title: Patch-clamp recordings and calcium imaging followed by single-cell PCR reveal the developmental profile of 13 genes in iPSC-derived human neurons.
abstract: Molecular genetic studies are typically performed on homogenized biological samples, resulting in contamination from non-neuronal cells. To improve expression profiling of neurons we combined patch recordings with single-cell PCR. Two iPSC lines (healthy subject and 22q11.2 deletion) were differentiated into neurons. Patch electrode recordings were performed on 229 human cells from Day-13 to Day-88, followed by capture and single-cell PCR for 13 genes: ACTB, HPRT, vGLUT1, βTUBIII, COMT, DISC1, GAD1, PAX6, DTNBP1, ERBB4, FOXP1, FOXP2, and GIRK2. Neurons derived from both iPSC lines expressed βTUBIII, fired action potentials, and experienced spontaneous depolarizations (UP states) ~2 weeks before vGLUT1, GAD1 and GIRK2 appeared. Multisite calcium imaging revealed that these UP states were not synchronized among hESC-H9-derived neurons. The expression of FOXP1, FOXP2 and vGLUT1 was lost after 50 days in culture, in contrast to other continuously expressed genes. When gene expression was combined with electrophysiology, two subsets of genes were apparent; those irrelevant to spontaneous depolarizations (including vGLUT1, GIRK2, FOXP2 and DISC1) and those associated with spontaneous depolarizations (GAD1 and ERBB4). The results demonstrate that in the earliest stages of neuron development, it is useful to combine genetic analysis with physiological characterizations, on a cell-to-cell basis.
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Glenn S
authors.initials: GS
authors.lastname: Belinsky
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Matthew T
authors.initials: MT
authors.lastname: Rich
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Carissa L
authors.initials: CL
authors.lastname: Sirois
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA.
authors.forename: Shaina M
authors.initials: SM
authors.lastname: Short
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Erika
authors.initials: E
authors.lastname: Pedrosa
authors.affiliation: Department of Psychiatry and Behavioral Sciences, Albert Einstein College of Medicine, Bronx, New York, USA.
authors.forename: Herbert M
authors.initials: HM
authors.lastname: Lachman
authors.affiliation: Department of Neuroscience, University of Connecticut Health Center, Farmington, CT, USA. Electronic address: antic@neuron.uchc.edu.
authors.forename: Srdjan D
authors.initials: SD
authors.lastname: Antic
mesh_terms: D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002454:Cell Differentiation; D002460:Cell Line; D020552:Comet Assay; D055724:Electrophysiological Phenomena; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry; D017353:Gene Deletion; D018507:Gene Expression Regulation, Developmental; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D016133:Polymerase Chain Reaction; D066247:Receptor, ErbB-4; D059010:Single-Cell Analysis; D013997:Time Factors; D059467:Transcriptome
chemical_list: D005456:Fluorescent Dyes; C587689:ERBB4 protein, human; D066246:ErbB Receptors; C587690:Erbb4 protein, mouse; D066247:Receptor, ErbB-4; D005968:Glutamate Decarboxylase; C402565:glutamate decarboxylase 1; D002118:Calcium
keywords: AP; CC; EB; MEFs; NE; R(IN); V(R); VC; action potential; current clamp; embryoid bodies; input resistance.; mouse embryonic fibroblasts; neuroepithelial (rosettes); resting membrane potential; voltage clamp