Understanding the complexities of the human brain and its associated disorders poses a significant challenge in neuroscience. Traditional research methods have limitations in replicating its intricacies, necessitating the development of in vitro models that can simulate its structure and function. Three-dimensional in vitro models, including organoids, cerebral organoids, bioprinted brain models, and functionalized brain organoids, offer promising platforms for studying human brain development, physiology, and disease. These models accurately replicate key aspects of human brain anatomy, gene expression, and cellular behavior, enabling drug discovery and toxicology studies while providing insights into human-specific phenomena not easily studied in animal models. The use of human-induced pluripotent stem cells has revolutionized the generation of 3D brain structures, with various techniques developed to generate specific brain regions. These advancements facilitate the study of brain structure development and function, overcoming previous limitations due to the scarcity of human brain samples. This technical review provides an overview of current 3D in vitro models of the human cortex, their development, characterization, and limitations, and explores the state of the art and future directions in the field, with a specific focus on their applications in studying neurodevelopmental and neurodegenerative disorders.

Background: Single-cell atlases have described diverse stem cell states in colorectal cancer (CRC), however, the overarching trajectories of those states and the underlying functional mechanisms, including their relevance for drug sensitivity, need better understanding. Methods: We established 64 patient-derived organoids from microsatellite-stable colorectal cancers, characterized their transcriptomes and genomes, and performed drug screening with 62-140 clinically approved substances. We analyzed additional published transcriptome data from patient-derived organoids (72 patients from three independent datasets), TCGA-CRC data (466 patients), and single-cell transcriptomes of tumor biopsies (123,000 cells from six independent cohorts) to establish a functional and molecular landscape of CRC stem cells. We performed mechanistic follow-up analyses by mass-spectrometry-based proteomics, large-scale kinase inhibition assays and immunofluorescence analyses. Results: We find a continuous landscape of CRC stem cells that is characterized by distinct developmental programs: adult stem cell- to fetal-like regenerative states and transition between differentiation programs. By large-scale drug perturbations and multi-omics modeling, we identify a regenerative/fetal-like stem cell trajectory characterized by PI3K/mTOR dependency. We find the identified developmental axes conserved in organoid, clinical, as well as single-cell data, and the fetal-like PI3K/mTOR-dependent state to be associated with poor clinical prognosis. Mechanistically, PI3K/mTOR vulnerability is linked to a lack of adaptive capability due to suppressed mRNA translation and associated with an upregulated SRC signaling network. Conclusions: Our work moves beyond a molecular CRC landscape by combined functional perturbation analyses in organoids. This enables mechanistic modeling of stem cell state regulation and identifies an SRC/mTOR-dependent regenerative state in CRC, which might allow improved therapeutic targeting in the future.