Isolation of single crypt cellsMice were killed at 3–5 weeks of age, and duodenum and jejunum (10 cm from the stomach) were harvested and rinsed three times by using a jet of cold 1 × phosphatebuffered saline (PBS). Then, the intestinal tubes were opened longitudinally and the villi were scraped using a coverslip, followed by washing three times in cold 1× PBS. The intestinal tubes were then cut into 2–3 mm pieces and suspended in 1× PBS + 2% fetal bovine serum (FBS). The 2–3-mm pieces were extensively washed (10–15 times) with 1× PBS + 2% FBS until the supernatant became clear. Then, the 2–3-mm pieces were treated with 50 mM EDTA/ 1× PBS for 30 min at 4°C on a rocking platform to dissociate the crypts from the intestinal tubes. The dissociated crypts were passed through a 70-μm cell strainer, washed once with 1× PBS−, and treated with TrypLE Express (Life Technologies, Carlsbad, CA, USA) for 30 min at 37°C to dissociate cell-to-cell attachment. Then, the dissociated cells were passed through a 40-μm strainer and subsequently, through a 20-μm strainer. The strained cells were pelleted, resuspended in 1× PBS + 2% FBS, and used as single crypt cells.

Epithelium dissociation/FACSTo isolate intestinal crypt cells for fluorescence-activated cell sorting (FACS), small intestine epithelium was dissociated into single cells essentially as previously described (13) with slight modifications. For FACS experiments, mouse intestine were flushed with cold PBS, cut open lengthwise in 10-cm-long pieces, and immersed in PBS/30 mM EDTA/1.5 mM DTT over ice for 20 min. The solution was disposed of, and the tissue was shaken vigorously in fresh PBS/30mMEDTA for 30 s before being incubated at 37°C for 10 min. Intact tissue was discarded, and dissociated crypts and villi were pelleted at 2,500 revolution/min for 5 min. The cells were washed twice with cold PBS, resuspended in HBSS/0.3 U/ml dispase at 37°C, and shaken approximately every 2 min for 10 min. Then, (5%) and 100 µg DNaseI was added before the cells were passed through a 70-µm filter. Cells were pelleted at 2,500 revolution/min for 5 min and resuspended in 4 ml HBSS with 5% FBS, then passed through a 100-µm filter and combined with an additional 100 µg DNaseI.

Cell preparation and SP staining/analysisEpithelial cells were isolated from the jejunum of mice using our previous published EDTA method (von Furstenberg et al., 2011). For SP analysis, cells from the preparation were incubated in “SP buffer” made with 2% FBS, and 10 mM HEPES (Life Technologies) in HBSS (Life Technologies) and either Hoechst 33342 (10 μg/ml equal to 17 μM) (Sigma-Aldrich) for 90 min at 37 °C. Following the incubation, the cells were washed with ice cold HBSS and labeled with propidium iodide (Sigma-Aldrich) and CD45-FITC (Biolegend) at 0.5 μg/106 cells for removal of cells that were dead and/or of hematopoietic origin. For validation purposes, in order to block vital dye efflux, cells were pre-incubated with 100 μM verapamil (Sigma-Aldrich) for 20 min at 37 °C and incubated with the above SP solution for 90 min at 37 °C with the addition of 100 μM verapamil. To generate the SP fluorescent phenotype, Hoechst 33342 samples were excited using a UV laser on a MoFlo (Beckman Coulter) fluorescence activated cell sorting (FACS) machine. Corresponding band-pass filter sets were: MoFlo (blue = 450–50 nm, red = 670-30 nm). On each machine, the SP was defined on the redlo population which was eliminated by verapamil. For analyses and cell collections, the SP was subdivided into two regions: upper SP (USP) and lower SP (LSP).

Control Verapamil

Epithelial isolation and dissociation:A segment of the proximal intestine representing a 10-cm region from 2 to 12 cm distal to the pyloric sphincter was used for cell isolation (Fig. 1, n 3 mice). The intestinal segment was flushed with cold PBS, longitudinally cut open and lightly rinsed to remove residual luminal contents. Tissue was incubated in PBS containing 30 mmol/l EDTA and 1.5 mmol/l dithiothreitol on ice for 20 min. Intestinal tissue was transferred to a 15-ml conical tube containing 5 ml of 30 mmol/l EDTA made in PBS, incubated at 37°C for 8 min, then shaken by hand along the tube’s long axis. Shaking frequency and duration were standardized to 2.5–3 shake cycles per second for 30 s.After shaking, the remnant muscle layer was removed and the dissociated cells were pelleted at 1,000 g, resuspended in PBS 10% FBS, and repelleted. The cells were then incubated in 10 ml of modified HBSS containing 0.3 U/ml of dispase (Roche, San Francisco, CA) at 37°C for 10 min with intermittent shaking (3–3.5 cycles per s for 15 s) every 2 min for 15 s to dissociate epithelial sheets to single cells. After 10 min, a 10-l sample of the cell solution was collected and viewed under light microscopy to monitor dissociation to single cells. If less than 50% of the tissue was single cells, the tissue underwent a second round of 30 s shaking and 2-min incubation at 37°C. To enhance cell viability and decrease cell clumping, 1 ml of FBS and 1,000 U DNase I (Roche) were added to cell suspension, which was sequentially passed through 70-m then 40-m filters (BD Falcon, Bedford, MA). Cells were pelleted, then resuspended in 10 ml of HBSS to inactivate the dispase. Cells were repelleted and resuspended at a concentration of 2 107 cells/ml in IESC media [Advanced DMEM/F12 (GIBCO, Grand Island, NY) supplemented with 1 N2 (Invitrogen), 1 B27 without vitamin A (Invitrogen), 10 mM HEPES (GIBCO), 10 M Y27632 (Sigma), 100 g/ml penicillin-streptomycin, and 2 mM L-glutamine]. Cell viability was assessed by Trypan blue (Invitrogen) staining; a 75% cell viability was the threshold to proceed forward. An