Human induced pluripotent stem cells (iPSCs) have great potential for disease modelling. However, generating iPSC-derived models to study brain diseases remains a challenge. In particular, the ability to differentiate cerebellar neurons from pluripotent stem cells is still limited. Recently, we described the long-term culture of functional cerebellar neurons from human iPSCs, which were maintained in cultures for up to 145 days without the need for co-culturing when re-plated on laminin. Dendritic spine morphology analyses, calcium imaging, and electrophysiological evaluation strongly indicated an efficient maturation of cerebellar precursors, both at the individual cell level and at the level of network connectivity. Nevertheless, defining protocols that allow the production of large numbers of organoids and a high yield of mature neurons maintaining the 3D structure is still difficult. Here, we present a new approach for the reproducible and scalable generation of cerebellar organoids under chemically defined conditions by using the novel PBS Vertical-Wheel single-use bioreactors. RNA sequencing (RNA-seq) was performed to evaluate the global gene expression profiles for different time points of cerebellar differentiation in the dynamic system and compared with the static 3D cerebellar differentiation. Gene expression profiles showed no impact of agitation in the efficacy of cerebellar differentiation. Additionally, the data suggests that cerebellar organoids obtained in bioreactors induced a 3D microenvironment enriched in extracellular matrix when compared with static conditions. Furthermore, different types of functional cerebellar neurons, including Granule cells (PAX6+ and MAP2+) and Purkinje cells (Calbindin+) were readily identified in cryosections of the organoids generated from this dynamic culture system. Thus, we describe for the first time the continuous and scalable generation of human iPSC-derived neural organoids that display cerebellar identity, as well as the generation of functional cerebellar neurons that were maintained in suspension for as long as 3 months, without feeder layers. This culture system increased reproducibility between experiments and allowed large-scale production of cerebellar organoids, which is crucial for future applications in drug screening and toxicological testing, as well as to study the pathological pathways involved in cerebellar dysfunction. 

Large scale production of cerebellar organoids from human pluripotent stem cells using the novel vertical-wheel single-use bioreactors

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.