2026 ECNP Congress

PS01-1157

Human iPSC-derived guided cortical organoids as a platform to study C4A-associated mechanisms in schizophrenia

Nadire Azizaj — J. Bitraj, B. Lana, G. Piovani, C. Campanelli, M. Fiore, G. Kikidis, G. Pergola, A. Rampino, E. Domenici, G. Collo · University of Brescia, Department of Molecular and Translational Medicine, Brescia, Italy

2026 ECNP Congress

Background

Schizophrenia is a complex, chronic, and highly heritable neuropsychiatric disorder characterized by alterations in cognition, perception, and behaviour. Its underlying biological mechanisms remain only partially understood, reflecting the intricate interplay between genetic susceptibility and neurodevelopmental processes. Among the strongest genetic risk factors, variation in complement component 4A (C4A) has emerged as a critical contributor [1]. Increased C4A gene expression has been associated with dysregulation of the complement cascade, a pathway traditionally linked to immune function but also involved in synaptic pruning during brain development. Abnormal activation of this system may lead to excessive synapse elimination, a feature implicated in the pathophysiology of schizophrenia.

Understanding how C4A variation contributes to disease-related molecular alterations remains a major challenge. In this context, induced pluripotent stem cell (iPSC) technology provides a powerful and versatile platform for modelling neuropsychiatric disorders in a patient-specific manner. Among iPSC-derived systems, guided 3D cortical organoids (gCOs) represent an advanced and physiologically relevant model. gCOs exhibit multicellular complexity, interconnected neuronal–glial networks, and partial laminar organization that recapitulates key stages of cortical development [2].

Aims

The aim of this study was to generate and characterize iPSC lines and gCOs derived from schizophrenia patients stratified by high and low C4A expression levels, to establish a robust platform for the investigation of C4A-related molecular signatures in cortical development.

Methods

iPSC lines were generated by reprogramming peripheral blood mononuclear cells from two patients with high C4A expression and two patients with low C4A expression. Selected clones were validated for pluripotency through morphological assessment, expression of pluripotency markers, trilineage differentiation potential and karyotype stability. iPSCs were differentiated into gCOs up to day 90 following an established protocol [3]. RT-qPCR and immunofluorescence assays were performed from day 15 to day 90 to assess cortical phenotype induction. Immunofluorescence studies were carried out with confocal microscopy (Zeiss LSM880 with Zeiss ZEN software).

Results

All generated iPSC lines displayed typical stem cell morphology and expressed key pluripotency markers such as OCT3/4, SOX2, SSEA4, and TRA-1-60 assessed via RT-qPCR and immunofluorescence. In addition, each line demonstrated capacity for trilineage differentiation, and maintained normal karyotype, confirming successful reprogramming. Upon directed cortical differentiation iPSC lines generated gCOs exhibiting progressive maturation over time with structural and molecular features consistent with early cortical development assessed by RT-qPCR and immunofluorescence in different timepoints. At day 15, early dorsal forebrain patterning was confirmed by expression of the neural progenitor and cortical markers PAX6, FOXG1, and TBR2. By day 25, sustained PAX6 and TBR2 expression indicated continued radial glia and intermediate progenitor activity. At day 50, the emergence of MAP2, TBR1, and VGLUT2 marked the transition toward deep-layer glutamatergic neuronal identity. By day 90, mature neuronal and early glial features were evidenced by expression of MAP2, BRN2, VGLUT1, and GFAP.

ConclusionsThis work establishes a characterized panel of patient-specific iPSC-derived gCOs with distinct C4A expression profiles. This platform provides a robust foundation for in vitro modelling of schizophrenia and for investigating complement-mediated mechanisms underlying disease pathophysiology.

References

  1. Sekar A, Bialas AR, de Rivera H, et al. Schizophrenia risk from complex variation of complement component 4. Nature. 2016;530(7589):177-183.
  2. Whiteley JT, Fernandes S, Sharma A, Mendes APD, Racha V, Benassi SK, Marchetto MC. Reaching into the toolbox: Stem cell models to study neuropsychiatric disorders. Stem Cell Reports. 2022;17:187-210.
  3. Yoon SJ, Elahi LS, Pașca AM, Marton RM, Gordon A, Revah O, Miura Y, Walczak EM, Holdgate GM, Fan HC, Huguenard JR, Geschwind DH, Pașca SP. Reliability of human cortical organoid generation. Nat Methods. 2019;16:75-78.