A simplified protocol for the generation of cortical brain organoids

Abstract

Human brain organoid technology has the potential to generate unprecedented insight into normal and aberrant brain development. It opens up a developmental time window in which the effects of gene or environmental perturbations can be experimentally tested. However, detection sensitivity and correct interpretation of phenotypes are hampered by notable batch-to-batch variability and low reproducibility of cell and regional identities. Here, we describe a detailed, simplified protocol for the robust and reproducible generation of brain organoids with cortical identity from feeder-independent induced pluripotent stem cells (iPSCs). This self-patterning approach minimizes media supplements and handling steps, resulting in cortical brain organoids that can be maintained over prolonged periods and that contain radial glial and intermediate progenitors, deep and upper layer neurons, and astrocytes.

Brain organoid as a model to study the role of mitochondria in neurodevelopmental disorders: achievements and weaknesses

“…Different extrinsic patterning factors can differentiate these NPCs into different cerebral lineages, resulting in the formation of region-specific brain organoids ( Susaimanickam et al, 2022 ). Specifically, several guided differentiation methodologies have been described for obtaining forebrain lineage organoids [as cortical organoids ( Paşca et al, 2015 ; Sloan et al, 2018 ; Trujillo et al, 2019 ; Qian et al, 2020 ; Urresti et al, 2021 ; Eigenhuis et al, 2023 ); hippocampal organoids ( Sakaguchi et al, 2015 ; Ciarpella et al, 2023 ); retinal organoids ( Völkner et al, 2016 ; Fligor et al, 2018 ; Wahle et al, 2023 ); thalamic organoids ( Xiang et al, 2019 ; Kiral et al, 2023 ); hypothalamic organoids ( Ozone et al, 2016 ; Qian et al, 2016 ; Huang et al, 2021 ); and choroid plexus organoids ( Pellegrini et al, 2020 )], midbrain linage organoids ( Jo et al, 2016 ; Smits et al, 2019 ; Nickels et al, 2020 ; Smits and Schwamborn, 2020 ; Sabate-Soler et al, 2022 ), and hindbrain linage organoids [as cerebellar organoids ( Muguruma et al, 2015 ; Chen et al, 2023 ; Atamian et al, 2024 )]. Guided protocols can also be used to generate two or more brain-region-specific organoids separately and subsequently fuse them, obtaining the “assembloids,” which model the interactions between different brain regions ( Jang et al, 2022 ).…”

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.…”

Development of brain organoid technology derived from iPSC for the neurodegenerative disease modelling: a glance through

“…Most typical processes to create brain organoids derived from iPSCs are firstly self-aggregating the iPSC into embryoid bodies (EB) which then will be embedded the growing organoids in supporting extracellular matrix (Matrigel) inside the appropriate medium supplemented with specific growth factors under agitating conditions ( Hopkins et al, 2021 ). Eigenhuis et al (2023) had recently published the simplified and optimized protocols to generate robust dorsal forebrain organoids from iPSC-neural induction. They demonstrated an effective strategy by bypassing the initial steps which are embryoid body formation, neural induction, and cortical differentiation that are time-consuming into a new approaches which lessen the uses of media supplements and Matrigel ( Eigenhuis et al, 2023 ).…”

“… Eigenhuis et al (2023) had recently published the simplified and optimized protocols to generate robust dorsal forebrain organoids from iPSC-neural induction. They demonstrated an effective strategy by bypassing the initial steps which are embryoid body formation, neural induction, and cortical differentiation that are time-consuming into a new approaches which lessen the uses of media supplements and Matrigel ( Eigenhuis et al, 2023 ).…”