Evolutionary development of the human brain is characterized by the expansion of various brain regions. Here, we show that developmental processes specific to humans are responsible for malformations of cortical development (MCDs), which result in developmental delay and epilepsy in children. We generated a human cerebral organoid model for tuberous sclerosis complex (TSC) and identified a specific neural stem cell type, caudal late interneuron progenitor (CLIP) cells. In TSC, CLIP cells over-proliferate, generating excessive interneurons, brain tumors, and cortical malformations. Epidermal growth factor receptor inhibition reduces tumor burden, identifying potential treatment options for TSC and related disorders. The identification of CLIP cells reveals the extended interneuron generation in the human brain as a vulnerability for disease. In addition, this work demonstrates that analyzing MCDs can reveal fundamental insights into human-specific aspects of brain development.

The current understanding of neurological diseases is derived mostly from direct analysis of patients and from animal models of disease. However, most patient studies do not capture the earliest stages of disease development and offer limited opportunities for experimental intervention, so rarely yield complete mechanistic insights. The use of animal models relies on evolutionary conservation of pathways involved in disease and is limited by an inability to recreate human-specific processes. In vitro models that are derived from human pluripotent stem cells cultured in 3D have emerged as a new model system that could bridge the gap between patient studies and animal models. In this Review, we summarize how such organoid models can complement classical approaches to accelerate neurological research. We describe our current understanding of neurodevelopment and how this process differs between humans and other animals, making human-derived models of disease essential. We discuss different methodologies for producing organoids and how organoids can be and have been used to model neurological disorders, including microcephaly, Zika virus infection, Alzheimer disease and other neurodegenerative disorders, and neurodevelopmental diseases, such as Timothy syndrome, Angelman syndrome and tuberous sclerosis. We also discuss the current limitations of organoid models and outline how organoids can be used to revolutionize research into the human brain and neurological diseases.

Organoids enable in vitro modeling of complex developmental processes and disease pathologies. Like most 3D cultures, organoids lack sufficient oxygen supply and therefore experience cellular stress. These negative effects are particularly prominent in complex models, such as brain organoids, and can affect lineage commitment. Here, we analyze brain organoid and fetal single‐cell RNA sequencing (scRNAseq) data from published and new datasets, totaling about 190,000 cells. We identify a unique stress signature in the data from all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined subpopulation of cells that is unique to organoids and does not affect neuronal specification or maturation. We have developed a computational algorithm, Gruffi, which uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. We validated our method using six additional datasets from different organoid protocols and early brains, and show its usefulness to other organoid systems including retinal organoids. Our data show that the adverse effects of cell stress can be corrected by bioinformatic analysis for improved delineation of developmental trajectories and resemblance to in vivo data.

29Although the intricate and prolonged development of the human brain critically 30 distinguishes it from other mammals 1 , our current understanding of 31 neurodevelopmental diseases is largely based on work using animal models. Recent 32 studies revealed that neural progenitors in the human brain are profoundly different 33 from those found in rodent animal models [2][3][4][5] . Moreover, post-mortem studies 34 revealed extensive migration of interneurons into the late-gestational and post-natal 35 human prefrontal cortex that does not occur in rodents 6 . Here, we use cerebral 36 organoids to show that overproduction of mid-gestational human interneurons 37 causes Tuberous Sclerosis Complex (TSC), a severe neuro-developmental disorder 38 associated with mutations in TSC1 and TSC2. We identify a previously 39 uncharacterized population of caudal late interneuron progenitors, the CLIP-cells. In 40 organoids derived from patients carrying heterozygous TSC2 mutations, 41 dysregulation of mTOR signaling leads to CLIP-cell over-proliferation and formation 42 of cortical tubers and subependymal tumors. Surprisingly, second-hit events 43 resulting from copy-neutral loss-of-heterozygosity (cnLOH) are not causative for but 44 occur during the progression of tumor lesions. Instead, EGFR signaling is required 45 for tumor proliferation, opening up a promising approach to treat TSC lesions. Our 46 study demonstrates that the analysis of developmental disorders in organoid models 47 can lead to fundamental insights into human brain development and neuropsychiatric 48 disorders. 49Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder 50 characterized by pathological malformations in multiple organs 7 . Among those, brain 51 defects leading to severe neuropsychiatric symptoms like autism spectrum disorder 52 (ASD), intractable seizures and intellectual disability (ID) are most debilitating and 53 seen in the majority of patients 8 . Most patients have cortical tubers 9 , focal dysplastic 54 regions in the cortex that are diagnosed by MRI and consist of dysmorphic neurons 55 and giant cells. In addition, 80% of the patients display subependymal nodules 56 (SEN) that form along the lateral ventricle and can develop into subependymal giant 57 cell astrocytomas (SEGAs) in 10-15% of the patients 7,10 . It was thought that TSC 58 pathogenesis is initiated by constitutive mTOR activity resulting from inactivation of 59 the second allele 11 along the lines of the classic Knudson two-hit hypothesis of 60 tumorigenesis 12 . This is supported by existing mouse models and a spheroid model 61 for TSC, as characteristic brain alterations are observed exclusively in Tsc1 or Tsc2 62 homozygous mutant mice and spheroids [13][14][15][16][17][18] . Genetic analysis in patients, however, 63 revealed that loss of the second allele is frequent in SEN/SEGA, but rare in cortical 64 tubers 19-22 , conflicting with the two-hit hypothesis. In addition, the cellular origins of 65 cortical tubers and SEN/SEGAs remain unclear. Interesting...

Organoids enable disease modeling in complex and structured human tissue, in vitro. Like most 3D models, they lack sufficient oxygen supply, leading to cellular stress. These negative effects are particularly prominent in complex models, like brain organoids, where they can prevent proper lineage commitment. Here, we analyze brain organoid and fetal single cell RNA sequencing (scRNAseq) data from published and new datasets totaling over 190,000 cells. We describe a unique stress signature found in all organoid samples, but not in fetal samples. We demonstrate that cell stress is limited to a defined organoid cell population, and present Gruffi, an algorithm that uses granular functional filtering to identify and remove stressed cells from any organoid scRNAseq dataset in an unbiased manner. Our data show that adverse effects of cell stress can be corrected by bioinformatic analysis, improving developmental trajectories and resemblance to fetal data.

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Radiotherapy can act as an in situ vaccine thereby activating tumor-specific immune responses that prevent tumor outgrowth in treated patients. While carbon ion radiotherapy has shown superior biophysical properties over conventional photon irradiation, the immunological effects induced have remained largely uncovered. The combination of radiotherapy with immune checkpoint inhibition (radioimmunotherapy) aims at further enhancement of anti-tumor immunity; however, studies on the immune cell composition in irradiated and distant tumors following radioimmunotherapy with carbon ions are scarce. We have established a bilateral tumor model by time shifted transplantation of murine, Her2+ EO771 tumor cells onto the flanks of immune competent mice followed by selective irradiation of the primal tumor, while sparing the consecutive tumor. We demonstrate that αCTLA4- but not αPD-L1-based radioimmunotherapy induces complete tumor rejection in our model. Intriguingly, local tumor control caused in situ immunization resulting even in eradication of non-irradiated, distant tumors. Moreover, cured mice were protected against EO771 rechallenge indicative of long lasting, tumor-protective immunological memory. Deconvolution of the treatment induced immunological effects by single cell RNA-sequencing (scRNA-seq) and concomitant flow cytometric analyses revealed in irradiated tumors predominating myeloid cells that developed into distinct tumor-associated macrophage clusters with upregulated expression of TNF and IL1 responsive genes, as well as activation of NK cells. Non-irradiated tumors showed higher frequencies of naïve T cells in irradiated mice, which were activated when combined with CTLA4 blockade. In conclusion, radioimmunotherapy with carbon ions plus CTLA4 inhibition reshapes the tumor-infiltrating immune cell composition and can induce complete rejection even of non-irradiated tumors. Our data present a rationale to combine radiotherapy approach with CTLA4 blockade to achieve durable anti-tumor immunity. Evaluation of future radioimmunotherapy approaches should thus not only focus on the immunological impacts at the site of irradiation but should also consider systemic immunological effects that might affect outgrowth of non-irradiated tumors.