Fig. 1. Ventricular progenitors are disrupted in Scz patient-derived organoids.

a Schematic of 3D cerebral organoid generation pipeline. Briefly, two-dimensional iPSC colonies were dissociated, cultured into 3D embryoid body aggregates, differentiated towards a neuroectodermal fate, embedded in a Matrigel droplet to provide a matrix for expansion, and maturated upon an orbital shaker. b Representative images of neural induction in 3D cerebral organoids. Shown is a representative image of a single ventricular zone from each group, exhibiting filamentous processes of neural stem cells (NESTIN+ cells), ventricular neural progenitors (SOX2+ cells), and their radially organized fibers which lead to dense neuronal fields (β-tubulin III; β3). c Scz ventricular zone neural progenitors exhibit increased apoptosis. Analysis of co-localization of cleaved (activated) CAS3+ with SOX2+ cells revealed that neural progenitor apoptosis was significantly elevated in Scz organoids compared to Ctrl organoids (total n = 20 iPSC donors; Ctrl n = 5 and Scz n = 15). Scz organoids exhibited increased progenitor death irrespective of ventricular zone size and exhibited stable rates of error across ventricular zones (data not shown). In follow-up orthogonal validation experiments, increased death was replicated in an independent cohort via single-cell analysis of DNA fragmentation, DNA damage induction, and expression of cleaved PARP (see Fig. S2). Together, these data establish progenitor entropy as an intrinsic Scz phenotype in cultures of 3D patient-derived neural tissue. d Schematic of progenitor neuropathology in Scz organoids. Cortical development is defined by the sequential differentiation of neural progenitors into neurons that reside within the developing cortical plate. Due to a disruption in the survival of ventricular progenitors, we next assessed neuron numbers in Ctrl and Scz organoids. Scz organoids exhibited a decrease in the numbers of MAP2+ neurons relative to Ctrls (total n = 21 iPSC donors; Ctrl n = 4 and Scz n = 17). e Neuronal differentiation is disrupted in Scz organoids. Last, we sought to determine if disrupted differentiation also contributes to neuronal depletion in Scz organoids. To label dividing progenitor cells and assess their fate, we used a 24hr BrdU pulse and 7d chase paradigm in a pseudorandomly selected cross-sectional cohort. Data is provided as a Sankey flow visualization so that differentiation can be evaluated as the proportion of all BrdU+ cells within a field. Approximately half of the BrdU+ cells differentiated into CTIP2+ early-born cortical neurons (48.6%) in Ctrl organoids. Contrary to this, Scz organoids exhibited a disrupted developmental trajectory whereby only 16.1% of cells differentiated into early-born CTIP2+ neurons (total n = 8 iPSC donors; n = 4 Ctrl and n = 4 Scz). While a trend for increased self-renewal of PAX6+ progenitors was detected in Scz organoids (43.7% BrdU+ cells) relative to Ctrls (34.4% BrdU+ cells), this comparison was not significant. Thus, in addition to progenitor death, Scz organoids exhibit fewer neurons in developing cortical fields due to a mechanism that restrained neurogenesis by altering the differentiation trajectory of neural progenitors. *p < 0.05, ****p < 0.0001. Error bars reflect Standard Error of the Mean. Scale bar: b and d = 60 µm, whole organoid panel in c = 60 µm, far right panel of c = 100 µm. Ctrl control, Scz schizophrenia, VZ ventricular zone.