Dokument-ID Dokumenttyp Verfasser/Autoren Herausgeber Haupttitel Abstract Auflage Verlagsort Verlag Erscheinungsjahr Seitenzahl Schriftenreihe Titel Schriftenreihe Bandzahl ISBN Quelle der Hochschulschrift Konferenzname Quelle:Titel Quelle:Jahrgang Quelle:Heftnummer Quelle:Erste Seite Quelle:Letzte Seite URN DOI Abteilungen OPUS4-26033 Wissenschaftlicher Artikel Vitale, Maria Rosaria; Zöller, Johanna Eva Maria; Jansch, Charline; Janz, Anna; Edenhofer, Frank; Klopocki, Eva; van den Hove, Daniel; Vanmierlo, Tim; Rivero, Olga; Kasri, Nael Nadif; Ziegler, Georg Christoph; Lesch, Klaus-Peter Generation of induced pluripotent stem cell (iPSC) lines carrying a heterozygous (UKWMPi002-A-1) and null mutant knockout (UKWMPi002-A-2) of Cadherin 13 associated with neurodevelopmental disorders using CRISPR/Cas9 Fibroblasts isolated from a skin biopsy of a healthy 46-year-old female were infected with Sendai virus containing the Yamanaka factors to produce transgene-free human induced pluripotent stem cells (iPSCs). CRISPR/Cas9 was used to generate isogenic cell lines with a gene dose-dependent deficiency of CDH13, a risk gene associated with neurodevelopmental and psychiatric disorders. Thereby, a heterozygous CDH13 knockout (CDH13\(^{+/-}\)) and a CDH13 null mutant (CDH13\(^{-/-}\)) iPSC line was obtained. All three lines showed expression of pluripotency-associated markers, the ability to differentiate into cells of the three germ layers in vitro, and a normal female karyotype. 2021 Stem Cell Research 51 urn:nbn:de:bvb:20-opus-260331 10.1016/j.scr.2021.102169 Institut für Humangenetik OPUS4-19086 Wissenschaftlicher Artikel Scognamiglio, Roberta; Cabezas-Wallscheid, Nina; Thier, Marc Christian; Altamura, Sandro; Reyes, Alejandro; Prendergast, Áine M.; Baumgärtner, Daniel; Carnevalli, Larissa S.; Atzberger, Ann; Haas, Simon; von Paleske, Lisa; Boroviak, Thorsten; Wörsdörfer, Philipp; Essers, Marieke A. G.; Kloz, Ulrich; Eisenman, Robert N.; Edenhofer, Frank; Bertone, Paul; Huber, Wolfgang; van der Hoeven, Franciscus; Smith, Austin; Trumpp, Andreas Myc depletion induces a pluripotent dormant state mimicking diapause Mouse embryonic stem cells (ESCs) are maintained in a naive ground state of pluripotency in the presence of MEK and GSK3 inhibitors. Here, we show that ground-state ESCs express low Myc levels. Deletion of both c-myc and N-myc (dKO) or pharmacological inhibition of Myc activity strongly decreases transcription, splicing, and protein synthesis, leading to proliferation arrest. This process is reversible and occurs without affecting pluripotency, suggesting that Myc-depleted stem cells enter a state of dormancy similar to embryonic diapause. Indeed, c-Myc is depleted in diapaused blastocysts, and the differential expression signatures of dKO ESCs and diapaused epiblasts are remarkably similar. Following Myc inhibition, pre-implantation blastocysts enter biosynthetic dormancy but can progress through their normal developmental program after transfer into pseudo-pregnant recipients. Our study shows that Myc controls the biosynthetic machinery of stem cells without affecting their potency, thus regulating their entry and exit from the dormant state. 2016 668-680 Cell 164 4 urn:nbn:de:bvb:20-opus-190868 10.1016/j.cell.2015.12.033 Institut für Anatomie und Zellbiologie OPUS4-11974 Wissenschaftlicher Artikel Peitz, Michael; Münst, Bernhard; Thummer, Rajkumar P.; Helfen, Martina; Edenhofer, Frank Cell-permeant recombinant Nanog protein promotes pluripotency by inhibiting endodermal specification A comprehensive understanding of the functional network of transcription factors establishing and maintaining pluripotency is key for the development of biomedical applications of stem cells. Nanog plays an important role in early development and is essential to induce natural pluripotency in embryonic stem cells (ESCs). Inducible gain-of-function systems allowing a precise control over time and dosage of Nanog activity would be highly desirable to study its vital role in the establishment and maintenance of pluripotency at molecular level. Here we engineered a recombinant cell permeable version of Nanog by fusing it with the cell penetrating peptide TAT. Nanog-TAT can be readily expressed in and purified from E. coli and binds to a consensus Nanog DNA sequence. At cellular level it enhances proliferation and self-renewal of ESCs in the absence of leukemia inhibitory factor (LIF). Nanog-TAT together with LIF acts synergistically as judged by enhanced clonogenicity and activation of an Oct4-promoter-driven GFP reporter gene. Furthermore Nanog-TAT, in the absence of LIF, promotes pluripotency by inhibiting endodermal specification in a Stat3-independent manner. Our results demonstrate that Nanog protein transduction is an attractive tool allowing control over dose and time of addition to the cells for studying the molecular control of pluripotency without genetic manipulation. 2014 680–689 Stem Cell Research 12 3 urn:nbn:de:bvb:20-opus-119740 10.1016/j.scr.2014.02.006 Institut für Anatomie und Zellbiologie OPUS4-22007 Wissenschaftlicher Artikel Palladino, Viola Stella; Chiocchetti, Andreas G.; Frank, Lukas; Haslinger, Denise; McNeill, Rhiannon; Radtke, Franziska; Till, Andreas; Haupt, Simone; Brüstle, Oliver; Günther, Katharina; Edenhofer, Frank; Hoffmann, Per; Reif, Andreas; Kittel-Schneider, Sarah Energy metabolism disturbances in cell models of PARK2 CNV carriers with ADHD The main goal of the present study was the identification of cellular phenotypes in attention-deficit-/hyperactivity disorder (ADHD) patient-derived cellular models from carriers of rare copy number variants (CNVs) in the PARK2 locus that have been previously associated with ADHD. Human-derived fibroblasts (HDF) were cultured and human-induced pluripotent stem cells (hiPSC) were reprogrammed and differentiated into dopaminergic neuronal cells (mDANs). A series of assays in baseline condition and in different stress paradigms (nutrient deprivation, carbonyl cyanide m-chlorophenyl hydrazine (CCCP)) focusing on mitochondrial function and energy metabolism (ATP production, basal oxygen consumption rates, reactive oxygen species (ROS) abundance) were performed and changes in mitochondrial network morphology evaluated. We found changes in PARK2 CNV deletion and duplication carriers with ADHD in PARK2 gene and protein expression, ATP production and basal oxygen consumption rates compared to healthy and ADHD wildtype control cell lines, partly differing between HDF and mDANs and to some extent enhanced in stress paradigms. The generation of ROS was not influenced by the genotype. Our preliminary work suggests an energy impairment in HDF and mDAN cells of PARK2 CNV deletion and duplication carriers with ADHD. The energy impairment could be associated with the role of PARK2 dysregulation in mitochondrial dynamics. 2020 Journal of Clinical Medicine 9 12 urn:nbn:de:bvb:20-opus-220074 10.3390/jcm9124092 Klinik und Poliklinik für Kinder- und Jugendpsychiatrie, Psychosomatik und Psychotherapie OPUS4-16332 unpublished Nose, Naoko; Werner, Rudolf A.; Ueda, Yuichiro; Günther, Katharina; Lapa, Constantin; Javadi, Mehrbod S.; Fukushima, Kazuhito; Edenhofer, Frank; Higuchi, Takahiro Metabolic substrate shift in human induced pluripotent stem cells during cardiac differentiation: Functional assessment using in vitro radionuclide uptake assay Background: Recent developments in cellular reprogramming technology enable the production of virtually unlimited numbers of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Although hiPSC-CM share various characteristic hallmarks with endogenous cardiomyocytes, it remains a question as to what extent metabolic characteristics are equivalent to mature mammalian cardiomyocytes. Here we set out to functionally characterize the metabolic status of hiPSC-CM in vitro by employing a radionuclide tracer uptake assay. Material and Methods: Cardiac differentiation of hiPSC was induced using a combination of well-orchestrated extrinsic stimuli such as WNT activation (by CHIR99021) and BMP signalling followed by WNT inhibition and lactate based cardiomyocyte enrichment. For characterization of metabolic substrates, dual tracer uptake studies were performed with \(^{18}\)F-2-fluoro-2-deoxy-D-glucose (\(^{18}\)F-FDG) and \(^{125}\)I-β-methyl-iodophenyl-pentadecanoic acid (\(^{125}\)I-BMIPP) as transport markers of glucose and fatty acids, respectively. Results: After cardiac differentiation of hiPSC, in vitro tracer uptake assays confirmed metabolic substrate shift from glucose to fatty acids that was comparable to those observed in native isolated human cardiomyocytes. Immunostaining further confirmed expression of fatty acid transport and binding proteins on hiPSC-CM. Conclusions: During in vitro cardiac maturation, we observed a metabolic shift to fatty acids, which are known as a main energy source of mammalian hearts, suggesting hi-PSC-CM as a potential functional phenotype to investigate alteration of cardiac metabolism in cardiac diseases. Results also highlight the use of available clinical nuclear medicine tracers as functional assays in stem cell research for improved generation of autologous differentiated cells for numerous biomedical applications. 2018 International Journal of Cardiology urn:nbn:de:bvb:20-opus-163320 10.1016/j.ijcard.2018.06.089 Klinik und Poliklinik für Nuklearmedizin OPUS4-17069 Wissenschaftlicher Artikel Nose, Naoko; Werner, Rudolf A.; Ueda, Yuichiro; Günther, Katharina; Lapa, Constantin; Javadi, Mehrbod S.; Fukushima, Kazuhito; Edenhofer, Frank; Higuchi, Takahiro Metabolic substrate shift in human induced pluripotent stem cells during cardiac differentiation: Functional assessment using in vitro radionuclide uptake assay BACKGROUND: Recent developments in cellular reprogramming technology enable the production of virtually unlimited numbers of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Although hiPSC-CM share various characteristic hallmarks with endogenous cardiomyocytes, it remains a question as to what extent metabolic characteristics are equivalent to mature mammalian cardiomyocytes. Here we set out to functionally characterize the metabolic status of hiPSC-CM in vitro by employing a radionuclide tracer uptake assay. MATERIAL AND METHODS: Cardiac differentiation of hiPSC was induced using a combination of well-orchestrated extrinsic stimuli such as WNT activation (by CHIR99021) and BMP signalling followed by WNT inhibition and lactate based cardiomyocyte enrichment. For characterization of metabolic substrates, dual tracer uptake studies were performed with \(^{18}\)F‑2‑fluoro‑2‑deoxy‑d‑glucose (\(^{18}\)F-FDG) and \(^{125}\)I‑β‑methyl‑iodophenyl‑pentadecanoic acid (\(^{125}\)I-BMIPP) as transport markers of glucose and fatty acids, respectively. RESULTS: After cardiac differentiation of hiPSCs, in vitro tracer uptake assays confirmed metabolic substrate shift from glucose to fatty acids that was comparable to those observed in native isolated human cardiomyocytes. Immunostaining further confirmed expression of fatty acid transport and binding proteins on hiPSC-CM. CONCLUSIONS: During in vitro cardiac maturation, we observed a metabolic shift to fatty acids, which are known as a main energy source of mammalian hearts, suggesting hi-PSC-CM as a potential functional phenotype to investigate alteration of cardiac metabolism in cardiac diseases. Results also highlight the use of available clinical nuclear medicine tracers as functional assays in stem cell research for improved generation of autologous differentiated cells for numerous biomedical applications. 2018 229-234 International Journal of Cardiology 269 urn:nbn:de:bvb:20-opus-170699 Klinik und Poliklinik für Nuklearmedizin OPUS4-19076 Wissenschaftlicher Artikel Münst, Bernhard; Thier, Marc Christian; Winnemöller, Dirk; Helfen, Martina; Thummer, Rajkumar P.; Edenhofer, Frank Nanog induces suppression of senescence through downregulation of p27\(^{KIP1}\) expression A comprehensive analysis of the molecular network of cellular factors establishing and maintaining pluripotency as well as self renewal of pluripotent stem cells is key for further progress in understanding basic stem cell biology. Nanog is necessary for the natural induction of pluripotency in early mammalian development but dispensable for both its maintenance and its artificial induction. To gain further insight into the molecular activity of Nanog, we analyzed the outcomes of Nanog gain-of-function in various cell models employing a recently developed biologically active recombinant cell-permeant protein, Nanog-TAT. We found that Nanog enhances the proliferation of both NIH 3T3 and primary fibroblast cells. Nanog transduction into primary fibroblasts results in suppression of senescence-associated beta-galactosidase activity. Investigation of cell cycle factors revealed that transient activation of Nanog correlates with consistent downregulation of the cell cycle inhibitor p27\(^{KIP1}\) (also known as CDKN1B). By performing chromatin immunoprecipitation analysis, we confirmed bona fide Nanog-binding sites upstream of the p27\(^{KIP1}\) gene, establishing a direct link between physical occupancy and functional regulation. Our data demonstrates that Nanog enhances proliferation of fibroblasts through transcriptional regulation of cell cycle inhibitor p27 gene. 2016 912-920 Journal of Cell Science 129 5 urn:nbn:de:bvb:20-opus-190761 10.1242/jcs.167932 Institut für Anatomie und Zellbiologie OPUS4-12057 Wissenschaftlicher Artikel Kadari, Asifiqbal; Lu, Min; Li, Ming; Sekaran, Thileepan; Thummer, Rajkumar P.; Guyette, Naomi; Chu, Vi; Edenhofer, Frank Excision of viral reprogramming cassettes by Cre protein transduction enables rapid, robust and efficient derivation of transgene-free human induced pluripotent stem cells Integrating viruses represent robust tools for cellular reprogramming; however, the presence of viral transgenes in induced pluripotent stem cells (iPSCs) is deleterious because it holds the risk of insertional mutagenesis leading to malignant transformation. Here, we combine the robustness of lentiviral reprogramming with the efficacy of Cre recombinase protein transduction to derive iPSCs devoid of transgenes. By genome-wide analysis and targeted differentiation towards the cardiomyocyte lineage, we show that transgene-free iPSCs are superior to iPSCs before Cre transduction. Our study provides a simple, rapid and robust protocol for the generation of clinical-grade iPSCs suitable for disease modeling, tissue engineering and cell replacement therapies. 2014 47 Stem Cell Research & Therapy 5 2 urn:nbn:de:bvb:20-opus-120578 10.1186/scrt435 Institut für Anatomie und Zellbiologie OPUS4-25984 Wissenschaftlicher Artikel Janz, Anna; Zink, Miriam; Cirnu, Alexandra; Hartleb, Annika; Albrecht, Christina; Rost, Simone; Klopocki, Eva; Günther, Katharina; Edenhofer, Frank; Ergün, Süleyman; Gerull, Brenda CRISPR/Cas9-edited PKP2 knock-out (JMUi001-A-2) and DSG2 knock-out (JMUi001-A-3) iPSC lines as an isogenic human model system for arrhythmogenic cardiomyopathy (ACM) Arrhythmogenic cardiomyopathy (ACM) is characterized by fibro-fatty replacement of the myocardium, heart failure and life-threatening ventricular arrhythmias. Causal mutations were identified in genes encoding for proteins of the desmosomes, predominantly plakophilin-2 (PKP2) and desmoglein-2 (DSG2). We generated gene-edited knock-out iPSC lines for PKP2 (JMUi001-A-2) and DSG2 (JMUi001-A-3) using the CRISPR/Cas9 system in a healthy control iPSC background (JMUi001A). Stem cell-like morphology, robust expression of pluripotency markers, embryoid body formation and normal karyotypes confirmed the generation of high quality iPSCs to provide a novel isogenic human in vitro model system mimicking ACM when differentiated into cardiomyocytes. 2021 102256 Stem Cell Research 53 urn:nbn:de:bvb:20-opus-259846 10.1016/j.scr.2021.102256 Institut für Humangenetik OPUS4-26851 Wissenschaftlicher Artikel Jansch, Charline; Ziegler, Georg C.; Forero, Andrea; Gredy, Sina; Wäldchen, Sina; Vitale, Maria Rosaria; Svirin, Evgeniy; Zöller, Johanna E. M.; Waider, Jonas; Günther, Katharina; Edenhofer, Frank; Sauer, Markus; Wischmeyer, Erhard; Lesch, Klaus-Peter Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly Human induced pluripotent stem cells (hiPSCs) have revolutionized the generation of experimental disease models, but the development of protocols for the differentiation of functionally active neuronal subtypes with defined specification is still in its infancy. While dysfunction of the brain serotonin (5-HT) system has been implicated in the etiology of various neuropsychiatric disorders, investigation of functional human 5-HT specific neurons in vitro has been restricted by technical limitations. We describe an efficient generation of functionally active neurons from hiPSCs displaying 5-HT specification by modification of a previously reported protocol. Furthermore, 5-HT specific neurons were characterized using high-end fluorescence imaging including super-resolution microscopy in combination with electrophysiological techniques. Differentiated hiPSCs synthesize 5-HT, express specific markers, such as tryptophan hydroxylase 2 and 5-HT transporter, and exhibit an electrophysiological signature characteristic of serotonergic neurons, with spontaneous rhythmic activities, broad action potentials and large afterhyperpolarization potentials. 5-HT specific neurons form synapses reflected by the expression of pre- and postsynaptic proteins, such as Bassoon and Homer. The distribution pattern of Bassoon, a marker of the active zone along the soma and extensions of neurons, indicates functionality via volume transmission. Among the high percentage of 5-HT specific neurons (~ 42%), a subpopulation of CDH13 + cells presumably designates dorsal raphe neurons. hiPSC-derived 5-HT specific neuronal cell cultures reflect the heterogeneous nature of dorsal and median raphe nuclei and may facilitate examining the association of serotonergic neuron subpopulations with neuropsychiatric disorders. 2021 225-241 Journal of Neural Transmission 128 2 urn:nbn:de:bvb:20-opus-268519 10.1007/s00702-021-02303-5 Physiologisches Institut