%0 journal article %@ 2162-2531 %A Moradian, H.,Roch, T.,Anthofer, L.,Lendlein, A.,Gossen, M. %D 2022 %J Molecular Therapy Nucleic Acids %N %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 %@ 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 %@ 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 %N %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 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 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 %@ 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 %@ 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 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 journal article %@ 1479-5876 %A Liu, Z.,Klose, K.,Neuber, S.,Jiang, M.,Gossen, M.,Stamm, C. %D 2020 %J Journal of Translational Medicine %N %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 Background,Vectors derived from adeno-associated viruses (AAVs) are widely used for gene transfer both in vitro and in vivo and have gained increasing interest as shuttle systems to deliver therapeutic genes to the heart. However, there is little information on their tissue penetration and cytotoxicity, as well as the optimal AAV serotype for transferring genes to diseased hearts. Therefore, we aimed to establish an organotypic heart slice culture system for mouse left ventricular (LV) myocardium and use this platform to analyze gene transfer efficiency, cell tropism, and toxicity of different AAV serotypes.,Methods,LV tissue slices, 300 µm thick, were prepared from 15- to 17-day-old transgenic alpha-myosin heavy-chain-mCherry mice using a vibrating microtome. Tissue slice viability in air-liquid culture was evaluated by calcein-acetoxymethyl ester staining, mCherry fluorescence intensity, and the tetrazolium assay. Four recombinant AAV serotypes (1, 2, 6, 8) expressing green fluorescent protein (GFP) under the CAG promoter were added to the slice surface. Gene transfer efficiency was quantified as the number of GFP-positive cells per slice. AAV cell tropism was examined by comparing the number of GFP-positive cardiomyocytes (CMs) and fibroblasts within heart slices.,Results,Slices retained viability in in vitro culture for at least 5 days. After adding AAV particles, AAV6-infected slices showed the highest number of GFP-expressing cells, almost exclusively CMs. Slice incubation with AAV1, 2, and 8 resulted in fewer GFP-positive cells, with AAV2 having the lowest gene transfer efficiency. None of the AAV serotypes tested caused significant cytotoxicity when compared to non-infected control slices.,Conclusions,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 %N %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 %N %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 %N %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 %@ 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 %N %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 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 %@ 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 %@ 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 %N %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 %@ 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 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 journal article %@ 0898-6568 %A Hildebrand, L.,Stange, K.,Deichsel, A.,Gossen, M.,Seemann, P. %D 2017 %J Cellular Signalling %N %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 Patients with Fibrodysplasia Ossificans Progressiva (FOP) suffer from ectopic bone formation, which progresses during life and results in dramatic movement restrictions. Cause of the disease are point mutations in the Activin A receptor type 1 (ACVR1), with p.R206H being most common.,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 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 %N %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 %@ 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 Fibrodysplasia ossificans progressiva (FOP) is an extremely rare, autosomal dominant transmitted genetic disease. Patients experience progressive bone formation replacing tendons, ligaments, muscle and soft tissue. Cause of FOP are gain-of-function mutations in the Bone Morphogenetic Protein (BMP) receptor Activin A receptor type 1 (ACVR1) (Kaplan et al., 2008). The most common mutation is R206H, which leads to the substitution of codon 206 from arginine to histidine (Shore et al., 2006).,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 %@ 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 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 Background aims,In Parkinson's disease (PD), neurogenesis in the subventricular zone (SVZ)–olfactory bulb (OB) axis is affected as the result of the lack of dopaminergic innervations reaching the SVZ. This aberrant network has been related to the hyposmia of PD patients, which is an early diagnostic marker of the disease. Consequently, much interest arose in finding mechanisms to modulate the SVZ-OB axis. Direct modulation of this axis could be achieved by transplantation of mesenchymal stromal cells (MSC), as it has been shown in rat and mouse PD models. However, the neurogenic effect of MSC in PD was thus far only analyzed weeks after transplantation, and little is known about effects immediately after transplantation.,Methods,We assessed the acute neuroprotective and neurogenic effects of adipose-derived MSC transplanted into the rat substantia nigra in the 6-hydroxydopamine model of PD.,Results,Three days after transplantation, subventricular neurogenesis was significantly increased in MSC-transplanted versus non-transplanted animals. Most MSC were found in the region of the substantia nigra and the surrounding arachnoid mater, expressing S100β and brain-derived neurotrophic factor, whereas some MSC showed an endothelial phenotype and localized around blood vessels.,Conclusions,The acute neurogenic effects and neurotrophic factor expression of MSC could help to restore the SVZ-OB axis in PD. %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 %@ 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 %@ 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.