30943
High Throughput Screening of Human Induced Pluripotent Stem Cell (hiPSC)-Derived Organoids Reveals Cellular and Electrophysiological Phenotypes in Idiopathic Autism
Human induced pluripotent stem cells (hiPSCs) are an important tool for understanding the interaction between genetics and cellular function in autism. hiPSC-derived three dimensional (3D) organoid cultures, such as serum free embryoid bodies (SFEB), can mimic aspects of early corticogenesis, and therefore provide insights regarding potential neurodevelopmental perturbations with respect to autism. However, the utility of 3D organoids is restricted by variability and reproducibility, and require large sample sizes for sufficient statistical power. This can be addressed by application of high-throughput methods in screening for autism-relevant phenotypes.
Objectives:
We aimed to apply assays such as high-content imaging and multielectrode array (MEA) to screen for autism-relevant phenotypes in hiPSC-derived organoids. Our goal is to develop a platform to establish neuronal phenotypes in 3D cultures, and use these methodologies for phenotypic drug screening and translational medicine.
Methods:
We used the SFEB model optimized by Nestor et.al. (2013). SFEBs were differentiated from autism and control iPSCs by plating in a 96-well V-bottom plate for 14 days, after which the SFEBs were transferred to cell culture inserts and grown until 60 days in vitro (DIV). All imaging for immunocytochemistry was done using the ThermoFisher ArrayScan XTI. For electrophysiology, SFEBs were transferred to 48-well MEA plates coated with polyethylenimine and laminin. Baseline spontaneous activity as well as response to pharmacological agents was measured for 20 minutes. Calcium imaging was performed by transducing SFEBs with AAV9-GCamp6 and imaging for 2 minutes per well 7 days post transduction.
Results:
First, we determined if there were differences in cellular composition and morphology between autism and control SFEBs. Because excitatory/inhibitory balance has emerged as a possible hypothesis underlying autism neurobiology, we examined ratio of γ-aminobutyric acid (GABA)ergic and glutamategic populations within SFEBs. Using high content imaging, we found a decrease in GABAergic neurons in the autism cohort (n=270 SFEBs/line). Consistent with this finding some of our autism lines showed increased baseline spontaneous network-level synaptic activity (n = 72 SFEBs/line).dditionally, we found a potential deficit in synaptic plasticity based on the lack of response to induction of long-term potentiation with glycine. We have also demonstrated that calcium transients from individual neurons can be detected in control and autism-derived SFEBs using high content live-cell imaging. Activity from individual neurons will be correlated with field recordings from the MEA, as well as analysis of microcircuitry around active electrodes, to determine the contribution of individual neurons to network activity.
Conclusions:
In this study, we addressed the issue of experimental variability in 3D-organoids by adapting high-throughput approaches. Future studies would benefit from additional refinement of high-throughput methods, as well as an increased number of cell lines. Our approach increases the efficiency of screening for autism-relevant phenotypes, and provides a promising starting point for building phenotypic drug screening platforms.