{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,29]],"date-time":"2026-09-29T12:16:11Z","timestamp":1790684171599,"version":"4.1.0"},"reference-count":59,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2022,3,23]],"date-time":"2022-03-23T00:00:00Z","timestamp":1647993600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["FOR 2488"],"award-info":[{"award-number":["FOR 2488"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Cell. Neurosci."],"abstract":"<jats:p>Induced pluripotent stem cell (iPSC)-based generation of tyrosine hydroxylase-positive (TH<jats:sup>+<\/jats:sup>) dopaminergic neurons (DNs) is a powerful method for creating patient-specific <jats:italic>in vitro<\/jats:italic> models to elucidate mechanisms underlying Parkinson\u2019s disease (PD) at the cellular and molecular level and to perform drug screening. However, currently available differentiation paradigms result in highly heterogeneous cell populations, often yielding a disappointing fraction (&amp;lt;50%) of the PD-relevant TH<jats:sup>+<\/jats:sup> DNs. To facilitate the targeted analysis of this cell population and to characterize their electrophysiological properties, we employed CRISPR\/Cas9 technology and generated an mCherry-based human TH reporter iPSC line. Subsequently, reporter iPSCs were subjected to dopaminergic differentiation using either a \u201cfloor plate protocol\u201d generating DNs directly from iPSCs or an alternative method involving iPSC-derived neuronal precursors (NPC-derived DNs). To identify the strategy with the highest conversion efficiency to mature neurons, both cultures were examined for a period of 8 weeks after triggering neuronal differentiation by means of immunochemistry and single-cell electrophysiology. We confirmed that mCherry expression correlated with the expression of endogenous TH and that genetic editing did neither affect the differentiation process nor the endogenous TH expression in iPSC- and NPC-derived DNs. Although both cultures yielded identical proportions of TH<jats:sup>+<\/jats:sup> cells (\u224830%), whole-cell patch-clamp experiments revealed that iPSC-derived DNs gave rise to larger currents mediated by voltage-gated sodium and potassium channels, showed a higher degree of synaptic activity, and fired trains of mature spontaneous action potentials more frequently compared to NPC-derived DNs already after 2 weeks in differentiation. Moreover, spontaneous action potential firing was more frequently detected in TH<jats:sup>+<\/jats:sup> neurons compared to the TH<jats:sup>\u2013<\/jats:sup> cells, providing direct evidence that these two neuronal subpopulations exhibit different intrinsic electrophysiological properties. In summary, the data reveal substantial differences in the electrophysiological properties of iPSC-derived TH<jats:sup>+<\/jats:sup> and TH<jats:sup>\u2013<\/jats:sup> neuronal cell populations and that the \u201cfloor plate protocol\u201d is particularly efficient in generating electrophysiologically mature TH<jats:sup>+<\/jats:sup> DNs, which are the most vulnerable neuronal subtype in PD.<\/jats:p>","DOI":"10.3389\/fncel.2022.817198","type":"journal-article","created":{"date-parts":[[2022,3,30]],"date-time":"2022-03-30T18:27:47Z","timestamp":1648664867000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":21,"title":["Electrophysiological Properties of Induced Pluripotent Stem Cell-Derived Midbrain Dopaminergic Neurons Correlate With Expression of Tyrosine Hydroxylase"],"prefix":"10.3389","volume":"16","author":[{"given":"Aleksandar","family":"Rakovic","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorothea","family":"Vo\u00df","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Franca","family":"Vulinovic","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Britta","family":"Meier","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ann-Katrin","family":"Hellberg","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carla","family":"Nau","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Christine","family":"Klein","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Enrico","family":"Leipold","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2022,3,23]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0006286","article-title":"Neural differentiation of embryonic stem cells in vitro: a road map to neurogenesis in the embryo.","volume":"4","author":"Abranches","year":"2009","journal-title":"PLoS One"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1117\/1.NPh.7.1.011402","article-title":"Interpreting in vivo calcium signals from neuronal cell bodies, axons, and dendrites: a review.","volume":"7","author":"Ali","year":"2020","journal-title":"Neurophotonics"},{"key":"B3","doi-asserted-by":"publisher","DOI":"10.3390\/ijms22073381","article-title":"Current State-of-the-Art and Unresolved Problems in Using Human Induced Pluripotent Stem Cell-Derived Dopamine Neurons for Parkinson\u2019s Disease Drug Development.","volume":"22","author":"Antonov","year":"2021","journal-title":"Int. J. Mol. Sci."},{"key":"B4","doi-asserted-by":"publisher","first-page":"1853","DOI":"10.1016\/j.stemcr.2017.10.029","article-title":"Altered Differentiation Potential of Gaucher\u2019s Disease iPSC Neuronal Progenitors due to Wnt\/beta-Catenin Downregulation.","volume":"9","author":"Awad","year":"2017","journal-title":"Stem Cell Rep."},{"key":"B5","doi-asserted-by":"publisher","first-page":"1573","DOI":"10.1038\/mp.2016.158","article-title":"Predicting the functional states of human iPSC-derived neurons with single-cell RNA-seq and electrophysiology.","volume":"21","author":"Bardy","year":"2016","journal-title":"Mol. Psychiatry"},{"key":"B6","doi-asserted-by":"publisher","first-page":"587","DOI":"10.1016\/j.stemcr.2017.06.005","article-title":"MAPT Genetic Variation and Neuronal Maturity Alter Isoform Expression Affecting Axonal Transport in iPSC-Derived Dopamine Neurons.","volume":"9","author":"Beevers","year":"2017","journal-title":"Stem Cell Rep."},{"key":"B7","doi-asserted-by":"publisher","first-page":"1733","DOI":"10.1089\/scd.2010.0501","article-title":"Physiological properties of neurons derived from human embryonic stem cells using a dibutyryl cyclic AMP-based protocol.","volume":"20","author":"Belinsky","year":"2011","journal-title":"Stem Cells Dev."},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.3389\/fnins.2016.00269","article-title":"Parkinson\u2019s Disease and Neurodegeneration: GABA-Collapse Hypothesis.","volume":"10","author":"Blaszczyk","year":"2016","journal-title":"Front. Neurosci."},{"key":"B9","doi-asserted-by":"crossref","first-page":"2284","DOI":"10.1016\/S0140-6736(21)00218-X","article-title":"Parkinson\u2019s disease.","volume":"397","author":"Bloem","year":"2021","journal-title":"Lancet"},{"key":"B10","doi-asserted-by":"publisher","first-page":"955","DOI":"10.1002\/stem.408","article-title":"Inhibition of notch signaling in human embryonic stem cell-derived neural stem cells delays G1\/S phase transition and accelerates neuronal differentiation in vitro and in vivo.","volume":"28","author":"Borghese","year":"2010","journal-title":"Stem Cells"},{"key":"B11","doi-asserted-by":"publisher","first-page":"899","DOI":"10.4252\/wjsc.v7.i6.899","article-title":"Importance of being Nernst: synaptic activity and functional relevance in stem cell-derived neurons.","volume":"7","author":"Bradford","year":"2015","journal-title":"World J. Stem Cells"},{"key":"B12","doi-asserted-by":"publisher","DOI":"10.1038\/s41598-019-43080-2","article-title":"CRISPR\/Cas9-mediated generation of a tyrosine hydroxylase reporter iPSC line for live imaging and isolation of dopaminergic neurons.","volume":"9","author":"Calatayud","year":"2019","journal-title":"Sci. Rep."},{"key":"B13","doi-asserted-by":"publisher","first-page":"275","DOI":"10.1038\/nbt.1529","article-title":"Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling.","volume":"27","author":"Chambers","year":"2009","journal-title":"Nat. Biotechnol."},{"key":"B14","doi-asserted-by":"publisher","DOI":"10.1038\/nature12354","article-title":"Ultrasensitive fluorescent proteins for imaging neuronal activity.","volume":"499","author":"Chen","year":"2013","journal-title":"Nature"},{"key":"B15","doi-asserted-by":"publisher","first-page":"258","DOI":"10.1016\/j.mcn.2010.06.017","article-title":"Differentiation of human ES and Parkinson\u2019s disease iPS cells into ventral midbrain dopaminergic neurons requires a high activity form of SHH, FGF8a and specific regionalization by retinoic acid.","volume":"45","author":"Cooper","year":"2010","journal-title":"Mol. Cell. Neurosci."},{"key":"B16","doi-asserted-by":"publisher","first-page":"1218","DOI":"10.1126\/science.1158799","article-title":"Induced pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons.","volume":"321","author":"Dimos","year":"2008","journal-title":"Science"},{"key":"B17","doi-asserted-by":"publisher","first-page":"337","DOI":"10.1016\/j.stemcr.2014.01.013","article-title":"Isolation of human induced pluripotent stem cell-derived dopaminergic progenitors by cell sorting for successful transplantation.","volume":"2","author":"Doi","year":"2014","journal-title":"Stem Cell Rep."},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.04059","article-title":"Non-linear developmental trajectory of electrical phenotype in rat substantia nigra pars compacta dopaminergic neurons.","volume":"3","author":"Dufour","year":"2014","journal-title":"Elife"},{"key":"B19","doi-asserted-by":"publisher","first-page":"277","DOI":"10.1038\/nature07677","article-title":"Induced pluripotent stem cells from a spinal muscular atrophy patient.","volume":"457","author":"Ebert","year":"2009","journal-title":"Nature"},{"key":"B20","doi-asserted-by":"publisher","DOI":"10.4155\/fmc-2018-0520","article-title":"Use of human pluripotent stem cell-derived cells for neurodegenerative disease modeling and drug screening platform.","volume":"11","author":"Garcia-Leon","year":"2019","journal-title":"Future Med. Chem."},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.3389\/fnsyn.2019.00020","article-title":"Approaches and Limitations in the Investigation of Synaptic Transmission and Plasticity.","volume":"11","author":"Glasgow","year":"2019","journal-title":"Front. Synaptic Neurosci."},{"key":"B22","doi-asserted-by":"publisher","first-page":"2161","DOI":"10.1152\/jn.00160.2009","article-title":"Membrane capacitance measurements revisited: dependence of capacitance value on measurement method in nonisopotential neurons.","volume":"102","author":"Golowasch","year":"2009","journal-title":"J. Neurophysiol."},{"key":"B23","doi-asserted-by":"publisher","DOI":"10.3389\/fnmol.2018.00277","article-title":"Generation of iPSC-Derived Human Peripheral Sensory Neurons Releasing Substance P Elicited by TRPV1 Agonists.","volume":"11","author":"Guimaraes","year":"2018","journal-title":"Front. Mol. Neurosci."},{"key":"B24","doi-asserted-by":"publisher","first-page":"1336","DOI":"10.1038\/mp.2017.56","article-title":"A simplified protocol for differentiation of electrophysiologically mature neuronal networks from human induced pluripotent stem cells.","volume":"23","author":"Gunhanlar","year":"2018","journal-title":"Mol. Psychiatry"},{"key":"B25","doi-asserted-by":"publisher","first-page":"11011","DOI":"10.1523\/JNEUROSCI.2519-09.2009","article-title":"Robust pacemaking in substantia nigra dopaminergic neurons.","volume":"29","author":"Guzman","year":"2009","journal-title":"J. Neurosci."},{"key":"B26","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0087388","article-title":"Physiological characterisation of human iPS-derived dopaminergic neurons.","volume":"9","author":"Hartfield","year":"2014","journal-title":"PLoS One"},{"key":"B27","doi-asserted-by":"publisher","first-page":"119","DOI":"10.1007\/978-1-4939-9007-8_9","article-title":"Generating Neural Stem Cells from iPSCs with Dopaminergic Neurons Reporter Gene.","volume":"1919","author":"Hong","year":"2019","journal-title":"Methods Mol. Biol."},{"key":"B28","doi-asserted-by":"publisher","first-page":"4335","DOI":"10.1073\/pnas.0910012107","article-title":"Neural differentiation of human induced pluripotent stem cells follows developmental principles but with variable potency.","volume":"107","author":"Hu","year":"2010","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B29","doi-asserted-by":"publisher","first-page":"5188","DOI":"10.1093\/hmg\/ddw339","article-title":"Genetic and pharmacological correction of aberrant dopamine synthesis using patient iPSCs with BH4 metabolism disorders.","volume":"25","author":"Ishikawa","year":"2016","journal-title":"Hum. Mol. Genet."},{"key":"B30","doi-asserted-by":"publisher","first-page":"14063","DOI":"10.1073\/pnas.0506029102","article-title":"Imaging input and output of neocortical networks in vivo.","volume":"102","author":"Kerr","year":"2005","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B31","doi-asserted-by":"publisher","first-page":"703","DOI":"10.1016\/j.celrep.2012.04.009","article-title":"Generation of regionally specified neural progenitors and functional neurons from human embryonic stem cells under defined conditions.","volume":"1","author":"Kirkeby","year":"2012","journal-title":"Cell Rep."},{"key":"B32","doi-asserted-by":"publisher","DOI":"10.3390\/ijms21197113","article-title":"Patient-Derived Induced Pluripotent Stem Cell-Based Models in Parkinson\u2019s Disease for Drug Identification.","volume":"21","author":"Kouroupi","year":"2020","journal-title":"Int. J. Mol. Sci."},{"key":"B33","doi-asserted-by":"publisher","first-page":"547","DOI":"10.1038\/nature10648","article-title":"Dopamine neurons derived from human ES cells efficiently engraft in animal models of Parkinson\u2019s disease.","volume":"480","author":"Kriks","year":"2011","journal-title":"Nature"},{"key":"B34","doi-asserted-by":"publisher","first-page":"1459","DOI":"10.1016\/j.cub.2012.06.007","article-title":"Dissection of cortical microcircuits by single-neuron stimulation in vivo.","volume":"22","author":"Kwan","year":"2012","journal-title":"Curr. Biol."},{"key":"B35","doi-asserted-by":"publisher","first-page":"289","DOI":"10.1038\/s41591-019-0739-1","article-title":"iPSC modeling of young-onset Parkinson\u2019s disease reveals a molecular signature of disease and novel therapeutic candidates.","volume":"26","author":"Laperle","year":"2020","journal-title":"Nat. Med."},{"key":"B36","doi-asserted-by":"publisher","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"B37","doi-asserted-by":"publisher","DOI":"10.1155\/2016\/9832839","article-title":"Pathophysiology of Motor Dysfunction in Parkinson\u2019s Disease as the Rationale for Drug Treatment and Rehabilitation.","volume":"2016","author":"Magrinelli","year":"2016","journal-title":"Parkinsons Dis."},{"key":"B38","doi-asserted-by":"publisher","DOI":"10.1038\/s41419-019-2133-9","article-title":"Homogenous generation of dopaminergic neurons from multiple hiPSC lines by transient expression of transcription factors.","volume":"10","author":"Mahajani","year":"2019","journal-title":"Cell Death Dis."},{"key":"B39","doi-asserted-by":"publisher","first-page":"1027","DOI":"10.1073\/pnas.0610155104","article-title":"Controlled expression of transgenes introduced by in vivo electroporation.","volume":"104","author":"Matsuda","year":"2007","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"B40","doi-asserted-by":"publisher","first-page":"423","DOI":"10.1038\/nrn2379","article-title":"Presynaptic glutamate receptors: physiological functions and mechanisms of action.","volume":"9","author":"Pinheiro","year":"2008","journal-title":"Nat. Rev. Neurosci."},{"key":"B41","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0103418","article-title":"A time course analysis of the electrophysiological properties of neurons differentiated from human induced pluripotent stem cells (iPSCs).","volume":"9","author":"Pre","year":"2014","journal-title":"PLoS One"},{"key":"B42","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0059252","article-title":"Derivation and expansion using only small molecules of human neural progenitors for neurodegenerative disease modeling.","volume":"8","author":"Reinhardt","year":"2013","journal-title":"PLoS One"},{"key":"B43","doi-asserted-by":"publisher","first-page":"1687","DOI":"10.1002\/stem.2634","article-title":"Genetic Rescue of Mitochondrial and Skeletal Muscle Impairment in an Induced Pluripotent Stem Cells Model of Coenzyme Q(10) Deficiency.","volume":"35","author":"Romero-Moya","year":"2017","journal-title":"Stem Cells"},{"key":"B44","doi-asserted-by":"publisher","DOI":"10.1038\/srep05400","article-title":"Multiplex genome engineering in human cells using all-in-one CRISPR\/Cas9 vector system.","volume":"4","author":"Sakuma","year":"2014","journal-title":"Sci. Rep."},{"key":"B45","doi-asserted-by":"publisher","first-page":"56","DOI":"10.1089\/hum.2011.054","article-title":"Efficient Generation of A9 Midbrain Dopaminergic Neurons by Lentiviral Delivery of LMX1A in Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.","volume":"23","author":"Sanchez-Danes","year":"2012","journal-title":"Hum. Gene Ther."},{"key":"B46","doi-asserted-by":"publisher","first-page":"671","DOI":"10.1038\/nmeth.2089","article-title":"NIH Image to ImageJ: 25 years of image analysis.","volume":"9","author":"Schneider","year":"2012","journal-title":"Nat. Methods"},{"key":"B47","doi-asserted-by":"publisher","DOI":"10.1002\/brb3.491","article-title":"Using iPSC-Derived Human DA Neurons From Opioid-Dependent Subjects to Study Dopamine Dynamics.","volume":"26","author":"Sheng","year":"2017","journal-title":"Cell Transplant."},{"key":"B48","doi-asserted-by":"publisher","first-page":"115","DOI":"10.1038\/nrd.2016.245","article-title":"Induced pluripotent stem cell technology: a decade of progress.","volume":"16","author":"Shi","year":"2017","journal-title":"Nat. Rev. Drug Discov."},{"key":"B49","doi-asserted-by":"publisher","first-page":"964","DOI":"10.1016\/j.cell.2009.02.013","article-title":"Parkinson\u2019s disease patient-derived induced pluripotent stem cells free of viral reprogramming factors.","volume":"136","author":"Soldner","year":"2009","journal-title":"Cell"},{"key":"B50","doi-asserted-by":"publisher","DOI":"10.1186\/scrt423","article-title":"Functional differentiation of midbrain neurons from human cord blood-derived induced pluripotent stem cells.","volume":"5","author":"Stanslowsky","year":"2014","journal-title":"Stem Cell Res. Ther."},{"key":"B51","doi-asserted-by":"publisher","first-page":"1548","DOI":"10.1002\/stem.1415","article-title":"Improved Cell Therapy Protocols for Parkinson\u2019s Disease Based on Differentiation Efficiency and Safety of hESC-, hiPSC-, and Non-Human Primate iPSC-Derived Dopaminergic Neurons.","volume":"31","author":"Sundberg","year":"2013","journal-title":"Stem Cells"},{"key":"B52","doi-asserted-by":"publisher","first-page":"861","DOI":"10.1016\/j.cell.2007.11.019","article-title":"Induction of pluripotent stem cells from adult human fibroblasts by defined factors.","volume":"131","author":"Takahashi","year":"2007","journal-title":"Cell"},{"key":"B53","doi-asserted-by":"publisher","DOI":"10.1038\/nmeth.1398","article-title":"Imaging neural activity in worms, flies and mice with improved GCaMP calcium indicators.","volume":"6","author":"Tian","year":"2009","journal-title":"Nat. Methods"},{"key":"B54","doi-asserted-by":"publisher","DOI":"10.1016\/j.scr.2019.101656","article-title":"Application of CRISPR\/Cas9 editing and digital droplet PCR in human iPSCs to generate novel knock-in reporter lines to visualize dopaminergic neurons.","volume":"41","author":"Uberbacher","year":"2019","journal-title":"Stem Cell Res."},{"key":"B55","doi-asserted-by":"publisher","first-page":"561","DOI":"10.1111\/ejn.14345","article-title":"Induced pluripotent stem cell-based modeling of mutant LRRK2-associated Parkinson\u2019s disease.","volume":"49","author":"Weykopf","year":"2019","journal-title":"Eur. J. Neurosci."},{"key":"B56","doi-asserted-by":"publisher","DOI":"10.1002\/stem.1152","article-title":"Specification of midbrain dopamine neurons from primate pluripotent stem cells.","volume":"30","author":"Xi","year":"2012","journal-title":"Stem Cells"},{"key":"B57","doi-asserted-by":"publisher","DOI":"10.1016\/j.celrep.2017.02.023","article-title":"A Knockin Reporter Allows Purification and Characterization of mDA Neurons from Heterogeneous Populations.","volume":"18","author":"Xia","year":"2017","journal-title":"Cell Rep."},{"key":"B58","doi-asserted-by":"crossref","DOI":"10.1126\/science.1151526","article-title":"Induced pluripotent stem cell lines derived from human somatic cells.","volume":"318","author":"Yu","year":"2007","journal-title":"Science"},{"key":"B59","doi-asserted-by":"publisher","first-page":"2412","DOI":"10.1093\/hmg\/ddx132","article-title":"SLP-2 interacts with Parkin in mitochondria and prevents mitochondrial dysfunction in Parkin-deficient human iPSC-derived neurons and Drosophila.","volume":"26","author":"Zanon","year":"2017","journal-title":"Hum. Mol. Genet."}],"container-title":["Frontiers in Cellular Neuroscience"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fncel.2022.817198\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,3,30]],"date-time":"2022-03-30T18:27:52Z","timestamp":1648664872000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fncel.2022.817198\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,3,23]]},"references-count":59,"alternative-id":["10.3389\/fncel.2022.817198"],"URL":"https:\/\/doi.org\/10.3389\/fncel.2022.817198","relation":{},"ISSN":["1662-5102"],"issn-type":[{"value":"1662-5102","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,3,23]]},"article-number":"817198"}}