Neurons derived from induced pluripotent stem cells on microelectrode arrays: a human model for neurodevelopmental disorders

Abstract

Event Abstract Back to Event Neurons derived from induced pluripotent stem cells on microelectrode arrays: a human model for neurodevelopmental disorders Monica Frega1, 2*, Katrin Linda1, 2, Britt Mossink1, 2, Jon-Ruben Van Rhijn1, 2, Jason Keller1, 2, Sebastian Van Gestel1, Dirk Schubert2, 3 and Nael Nadif Kasri1, 2, 3* 1 Radboud University Nijmegen Medical Centre, Netherlands 2 Donders Institute for Brain, Cognition and Behaviour, Radboud University, Netherlands 3 Radboud University Nijmegen, Deparment of Cognitive Neuroscience, Netherlands Motivation A major challenge in clinical genetics and medicine is represented by genetically and phenotypically highly diverse neurodevelopmental disorders (NDDs) affecting about 1–2% of the population, like intellectual disability (ID) and autism [1, 2]. Depending on specific etiology of different disorders, developmental deviations result in major impairments in cognition, communication, behavior and/or motor skills [1, 3]. Unfortunately, no effective treatments are yet available for NNDs. Identification of underlying genetic defects became increasingly efficient having great benefits for diagnostic and prognostic purposes. However, for the vast majority of NNDs there is little conceptual knowledge about neurobiological mechanisms the causing genes control at the cellular and network level. Understanding the neural circuit basis of NDDs is therefore imperial for a better understanding of these disorders and for the development of better treatment strategies. Here, we studied the effect of NDD genes-deficiency on neuronal activity in-vitro using neurons derived from induced Pluripotent Stem Cells (iPSC). Using micro-electrode arrays (MEAs) technology and whole-cell patch-clamp recordings we characterized the electrophysiological activity of neuronal cultures derived from healthy subjects and NDDs patients on both network- and single-cell level in vitro. In particular, here we focused on Kleefstra syndrome (KS), NND characterized by moderate-to-severe ID, autistic behaviour, microcephaly and dysmorphic features [4]. It is caused by heterozygous loss of Euchromatin Histone Methyltransferase 1 (EHMT1). To further corroborate our results, we also monitored the electrophysiological activity of neuronal cultures dissociated from rodent coupled to MEAs. Material and Methods Neurons derived from iPSC. iPSCs (reprogrammed from somatic cells of healthy subjects and NDDs patients) were directly derived into upper-layer cortical neurons by overexpressing the neuronal determinant neurogenin 2 (NGN2) upon doxycycline treatment [5]. The derived neurons were plated onto MEAs (Multi Channel Systems - MCS, Reutlingen, Germany) at a final density of 1200 cells/mm2. Rodent dissociated neuronal cultures. Dissociated neuronal cultures were obtained from cortex of embryonic mice and plated onto MEAs at a final density of 1200 cells/mm2. Analysis and statistics. Data were analysed using a custom software package developed in MATLAB© (The Mathworks, Natick, MA, USA) [6]. Data are expressed as mean ± standard error of the mean. Statistical analysis were performed with Mann–Whitney test. Results Here, we recorded the electrophysiological network activity of human neurons derived from healthy subjects (i.e. control) and KS patients grown on MEAs (n=12 for each line). In total, four control (2 independent and 2 isogenic) and four KS (2 independent and 2 isogenic) iPSCs lines were used for this study. Few days after plating on MEAs, the neurons derived from healthy subject formed functionally active neuronal networks, showing spontaneous events already during the second week in vitro. Late in development (i.e. fourth week in vitro) the neuronal network showed high level of spontaneous activity as well as regular network burst (i.e. synchronous events involving almost all channels of the MEAs). Neurons derived from KS patients established spiking activity during early network development as well as synchronous events involving most of the channels of the MEAs later in development, too (see raster plot in Figure 1A). During the fourth week in vitro, KS patient-derived neuronal networks exhibited a global level of activity compared to controls. However, the pattern of synchronous activity exhibited by KS patient-derived neuronal networks was very different compared to control condition. The number of synchronous events exhibited by all the KS-deficient neuronal networks was statistically lower than controls (Control: 4.29±0.33 burst/min; KS: 1,6±0.09 burst/min, Figure 1B). Furthermore, the synchronous events appeared with longer durations compared to controls (Control: 1.39±0.07 s; KS: 3.79±0.24 s). Finally, KS patient-derived neuronal networks exhibited an irregular bursting pattern. The results indicate that loss of function of EHMT1 leads to altered pattern of activity in neuronal networks composed by glutamatergic neurons. In addition, we found that homeostatic plasticity was impaired in neurons derived from KS patients iPSCs, indicating the inability of the neurons to maintain the stability of the neuronal network activity. Finally, we investigated whether the rescue of the phenotype exhibited by KS patient-derived neuronal networks was possible. After treatment, we found that the number of synchronous events exhibited by KS patient-derived neuronal network increased, approaching the frequency of the control cultures, together with a decrease in the burst duration and interval. To corroborate our results on KS neuronal network dysfunction, we monitored the activity cultures from wild-type (WT, n=12) and Ehmt1+/- mice (n=10) on MEAs. During the fourth week in vitro, the EHMT1-deficient network showed a level of activity similar to the control (WT, 2.0±0.2 spike/s; Ehmt1, 1.6±0.2 spike/s). However, the synchronized bursting activity remained impaired by means of significantly different bursting rates (WT 8.2±1.1 burst/min; Ehmt1 2.6±0.7 burst/min). Discussion In this study, we made use of recent advances in iPSCs technology and developed a human in vitro model of NDDs that enables us to study neuronal network deficits and to perform drug screening assays. In particular, here we showed neuronal networks and mechanistic (i.e. homeostatic plasticity) impairments in a human model for KS and we showed that it is possible to rescue a neuronal phenotype on MEAs. As our models produce reliable functional data on disease related network activity both on animal and human material, it allow us to perform disease specific pharmacological and genetic rescue experiments. Using this approach, we also found neuronal network impairments in other NDDs. We observed that each NND showed a particular neuronal network dysfunction, indicating a genotype-phenotype relationship. Conclusion In conclusion, our results indicate that iPSCs-derived neuronal networks coupled to MEAs is a powerful platform to study NNDs: it is a robust and sensitive method to perform genotype-phenotype analyses and drug screening assays. Figure 1 References [1] Mullin, A., Gokhale, A., Moreno-De-Luca, A., Sanyal, S., Waddington, J., & Faundez, V. “Neurodevelopmental disorders: mechanisms and boundary definitions from genomes, interactomes and proteomes”. Translational psychiatry, 3(12), e329, 2013. [2] Telias, M., & Ben-Yosef, D. “Modeling neurodevelopmental disorders using human pluripotent stem cells”. Stem Cell Reviews and Reports, 10(4), 494-511, 2014. [3] Kim, Y. S. “Recent challenges to the psychiatric diagnostic nosology: a focus on the genetics and genomics of neurodevelopmental disorders”. International journal of epidemiology, 43(2), 465-475, 2014. [4] Kleefstra, T. et al. “Loss-of-function mutations in euchromatin histone methyl transferase 1 (EHMT1) cause the 9q34 subtelomeric deletion syndrome”. The American Journal of Human Genetics, 79(2), 370-377, 2006. [5] Frega, M. et al. “Rapid neuronal differentiation of induced pluripotent stem cells for measuring network activity on micro-electrode arrays”. Journal of visualized experiments: JoVE, (119), 2017. [6] Bologna, L. L. et al. “Investigating neuronal activity by SPYCODE multi-channel data analyzer”. Neural networks: the official journal of the International Neural Network Society, 23, 685-697, (2010). Keywords: Human Induced Pluripotent Stem Cells, derived neurons, neurodevelopmental disorder, activity impairments, neuronal networks Conference: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays, Reutlingen, Germany, 4 Jul - 6 Jul, 2018. Presentation Type: Oral Presentation Topic: Stem cell-derived applications Citation: Frega M, Linda K, Mossink B, Van Rhijn J, Keller J, Van Gestel S, Schubert D and Nadif Kasri N (2019). Neurons derived from induced pluripotent stem cells on microelectrode arrays: a human model for neurodevelopmental disorders. Conference Abstract: MEA Meeting 2018 | 11th International Meeting on Substrate Integrated Microelectrode Arrays. doi: 10.3389/conf.fncel.2018.38.00011 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. 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Received: 18 Mar 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Monica Frega, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, monica.frega@radboudumc.nl Dr. Nael Nadif Kasri, Radboud University Nijmegen Medical Centre, Nijmegen, Netherlands, N.Nadif@donders.ru.nl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Monica Frega Katrin Linda Britt Mossink Jon-Ruben Van Rhijn Jason Keller Sebastian Van Gestel Dirk Schubert Nael Nadif Kasri Google Monica Frega Katrin Linda Britt Mossink Jon-Ruben Van Rhijn Jason Keller Sebastian Van Gestel Dirk Schubert Nael Nadif Kasri Google Scholar Monica Frega Katrin Linda Britt Mossink Jon-Ruben Van Rhijn Jason Keller Sebastian Van Gestel Dirk Schubert Nael Nadif Kasri PubMed Monica Frega Katrin Linda Britt Mossink Jon-Ruben Van Rhijn Jason Keller Sebastian Van Gestel Dirk Schubert Nael Nadif Kasri Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.