LAUSR

Background Characteristics of schizophrenia include the deregulation of the glutamatergic and the GABAergic neurotransmission and the degeneration of predominantly inhibitory interneurons within the cortex of the human brain. Cortical development was extensively studied in rodents, but the unique and complex architecture of the human cortex differs dramatically from brain of established animal models. Accordingly, the analysis of schizophrenia animal models is limited by the species differences, which requires the development of adequate human in vitro models mimicking the human cortical brain development. Schizophrenia is associated with DNA variations suggesting that genetic factors contribute to onset, progression and treatment of the disease. We generated induced pluripotent stem cells (iPS cells), which allow the generation of disease- and patient-specific neuronal cultures for the analysis of genetic factors. However, the functional characterization of genetic factors requires the analysis of advanced tissue-based 3D disease models. Therefore, we generated cerebral organoids for mimicking aspects of the human cortical development. Methods Human iPS cells from healthy donors and patient-specific iPS cells from schizophrenia patients were differentiated into permanent Neural Stem Cells (NSCs). Transcript and protein analysis confirmed the differentiation status of iPS cells and NSCs. NSCs were characterized in cellular 2D differentiation models. NSCs were further differentiated as free-floating neurospheres to acquire functional 3D organoids. Cells were analyzed by transcript and protein analysis. Cryosectioning of organoids was applied to visualize the distribution of different neuronal subtypes. Results Protein and transcript analysis of specific markers including OCT4 and the activity of alkaline phosphatases verified pluripotency in patient-derived iPS cells. The analysis of several NSC markers such as SOX2 and PAX6 indicated stable generation of permanent NSCs from patient-specific iPS cells. Patient-specific iPS cells were successfully differentiated into progenitor cells expressing a variety of neural lineage markers including neural makers such as TUBB3 and STX as wells as glia cell markers such as GFAP and O4. Immunostaining and whole-cell patch-clamp recordings of mature neurons showed the presence of different neuronal subtypes including inhibitory GABAergic neurons. NSCs were also successfully applied to the generation of 3D cerebral organoids. The 3D organoid differentiation model showed the RNA and protein expression of neural lineage markers similar to cells obtained from the 2D monolayer cultures. The expression pattern of cortical markers such as TBR1 observed in cryosections of organoids revealed the induction of cortical layers mimicking the developing human cortex. Discussion We successfully generated 3D cerebral organoids mimicking aspects of the early human brain development. These culture systems enable functional studies of healthy and diseased human cortical development for the analysis of neurodevelopmental diseases including schizophrenia.