Fig. 2: Application of SPACECAT for physical isolation of stem cell niches from human small intestinal organoid models.
a Schematic of organoid crypt selection with the SPACECAT protocol. Organoid graphic adapted with permission from ref. 67. b Representative images of cultured organoids and example selected regions for photoactivation and selection, as well as changes in fluorescence normalized to baseline fluorescence. Dashed line: photoactivated region boundary; scale bar = 100 μm. n = ~70 regions from the periphery of ~20 organoids, targeting 10–20 regions per protrusion. c UMAP of 3,588 single cells in total, from non-photoactivated control organoids (n = 266 cells), sorted calcein NVOC+ cells from photoactivated organoids (n = 187 cells), and an internal reference dataset of organoids derived from the same patient and cultured identically (n = 3135 cells); cells colored by annotated cell type. d Scoring of cell types for expression of a module of stem-associated genes (LGR5, AXIN2, FZD7, OLFM4, LRIG1), following the approach of Tirosh et al.68. e UMAP of organoid dataset, colored by experimental condition (non-photoactivated controls: black; calcein NVOC+: green; reference dataset: gray). f Cell cycle stage by isolation condition as determined by gene module scoring (Tirosh et al.68), p value < 0.001 when comparing Calcein NVOC+ cells to either other cell population by Chi-square test (Calcein NVOC+: 187 cells, green; non-photoactivated control: 266 cells, black; reference dataset: 3,135 cells, gray; p value = 7.404 × 10−8 for calcein NVOC+ vs. non-photoactivated control, p value = 3.436 × 10−12 for calcein NVOC+ vs. reference). g Selected differentially expressed genes marking each cell type. Circle size denotes percentage of each cluster expressing a given gene, circle color denotes relative digital gene expression compared to other clusters. h Cluster membership by experimental condition, p value < 0.001 when comparing calcein NVOC+ cells to either other cell population by Chi-square test (p value = 3.587 × 10−8 for green vs. black, p value < 2.2 × 10−16 for green vs. gray). Source data are available as a Source data file.
