Development of a psychopharmacological screening platform for bipolar disorder using iPSC-derived cortical organoids – a personalized medicine approach.

Development of psychopharmacological screening platform using iPSC-derived cortical organoids; personalized medicine approach- Published findings on Lithium transcriptional and functional effects in cortical organoids (hCS). - Published findings on Longitudinal transcriptional analysis of SCZ organoids - Performing investigations on Clozapine transcriptional and functional effects in cortical organoids - Comparison of reprogramming methods-Lithium transcriptional and functional effects in cortical organoids (hCS). The manuscript was prepared and submitted to Molecular Psychiatry (IF 15.992), and successfully published (Osete et al., 2023). Briefly, we differentiated induced pluripotent stem cells (iPSCs) from 10 healthy controls (CTRL) and 11 bipolar disorder (BD) patients to 3-dimensional (3D) cortical spheroids (hCS). The hCS were differentiated in vitro for 5 months, and then treated for 1 month with lithium (Li) from day 150 until day 180. -Longitudinal transcriptional analysis of SCZ organoids. As we had established the protocol for hCS differentiation in our laboratory, we expanded the scope of our research from BD to schizophrenia (SCZ) and differentiated iPSCs from 14 CTRL and 14 SCZ donors to hCS. For this project, we collected RNA samples at different time points (days 0, 30, 90 and 150) to longitudinally assess the gene co-expression network analysis and carry out a comprehensive molecular characterization across 4 time points. The study has been recently published in the journal of Biological Psychiatry (IF10.6) (Akkouh et al., 2023). Clozapine (CLZ) transcriptional and functional effects in cortical organoids (hCS). Based on these accomplishments, we are now performing follow-up experiments with our cellular platform where we are extending our research to a more translational paradigm. In particular, we are using our IPSC-hCS model as a screening platform to investigate the molecular effects of the antipsychotic clozapine (CLZ). We anticipate that the results of this project will have academic and societal impact, as CLZ is the gold-standard drug of choice for treatment resistant SCZ (TRS) patients. The experimental design will consist of treatment of hCS from 14 SCZ and 14 CTRL lines with different relevant concentrations of CLZ (1µM, 100nM and 10nM). -Comparison of reprogramming methods (Epi, SeV and mRNA). We have successfully reprogrammed fibroblasts from 5 donors with 3 different reprogramming methods (messenger RNA transfections, Sendai virus transductions and Episomal vectors electroporation). Now we will start the differentiation of these iPSCs to midbrain dopaminergic neurons (mDA). Our goal will be to compare the differentiated dopaminergic neurons by means of RNA-sequencing, proteome and methylome analyses in order to identify protein, genetic and/or epigenetic aberrations associated with any of the methods in order to identify the clinically most suitable reprogramming method for clinical application.

Development of a psychopharmacological screening platform for bipolar disorder using iPSC-derived cortical organoids- Study on Lithium transcriptional and functional effects in cortical organoids from healthy controls patients with bipolar disorder (both Lithium responders and Lithium non-treated) - International collaborations established - Further attempts to use this psychopharmacological screening platform to investigate the molecular effects of other drugs-Lithium transcriptional and functional effects in cortical organoids (hCS). We recruited 21 donors: 10 healthy controls (CTRL) and 11 bipolar disorder (BD) patients (6 lithium responders, Li-R, and 5 lithium non-treated, Li-N). Fibroblasts were isolated from skin biopsies, and cells were reprogrammed to induced pluripotent stem cells (iPSCs). We differentiated those iPSCs to 3-dimensional (3D) cortical spheroids (hCS) following a highly reproducible protocol published in Nature Methods (Yoon et al., 2019). The hCS were differentiated in vitro during 5 months, and then treated for 1 month with lithium (Li) from day 150 until day 180. Interestingly, spatiotemporal gene expression analysis comparing the gene expression profiles of our hCS with the BrainSpan dataset showed resemblance of our in vitro cells with in vivo cortical tissue. Moreover, by performing differential expression analysis we identified novel genes associated with BD disease and with Li-treatment. Additionally, we recorded data for electrophysiological and mitochondrial characterization of the cells. Whole cell patch-clamp recordings identified decreased excitability in BD hCS compared with CTRL hCS. Moreover, by measuring mitochondrial activity with a Seahorse XFe24 extracellular flux analyser (Agilent), we found that Li increased mitochondrial reserve capacity in BD hCS. Lastly, we also identified that Li treatment regulates the secretion of pro-inflammatory cytokines differently in CTRL and BD cells. The manuscript has been submitted to Molecular Psychiatry (IF 2021-2022: 15.992). -Using this approach, we are currently making further attempts to use this psychopharmacological screening platform to investigate the molecular effects of other drugs such as clozapine, which is used in the clinic treatment-resistant schizophrenia patients. -Exosomal microRNA sequencing of lithium treated cortical organoids (hCS). As a follow-up of the BD hCS lithium treatment project, we are currently dealing with the isolation of microRNA from exosomes contained in supernatants that we collected from all 21 donor hCS without treatment at day 150, and with and without treatment at day 180. Our goal is to determine whether microRNA expression patterns can be associated with Li- treatment, and use it to generate a potential prediction tool of clinical outcome with direct clinical application for precision medicine. -Monitoring of human organoids implanted in mice reveal functional connection with visual cortex. In an international collaboration with researchers from University of San Diego, we have recently published a study in Nature Communications (Wilson et al., 2022). We show that chimeric transplantation of human organoids in adult mice can functionally connect to the sensory network. Combining transparent microelectrode arrays and two-photon imaging we established a longitudinal and multimodal monitoring system for organoids transplanted into the cortex of adult mice. -In another international collaboration, we demonstrated a link between SETD1A and the cAMP-dependent pathway in human neurons (Wang et al., 2022), by using CRISPR-Cas9 and generating excitatory/inhibitory neuronal networks. -We have established a novel collaboration with University of San Diego where we aim to: i) develop organoids from selected cohort of SCZ patients, test the expression of TCF4, and investigate if we can restore tCF4 expression using viral vectors and CRISPR editing technology.