Fig. 3: Optogenetic stimulation of SHH in human stem cells and organoids.

a, Optogenetic stimulation of SHH coupled with spatial readouts. b, Representative (n = 3) imaging of DAPI, SHH-expressing cells (NG) and FOXA2 (immunofluorescence) in hiPSCs 6 days poststimulation. SHH was induced in two ROI (left and in magenta) with a DMD. Scale bars, 100 μm. c, Representative (n = 3) image of hiPSCs Cre–Lox SHH cultured on a PET membrane, stimulated through a 500 μm-wide circular photomask (left). The right shows NG. Scale bar, 500 μm. d, The left shows representative (n = 4) hematoxylin and eosin (H&E) staining of a hiPSC layer cultured on a membrane and transferred onto a Visium slide. The right shows spatial subsetting of Visium spots into seven concentric circles, centered on the SHH-induced area. Scale bar, 500 μm. e, The left shows normalized (Norm.) counts of an SHH gene set in the seven concentric circles (c1–7), color coded as in d, in hiPSCs stimulated for 48 h (n = 1; other samples in Extended Data Fig. 3). The middle shows the same as the left, sampling 1,000 times a random spot as center. The right is the same as the left, sampling 1,000 times a random gene set. Exact P < 0.05 are indicated, computed as the fraction of values exceeding the tested value from random sampling of 1,000 centers and 1,000 gene sets. f. Optogenetic patterning of neural organoids. g, Representative (n = 5) imaging of DAPI, SHH-expressing cells marked by NG and FOXA2/OLIG2/NKX6-1 in adjacent cryo-sections of neural organoids with laser induction of SHH in the north-west pole. The signal is in gray scale for each target separately, and merged in green and magenta (right). Scale bars, 100 μm. The experiment was performed four times and the representative images are shown here. h, Molecular Cartography spatial transcriptomic data of control (left, dark) and SHH-induced (right, lit) organoids, with the indicated transcripts colored according to the legend (right). Experiment was performed four times per condition and three examples are shown here (one dark and two lit). Scale bar, 100 μm. i, Distance distribution (μm) of cells expressing the indicated transcripts from the nearest SHH⁺ cell, in the most left induced organoid of h. In all boxplots, the center and bounds represent the median, 25 and 75% quantiles. Whiskers, if present, represent 1.5× interquartile ranges.