A specific, non-immune system-related isoform of the human inducible nitric oxide synthase is expressed during differentiation of human stem cells into various cell types.

BACKGROUND: NOS2 expression is mostly found in bacteria-exposed or cytokine-treated tissues and is mostly connected to innate immune reactions. There are three isoforms of NOS2 (NOS2-1 to -3). In RNA-seq data sets, analyzing inflammatory gene expression, only expression of the NOS2-1 mRNA isoform is detected. However, the expression of NOS2 in differentiating human pluripotent stems (hPSCs) has not been analyzed yet. METHODS: Public available RNA-seq databases were screened for data of hPSCs during differentiation to different target cells. An isoform specific algorithm was used to analyze NOS2 mRNA isoform expression. In addition, we differentiated four different human iPSC cell lines toward cortical neurons and analyzed NOS2 mRNA expression by qRT-PCR and 5'-RACE. The functionality of the NOS2-2 protein was analyzed by transient transfection of expression clones in human DLD1 cells and nitrate measurement in the supernatant of these cells. RESULTS: In RNA-seq databases we detected a transient expression of the NOS2 mRNA during the differentiation of hPSCs to cardiomyocytes, chondrocytes, mesenchymal stromal cells, neurons, syncytiotrophoblast cells, and trophoblasts. NOS2 mRNA isoform specific analyses showed, that the transiently expressed NOS2 mRNA in differentiating hPSC (NOS2-2; "diff-iNOS") differ remarkably from the already described NOS2 transcript found in colon or induced islets (NOS2-1; "immuno-iNOS"). Also, analysis of the NOS2 mRNA- and protein expression during the differentiation of four different hiPSC lines towards cortical neurons showed a transient expression of the NOS2 mRNA and NOS2 protein on day 18 of the differentiation course. 5'-RACE experiments and isoform specific qRT-PCR analyses revealed that only the NOS2-2 mRNA isoform was expressed in these experiments. To analyze the functionality of the NOS2-2 protein, we transfected human DLD-1 cells with tetracycline inducible expression clones encoding the NOS2-1- or -2 coding sequence. After induction of the NOS2-1 or -2 mRNA expression by tetracycline a similar nitrate production was measured proofing the functionality of the NOS2-2 protein isoform. CONCLUSIONS: Our data show that a differentiation specific NOS2 isoform (NOS2-2) is transiently expressed during differentiation of hPSC. Video Abstract.

Protocol for the Standardized Generation of Forward Programmed Cryopreservable Excitatory and Inhibitory Forebrain Neurons.

This protocol describes a highly standardized pipeline for transcription factor-mediated forward programming of human pluripotent stem cells into highly enriched glutamatergic or GABAergic neurons followed by a cryopreservation step that enables the generation of large quality-controlled batches. This approach is particularly useful for reducing interexperimental variability in the context of collaborative studies across different locations and time points. For complete details on the use and execution of this protocol, please refer to Meijer et al. (2019) and Rhee et al. (2019).

Human stem cell-based models for studying autism spectrum disorder-related neuronal dysfunction.

The controlled differentiation of pluripotent stem cells (PSCs) into neurons and glia offers a unique opportunity to study early stages of human central nervous system development under controlled conditions in vitro. With the advent of cell reprogramming and the possibility to generate induced pluripotent stem cells (iPSCs) from any individual in a scalable manner, these studies can be extended to a disease- and patient-specific level. Autism spectrum disorder (ASD) is considered a neurodevelopmental disorder, with substantial evidence pointing to early alterations in neurogenesis and network formation as key pathogenic drivers. For that reason, ASD represents an ideal candidate for stem cell-based disease modeling. Here, we provide a concise review on recent advances in the field of human iPSC-based modeling of syndromic and non-syndromic forms of ASD, with a particular focus on studies addressing neuronal dysfunction and altered connectivity. We further discuss recent efforts to translate stem cell-based disease modeling to 3D via brain organoid and cell transplantation approaches, which enable the investigation of disease mechanisms in a tissue-like context. Finally, we describe advanced tools facilitating the assessment of altered neuronal function, comment on the relevance of iPSC-based models for the assessment of pharmaceutical therapies and outline potential future routes in stem cell-based ASD research.

Accuracy of expert predictions of seizure freedom after epilepsy surgery.

PURPOSE: To evaluate the accuracy of expert estimations of achieving seizure freedom after epilepsy surgery in the context of presurgical patient counseling. METHOD: The retrospective study comprised a random sample of 200 patients who underwent any type of resective epilepsy surgery at the University of Bonn Epilepsy Center and the routine 1-year postoperative control visit in the years from 2008-2016. The prediction by a team of epileptologists and the actual seizure outcome were extracted from the pre- and postsurgical medical files, respectively. A deviation of >10% was a priori defined as a relevant discrepancy. RESULTS: Estimated chances of achieving seizure freedom ranged from 30 to 90% (mean: 67%). The actual seizure freedom rate was 66% (Engel Ia/ ILAE 1a). Nine of 12 estimation categories showed a tolerable deviation of ≤10%, none of these with a worse than expected outcome. Two estimation categories (40-50%, and 80%) showed a worse actual seizure outcome with deviations of -40% (n = 3); and -17% (n = 30), respectively. All in all, for 83% of the patients a correct prediction was provided. CONCLUSIONS: For the vast majority of surgical patients, the expert prediction of postsurgical seizure freedom at the 1-year follow-up was accurate despite the heterogeneity of patients and surgical procedures.

An Autaptic Culture System for Standardized Analyses of iPSC-Derived Human Neurons.

iPSC-derived human neurons are expected to revolutionize studies on brain diseases, but their functional heterogeneity still poses a problem. Key sources of heterogeneity are the different cell culture systems used. We show that an optimized autaptic culture system, with single neurons on astrocyte feeder islands, is well suited to culture, and we analyze human iPSC-derived neurons in a standardized, systematic, and reproducible manner. Using classically differentiated and transcription factor-induced human glutamatergic and GABAergic neurons, we demonstrate that key features of neuronal morphology and function, including dendrite structure, synapse number, membrane properties, synaptic transmission, and short-term plasticity, can be assessed with substantial throughput and reproducibility. We propose our optimized autaptic culture system as a tool to study functional features of human neurons, particularly in the context of disease phenotypes and experimental therapy.

A Single-Cell Model for Synaptic Transmission and Plasticity in Human iPSC-Derived Neurons.

Synaptic dysfunction is associated with many brain disorders, but robust human cell models to study synaptic transmission and plasticity are lacking. Instead, current in vitro studies on human neurons typically rely on spontaneous synaptic events as a proxy for synapse function. Here, we describe a standardized in vitro approach using human neurons cultured individually on glia microdot arrays that allow single-cell analysis of synapse formation and function. We show that single glutamatergic or GABAergic forebrain neurons differentiated from human induced pluripotent stem cells form mature synapses that exhibit robust evoked synaptic transmission. These neurons show plasticity features such as synaptic facilitation, depression, and recovery. Finally, we show that spontaneous events are a poor predictor of synaptic maturity and do not correlate with the robustness of evoked responses. This methodology can be deployed directly to evaluate disease models for synaptic dysfunction and can be leveraged for drug development and precision medicine.