Figure 7. smNPC-derived AMOs are morphologically, structurally, and functionally more homogeneous than automated hiPSC-derived organoids.

(a/b) Light microscopy images of hiPSC-derived organoids (a) and AMOs (b) generated from the same cell line demonstrating the higher morphological homogeneity of AMOs at day 30 of differentiation.(c/d) Single optical confocal slices of either hiPSC-derived organoids (c) or AMOs (d) at day 30 stained for DAPI, the astrocyte marker GFAP, the neural precursor marker Sox2, and the neuronal marker Map2. The direct comparison illustrates the higher level of structural homogeneity as well as accelerated maturation, especially the earlier emergence of GFAP+ astrocytes in AMOs. Rows depict three samples from one batch. (e/f) Size (area of the largest cross section) and cell viability measurements of individual organoids from three independent batches (per cell line/differentiation protocol) illustrating the high homogeneity of AMOs compared to standard hiPSC organoids. (g/h) Coefficients of variation calculated based on the data shown in (e) and (f). (i/j) Quantitative whole mount staining (see also Figure 6) for Sox2 (i), and Map2 (j) showing the higher variability of hiPSC organoids compared to AMOs from the same iPSC line even after normalization to the organoid area. All data gathered from organoids at day 30 of differentiation. Scale bars: 300 μm (a), 200 μm (b/c), 100 μm (d). Also see Figure 7—figure supplements 1 and 2.

Figure 7—figure supplement 1. Overview of the protocol for the automated generation of hiPSC-based organoids and modifications from the published original.

Schematic overview of the automated HTS workflow for the generation and maintenance of the automated hiPSC-organoids. The right half of the Figure details the modifications made from the original protocol published by Paşca et al., 2015 (and described in more detail by Sloan et al., 2018).

Figure 7—figure supplement 2. The expression of typical neural and cortical markers confirms the correct differentiation of automated hiPSC-derived organoids.

(a/b) Single optical confocal slices of whole mount stained and tissue-cleared organoids showing the expression of the early cortical neuron marker CTIP2, precursor marker Pax6, and general neuronal marker Map2 at day 40. Expression of the different markers is generally confined to specific regions of the organoid. While Pax6 expression is highest in the neural rosette-like structures, CTIP2 is mostly found surrounding these regions (see (b) for an enlarged view of a rosette). The expression pattern of the general neuronal marker Map2 indicates that most neurons are located within the outer regions of the organoid with lower expression toward the center. (c) At day 30, staining for the forebrain precursor marker FoxG1 combined with Map2 further confirms the correct differentiation toward a cortical fate. (d/e) Consistently, the expression of the precursor markers Brn2 (d) and Sox2 (e) is mostly confined to the neural rosette-like structures, while the neural marker DCX (d) shows a distribution similar to that of Map2 in 30 days old organoids. (f/g) The precursor marker TBR2 is also mainly expressed in confined regions of the organoid surrounded by more mature cortical markers like TBR1 (f) and CTIP2 (g) at day 30 of differentiation. (h) Late-born SATB2+ neurons arise starting day 50. (i) At day 40, few synapsin-positive punctae on Map2-positive neurites indicate formation of first synapses. (j) Maximum intensity projection (MIP) showing the network of β-Tubulin III (TUBB3) positive neurons as well as Sox2-positive precursors throughout a 30 days old organoid. (k) While the mostly unguided nature of the differentiation protocol does allow for the emergence of other cell types, expression of TH is confined to a small area and limited to few cells after 40 days of differentiation. Organoids shown were at either day 30 (c, d, e, f, g, j), 40 (a, b, i, k), or 50 (h) of differentiation. Scale bars: 200 μm (a, c, d, e, f, g, h), 20 μm (b, g).