A beginner’s guide on the use of brain organoids for neuroscientists: a systematic review

Abstract

Background The first human brain organoid protocol was presented in the beginning of the previous decade, and since then, the field witnessed the development of many new brain region-specific models, and subsequent protocol adaptations and modifications. The vast amount of data available on brain organoid technology may be overwhelming for scientists new to the field and consequently decrease its accessibility. Here, we aimed at providing a practical guide for new researchers in the field by systematically reviewing human brain organoid publications. Methods Articles published between 2010 and 2020 were selected and categorised for brain organoid applications. Those describing neurodevelopmental studies or protocols for novel organoid models were further analysed for culture duration of the brain organoids, protocol comparisons of key aspects of organoid generation, and performed functional characterisation assays. We then summarised the approaches taken for different models and analysed the application of small molecules and growth factors used to achieve organoid regionalisation. Finally, we analysed articles for organoid cell type compositions, the reported time points per cell type, and for immunofluorescence markers used to characterise different cell types. Results Calcium imaging and patch clamp analysis were the most frequently used neuronal activity assays in brain organoids. Neural activity was shown in all analysed models, yet network activity was age, model, and assay dependent. Induction of dorsal forebrain organoids was primarily achieved through combined (dual) SMAD and Wnt signalling inhibition. Ventral forebrain organoid induction was performed with dual SMAD and Wnt signalling inhibition, together with additional activation of the Shh pathway. Cerebral organoids and dorsal forebrain model presented the most cell types between days 35 and 60. At 84 days, dorsal forebrain organoids contain astrocytes and potentially oligodendrocytes. Immunofluorescence analysis showed cell type-specific application of non-exclusive markers for multiple cell types. Conclusions We provide an easily accessible overview of human brain organoid cultures, which may help those working with brain organoids to define their choice of model, culture time, functional assay, differentiation, and characterisation strategies.

Brain organoid protocols and limitations

“…Furthermore, the strength and exposure time of diffusible morphogens and crosstalk among different signaling pathways are also critical for precise pattern formation (Fattah et al, 2023 ). Various combinations of morphogens from each signaling pathway were applied to generate region-specific organoids, such as dorsal (Pasca et al, 2015 ; Sebastian et al, 2023 ), ventral (Sloan et al, 2018 ; Eigenhuis et al, 2023 ; Mulder et al, 2023 ), hippocampal (Jacob et al, 2020 ), cerebellum (Silva et al, 2020 ; Atamian et al, 2024 ), hindbrain (Valiulahi et al, 2021 ), and spinal cord (Lee et al, 2022 ) brain regions. As shown in Supplementary Table 1 , cortical and dorsal forebrain organoids often use BMP and TGF inhibitors; in contrast, caudal parts of the brain, such as hindbrain and spinal cord organoids, frequently use WNT, RA, and FGF activators instead.…”

“…The current ECMs used in the cortical organoid culture (Heo et al, 2022 ) include natural scaffolds such as matrigel (Lancaster et al, 2013 ), decellularized tissue-derived scaffolds (Cho et al, 2021 ; Simsa et al, 2021 ) and synthetic polymer-based scaffolds (Lancaster et al, 2017 ; Oksdath et al, 2018 ; Hofer and Lutolf, 2021 ). Matrigel is the most used ECM preparation in the organoid protocols (Mulder et al, 2023 ). Matrigel is extracted from murine Engelbreth-Holm-Swarm sarcomas cells containing more than 1800 unique proteins (Hughes et al, 2010 ).…”

Lipid Rafts: The Maestros of Normal Brain Development

“…ESCs are pluripotent cells that can differentiate into all cell types of the body, whereas iPSCs are derived from adult cells that have been reprogrammed to a pluripotent state and can be harvested noninvasively from human donors. NPCs are multipotent cells that can give rise to neurons, astrocytes, and oligodendrocytes [ 251 ]. Brain organoids have been instrumental in elucidating the molecular signals that regulate NPC proliferation, differentiation, and migration, providing insights into how these processes are perturbed in various neurodevelopmental disorders.…”

Human In Vitro Models of Neuroenergetics and Neurometabolic Disturbances: Current Advances and Clinical Perspectives

“…Human brain organoids generate and mature through a self-guided organization and differentiation from pluripotent stem cells; they contain heterogeneous cell populations, which can dynamically communicate and interact with one another, as well with the extracellular matrix, creating a physiological microenvironment. They recapitulate certain aspects of brain development and physiology, including brain regionality 34 , 35 (comprehensively reviewed by Mulder et al 36 ). Cerebral organoids, for instance, revealed to be an adequate model to study embryonic brain remodeling and disrupted energy metabolism when generated from iPSCs derived from Leigh syndrome- 37 or autism spectrum disorder patients.…”