Project description:Neurons within the cerebellum form temporal-spatial connections through the cerebellum, and the entire brain. Organoid models provide an opportunity to model the early differentiation of the developing human cerebellum, which is difficult to study in vivo, and affords the opportunity to study neurodegenerative and neurodevelopmental diseases of the cerebellum. Previous cerebellar organoid models focused on early neuron generation and single cell activity. Here, we modify previous protocols to generate more mature cerebellar organoids that allow for the establishment of several classes of mature neurons during cerebellar differentiation and development, including the establishment of neural networks during whole organoid maturation. This will provide a means to study the generation of several more mature cerebellar cell types, including Purkinje cells, granule cells, interneurons expression as well as neuronal communication for biomedical, clinical, and pharmaceutical application.
Project description:Cerebellar organoids differentiated from human induced pluripotent stem cells (iPSCs) contain key cerebellar cell types and are increasingly used to study cerebellar diseases. In this study, we demonstrate the potential of cerebellar organoids for studying features of early human cerebellar development. Forkhead box protein P2 (FOXP2) is a transcription factor associated with speech and language development that is highly expressed in the developing brain. However, little attention has been directed to the study of FOXP2 in the early developing cerebellum. Here we used CRISPR gene editing in human iPSCs to generate a fluorescent FOXP2-reporter line. By combining transcriptomic analysis of iPSC-derived cerebellar organoids with published cerebellar datasets, we describe the expression and identify potential downstream targets of FOXP2 in the early developing human cerebellum. Our results highlight expression of FOXP2 in early human Purkinje cells and cerebellar nuclei neurons, and the vulnerability of these cell populations to neurodevelopmental disorders. Our study demonstrates the power of cerebellar organoids to model early human developmental processes and disorders.
Project description:Human induced pluripotent stem cells (iPSCs) have great potential for disease modeling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to recapitulate cerebellar development in vitro is still limited. We presented for the first time a reproducible and scalable production of cerebellar organoids by using the novel Vertical-Wheel single-use bioreactors, in which functional cerebellar neurons were obtained. Here, we evaluate the global gene expression profiles by RNA sequencing (RNA-seq) across cerebellar differentiation, demonstrating a faster cerebellar commitment in this novel dynamic differentiation protocol. Furthermore, transcriptomic profiles suggest a significant enrichment of extracellular matrix (ECM) in dynamic-derived cerebellar organoids, which can better mimic the neural microenvironment and support a consistent neuronal network. The presence of factors that favors angiogenesis onset was detected in dynamic condition, which can enhance functional maturation of cerebellar organoids. We anticipate that large-scale production of cerebellar organoids may help developing models for drug screening, toxicological tests and studying pathological pathways involved in cerebellar degeneration.
Project description:Faithful in vivo models of Group 4 medulloblastoma (G4 MB) are currently unavailable and recent studies showed the unique role of human-specific progenitors in the development of G3 and G4 MB. Here, we obtained cerebellar organoid (CbO) from expanded potential stem cells (EPSC) and developed them in vitro as 3D structures. We performed extensive characterisation of the epigenetic and transcriptomic profile of the mature CbO confirming the presence of sub compartment-specific cerebellar lineages linked to MB formation, including populations expressing signature genes reminiscent of the G3 and G4 MB cell-of-origin never described before. In parallel we have established a protocol to co-culture primary MB cells with our CbO to generate CbO-MB models. We confirmed that CbO sustains MB cells proliferation and invasion while preserving their molecular identity, and that CbO-MB recapitulated the efficacy of in vivo anti-tumour therapy, hence representing a suitable model to gain insight into G3/G4 MB pathogenesis and to set up pre-clinical drug screening. DNAme analysis of cerebellar organoids derived from 2 EPSC lines during development, then comparison of DNAme profiles of cerebellar organoids to human foetal cerebellum during development
Project description:Microcephaly and medulloblastoma result from mutations that compromise genomic stability. We report that Atr, which is mutated in the microcephalic disorder Seckel syndrome, is required to maintain chromosomal integrity during postnatal cerebellar neurogenesis. Atr deletion in cerebellar granule neuron progenitors (CGNPs) induced proliferation-associated DNA damage, p53 activation, apoptosis, and cerebellar hypoplasia. Co-deletions of either Bax and Bak or p53 prevented apoptosis in Atr-deleted CGNPs, but failed to fully rescue cerebellar growth. Atr-deficient CGNPs showed impaired cell cycle checkpoint function and continued to proliferate, accumulating chromosomal abnormalities. RNA-Seq demonstrated that the transcriptional response to Atr-deficient proliferation was p53-driven. Acute Atr inhibition in vivo by nanoparticle-formulated VE-822 reproduced the disruptions seen with Atr deletion. Our data show that p53-driven apoptosis and senescence, and non-apoptotic cell death redundantly limit growth in Atr-deficient progenitors. These overlapping mechanisms that suppress growth in Atr-disrupted CGNPs may be exploited for treatment of CGNP-derived medulloblastoma using Atr inhibition.