title: Upscaling of hiPS Cell-Derived Neurons for High-Throughput Screening.
abstract: The advent of human-induced pluripotent stem (hiPS) cell-derived neurons promised to provide better model cells for drug discovery in the context of the central nervous system. This work demonstrates both the upscaling of cellular expansion and the acceleration of neuronal differentiation to accommodate the immense material needs of a high-throughput screening (HTS) approach. Using GRowth factor-driven expansion and INhibition of NotCH (GRINCH) during maturation, the derived cells are here referred to as GRINCH neurons. GRINCH cells displayed neuronal markers, and their functional activity could be demonstrated by electrophysiological recordings. In an application of GRINCH neurons, the brain-derived neurotrophic factor (BDNF)-mediated activation of tropomyosin receptor kinase (TrkB) was investigated as a promising drug target to treat synaptic dysfunctions. To assess the phosphorylation of endogenous TrkB in the GRINCH cells, the highly sensitive amplified luminescent proximity homogeneous assay LISA (AlphaLISA) format was established as a primary screen. A high-throughput reverse transcription (RT)-PCR format was employed as a secondary assay to analyze TrkB-mediated downstream target gene expression. In summary, an optimized differentiation protocol, highly efficient cell upscaling, and advanced assay miniaturization, combined with increased detection sensitivity, pave the way for a new generation of predictive cell-based drug discovery.
authors.affiliation: 1 Lead Identification and Optimization Support, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Traub
authors.affiliation: 2 Immunological and Respiratory Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
authors.forename: Heiko
authors.initials: H
authors.lastname: Stahl
authors.affiliation: 3 CNS Diseases Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
authors.forename: Holger
authors.initials: H
authors.lastname: Rosenbrock
authors.affiliation: 4 Target Discovery Research, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
authors.forename: Eric
authors.initials: E
authors.lastname: Simon
authors.affiliation: 1 Lead Identification and Optimization Support, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany.
authors.forename: Ralf
authors.initials: R
authors.lastname: Heilker
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D019208:Brain-Derived Neurotrophic Factor / Q000235:genetics* / Q000378:metabolism / Q000494:pharmacology; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000187:drug effects; D003959:Diamines / Q000494:pharmacology; D055808:Drug Discovery / Q000295:instrumentation / Q000379:methods*; D016222:Fibroblast Growth Factor 2 / Q000494:pharmacology; D005786:Gene Expression Regulation; D057166:High-Throughput Screening Assays; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D008163:Luminescent Measurements; D008562:Membrane Glycoproteins / Q000235:genetics* / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D018408:Patch-Clamp Techniques; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D020813:Receptor, trkB / Q000235:genetics* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods; D054852:Small Molecule Libraries / Q000494:pharmacology*; D013844:Thiazoles / Q000494:pharmacology
chemical_list: C000612000:24-diamino-5-phenylthiazole; D015415:Biomarkers; D019208:Brain-Derived Neurotrophic Factor; D003959:Diamines; D008562:Membrane Glycoproteins; D054852:Small Molecule Libraries; D013844:Thiazoles; D016222:Fibroblast Growth Factor 2; C579455:BDNF protein, human; D020813:Receptor, trkB; C000591259:tropomyosin-related kinase-B, human
keywords: human-induced pluripotent stem (hiPS) cells; neuronal model cells; tropomyosin receptor kinase B (TrkB); upscaling of cell culture