@phdthesis{Guenther2018,
  author    = {G{\"u}nther, Katharina},
  title     = {Generation of early human neuroepithelial progenitors from primary cells for biomedical applications},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-150348},
  school      = {Universit{\"a}t W{\"u}rzburg},
  year      = {2018},
  abstract  = {Patient-specific induced pluripotent stem cells (iPSCs) emerged as a promising cell source for disease modeling and drug screening as well as a virtually unlimited source for restorative therapy. The thesis deals with three major topics to help realizing biomedical applications with neural stem cells. To enable the generation of transgene-free iPSCs, alternatives to retroviral reprogramming were developed. Hence, the adaptation and evaluation of reprogramming using excisable lentiviral constructs, Sendai virus (SeV) and synthetic mRNA-based methods was assessed in the first part of this thesis. hiPSCs exhibit the pluripotency markers OCT4, SSEA-4, TRA1-60 which were confirmed by immunofluorescence and flow cytometry. Besides, the potential to differentiate in cell types of all three germ layers was detected, confirming pluripotent identity of proliferating colonies resulting from various reprogramming strategies. However, major differences such as high efficiency with SeV in contrast to a relatively low efficiency with mRNA in regard to passage number and the phenotype of starting fibroblasts were observed. Furthermore, a prolonged clone- and passage-dependent residual presence of viral RNA genes was identified in SeV-iPSCs for up to 23 passages using RT-PCR underlining the importance of careful monitoring of clone selection. In contrast, viral-free reprogramming by synthetic mRNA represents a fully non-integrative approach but requires further refinement to be efficiently applicable to all fibroblasts. The second part of this thesis deals with the establishment of a rapid monolayer approach to differentiate neural progenitor cells from iPSCs. To achieve this, a two-step protocol was developed allowing first the formation of a stable, primitive NPC line within 7 days which was expanded for 2-3 passages. In a second step, a subsequent adaptation to conditions yielding neural rosette-like NPCs followed. Both neural lines were demonstrated to be expandable, cryopreservable and negative for the pluripotency marker OCT4. Furthermore, a neural precursor identity including SOX1, SOX2, PAX6, Nestin was confirmed by immunofluorescence and quantitative RT-PCR. Moreover, the differentiation resulted in TUJ1-positive neurons and GFAP-positive astrocytes. Nonetheless, the outcome of glial differentiation from primitive NSCs remained low, whereas FGF/EGF-NPCs were efficiently differentiated into GFAP-positive astrocytes which were implicated in a cellular model of the blood brain barrier. The third and major objective of this study was to generate human early neural progenitor cells from fetal brain tissue with a wide neural differentiation capacity. Therefore, a defined medium composition including small molecules and growth factors capable of modulation of crucial signaling pathways orchestrating early human development such as SHH and FGF was assessed. Indeed, specific culture conditions containing TGFβ inhibitor SB431542, SHH agonist Purmorphamine, GSK3β inhibitor CHIR99021 and basic FGF, but no EGF enabled robust formation of early neuroepithelial progenitor (eNEP) colonies displaying a homogeneous morphology and a high proliferation rate. Moreover, primary eNEPs exhibit a relatively high clonogenicity of more than 23 \% and can be monoclonally expanded for more than 45 passages carrying a normal karyotype. Characterization by immunofluorescence, flow cytometry and quantitative RT-PCR revealed a distinct NPC profile including SOX1, PAX6, Nestin and SOX2 and Prominin. Furthermore, primary eNEPs show NOTCH and HES5 activation in combination with non-polarized morphology, indicative of an early neuroepithelial identity. Microarray analysis unraveled SOX11, BRN2 and other HES-genes as characteristic upregulated genes. Interestingly, eNEPs were detected to display ventral midbrain/hindbrain regional identity. The validation of yielded cell types upon differentiation indicates a strong neurogenic potential with more than 90 \% of TUJ1-positive neurons. Moreover, astrocytes marked by GFAP and putative myelin structures indicating oligodendrocytes were identified. Electrophysiological recordings revealed functionally active neurons and immunofluorescence indicate GABAergic, glutamatergic, dopaminergic and serotonergic subtypes. Additionally, putative physiological synapse formation was observed by the presence of Synapsin and PSD-95 as well as by ultrastructural examination. Notably, rare neurons stained positive for the peripheral neuronal marker Peripherin suggesting the potential of eNEPS to give rise to cells of neural tube and neural crest origin. By the application of specific differentiation protocols an increase of TH-positive neurons or neural crest-derivatives such as putative A- and C-sensory neurons and mesenchymal cells was identified. Taken together, primary eNEPs might help to elucidate mechanisms of early human neurodevelopment and will serve as a novel source for cell replacement and further biomedical applications.},
  subject      = {progenitors},
  language  = {en}
}
@article{AppeltMenzelCubukovaGuentheretal.2017,
  author    = {Appelt-Menzel, Antje and Cubukova, Alevtina and G{\"u}nther, Katharina and Edenhofer, Frank and Piontek, J{\"o}rg and Krause, Gerd and St{\"u}ber, Tanja and Walles, Heike and Neuhaus, Winfried and Metzger, Marco},
  title     = {Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells},
  series = {Stem Cell Reports},
  volume    = {8},
  journal   = {Stem Cell Reports},
  number    = {4},
  doi       = {10.1016/j.stemcr.2017.02.021},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170982},
  pages     = {894-906},
  year      = {2017},
  abstract  = {In vitro models of the human blood-brain barrier (BBB) are highly desirable for drug development. This study aims to analyze a set of ten different BBB culture models based on primary cells, human induced pluripotent stem cells (hiPSCs), and multipotent fetal neural stem cells (fNSCs). We systematically investigated the impact of astrocytes, pericytes, and NSCs on hiPSC-derived BBB endothelial cell function and gene expression. The quadruple culture models, based on these four cell types, achieved BBB characteristics including transendothelial electrical resistance (TEER) up to 2,500 Ω cm\(^{2}\) and distinct upregulation of typical BBB genes. A complex in vivo-like tight junction (TJ) network was detected by freeze-fracture and transmission electron microscopy. Treatment with claudin-specific TJ modulators caused TEER decrease, confirming the relevant role of claudin subtypes for paracellular tightness. Drug permeability tests with reference substances were performed and confirmed the suitability of the models for drug transport studies.},
  language  = {en}
}
@article{JanschGuentherWaideretal.2018,
  author    = {Jansch, Charline and G{\"u}nther, Katharina and Waider, Jonas and Ziegler, Georg C. and Forero, Andrea and Kollert, Sina and Svirin, Evgeniy and P{\"u}hringer, Dirk and Kwok, Chee Keong and Ullmann, Reinhard and Maierhofer, Anna and Flunkert, Julia and Haaf, Thomas and Edenhofer, Frank and Lesch, Klaus-Peter},
  title     = {Generation of a human induced pluripotent stem cell (iPSC) line from a 51-year-old female with attention-deficit/hyperactivity disorder (ADHD) carrying a duplication of SLC2A3},
  series = {Stem Cell Research},
  volume    = {28},
  journal   = {Stem Cell Research},
  doi       = {10.1016/j.scr.2018.02.005},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-176654},
  pages     = {136-140},
  year      = {2018},
  abstract  = {Fibroblasts were isolated from a skin biopsy of a clinically diagnosed 51-year-old female attention-deficit/hyperactivity disorder (ADHD) patient carrying a duplication of SLC2A3, a gene encoding neuronal glucose transporter-3 (GLUT3). Patient fibroblasts were infected with Sendai virus, a single-stranded RNA virus, to generate transgene-free human induced pluripotent stem cells (iPSCs). SLC2A3-D2-iPSCs showed expression of pluripotency-associated markers, were able to differentiate into cells of the three germ layers in vitro and had a normal female karyotype. This in vitro cellular model can be used to study the role of risk genes in the pathogenesis of ADHD, in a patient-specific manner.},
  language  = {en}
}
@unpublished{NoseWernerUedaetal.2018,
  author    = {Nose, Naoko and Werner, Rudolf A. and Ueda, Yuichiro and G{\"u}nther, Katharina and Lapa, Constantin and Javadi, Mehrbod S. and Fukushima, Kazuhito and Edenhofer, Frank and Higuchi, Takahiro},
  title     = {Metabolic substrate shift in human induced pluripotent stem cells during cardiac differentiation: Functional assessment using in vitro radionuclide uptake assay},
  series = {International Journal of Cardiology},
  journal   = {International Journal of Cardiology},
  issn      = {0167-5273},
  doi       = {10.1016/j.ijcard.2018.06.089},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-163320},
  year      = {2018},
  abstract  = {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.},
  subject      = {Stammzelle},
  language  = {en}
}
@article{JanzZinkCirnuetal.2021,
  author    = {Janz, Anna and Zink, Miriam and Cirnu, Alexandra and Hartleb, Annika and Albrecht, Christina and Rost, Simone and Klopocki, Eva and G{\"u}nther, Katharina and Edenhofer, Frank and Erg{\"u}n, S{\"u}leyman and Gerull, Brenda},
  title     = {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)},
  series = {Stem Cell Research},
  volume    = {53},
  journal   = {Stem Cell Research},
  doi       = {10.1016/j.scr.2021.102256},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-259846},
  pages     = {102256},
  year      = {2021},
  abstract  = {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.},
  language  = {en}
}
@article{NoseWernerUedaetal.2018,
  author    = {Nose, Naoko and Werner, Rudolf A. and Ueda, Yuichiro and G{\"u}nther, Katharina and Lapa, Constantin and Javadi, Mehrbod S. and Fukushima, Kazuhito and Edenhofer, Frank and Higuchi, Takahiro},
  title     = {Metabolic substrate shift in human induced pluripotent stem cells during cardiac differentiation: Functional assessment using in vitro radionuclide uptake assay},
  series = {International Journal of Cardiology},
  volume    = {269},
  journal   = {International Journal of Cardiology},
  url       = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-170699},
  pages     = {229-234},
  year      = {2018},
  abstract  = {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.},
  subject      = {Stammzelle},
  language  = {en}
}