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. 2024 Jan 4;19(2):254–269. doi: 10.1016/j.stemcr.2023.12.001

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© 2023 The Author(s)

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

PMC Copyright notice

Characteristic spheroid morphologies after re-aggregation of the cells sorted by LF-GC-based sorting in Crx::Venus cell line

(A) Representative phenotypes of Crx::Venus-derived organoids (Crx#03). (Top) Bright-field image. (Bottom) Venus-fluorescent image.

(B) FSC and Venus profiles to evaluate fluorescence intensity (Crx#03).

(C) Scatterplot of FSC and BSC (Crx#03). SVM score histograms for the classification of target cells and others were calculated for the cells within the live-cell gate (green).

(D) SVM score of the classifier and the number of cells (Crx#03). Cells with SVM scores of ≥1.0 (red) were sorted.

(E) Scatterplot of FSC and BSC for the cells in the live-cell gate (C). Blue indicated the cells predicted to be positive in the LF-GC-based sorting system, and orange indicated the negative cells.

(F) Characteristics of Sorted, Waste, and Control cells after LF-GC-based sorting experiments for Crx::Venus cell line (Crx#01-#03). Cell viability and average cell size (top), and gene expressions of each sample (bottom). Changes in gene expressions are shown as a fold change in gene expressions of Sorted cells (blue) and Waste cells (orange) compared with those of Control cells (black).

(G and H) Summary of phenotypes in spheroid assays of Crx::Venus cell line on DD60-70. One box corresponds with one spheroid (G) and each color box (G) and bar (H) represent the samples with the same color. C-spheroids and P-spheroids were significantly more abundant in the Sorted group. ^∗∗∗∗p < 0.0001; ns, not significant. Fisher’s exact test with Bonferroni correction.

(I and J) Immunostaining images of C-spheroids and P-spheroids with low (left) and high (right) magnification on DD60–70. PAX6 (white) and BRN3 (magenta, bottom) were expressed on the basal side, while VSX2 (magenta, top) and CRX (green) were expressed mainly on the apical side.

(K) Immunostaining images of N-spheroids on DD60–70. Some cells are positive for PAX6 (white, top left) and EMX2 (white, top right).

Scale bars, 500 μm (A and H), 200 μm (left, I, left, J, and K), and 100 μm (right, I and J).