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Studying Neuronal Network Formation in Dissociated Cerebral Organoid Cultures

Review of “Self-Organized Synchronous Calcium Transients in a Cultured Human Neural Network Derived from Cerebral Organoids” from Stem Cell Reports by Stuart P. Atkinson

The generation of three-dimensional cerebral organoids from human pluripotent stem cells has enabled the modeling of both cerebral development and the pathogenesis of related diseases; however, limitations in this field include the inability to functionally evaluate neural network activity. Multiple studies have employed calcium imaging to characterize cerebral organoids, and the application of high-density silicon microelectrodes has provided the first evidence of network activity in organoids [1, 2]. In a recent Stem Cell Reports article, researchers led by Jun Takahashi (Kyoto University, Japan) employed dissociated human cerebral organoids to evaluate individual and synchronized patterns of neural network activity in the hope of implementing this approach to fully investigate the functionality of human neuronal networks [3].

Sakaguchi et al. employed a modified serum-free floating culture of embryoid body-like aggregates with quick reaggregation method [4] to successfully generate cerebral organoids with elongated epithelium and the spatially appropriate expression of marker proteins from human pluripotent stem cells. Subsequent three-dimensional immunohistochemistry and imaging of whole organoids through light-sheet microscopy confirmed the effective generation of cerebral organoids and intracellular calcium dynamics analysis using two-photon microscopy revealed spontaneous individual activity within organoids although little synchronized activity.

The authors then created dissociated cerebral organoid cultures for the more prolonged study of neural network activity and observed the generation of a self-organizing neuronal network that contained mature glutamatergic neurons and GABAergic neurons and synapse formation. Excitingly, analyses of these cultures by calcium imaging revealed both individual and synchronized activity of cerebral organoid-derived neural networks following extended time in culture. While glutamate represented the major excitatory transmitter, GABA acted as the major inhibitory transmitter within the network, and non-NMDA receptor activation generated synchronized bursts. Finally, treatment with a non-N-methyl-D-aspartic acid receptor inhibitor known to block electrical synaptic transmissions permitted the study of dynamic changes in the self-organizing activity of cerebral organoid-derived human neural networks.

Overall, the authors of this fascinating study anticipate that the generation in vitro active neuronal networks from human pluripotent stem cell-derived cerebral organoids will allow for advanced drug screening efforts and contribute to the development of psychiatric disease models through the application of patient- and disease-specific induced pluripotent stem cells [5], thus heralding “a novel paradigm of neuroscientific and neuropharmaceutical research on human brain function and neuropsychiatric disorders”.

For more on cerebral organoids, in vitro neural networks, and the development of advanced screening and modeling techniques, stay tuned to the Stem Cells Portal!

References

  1. Mansour AA, Gonçalves JT, Bloyd CW, et al., An in vivo model of functional and vascularized human brain organoids. Nature Biotechnology 2018;36:432.
  2. Quadrato G, Nguyen T, Macosko EZ, et al., Cell diversity and network dynamics in photosensitive human brain organoids. Nature 2017;545:48.
  3. Sakaguchi H, Ozaki Y, Ashida T, et al., Self-Organized Synchronous Calcium Transients in a Cultured Human Neural Network Derived from Cerebral Organoids. Stem Cell Reports 2019;In Press.
  4. Kadoshima T, Sakaguchi H, Nakano T, et al., Self-organization of axial polarity, inside-out layer pattern, and species-specific progenitor dynamics in human ES cell–derived neocortex. Proceedings of the National Academy of Sciences 2013;110:20284.
  5. Vadodaria KC, Amatya DN, Marchetto MC, et al., Modeling psychiatric disorders using patient stem cell-derived neurons: a way forward. Genome Medicine 2018;10:1.