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.

Keywords: cortical brain organoids; dorsal identity; feeder-independent; human brain development; induced pluripotent stem cells; self-patterning.

Figure 1

Protocol timeline for a cortical organoid generation. For each developmental stage, the corresponding medium is displayed. iPSCs, induced pluripotent stem cells; EB, embryoid body; NI, neural induction; CDM, cortical differentiation medium; CMM, cortical maturation medium. This figure was created with BioRender.com.

Figure 2

Generating a single-cell iPS suspension. (a) Healthy iPS colonies at 70–80% confluency, without signs of differentiation. (b) After 0.5 mM EDTA incubation and subsequent incubation with accutase, all cells have dissociated into single cells or small groups of cells, and all cells are floating. (c) After gentle trituration of the dissociated cell suspension, a pure single-cell suspension is created.

Figure 3

Overview of different steps in embryoid body and cortical organoid development from PSCs. Healthy PSCs (a). The scalebar in panels (b–e) represents 500 μm; the scalebar in panels (f–i) represents 1 mm; the scalebar in panels (f′–i′) represents 500 μm.

Figure 4

Immunofluorescent staining for brain regions and neuronal cell types in 35-day old cortical organoids. (A) Multiple PAX6⁺ progenitor zones are distributed throughout the organoid in the shape of large folds or small rosettes, and surrounding ventricle-like cavities. Whereas progenitor zones are negative for TUJ1 (class-III tubulin), early neuronal differentiation outside of progenitor zones is marked by abundant TUJ1 expression. Early-born deep-layer neurons (CTIP2) reside outside of progenitor zones. Scalebar: 1 mm for all panels. (B) Apical radial glial cells (NESTIN⁺) occupy progenitor zones and extend from the apical to the basal surface (panel a″). Scalebar a, a′: 250 μm, a″: 50 μm. (C) Correctly formed organoids show structured progenitor zones (panel a), presence of deep-layer neurons (CTIP2, panel a′), and expression of dorsal forebrain markers FOXG1, TBR2, and PAX6 (panels a″, b, and c). Incorrect organoids lack organized progenitor zones (panel d), contain dispersed deep-layer neurons (panel d′), and show absence of dorsal forebrain progenitor markers (panels d″, e, and f). Scalebar: 250 μm for all panels. (D) Batch efficiency of brain organoid differentiation. (E) Efficiency of cortical identity acquisition.

Figure 5

Characterization of dorsal forebrain identity in cortical organoid brain regions. (A) A 35-day-old organoid showing multiple radial glial progenitor regions (indicated by dashed lines) marked by FOXG1-positive neural progenitor cells, and early neuronal differentiation (TUJ1) (panel a), proliferative progenitors (Ki-67, panel b), and IPCs (TBR2, panel b). Intermediate progenitor cells (TBR2) are generated in the proliferative zone and migrate to align at the basal side (indicated by arrows), where they generate neurons (TUJ1) (panel c). Magnification of a typically organized proliferative zone (TUJ1 negative) located next to a ventricle-like cavity with intermediate progenitors (TBR2) that give rise to deep-layer neurons (CTIP2) that form a layer right above (panel d). Scalebar a, b: 300 μm, c: 100 μm, d: 50 μm. (B) Non-dorsal forebrain identity regions can be recognized by aberrant TUJ1 morphology, lacking progenitor zones (panels a, c), or FOXG1 expression (panels a′ and c′). The presence of ventral forebrain progenitors (GSX2) and choroid plexus epithelia (TRR) surrounding ventricle-like cavities can be identified (panels b, b′, d, and d′). CBO, cortical brain organoid. Scalebar a, a′, b, c, c′, and d: 250 μm, b′ and d′: 50 μm.

Figure 6

Mature cortical organoids. Staining for upper-layer neurons (SATB2) and deep-layer neurons (CTIP2) in 70- and 120-day-old mature cortical organoids. After 70 days, progenitor zones are still present (indicated by dashed lines) and both types of neurons reside outside of these zones in a disorganized manner (A). In 120-day-old organoids, progenitor zones are absent and deep-layer neurons have compacted into a more organized layer, positioned below a layer of abundantly present upper-layer neurons (B). Staining for astrocytes (GFAP), neurons (FOXG1), and proliferating cells (Ki-67) in a 120-day-old cortical organoid cortex-like region (C). Scalebar: 150 μm in all images.