TY - THES A1 - Günther, Katharina T1 - Generation of early human neuroepithelial progenitors from primary cells for biomedical applications T1 - Generierung früher humaner neuroepithelialer Vorläufer aus primären Zellen für biomedizinische Anwendungen N2 - 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. N2 - Patientenspezifische induziert pluripotente Zellen (iPSZ) haben sich als eine vielversprechende Möglichkeit erwiesen Zellen zu gewinnen, die für Krankheitsmodellierung, Arzneimitteltests und Zellersatztherapie in Frage kommen. In dieser Arbeit wurden drei wichtige Fragestellungen adressiert, die für potenzielle biomedizinische Anwendungen von neuralen Stammzellen von großem Interesse sind. Um die Generierung von transgenfreien iPSZ zu ermöglichen, wurden Alternativen zur retroviralen Reprogrammierung entwickelt. Im ersten Teil dieser Arbeit wurden Reprogrammierungsmethoden, die auf deletierbaren, lentiviralen Konstrukten oder nichtintegrativen Verfahren wie Sendaivirus (SeV)-Transduktion und Transfektion synthetischer mRNA basieren, adaptiert und evaluiert. Die daraus resultierenden iPSZ exprimieren die Pluripotenzmarker OCT4, SSEA-4 und TRA1-60. Weiterhin wurde das Potenzial in Zelltypen aller drei Keimblätter zu differenzieren nachgewiesen. Dadurch konnte die pluripotente Identität der proliferativen Kolonien bestätigt werden. Beim Vergleich der angewandten Methoden fielen, bezüglich der generierten iPSZ-Linien, sowohl qualitative als auch quantitative Unterschiede auf. Bei der Verwendung von SeV-Partikeln wurde eine hohe Reprogrammierungseffizienz festgestellt. Bei der Transfektion von mRNAs hingegen war die Reprogrammierungseffizienz deutlich niedriger. Diese war darüber hinaus abhängig von der Passage und dem Genotyp der Ausgangsfibroblasten. Des Weiteren konnte eine klon- und passagenabhängige Präsenz viraler Gene in SeV-iPSZ bis zu 23 Passagen lang beobachtet werden, während bei der mRNA-Transfektion keine Spuren der genetischen Manipulation zurückblieben. Dies verdeutlicht die Bedeutung einer sorgfältigen Qualitätskontrolle bei der Klonselektion im Falle der SeV-iPSZ. Im Gegensatz dazu stellt die Reprogrammierung durch Transfektion synthetischer mRNAs eine völlig nicht-integrative Strategie dar, erfordert allerdings weitere Verfeinerung um das Verfahren effizient und vor allem für alle Fibroblastenpräparationen anwendbar zu machen. Der zweite Teil der Arbeit behandelt die Etablierung eines schnellen, adhärenten Protokolls, um neurale Vorläuferpopulation aus iPSZ zu differenzieren. Um dies zu erreichen, wurde ein zweiphasiges Protokoll entwickelt, welches zunächst die Generierung einer primitiven neuralen Vorläuferzellpopulation innerhalb von 7 Tagen erlaubt. In einem zweiten Schritt erfolgte die Adaptierung an Kulturbedingungen, die eine neurale, rosettenähnliche Zellpopulation induzieren. Beide neuralen Zellpopulationen konnten weiter expandiert und eingefroren werden und waren negativ für den Pluripotenz-assoziierten Transkriptionsfaktor OCT4. Darüber hinaus konnte die neurale Vorläuferidentität mittels positiver Expression von SOX1, SOX2, PAX6 und Nestin bestätigt werden. Eine weitere Differenzierung dieser Zellen resultierte in TUJ1-positiven Neuronen und GFAP-positiven Astrozyten, die die Verwendung der Zellpopulation beispielsweise in einem zellulären Modell der Blut-Hirn-Schranke erlaubten. Das Hauptprojekt dieser Dissertation war es, frühe humane neurale Vorläuferzellen aus fetalem Hirngewebe zu isolieren und in Kultur zu stabilisieren. Diese Population sollte eine breite Differenzierungskapazität aufweisen. Zu diesem Zweck wurde eine chemisch definierte Medienzusammensetzung gewählt, die zusätzlich pharmakologisch wirksame Verbindungen und Wachstumsfaktoren beinhaltet. Hierdurch konnten Signaltransduktionswege wie zum Beispiel der Sonic-Hedgehog- (SHH) oder FGF-Signalweg, die bei der frühen neuralen Entwicklung eine bedeutende Rolle spielen, moduliert werden. In der Tat ermöglichten spezifische Kultivierungsbedingungen, die den TGFβ-Inhibitor SB431542, den SHH-Agonisten Purmorphamin, den GSK3β-Inhibitor CHIR99021 und basisches FGF, jedoch kein EGF enthielten, die robuste Bildung einer früheren neuroepithelialen Vorläuferpopulation (eNEP). Die so stabilisierten Kolonien wiesen eine homogene Morphologie und eine hohe Proliferationsrate auf. Außerdem zeigten sie eine hohe Klonogenitätsrate von 23%, die es ermöglichte monoklonale Zelllinien zu isolieren und für mehr als 45 Passagen zu expandieren. Dabei blieb ein normaler Karyotyp erhalten. Die Zellen zeigten ein eindeutiges neurales Profil, gekennzeichnet durch SOX1, PAX6, Nestin, SOX2 und Prominin-Expression. Weiterhin wiesen eNEPs NOTCH und HES5-Aktivierung in Kombination mit nicht-polarisierter Morphologie auf, was auf eine frühe neuropitheliale Identität hinweist. Eine Microarray-Analyse demonstrierte weiterhin SOX11, BRN2 und einige HES-Gene als charakteristisch hochregulierte Gene. Interessanterweise zeigen eNEPs eine regionale Identität, die auf eine Mittelhirn/Hinterhirn-Regionalisierung hinweist. Die Validierung ungerichtet ausdifferenzierter Zelltypen offenbarte mit einem Kulturanteil von 90% TUJ1-positiven Neuronen ein stark neurogenes Potenzial. Zusätzlich konnten GFAPpositive Astrozyten sowie mögliche Myelinstrukturen, die auf Oligodendrozyten hinweisen, nachgewiesen werden. Elektrophysiologische Aufzeichnungen deuten auf funktionell aktive Neurone hin und Immunofluoreszenzfärbungen zeigten GABAerge, glutamaterge, dopaminerge und serotonerge neuronale Subtypen. Außerdem wurden mittels Immunfluoreszenzanalyse Synapsin- und PSD-95- positive synaptische Strukturen nachgewiesen. Ultrastrukturelle Analysen mittels Transmissionselektronenmikroskopie bestätigten das Ergebnis. Hervorzuheben ist, dass einige Neurone positiv für den peripheren Neuronenmarker Peripherin gefärbt wurden, was darauf hinweist, dass eNEPs das Potenzial besitzen, in Zellen der Neuralleiste zu differenzieren. Durch die Verwendung von spezifischen Differenzierungsprotokollen konnte das Vorkommen TH-positiver und auch möglicher A- und C-sensorischer Fasern, sowie mesenchymaler Zellen nachgewiesen werden. Zusammenfassend lässt sich sagen, dass primäre eNEPs dazu beitragen könnten, die frühe humane Gehirnentwicklung zu verstehen. Darüber hinaus stellen eNEPs eine potentielle zelluläre Quelle für Zellersatztherapien und weitere biomedizinische Anwendungen dar. KW - progenitors KW - stem cells KW - biomedicine KW - human primary cells KW - biomedical applications KW - neuroepithelial progenitors KW - neuroepitheliale Vorläufer KW - early neural precursors KW - frühe neurale Vorläufer Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-150348 ER - TY - JOUR A1 - Palladino, Viola Stella A1 - Chiocchetti, Andreas G. A1 - Frank, Lukas A1 - Haslinger, Denise A1 - McNeill, Rhiannon A1 - Radtke, Franziska A1 - Till, Andreas A1 - Haupt, Simone A1 - Brüstle, Oliver A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Hoffmann, Per A1 - Reif, Andreas A1 - Kittel-Schneider, Sarah T1 - Energy metabolism disturbances in cell models of PARK2 CNV carriers with ADHD JF - Journal of Clinical Medicine N2 - 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. KW - ADHD KW - hiPSC KW - PARK2 KW - mitochondria KW - disease modelling Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-220074 SN - 2077-0383 VL - 9 IS - 12 ER - TY - JOUR A1 - Jansch, Charline A1 - Ziegler, Georg C. A1 - Forero, Andrea A1 - Gredy, Sina A1 - Wäldchen, Sina A1 - Vitale, Maria Rosaria A1 - Svirin, Evgeniy A1 - Zöller, Johanna E. M. A1 - Waider, Jonas A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Sauer, Markus A1 - Wischmeyer, Erhard A1 - Lesch, Klaus-Peter T1 - Serotonin-specific neurons differentiated from human iPSCs form distinct subtypes with synaptic protein assembly JF - Journal of Neural Transmission N2 - 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. KW - neuropsychiatric disorders KW - human induced pluripotent stem cell (hiPSC) KW - serotonin-specific neurons KW - median and dorsal raphe KW - synapse formation KW - Cadherin-13 (CDH13) Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-268519 SN - 1435-1463 VL - 128 IS - 2 ER - TY - JOUR A1 - Appelt-Menzel, Antje A1 - Cubukova, Alevtina A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Piontek, Jörg A1 - Krause, Gerd A1 - Stüber, Tanja A1 - Walles, Heike A1 - Neuhaus, Winfried A1 - Metzger, Marco T1 - Establishment of a Human Blood-Brain Barrier Co-culture Model Mimicking the Neurovascular Unit Using Induced Pluri- and Multipotent Stem Cells JF - Stem Cell Reports N2 - 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. KW - blood-brain barrier (BBB) model KW - human induced pluripotent stem cells (hiPSCs)human induced pluripotent stem cells (hiPSCs) KW - multipotent fetal neural stem cells (fNSCs) KW - neurovascular unit in vitro Y1 - 2017 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170982 VL - 8 IS - 4 ER - TY - JOUR A1 - Jansch, Charline A1 - Günther, Katharina A1 - Waider, Jonas A1 - Ziegler, Georg C. A1 - Forero, Andrea A1 - Kollert, Sina A1 - Svirin, Evgeniy A1 - Pühringer, Dirk A1 - Kwok, Chee Keong A1 - Ullmann, Reinhard A1 - Maierhofer, Anna A1 - Flunkert, Julia A1 - Haaf, Thomas A1 - Edenhofer, Frank A1 - Lesch, Klaus-Peter T1 - 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 JF - Stem Cell Research N2 - 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. KW - ADHD KW - SLC2A3 KW - induced pluripotent stem cells Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-176654 VL - 28 ER - TY - INPR A1 - Nose, Naoko A1 - Werner, Rudolf A. A1 - Ueda, Yuichiro A1 - Günther, Katharina A1 - Lapa, Constantin A1 - Javadi, Mehrbod S. A1 - Fukushima, Kazuhito A1 - Edenhofer, Frank A1 - Higuchi, Takahiro T1 - Metabolic substrate shift in human induced pluripotent stem cells during cardiac differentiation: Functional assessment using in vitro radionuclide uptake assay T2 - International Journal of Cardiology N2 - 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. KW - tracer KW - Stammzelle KW - induced pluripotent stem cells KW - cardiomyocytes KW - fatty acid KW - stem cell therapy KW - hiPSC-CM Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-163320 SN - 0167-5273 ER - TY - JOUR A1 - Janz, Anna A1 - Zink, Miriam A1 - Cirnu, Alexandra A1 - Hartleb, Annika A1 - Albrecht, Christina A1 - Rost, Simone A1 - Klopocki, Eva A1 - Günther, Katharina A1 - Edenhofer, Frank A1 - Ergün, Süleyman A1 - Gerull, Brenda T1 - 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) JF - Stem Cell Research N2 - 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. KW - mutations Y1 - 2021 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-259846 VL - 53 ER - TY - JOUR A1 - Nose, Naoko A1 - Werner, Rudolf A. A1 - Ueda, Yuichiro A1 - Günther, Katharina A1 - Lapa, Constantin A1 - Javadi, Mehrbod S. A1 - Fukushima, Kazuhito A1 - Edenhofer, Frank A1 - Higuchi, Takahiro T1 - Metabolic substrate shift in human induced pluripotent stem cells during cardiac differentiation: Functional assessment using in vitro radionuclide uptake assay JF - International Journal of Cardiology N2 - 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. KW - tracer KW - Stammzelle KW - induced pluripotent stem cells KW - cardiomyocytes KW - fatty acid KW - stem cell therapy KW - hiPSC-CM Y1 - 2018 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170699 VL - 269 ER -