High-resolution transcriptional landscape of xeno-free human induced pluripotent stem cell-derived cerebellar organoids

Abstract

Current protocols for producing cerebellar neurons from human pluripotent stem cells (hPSCs) often rely on animal co-culture and mostly exist as monolayers, limiting their capability to recapitulate the complex processes in the developing cerebellum. Here, we employed a robust method, without the need for mouse co-culture to generate three-dimensional cerebellar organoids from hPSCs that display hallmarks of in vivo cerebellar development. Single-cell profiling followed by comparison to human and mouse cerebellar atlases revealed the presence and maturity of transcriptionally distinct populations encompassing major cerebellar cell types. Encapsulation with Matrigel aimed to provide more physiologically-relevant conditions through recapitulation of basement-membrane signalling, influenced both growth dynamics and cellular composition of the organoids, altering developmentally relevant gene expression programmes. We identified enrichment of cerebellar disease genes in distinct cell populations in the hPSC-derived cerebellar organoids. These findings ascertain xeno-free human cerebellar organoids as a unique model to gain insight into cerebellar development and its associated disorders.

Human cerebral organoids — a new tool for clinical neurology research

“…This process is relevant to developmental disorders, as rhombic lip dysfunction has been associated with cerebellar vermis hypoplasia, also known as Dandy–Walker malformation 71 , 72 , and has been identified as the source of medulloblastoma group 3 and 4 — a severe childhood brain tumour 73 , 74 . Human cerebellar organoid models 75 that include rhombic lip progenitors 76 have recently been developed, enabling investigation of cerebellar development in humans.…”

“…During the initial culture period, organoids can be patterned to develop into representations of specific brain regions. Various protocols enable patterning for dorsal 96 , 99 , 100 , 161 , 283 – 285 or ventral forebrain 63 – 65 , thalamus 101 , hypothalamus 161 , midbrain 161 , 240 , 286 , hindbrain 287 and cerebellum 75 , 76 . b | Mature organoids recapitulate developmental hallmarks of the human brain, including ventricular zone (VZ) structures that contain apical radial glia, subventricular zone (SVZ) areas that contain intermediate progenitors and outer radial glia, and an emerging cortical plate (CP) that contains neurons.…”

“…Besides improving culture conditions, a major focus in recent years has been to create increasingly precise organoid models of specific brain areas. Protocols have been developed for models of the forebrain, midbrain 197 , 240 , spinal cord 241 , cerebellum 75 , 76 and other brain regions 242 . These approaches are likely to improve the reproducibility and robustness of models by minimizing the inherent variability of the progenitor pool.…”

AUTS2 Syndrome: Molecular Mechanisms and Model Systems

“…Mouse models of AUTS2 deficiency show cerebellar hypoplasia, impaired dendrite maturation in Purkinje cells, increased parallel fiber synapse formation, and decreased number of excitatory presynaptic synapses from climbing fiber innervations ( Yamashiro et al, 2020 ). Previous work utilizing a polarized cerebellar organoid model identified similarities in human cerebellar ontogenesis regarding the layered neural-tube-like structure with dorsoventral and apicobasal polarities ( Muguruma et al, 2015 ; Silva et al, 2020 ; Nayler et al, 2021 ). Cerebellar organoids derived from AUTS2 syndrome patients could be generated to further understand multiple aspects of cerebellar development, such as cellular differentiation, gene expression changes within specific cell types (i.e., sc RNA-seq focusing on Purkinje cells, granular cells, etc.…”

Modeling Brain Tumors: A Perspective Overview of in vivo and Organoid Models

“…Finally, GEBOs might be a valid model to investigate the cell of origin of glioma as already shown to be feasible for Group 3 Medulloblastoma ( Ballabio et al, 2021 ). Generation of cerebellar as well as cerebral organoids from hiPSCs relies on well-established protocols ( Muguruma et al, 2015 ; Velasco et al, 2019 ) that generate structures resembling fetal cerebellum or brain also from molecular points of view ( Velasco et al, 2019 ; Nayler et al, 2021 ).…”