter, C., Berg, J., Kurtz, A., Akyuez, L., Steiner, B. %D 2015 %J Regenerative Medicine %N 4 %P 431-446 %R doi:10.2217/RME.15.17 %T Adipose-derived human mesenchymal stem cells induce long-term neurogenic and anti-inflammatory effects and improve cognitive but not motor performance in a rat model of Parkinson's disease %U https://doi.org/10.2217/RME.15.17 4 %X Background: Mesenchymal stem cells (MSC) are easily harvested, and possess anti-inflammatory and trophic properties. Furthermore, MSC promote neuroprotection and neurogenesis, which could greatly benefit neurodegenerative disorders, such as Parkinson's disease. Methods: MSC were transplanted one week after 6-hydroxydopamine lesioning and effects were evaluated after 6 months. Results: MSC localized around the substantia nigra and the arachnoid mater, expressing pericyte and endothelial markers. MSC protected dopamine levels and upregulated peripheral anti-inflammatory cytokines. Furthermore, adipose-derived MSC increased neurogenesis in hippocampal and subventricular regions, and boosted memory functioning. Conclusion: Considering that hyposmia and loss of memory function are two major nonmotor symptoms in Parkinson's disease, transplants with modulatory effects on the hippocampus and subventricular zone could provide a disease-modifying therapy. %0 journal article %@ 0305-1048 %A Werner, J., Gossen, M. %D 2014 %J Nucleic Acids Research %N 21 %P 13061-13073 %R doi:10.1093/nar/gku1124 %T Modes of TAL effector-mediated repression %U https://doi.org/10.1093/nar/gku1124 21 %X Engineered transcription activator-like effectors, or TALEs, have emerged as a new class of designer DNA-binding proteins. Their DNA recognition sites can be specified with great flexibility. When fused to appropriate transcriptional regulatory domains, they can serve as designer transcription factors, modulating the activity of targeted promoters. We created tet operator (tetO)-specific TALEs (tetTALEs), with an identical DNA-binding site as the Tet repressor (TetR) and the TetR-based transcription factors that are extensively used in eukaryotic transcriptional control systems. Different constellations of tetTALEs and tetO modified chromosomal transcription units were analyzed for their efficacy in mammalian cells. We find that tetTALE-silencers can entirely abrogate expression from the strong human EF1α promoter when binding upstream of the transcriptional control sequence. Remarkably, the DNA-binding domain of tetTALE alone can effectively counteract trans-activation mediated by the potent tettrans-activator and also directly interfere with RNA polymerase II transcription initiation from the strong CMV promoter. Our results demonstrate that TALEs can act as highly versatile tools in genetic engineering, serving as trans-activators, trans-silencers and also competitive repressors. %0 journal article %@ 1022-1360 %A Rijckaert, B., Neffe, A.T., Roch, T., Gebauer, T., Pierce, B.F., Goers, J., Smink, J.J., Gossen, M., Lendlein, A., Leutz, A. %D 2014 %J Macromolecular Symposia %N 1 %P 91-99 %R doi:10.1002/masy.201400147 %T A High Content Screening Assay for Evaluation of Biomaterial-Mediated Cell Fusion Processes %U https://doi.org/10.1002/masy.201400147 1 %X Biomaterials are of increasing importance in regenerative medicine and entail delivery systems in somatic cell therapies, matrices for tissue engineering or tissue regeneration. The evaluation of biomaterial induced biological effects remains a key issue in clinical application. Cell-based assays for potential cytotoxic and immunological responses have been developed but are often inadequate to address cell-type specific responses to biomaterials. To quantitatively monitor attachment, survival, proliferation and fusion-controlled differentiation of osteoclasts (bone resorbing cells), a High Content Screening (HCS) assay has been developed based on osteoclast differentiation of the murine monocytic cell line RAW 264.7. This assay was applied to investigate the influence of degradation products of polymers from gelatin and lysine diisocyanate, which display tailorable mechanical properties and have potential as biomaterials. The data show that the degradation products inhibit formation of multinuclear osteoclasts and suggest a potential support of bone regeneration by suppression of bone resorption. %0 conference lecture %@ %A Li, Z., Wang, W., Kratz, K., Xu, X., Roch, M., Kurtz, A., Gossen, M., jung, F., Ma, N., Lendlein, A. %D 2014 %J 6th Forum on New Materials: Symposium Smart Polymers for Biomedical Applications, CIMTEC 2014 %T Scaffold Roughness Regulates the Endothelial Differentiation of Human Adipose Derived Mesenchymal Stem Cells %U %X %0 conference lecture %@ %A Rijckaert, B., Neffe, A.T., Roch, T., Gebauer, T., Pierce, B.F., Goers, J., Smink, J.J., Gossen, M., Lendlein, A., Leutz, A. %D 2013 %J 12th International Polymers for Advanced Technologies Conference, PAT 2013 %T A High Content Screening Assay for Evaluation of Biomaterial-Mediated Cell Fusion Processes %U %X %0 conference lecture %@ %A Gossen, M. %D 2013 %J Helmholtz Graduate School for Macromolecular Bioscience, Summer School 2013 %T Genetic Engineering for the Analysis of Cell Interactions with their Environment (including Biomaterial) %U %X %0 conference poster %@ %A Rijckaert, B., Pierce, B.F., Neffe, A.T., Goers, J., Roch, T., Gebauer, T., Smink, J.J., Lendlein, A., Gossen, M., Leutz, A. %D 2013 %J 12th International Conference on Polymers for Advanced Technologies, PAT 2013 %T Evaluation of Gelatin Hydrogels in their Interaction with Monocytic Osteoclast Precursor Cells %U %X %0 conference lecture %@ %A Li, Z., Ma, N., Kratz, K., Wang, W., Xu, X., Gossen, M., Roch, T., Kurtz, A., Lendlein, A. %D 2013 %J 32. Jahrestagung der Deutschen Gesellschaft fuer klinische Mikrozirkulation und Haemorheologie %T Effects of Roughness on Angiogenesis of Human Adipose Derived Stem Cells %U