Figure 2. Automated midbrain organoids express typical neural and midbrain markers and show signs of structural organization.

(a) Expression of the dopaminergic midbrain marker TH as well as the precursor markers nestin and Sox2 is evenly distributed throughout the entire aggregate at day 25, as shown by single confocal microscopy slices of tissue-cleared samples. The dotted box indicates the area shown in (b). Here, higher magnification of the peripheral aggregate region reveals two different zones with few nuclei but dense, circumferentially oriented neurites distally from the core and radial organization of TH-positive neurons more proximally. (c) The expression patterns of DCX and Brn2 further illustrate the organization of neurons (DCX) and neural precursors (Brn2) in the core of AMOs into zones. (d) Enlargement (of the dotted box in c) highlighting the circumferential organization of neurons (DCX) surrounding the core. (e) Maximum intensity projection (MIP) of fluorescent confocal images showing a dense cellular network expressing the neural marker β-tubulin III (TUBB33) within the AMOs at d25. (f/g) Continuing maturation of AMOs is indicated by the presence of synapses marked by the colocalization of the presynaptic synaptophysin and postsynaptic homer on Map2-positive neurites at day 50 (f, top right corner showing enlargement of two synapses without the Map2 channel) and S100b/GFAP double-positive astrocytes at day 75 (g). Scale bars: 100 μm (a, c, e), 20 μm (b, d, f, g). Also see Figure 2—figure supplements 1–3.

Figure 2—figure supplement 1. Automated midbrain organoids express synaptic and midbrain markers.

Single optical confocal slices of whole mount stained and cleared AMOs. (a/b) AMOs highly express the synaptic protein Synapsin frequently colocalizing with the neural marker Map2 (day 75). Shown in an overview (a) and in more detail (b, enlargement of the box in a) to highlight the colocalization. (c) In contrast to the highly abundant dopaminergic neurons, AMOs contain low numbers of GABAeric (vGAT) and glutamatergic (vGLUT1) neurons. (d–g) Successful differentiation of the AMOs toward a dopaminergic midbrain-like phenotype is indicated by the coexpression of typical midbrain markers including Foxa2 (d, day 25), Lmx1a (e, day 25), Nurr1 (f, day 25), and Pitx3 (g, day 50) together with the dopaminergic marker TH. Scale bars: 100 μm (a), 20 μm (b–g).

Figure 2—figure supplement 2. Characterization of AMOs generated from a second, independent patient iPSC-derived smNPC line.

(a-i) AMOs derived from a second smNPC line (AMO line 2) express the same neural precursor, neuron/synaptic, and midbrain markers as AMO line 1 (compare to Figure 2 and Figure 2—figure supplement 1). Neural precursors: Nestin (a), Sox2 (b), Brn2 (c); neurons and synapses: Map2 (b, e), DCX (c), TUBB3 (d), synapsin (e); midbrain: TH (a, f–i), FoxA2 (f), Lmx1a (g), Nurr1 (h), Pitx3 (i). All stainings were carried out after 30 days of differentiation and show either single optical confocal slices from whole mount samples or in the case of (d) maximum intensity projections (MIP). Scale bars: 100 μm (a–d), 20 μm (e–i). (j) Representative line graph of spontaneous and periodic bursts of calcium activity indicating functional coupling of the entire aggregate of AMOs from line 2 (compare to Figure 4). Experiment carried out at day 40 of differentiation on five organoids with similar outcome, recorded on a standard plate reader.

Figure 2—figure supplement 3. Electron microscopy displays ultrastructural morphology of neuronal phenotypes.

(a/b) Ultrastructural analysis of AMOs reveals a dense lattice of neuronal cells. The neuronal cell bodies are frequently surrounded by cellular projections commensurate with nerve fibers. (c) Enlargement of the dashed box in (b). AMOs contain bundles of nerve fibers with regularly spaced neurofilaments and microtubules resembling those of dendrites. (d) Enlarged portion marked by the dashed box in (c) showing vesicles with the characteristic size and localization of synaptic vesicles within the nerve fibers. N: Nucleus of a neural cell, nf: nerve fiber, arrows indicate synaptic vesicles. (Representative views of one example from a total of n = 3 AMOs at day 32).