Abstract
Medulloblastoma and high-grade glioma represent the most aggressive and frequent lethal solid tumors affecting individuals during pediatric age. During the past years, several models have been established for studying these types of cancers. Human organoids have recently been shown to be a valid alternative model to study several aspects of brain cancer biology, genetics and test therapies. Notably, brain cancer organoids can be generated using genetically modified cerebral organoids differentiated from human induced pluripotent stem cells (hiPSCs). However, the protocols to generate them and their downstream applications are very rare. Here, we describe the protocols to generate cerebellum and forebrain organoids from hiPSCs, and the workflow to genetically modify them by overexpressing genes found altered in patients to finally produce cancer organoids. We also show detailed protocols to use medulloblastoma and high-grade glioma organoids for orthotopic transplantation and co-culture experiments aimed to study cell biology in vivo and in vitro, for lineage tracing to investigate the cell of origin and for drug screening. The protocol takes 60–65 d for cancer organoids generation and from 1–4 weeks for downstream applications. The protocol requires at least 3–6 months to become proficient in culturing hiPSCs, generating organoids and performing procedures on immunodeficient mice.
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Acknowledgements
This project has received funding from the Giovanni Armenise-Harvard Foundation, USA (Career Development Award 2016, to L.T.), Pezcoller Foundation, CARITRO (to L.T.), My First AIRC Grant, Italy (Project Code: 19921 to L.T.) and EMBO YIP small grant to L.T. F.A has received support by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska Curie grant agreement no. 844677 (to F.A.) and by the University of Trento under the action MSCA+1. F.A. was also supported by Fondazione Umberto Veronesi postdoctoral fellowship (il Dono di Rossana). M.G. has been supported by a doctoral fellowship funded by Fondazione Pezcoller. We thank S. Robbiati (MOF facility), M. Pancher (HTS facility), M. Cardano (Cell Technology facility), G. Scarduelli and M. Roccuzzo (AICF) of the University of Trento.
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F.A., C.L., M.G., L.S., G.L., C.B. and M.A. performed the described procedures. F.A., C.L., M.G. and L.T. wrote the manuscript with support from G.L. and L.S. All the authors prepared the figures. F.A. and L.T. supervised the work.
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Nature Protocols thanks Anna Golebiewska, Amanda Linkous and Hongjun Song for their contribution to the peer review of this work.
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Key references using this protocol
Ballabio, C. et al. Nat. Commun. 11, 583 (2020): https://doi.org/10.1038/s41467-019-13989-3
Ballabio, C. et al. Sci. Adv. 7, eabd2781 (2021): https://doi.org/10.1126/sciadv.abd2781
Antonica, F. et al. Nat. Commun. 13, 4767 (2022): https://doi.org/10.1038/s41467-022-32448-0
Extended data
Extended Data Fig. 1 Expected outcomes and protocol failures for cerebellum and forebrain organoids generation.
a, Representative images of good outcomes and failures of hiPSCs maintenance. The dotted line indicates the empty degenerated region. b, Representative images of good outcomes and failures of cerebellum organoids formation in 96 V-bottom well (days 0-21). c, Representative images of good outcomes and failures of cerebellum organoids maintenance in suspension before electroporation (days 21-35). d, Representative images of good outcomes and failures of MB organoids after electroporation (days 35 + 25). e, Representative images of good outcomes and failures of forebrain organoids formation in 96 V-bottom well (days 0-18). f, Representative images of good outcomes and failures of forebrain organoids maintenance in suspension before electroporation (days 18-35). g, Representative images of good outcomes and failures of HGG organoids after electroporation (days 35 + 30). Images were acquired using a Leica DM IL LED microscope equipped with 5× objective and a fluorescent lamp. Scale bar, 200 μm.
Extended Data Fig. 2 Necessary equipment for transplantation procedure.
a, Representative image of the necessary equipment to perform the transplantation procedure of organoids in immunocompromised mice (Step 36A). b, Representative image showing the equipment setup of Hamilton syringe and pups adaptor on the stereotactic apparatus.
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Lago, C., Gianesello, M., Santomaso, L. et al. Medulloblastoma and high-grade glioma organoids for drug screening, lineage tracing, co-culture and in vivo assay. Nat Protoc 18, 2143–2180 (2023). https://doi.org/10.1038/s41596-023-00839-2
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DOI: https://doi.org/10.1038/s41596-023-00839-2
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