A protocol for the generation of medulloblastoma and high-grade glioma organoids by electroporating, respectively, cerebellum and forebrain organoids, along with procedures for their use in drug screening, lineage tracing, co-culture and in vivo assays.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.

Medulloblastoma and high-grade glioma organoids for drug screening, lineage tracing, co-culture and in vivo assay / Lago, Chiara; Gianesello, Matteo; Santomaso, Lucia; Leva, Gloria; Ballabio, Claudio; Anderle, Marica; Antonica, Francesco; Tiberi, Luca. - In: NATURE PROTOCOLS. - ISSN 1750-2799. - 18:7(2023), pp. 2143-2180. [10.1038/s41596-023-00839-2]

Medulloblastoma and high-grade glioma organoids for drug screening, lineage tracing, co-culture and in vivo assay

Lago, Chiara;Gianesello, Matteo;Santomaso, Lucia;Leva, Gloria;Ballabio, Claudio;Anderle, Marica;Antonica, Francesco
;
Tiberi, Luca
2023-01-01

Abstract

A protocol for the generation of medulloblastoma and high-grade glioma organoids by electroporating, respectively, cerebellum and forebrain organoids, along with procedures for their use in drug screening, lineage tracing, co-culture and in vivo assays.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.
2023
7
Lago, Chiara; Gianesello, Matteo; Santomaso, Lucia; Leva, Gloria; Ballabio, Claudio; Anderle, Marica; Antonica, Francesco; Tiberi, Luca
Medulloblastoma and high-grade glioma organoids for drug screening, lineage tracing, co-culture and in vivo assay / Lago, Chiara; Gianesello, Matteo; Santomaso, Lucia; Leva, Gloria; Ballabio, Claudio; Anderle, Marica; Antonica, Francesco; Tiberi, Luca. - In: NATURE PROTOCOLS. - ISSN 1750-2799. - 18:7(2023), pp. 2143-2180. [10.1038/s41596-023-00839-2]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11572/391149
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