Human brain development requires generating diverse cell types, a process explored by single-cell transcriptomics. Through parallel meta-analyses of the human cortex in development (seven datasets) and adulthood (16 datasets), we generated over 500 gene co-expression networks that can describe mechanisms of cortical development, centering on peak stages of neurogenesis. These meta-modules show dynamic cell subtype specificities throughout cortical development, with several developmental meta-modules displaying spatiotemporal expression patterns that allude to potential roles in cell fate specification. We validated the expression of these modules in primary human cortical tissues. These include meta-module 20, a module elevated in FEZF2⁺ deep layer neurons that includes TSHZ3, a transcription factor associated with neurodevelopmental disorders. Human cortical chimeroid experiments validated that both FEZF2 and TSHZ3 are required to drive module 20 activity and deep layer neuron specification but through distinct modalities. These studies demonstrate how meta-atlases can engender further mechanistic analyses of cortical fate specification.

Epithelial membrane protein 3 (EMP3) is an N-glycosylated tetraspanin with a putative trafficking function. It is highly expressed in isocitrate dehydrogenase-wild-type glioblastoma (IDH-wt GBM), and its high expression correlates with poor survival. However, the exact trafficking role of EMP3 and how it promotes oncogenic signaling in GBM remain unclear. Here, we show that EMP3 promotes EGFR/CDK2 signaling by regulating the trafficking and enhancing the stability of EGFR. BioID2-based proximity labeling revealed that EMP3 interacts with endocytic proteins involved in the vesicular transport of EGFR. EMP3 knockout (KO) enhances epidermal growth factor (EGF)-induced shuttling of EGFR into RAB7 + late endosomes, thereby promoting EGFR degradation. Increased EGFR degradation is rescued by the RAB7 negative regulator and novel EMP3 interactor TBC1D5. Phosphoproteomic and transcriptomic analyses further showed that EMP3 KO converges into the inhibition of the cyclin-dependent kinase CDK2 and the repression of EGFR-dependent and cell cycle transcriptional programs. Phenotypically, EMP3 KO cells exhibit reduced proliferation rates, blunted mitogenic response to EGF, and increased sensitivity to the pan-kinase inhibitor staurosporine and the EGFR inhibitor osimertinib. Furthermore, EGFR-dependent patient-derived glioblastoma stem cells display a transcriptomic signature consistent with reduced CDK2 activity, as well as increased susceptibility to CDK2 inhibition upon EMP3 knockdown. Lastly, using TCGA data, we showed that GBM tumors with high EMP3 expression have increased total and phosphorylated EGFR levels. Collectively, our findings demonstrate a novel EMP3-dependent mechanism by which EGFR/CDK2 activity is sustained in GBM. Consequently, EMP3’s stabilizing effect provides an additional layer of tumor cell resistance against targeted kinase inhibition.

Epithelial membrane protein 3 (EMP3) is a tetraspan membrane protein overexpressed in isocitrate dehydrogenase-wild-type (IDH-wt) glioblastoma (GBM). Several studies reported high EMP3 levels as a poor prognostic factor in GBM patients. Experimental findings based on glioma and non-glioma models have demonstrated the role of EMP3 in the regulation of several membrane proteins known to drive IDH-wt GBM. In this review, we summarize what is currently known about EMP3 biology. We discuss the regulatory effects that EMP3 exerts on a variety of oncogenic receptors and discuss how these mechanisms may relate to IDH-wt GBM. Lastly, we enumerate the open questions towards EMP3 function in IDH-wt GBM.

Characterization of cell type emergence during human cortical development, which enables unique human cognition, has focused primarily on anatomical and transcriptional characterizations. Metabolic processes in the human brain that allow for rapid expansion, but contribute to vulnerability to neurodevelopmental disorders, remain largely unexplored. We performed a variety of metabolic assays in primary tissue and stem cell derived cortical organoids and observed dynamic changes in core metabolic functions, including an unexpected increase in glycolysis during late neurogenesis. By depleting glucose levels in cortical organoids, we increased outer radial glia, astrocytes, and inhibitory neurons. We found the pentose phosphate pathway (PPP) was impacted in these experiments and leveraged pharmacological and genetic manipulations to recapitulate these radial glia cell fate changes. These data identify a new role for the PPP in modulating radial glia cell fate specification and generate a resource for future exploration of additional metabolic pathways in human cortical development.

A major obstacle to identifying effective therapies for the aggressive brain tumor glioblastoma is the lack of human-specific, immunocompetent models that reflect the human tumor microenvironment. To address this, we developed the immune-Human Organoid Tumor Transplantation (iHOTT) model. This is an autologous co-culture platform that integrates patient-derived tumor cells and matched peripheral blood mononuclear cells (PBMCs) within human cortical organoids, enabling the study of the patient-specific immune response to the tumor and tumor-immune interactions. This platform preserves tumor and immune populations, immune signaling, and cell-cell interactions observed in patient tumors. Treatment of iHOTT with pembrolizumab, a checkpoint inhibitor, mirrored cell type shifts and cell interactions observed in patients. TCR sequencing further revealed pembrolizumab-driven expansion of stem-like CD4-T-cell clonotypes exhibiting patient-specific repertoires. These findings establish iHOTT as a physiologically relevant platform for exploring autologous tumor–immune interactions and underscore the critical need for antigen-targeted strategies to enhance immunotherapy in glioblastoma.

According to the protocortex hypothesis, extrinsic thalamic signaling is necessary for refining cortical areas and cell types, but the mechanism by which these inputs shape the development and expansion of the human cortex remains largely unexplored. We fuse cortical and thalamic organoids to study this process. Using single-nuclei RNA-sequencing and cellular imaging, we discover that thalamic signals during a critical period promote human cortical upper-layer neurogenesis. In assembloid models and human primary cortex, we find NRXN1 mediates thalamic axon contact with primate-enriched outer radial glia, driving developmental gene expression changes. Genetic perturbation of NRXN1 in thalamic neurons reduces these contacts and attenuates cortical upper-layer neurogenesis. These findings in human developmental models suggest a novel role for thalamic regulation of primate outer radial glia cell fate.