Transcriptome profiling of human pluripotent stem cell‐derived cerebellar organoids reveals faster commitment under dynamic conditions

© 2021 Wiley Periodicals LLC 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 a reproducible and scalable production of cerebellar organoids by using the novel single-use Vertical-Wheel 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. Thus, an efficient generation of organoids with cerebellar identity was achieved for the first time in a continuous process using a dynamic system without the need of organoids encapsulation in ECM-based hydrogels, allowing the possibility of large-scale production and application in high-throughput processes. The presence of factors that favors angiogenesis onset was also detected in dynamic conditions, 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. This study was supported by Fundação para a Ciência e a Tecnologia (FCT), Portugal (UIDB/04565/2020 through Programa Operacional Regional de Lisboa 2020, Project no. 007317, PD/BD/105773/2014 to Teresa P. Silva and SFRH/BD/147906/2019 to Rui Sousa-Luís), projects co-funded by FEDER (POR Lisboa 2020—Programa Operacional Regional de Lisboa PORTUGAL 2020) and FCT through Grant PAC-PRECISE LISBOA-01-0145-FEDER-016394 and CEREBEX Generation of Cerebellar Organoids for Ataxia Research Grant LISBOA-01-0145-FEDER-029298.