Fig. 1. BENO generation from human iPSCs embedded in a defined collagen type I environment.

a Scheme illustrating the five different protocols tested for their potential to yield the highest amount of neurons and glia in BENO cultures: (1) Dual SMAD inhibition (SB/noggin) in the presence of RA during culture days 0–10; (2) FGF-2 treatment to enhance neuronal progenitor number during culture days 10–15; (3) addition of TGFβ-1 to enhance gliogenesis during culture days 10–28; (4) addition of DAPT to enhance neuronal differentiation during culture days 15–28; (5) combination of protocols 1–4. Right, bright field images of representative BENOs at d6, d15, and d28 of differentiation. Scale bar: 1 mm. b Transcriptome analysis of neuronal markers PAX6, MAP2, GABBR2, GRIN1 during BENO development (d-1, d0, d3, d8), n = 3–5 organoids/time point, two independent experiments; all transcripts in protocols 4 and 5 were significantly higher than in protocol 1. *p < 0.0001 two-way ANOVA with Tukey’s multiple comparisons post hoc test. c Comparison of protocols 4 and 5 as to their potential to enhance gliogenesis and neuronal maturation. WmIF (whole-mount immune fluorescence) analysis. Scale bar: overview 500 µm, insert 20 µm GFAP was used as a glia marker; d transcriptome analysis in d60 BENOs. n = 3–5 organoids/time point, two independent experiments, p values for PAX6, GABBR2, GRIN1, GFAP were 0.0485, 0.005, 0.0061, 0.0289, respectively; unpaired two-tailed Student’s t test. e Comparison of protocols 4 and 5 as to their potential to generate organoids with homogenous distribution of functional neurons. Spontaneous calcium imaging after loading with Fluo-8-AM in different areas of BENOs was performed at d30 by confocal microscopy n = 3 organoids/group; Scale bar 500 µm. Data are presented as mean values ±  SEM.