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. Author manuscript; available in PMC: 2012 Dec 28.
Published in final edited form as: Nature. 2012 Jun 28;486(7404):490–495. doi: 10.1038/nature11163

Figure 1. Calorie restriction augments the capacity of Paneth cells to boost ISC function.

Figure 1

a. Olfm4+ ISCs and Cryptdin4+ Paneth cells were increased in CR mice (in situ hybridization, proximal jejunum, n=3). b. Crypt base columnar cells (CB-cells) showed a 2-fold increase in BrdU incorporation and TA-cells revealed a reduction after a 4-hour pulse in CR mice (n=3). c. CR-crypts were 2-fold more capable of forming organoids (n=8). Representative AL and CR-organoids are shown at 5 days (red arrowhead marks organoids and yellow asterisk indicates aborted crypts; scale bar = 50 μm). d–e. CR increased the number of surviving (d) and proliferating (e, Ki67+) crypts after irradiation induced damage (n=3 for d and e). f. Schematic demonstrating dark green Lgr5hi ISCs, red Paneth cells, and light green EGFPlow progenitors. CR increased ISCs (dark green) and Paneth cells (red) by 1.5-fold, and reduced EGFPlow (light green) progenitors by 20% (CR, n=27; AL, n=26). g. A schematic illustrating the mixing of ISCs with Paneth cells in matrigel. h. Organoid formation per Lgr5hi ISCs cocultured with Paneth cells from CR mice was significantly increased (n=5). Representative image of primary organoids at day 7. i. Dissociated organoids derived from CR-Paneth cells gave rise to larger secondary organoids at day 18 (n=5). j. Sorted ISC-Paneth cell doublets plated at clonal density (50–100 doublets per 30 ul droplet of matrigel) demonstrated that CR-doublets had nearly 3-fold more organoid potential (n=3). k. EGFPlow progenitors harbored little organoid potential (n=4). l. Subcloning of individual CR-Paneth derived organoids gave rise to 3-fold more secondary organoids (27 organoids from 3 independent mice per condition were analyzed, shades of grey or blue denote separate mice). m. Paneth cells isolated from mice that had been on CR, but were returned to an AL diet for 3 days, also retained an augmented capacity to promote organoid formation (n=3). (Unless other wise indicated, in all panels: values = mean; error bars = s.d.; scale bars= 50 μm; * indicates P<0.05; ** P<0.01; and *** P<0.001).