• hiPS
• in vitro culture
• neurodevelopment
• Schizophrenia

The study of human brain development and related alterations remains a great challenge. Animal models provide valuable insights into the molecular, cellular, and behavioral aspects of neurodevelopment; however, it is important to acknowledge their limitations in studying complex and polygenic disorders such as schizophrenia (SCZ). Indeed, in the last decades, in vitro models have revealed their potential to mimic and recapitulate human brain neurodevelopment and affections. In this PhD project, the induced pluripotent stem cell (hiPSC) platform was used to investigate the molecular basis of SCZ disorder, a severe psychiatric disorder with a world-wide prevalence of 1%. Although the characteristic symptoms of SCZ, such as hallucinations and psychosis, manifest late in adolescence, it has neurodevelopmental origins with a prominent genetic basis. Given the high heterogeneity of clinical symptoms in SCZ, focusing attention on a subgroup of patients that share a common, quantifiable, and hereditable trait (endophenotype) could be a valid strategy in studying SCZ disorder. In particular, sleep spindles, generated by the interplay of the thalamic reticular nucleus (TRN – wholly composed of GABAergic neurons) with the dorsal thalamus, have been identified as endophenotype for this disorder.
In this PhD Thesis, in collaboration with Prof. Armando D’Agostino (ASST Santi Paolo e Carlo) and Prof. Luciano Conti (Department of Cellular, Computational and Integrative Biology - CIBIO - University of Trento), the objective was to elucidate the cellular and molecular bases that interrelate the sleep endophenotype and alterations in hiPSCs derived from SCZ patients. SCZ patients recruited for this study were drug-naïve at the first episode of psychosis and presented sleep alteration as a common endophenotype. In high-density EEG analyses to provide topographic characterization of the sleep spindle, there was a significant reduction in the average frequency of spindles with centroparietal topography in SCZ patients with respect to controls. To model this alteration and elucidate mechanisms in a relevant neural population (TRN neurons), in collaboration with the Luciano Conti group, we used the Sendai virus method to generate a collection of hiPSCs from SCZ patients and respective controls (CTRL).
After obtaining fully characterized hiPSCs, we focused on optimization of the TRN differentiation protocol and identification of human TRN neurodevelopmental markers. Regarding the second point, there was a knowledge gap for specific TRN neurodevelopmental markers. Therefore, developmental trajectories of a panel of selected markers (described in animal models and predicted to be conserved in humans) were analyzed using human tissue from the Human Developmental Biology Resource (HDBR) biobank. Specific and shared expression patterns in various developing brain regions provided new knowledge regarding activation of genetic machinery during development. Indeed, immunohistochemical analyses of human brain sections provided a window into the complex and unique aspects of human development, confirming that conservation of markers is required for formation of complex networks (e.g., corticothalamic/thalamocortical tracts).
Meanwhile, a protocol for TRN differentiation was optimized using a commercial-Sendai virus reprogrammed hiPSC line and a combination of insulin and CHIR99021. This protocol properly induced the diencephalic fate, with generation of TRN-GABAergic neurons. This protocol was applied to CTRL and SCZ-hiPSC lines to directly analyze characteristics of and identify aberrant phenotypes that mirrored clinical profiles and contributed to cellular characterization of the neurophysiological endophenotype. Analyzing expression of TRN-neurodevelopmental markers, morphological and functional features of SCZ-hiPSC neuro-derivatives enabled assessment of differentiation, from early stages to mature neurons; time-specific expression alterations may underlie generation of an impaired GABAergic neural population. In that regard, there were alterations in neural activity but not in the morphology of GABAergic neurons that may explain the sleep spindle dysfunction in SCZ patients.
This in vitro approach facilitated analysis of the neural population directly involved in the clinical phenotype, contributing to a deep understanding of complex neurodevelopmental psychiatric disorders such as SCZ. It is expected that the new knowledge derived from this study will contribute to future tailored treatments.