Chimeric organotypic cultures of purified 11-dpc c-Kit^low(CD45/TER119)^– R4 cells obtained from C57BL/6.GFP mouse embryos, cocultured with 2′deo-treated, 12-dpc liver fragments from C57BL/6 mice were analyzed after 3–5 weeks, as described in Methods. H&E and direct GFP views of cell depleted-only (a and b) and R4 cell–seeded (c and d) liver organoids. GFP⁺ cells are in green and GFP^– cells show only the blue signal from DAPI (b and d) (×40). (e–p) R4 cell–seeded livers. (e and f) Livers stained with secondary Alexa 647 goat anti-rabbit and rhodamine, goat anti-mouse Ab’s. Merged views of blue and red channels (e) and green channel (f) (×40). (g and h) Livers stained with anti-GFP mAb and revealed with Alexa 647–conjugated mAb. Blue GFP signals (g) and no signals in the merged green and red channels (h) (×60). (i–p) Two representative areas of the liver organoids subjected to GFP/ALB/CK19 staining: GFP⁺ blue (i and m), ALB⁺ green (j and m), and CK19⁺ red channels (k and o), and overlayed green/red signals (l and p) (×60). Arrowhead, ALB⁺CK19⁺ cells; arrow, ALB⁺CK19^low cells; star, ALB^lowCK19⁺ cells. (q) Frequencies of both GFP⁺ (black bars) and GFP^– (gray bars) hepatic cell subsets detected in R4 cell–seeded livers (top) and in 2′deo-treated livers (bottom). The results shown are the mean frequencies ± SEM, related to the GFP⁺ + GFP^– total cells recovered from five R4-seeded liver fragments. Cells in 15 different photomicrographs were counted by three independent observers (3,000 total cells).