Volume 129, Issue 5, Pages 1567-1580 (November 2005) Isolation and Characterization of a Putative Intestinal Stem Cell Fraction From Mouse Jejunum  Christopher M. Dekaney, Jose M. Rodriguez, M. Colleen Graul, Susan J. Henning  Gastroenterology  Volume 129, Issue 5, Pages 1567-1580 (November 2005) DOI: 10.1053/j.gastro.2005.08.011 Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 1 Identification of SP cells from adult mouse jejunum. FACS analysis of Hoechst 33342–stained cells from either (A) mucosal digests prepared with collagenase/dispase or (B) epithelial digests prepared with EDTA/dispase. Top panels: representative plot based on blue and red Hoechst intensities. Boxed cells represent SP fraction. Bottom panels: the SP fractions were analyzed further by sorting with FITC-labeled CD45 antibodies to distinguish cells of hematopoietic origin. In all panels in all figures in this article, dead cells (which stain positive with PI) are excluded. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 2 Histologic evaluation of epithelial preparation and of CD45-negative SP cells. (A) Intact mouse jejunum before exposure to 30 mmol/L EDTA. (B) Remaining intestinal tissue after epithelium has been removed by EDTA treatment. Arrows indicate ghosts left by crypt removal. (C) Representative micrographs of cytospun CD45-negative SP cells. All 3 panels show H&E staining. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 3 Analysis of the intestinal SP (from a collagenase/dispase preparation) for known markers of hematopoietic stem cells. Intestinal SP cells were sorted by the presence/absence of CD45 (axis) and for the presence/absence of Sca-1, Thy-1.2, CD34, or c-Kit (ordinate). The number within each quadrant represents the percentage of cells in that quadrant. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 4 Expression of β1-integrin on the surface of intestinal SP cells. FACS analysis of jejunal SP cells showing presence/absence of CD45 (axis) and presence/absence of β1-integrin (ordinate). The number within each quadrant represents the percentage of SP cells in that quadrant. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 5 Verapamil sensitivity of the intestinal SP. Cells from a mucosal preparation were incubated in HBSS containing 5 μg/mL Hoechst 33342 either alone or with 100 μmol/L verapamil and then sorted based on Hoechst staining. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 6 Binding of annexin V to CD45-negative SP cells. Mucosal digests were incubated with an anti-CD45–PE antibody and annexin V–FITC to determine the percentage of cells entering the apoptotic pathway. The histogram shows the results of a single sort with negative cells (no fluorofore) in gray and CD45-negative SP cells in black. Inset graph shows the composite results (3 sorts) for annexin V–negative and –positive cells. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 7 Immunostaining of CD45-negative SP cells from a mucosal preparation for cytokeratins (pan-CK; green) and α-smooth muscle actin (αSMA; green). Nuclei are stained with DAPI (blue). Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 8 Relative abundance of mRNA for Msi-1 and other markers in CD45-negative SP fractions from adult mouse jejunum as compared with intact jejunal tissue. Gray bars show mean ± SE (n = 3, representing 3 separate cell preparations each from jejunums of 2 mice) for Msi-1, collagen IV (coll IV), sucrase–isomaltase (si), intestinal trefoil factor 3 (tff3), and lysozyme mRNA measured by quantitative reverse-transcription polymerase chain reaction in CD45-negative SP. The solid bar represents the intact tissue level of each marker (abundance = 1.0 on this scale). (A) CD45-negative SP cells from collagenase/dispase isolation. (B) CD45-negative SP cells from EDTA/dispase isolation. nd, not detectible. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 9 Identification of SP cells from neonatal mouse jejunum. (A) Hoechst 33342 staining of isolated jejunal mucosa was analyzed by flow cytometry. The SP fraction was analyzed further by sorting with FITC-labeled CD45 antibodies to distinguish cells of hematopoietic origin. (B) Percentage of viable cells that decrease in SP. (C) Percentage of SP cells that are CD45 negative. (D) Percentage of viable cells that are CD45-negative SP. Bar graphs show mean ± SEM, n = 40 for adult and n = 3 for neonatal. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions

Figure 10 CD45-negative SP cells in culture. (A) Micrograph of cells after 7 days in culture. Note cells have not attached by this time. Magnification is 4×. (B) Micrograph of cells after 7 days in culture. Magnification, 20×. (C) Percentage of cells alive at 0 (n = 2), 7 (n = 2), and 14 (n = 3) days in culture as assayed by Trypan blue dye exclusion. Data equal mean ± SEM. Gastroenterology 2005 129, 1567-1580DOI: (10.1053/j.gastro.2005.08.011) Copyright © 2005 American Gastroenterological Association Terms and Conditions