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. 2011 Feb;300(2):G345-56.
doi: 10.1152/ajpgi.00278.2010. Epub 2010 Nov 18.

A stem cell marker-expressing subset of enteroendocrine cells resides at the crypt base in the small intestine

Yoshitatsu Sei  1 , Xinping Lu, Alice Liou, Xilin Zhao, Stephen A Wank
Affiliations

A stem cell marker-expressing subset of enteroendocrine cells resides at the crypt base in the small intestine

Yoshitatsu Sei et al. Am J Physiol Gastrointest Liver Physiol. 2011 Feb.

Abstract

The spatial orientation of the enteroendocrine cells along the crypt-villus axis is closely associated with their differentiation in the intestine. Here we studied this relationship using primary duodenal crypts and an ex vivo organoid system established from cholecystokinin-green fluorescent protein (CCK-GFP) transgenic mice. In the primary duodenal crypts, GFP+ cells were found not only in the upper crypt but also at the crypt base, where the stem cells reside. Many GFP+ cells below +4 position were positive for the putative intestinal stem cell markers, leucine-rich repeat-containing G protein-coupled receptor 5, CD133, and doublecortin and CaM kinase-like-1, and also for the neuroendocrine transcription factor neurogenin 3. However, these cells were neither stem nor transient amplifying precursor cells because they were negative for both Ki-67 and phospho-Histone H3 and positive for the mature endocrine marker chromogranin A. Furthermore, these cells expressed multiple endocrine hormones. Tracking of GFP+ cells in the organoids from CCK-GFP mice indicated that GFP+ cells were first observed around the +4 position, some of which localized to the crypt base later in the culture period. These results suggest that a subset of enteroendocrine cells migrates down to the crypt base or stays localized at the crypt base, where they express stem and postmitotic endocrine markers. Further investigation of the function of this subset may provide novel insights into the genesis and development of enteroendocrine cells as well as enteroendocrine tumorigenesis.

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Figures

Fig. 1.
Fig. 1.
The numbers and positions of cholecystokinin-green fluorescent protein (CCK-GFP)+ cells along the duodenal primary crypt axis in CCK-GFP transgenic mice. A: representative confocal images of CCK-GFP+ cells in the crypts from different z-axis levels. The confocal fluorescence images were merged with the bright-field images using LSM510 Meta software. B: proportion of crypts based on the number of CCK-GFP+ cells per crypt. A total of 447 GFP+ cells were observed in 431 crypts, resulting in an average 1.04 GFP cells per crypt. C: distribution of CCK-GFP+ cells along the crypt axis. The frequency histogram indicates the proportion of GFP+ cells found in different cell positions along the crypt axis.
Fig. 2.
Fig. 2.
Expression of differentiation markers in the CCK-GFP+ cells at different cell positions in the duodenal primary crypts. The graphs indicate percent marker-positive cells in the GFP+ cells located at different cell positions in the crypt. The data for each marker were obtained from 50–80 crypts. NA, not applicable because no GFP+ cell was identified at the position in the preparation. P, positive, but the proportion bar was not displayed because n was <2. Lgr5, leucine-rich repeat-containing G protein-coupled receptor 5; DCAMKL1, doublecortin and CaM kinase-like-1; Ngn3, neurogenin 3; ChgA, chromogranin A; Ghln, ghrelin.
Fig. 3.
Fig. 3.
Expression of phenotypic markers in CCK-GFP+ cells in the primary duodenal crypts from CCK-GFP transgenic mice. A: confocal immunofluorescence images of CCK-GFP+ cells located above position 10 and stained for ChgA, ghrelin, and CCK. B: confocal immunofluorescence images of CCK-GFP+ cells located below position +4 and stained for Lgr5, DCAMKL1, CD133, Ngn3, ChgA, CCK, and ghrelin. The images of CCK-GFP (green), Alexa 633 or 577(red), and merged (orange) on transmitted light image are shown from left to right, respectively. Arrows indicate double-positive (i.e., GFP+/Immunostaining+) cells.
Fig. 4.
Fig. 4.
The confocal immunofluorescence images of Ki-67-stained primary crypts (A) and phospho-Histone H3-stained primary crypts (B) from CCK-GFP transgenic mice. The representative images of Ki-67-stained primary crypts from CCK-GFP transgenic mouse. CCK-GFP+ cells (left, green), Ki-67 (middle, red) and merged (right). Ki-67 was also negative for another 102 GFP+ cells from 60 crypts. The two pairs of the representative images of phospho-Histone H3-stained primary crypts from CCK-GFP transgenic mouse. CCK-GFP+ cells (left, green) and phospho-Histone H3 (right, red). Phospho-Histone H3 was also negative for another 77 GFP+ cells examined from 62 crypts. C: bar graph indicates frequency of phospho-Histone H3-stained cells (open bar) and the GFP+ cells (closed bar) located at different cell positions in the crypt. There were no double-positive cells.
Fig. 5.
Fig. 5.
Colocalization of Lgr5-GFP with CCK and ChgA in the primary duodenal crypts from Lgr5-GFP transgenic mice. A: expression of CCK in Lgr5-GFP+ cells at position 2′ (arrow) in the primary duodenal crypts from Lgr5-GFP transgenic mice. The confocal immunofluorescence images of Lgr5-GFP (green), Alexa 633-anti-CCK (red), and merged (orange) with DAPI staining (blue) are shown from left to right. The arrows point at a CCK staining-positive and Lgr5-GFP-positive cell at position 2′. B: expression of ChgA in Lgr5-GFP+ cells at position 1′ (arrow) in the primary duodenal crypts from Lgr5-GFP transgenic mice. The confocal immunofluorescence images of Lgr5-GFP (green), Alexa 633-anti-ChgA (red), and merged (orange) with DAPI staining (blue) are shown from left to right. The arrows point at a ChgA staining-positive and Lgr5-GFP-positive cell at position 1′. C: graph indicates percent of GFP-positive cells among the ChgA+ cells located at different cell positions in the crypt. A total of 119 ChgA+ cells from 90 crypts was analyzed. Either high or low intensity of GFP is relative, determined on the basis of the background fluorescence and the brightest GFP in the field and may be correlated with the classification by Sato et al. (26).
Fig. 6.
Fig. 6.
CCK-GFP organoid. A: immunofluorescence images of an organoid established from a CCK-GFP transgenic mouse. The transmitted light image of the organoid is shown in the center. Multiple images taken from the bottom to the top of the organoid are shown clockwise. CCK-GFP cells are seen throughout the organoid. The lumen shows high green fluorescence background presumably attributable to the accumulated GFP within or released from apoptotic or dead CCK-GFP cells. B: crypt that resembles the primary crypt and the CCK-GFP cells. The red dashed lines indicate locations of the CCK-GFP cells. A CCK-GFP cell (arrow) resides at position 2′ between Paneth cells, and two are at higher positions. C: whole-mount immunostaining for DCAMKL1 and ChgA. Left: merged images of CCK-GFP (green) and DCAMKL1 (red) on transmitted light image. Right: merged images of CCK-GFP (green) and ChgA3 (red) on transmitted light image. The orange dashed lines indicate the location of Paneth cells. The arrow points at an antibody staining-positive and GFP-positive cell.
Fig. 7.
Fig. 7.
A schematic summary of appearance, fate, and migration of CCK-GFP cells in the organoids established from the CCK-GFP transgenic mice. Oval-shape CCK-GFP+ cells in 17 crypts from three organoids (B3T, C3T, and C3Q) are shown. Solid oval represents CCK-GFP+ cell that was already present at the initiation of the recording. Open oval represents de novo CCK-GFP cells appearing during the 120-h observation period. Arrows indicate the direction of migration. No arrow means no movement. Oval with star inside in C3T5 represents a CCK-GFP cell that appeared and disappeared during the observation period. The shaded area indicates the area near position +4. Time-course images for B3T2 and C3T7 are shown in Figs. 8 and 9, respectively.
Fig. 8.
Fig. 8.
Appearance near position +4 and bidirectional migrations of CCK-GFP cells in the organoid. A CCK-GFP+ cell (arrow head), which was already present near position +4 at time 0, migrated upward and reached the crypt-villus border within 72 h. Following subsequent absence of GFP activity for 97 h, a new CCK-GFP cell (arrow) appeared near position +4 at time 165 h, migrated down, and stayed at the bottom of the crypt for at least 80 h. The time of the recording is indicated on top of each image. The red dashed line is a rough tracing of the crypt outline.
Fig. 9.
Fig. 9.
Appearance near position +4 and downward migration of the CCK-GFP cells in the organoid. Two CCK-GFP cells were present near the crypt base at time 0 and stayed in the base area. Two new CCK-GFP cells were observed. The first GFP cell (1) appeared near position +4 at time 23 h and stayed at the same location for the rest of the observation period. The second GFP+ cell (2) appeared near the first GFP cell and moved down to the crypt base within 48 h. The time of the recording is indicated on top of each image. The red dotted line is a rough tracing of the crypt outline. Note that several GFP+ cells seen in upper crypt area were from a neighboring crypt above this crypt.
Fig. 10.
Fig. 10.
Hypothetical scheme of the relationship between position and phenotypes of GFP+ enteroendocrine cells in CCK-GFP transgenic mice. The observations from the ex vivo organoid system indicated that GFP+ enteroendocrine cells appear near position +4 and then either migrate up to villus domains or migrate down to the crypt base. The observed correlation between the positions and expression of phenotypic markers in the primary crypts indicated that a subset that migrates upward differentiates into a mature CCK-producing cells and that a subset that migrates downward expresses undifferentiated stem cell markers in addition to enteroendocrine markers and multiple neuropeptides. GIP, glucose-dependent insulinotropic polypeptide.
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