Figure 1.

Discrete reporter marked intestinal cell populations showed distinct growth patterns ex vivo. (A) Lgr5- and (B) Bmi1-GFP reporter expression patterns within murine small intestine. Left: Reporter tissue sections stained with antibodies against GFP (green) and E-cadherin (red). White arrowheads indicate GFP⁺ cells within the crypt. White dashed lines represent the epithelial-mesenchymal boundary. White arrowhead denotes GFP⁺ cells. Fluorescent images were captured on a Zeiss Observer Z1 microscope with ApoTome. Scale bar: 25 μm. N = 5 total mice per genotype. Middle: FACS plot showing GFP⁺ cell populations isolated from reporter mice. Inset: GFP-negative control. FACS analyses were performed on a BD Influx cell sorter at the OHSU Flow Cytometry Core Facility. Data are representative of N = 6 independent experiments, N = 12 mice per genotype. Right: Low-magnification (inset) and high-magnification bright field and accompanying GFP images of resultant ex vivo 3D epithelial cultures from respective GFP⁺ populations after 12 days of growth. White arrowheads denote regions of GFP expression. White dashed lines outline the lumen. Asterisk denotes autofluorescent cells within the lumen. Images were acquired on a Leica DMIRB inverted microscope. N = 4 independent experiments, N = 8 mice per genotype. (C) Quantification of growth phenotypes from single sorted Lgr5^GFP or Bmi1^GFP cell populations. N = 4 experiments, N = 8 mice per genotype. (D) qRT-PCR analysis of gene expression within Bmi1^GFP-derived spheroids compared with Lgr5^GFP-derived enteroids. Representative data from technical replicates. Gene expression analyses were performed on N = 4 groups of cultures representing N = 8 mice, means ± SEM of triplicate fold change. (E) Images of Lgr5^GFP-derived enteroids (left panel) or Bmi1^GFP-derived spheroids (right panel, surface view) stained with antibodies against the epithelial marker epithelial cell adhesion molecule (EpCAM, white) and GFP (green), or cell lineage markers (red) ChgA (top panel) and Lyz (lower panel). Images were acquired on a Zeiss Observer Z1 fluorescent microscope equipped with ApoTome. N = 3 independent experiments, N = 6 total mice. (F) Representative images of established Lgr5^GFP-derived enteroids (upper panel) and Bmi1^GFP-derived spheroids (lower panel) cultured for 7 days with (+) or without (-) Rspo1. Lower magnification in far right panel. (G) Brightfield images of Bmi1^GFP-derived spheroids over multiple passages. Culture images were captured on an inverted Leica DMIRB microscope. N = 3 independent experiments, N = 6 total mice. Scale bar: 200 μm. ChgA, chromogranin A; Lyz, lysozyme; SSC, side-scattered light.