IGF1 stimulates crypt expansion via differential activation of 2 intestinal stem cell populations

Abstract

Insulin-like growth factor 1 (IGF1) has potent trophic effects on normal or injured intestinal epithelium, but specific effects on intestinal stem cells (ISCs) are undefined. We used Sox9-enhanced green fluorescent protein (EGFP) reporter mice that permit analyses of both actively cycling ISCs (Sox9-EGFP(Low)) and reserve/facultative ISCs (Sox9-EGFP(High)) to study IGF1 action on ISCs in normal intestine or during crypt regeneration after high-dose radiation-induced injury. We hypothesized that IGF1 differentially regulates proliferation and gene expression in actively cycling and reserve/facultative ISCs. IGF1 was delivered for 5 days using subcutaneously implanted mini-pumps in uninjured mice or after 14 Gy abdominal radiation. ISC numbers, proliferation, and transcriptome were assessed. IGF1 increased epithelial growth in nonirradiated mice and enhanced crypt regeneration after radiation. In uninjured and regenerating intestines, IGF1 increased total numbers of Sox9-EGFP(Low) ISCs and percentage of these cells in M-phase. IGF1 increased percentages of Sox9-EGFP(High) ISCs in S-phase but did not expand this population. Microarray revealed that IGF1 activated distinct gene expression signatures in the 2 Sox9-EGFP ISC populations. In vitro IGF1 enhanced enteroid formation by Sox9-EGFP(High) facultative ISCs but not Sox9-EGFP(Low) actively cycling ISCs. Our data provide new evidence that IGF1 activates 2 ISC populations via distinct regulatory pathways to promote growth of normal intestinal epithelium and crypt regeneration after irradiation.

Keywords: actively cycling ISCs; facultative/reserve ISCs; irradiation; tissue regeneration.

Figure 1.

IGF1R is enriched in Sox9-EGFP^Low ISCs. A) Quantitative RT-PCR for IGF1R in the 4 different FACS-isolated Sox9-EGFP cell populations. Data are expressed as means ± SEM of fold change in IGF1R gene expression relative to total (nonsorted) cells (n = 5; ANOVA; ^aP < 0.05 vs. total cells; ^bP < 0.05 vs. Sox9-EGFP^Negative IECs; ^cP < 0.05 vs. Sox9-EGFP^Sublow progenitors; ^dP < 0.05 vs. Sox9-EGFP^High cells). IGF1R mRNA was significantly enriched in Sox9-EGFP^Low ISCs compared with other Sox9-EGFP cells, and was also expressed at higher levels in Sox9-EGFP^Sublow progenitors and Sox9-EGFP^High cells vs. Sox9-EGFP^Negative differentiated IECs. B) IGF1R immunoreactivity in intestinal epithelium strongly localized to the base of the crypts within the progenitor/ISC compartment. Red arrowheads show IGF1R-positive cells in the ISC zone (scale bar, 100 µm).

Figure 2.

Five day IGF1 treatment has trophic effects in normal and regenerating intestinal epithelium. A) Hematoxylin and eosin staining in nonirradiated mice demonstrates that IGF1 induced significant changes in intestinal morphology including increased crypt density, crypt depth, and villus height (scale bar, 250 µm). B) Morphometry data were quantified on 10 zones of ∼1.5 mm spanning the entire jejunum and are expressed as means ± sem (n = 3 mice per group; n ≥ 41 villi/mouse; n ≥ 48 crypts/mouse; Student's t test; ^aP < 0.05 vs. vehicle-treated mice). C) Hematoxylin and eosin staining at day 5 after radiation shows hyper-regenerative crypts in IGF1-treated mice compared with vehicle-treated controls (scale bar, 250 µm). D) Quantitative morphometry was performed on 20 zones of ∼1.5 mm spanning the entire jejunum and confirmed that IGF1 induced a significant increase in crypt depth after radiation (n = 3 mice per group; n ≥ 79 crypts/mouse; Student's t test; ^aP < 0.05 vs. vehicle-treated mice).

Figure 3.

IGF1 treatment preferentially expands Sox9-EGFP^Low cells. A) Immunofluorescence illustrates that 5-day IGF1 therapy increased the number of Sox9-EGFP^Low ISCs per crypt section in both normal (Non-Irr) and regenerating crypts (Irr) compared with vehicle (nuclei staining, DAPI, blue; Sox9-EGFP, green; scale bar, 50 µm). White arrows show Sox9-EGFP^Low cells in nonirradiated crypts. White arrowheads show Sox9-EGFP^High cells. B, C) Quantitative data are expressed as means ± sem (n = 4; Student's t test; ^aP < 0.05 vs. nonirradiated vehicle-treated mice; ^bP < 0.05 vs. nonirradiated IGF1-treated mice, ^cP < 0.05 vs. irradiated vehicle-treated mice). B) Quantitative analysis revealed that IGF1 significantly increased the number of Sox9-EGFP^Low ISCs per crypt section in normal and regenerating crypts but did not affect the number of Sox9-EGFP^High cells per crypt section. Thirty crypts were analyzed per animal in blinded fashion. C) Flow cytometry demonstrated that IGF1 significantly increased the proportion of Sox9-EGFP^Low ISCs in both nonirradiated and irradiated regenerating crypts but did not alter percentages of Sox9-EGFP^High and Sox9-EGFP^Sublow cells in either nonirradiated or irradiated intestines.

Figure 4.

IGF1 increases the proportion of pH3-positive Sox9-EGFP^Low cells only during crypt regeneration. A) Immunofluorescence demonstrates increased number of pH3-positive cells per crypt section in IGF1-treated mice vs. vehicle-treated controls at 5 days after radiation (Irr), but no effect of IGF1 on pH3-stained cells in normal crypts (Non-Irr). White arrowheads show pH3-positive Sox9-EGFP^Low cells (nuclei staining, DAPI, blue; Sox9-EGFP, green; pH3, red; scale bar, 50 µm). B, C) Quantitative data are expressed as means ± sem (n = 3; Student's t test; ^aP < 0.05 vs. nonirradiated vehicle-treated mice; ^bP < 0.05 vs. nonirradiated IGF1-treated mice, ^cP < 0.05 vs. irradiated vehicle-treated mice). B) Histogram shows total numbers of pH3-labeled cells per crypt section and demonstrates an increase in pH3-positive cells in regenerating crypts vs. nonirradiated controls and that IGF1 significantly enhanced this effect. Thirty crypts were studied per animal in blinded fashion. C) Histogram shows the proportion of Sox9-EGFP^Low cells colabeled with pH3. IGF1 significantly increased the proportion of pH3-labeled Sox9-EGFP^Low ISCs in regenerating crypts but not in nonirradiated crypts. D) Immunofluorescence illustrates that rare pH3-positive Sox9-EGFP^High cells were only found in mice treated with IGF1 for 5 days after radiation.

Figure 5.

IGF1 increases proportions of EdU-positive Sox9-EGFP^High cells but not Sox9-EGFP^Low cells. A) Immunofluorescence illustrates that IGF1 increased the numbers of EdU-positive cells per crypt section at 5 days after irradiation (Irr), but not in nonirradiated controls (Non-Irr). White arrowheads show EdU-positive Sox9-EGFP^High cells (nuclei staining, DAPI, blue; Sox9-EGFP, green; EdU, red; scale bar, 50 µm). B, C) Quantitative data are expressed as means ± sem (n = 4; Student's t test; ^aP < 0.05 vs. nonirradiated vehicle-treated mice; ^bP < 0.05 vs. nonirradiated IGF1-treated mice, ^cP < 0.05 vs. irradiated vehicle-treated mice). B) Histogram demonstrates that at 5 days after radiation, there was a dramatic increase in total numbers of EdU-positive cells per crypt section compared with uninjured intestine and that IGF1 further increased the total number of EdU-labeled cells per crypt section in irradiated mice. Thirty crypts were studied per animal in blinded fashion. C) Colocalization of EdU staining and Sox9-EGFP assessed the proportion of Sox9-EGFP^Low and Sox9-EGFP^High cells in S-phase. IGF1 induced no significant change in the proportion of EdU-positive Sox9-EGFP^Low ISCs in either nonirradiated or irradiated regenerating crypts. IGF1 treatment significantly increased the percentages of EdU-labeled Sox9-EGFP^High cells per crypt section in nonirradiated and regenerating crypts.

Figure 6.

IGF1 exerts differential impact on the gene expression profiles of the 2 Sox9-EGFP ISC populations. A and B illustrate selected mRNAs significantly regulated by IGF1 selectively/exclusively in each Sox9-EGFP ISC population isolated from uninjured small intestine (A) and at day 5 after radiation (B).

Figure 7.

Exogenous IGF1 directly stimulates enteroid formation ability in Sox9-EGFP^High cells but not in Sox9-EGFP^Low cells. A) (Upper) Representative photographs of enteroids grown from Sox9-EGFP^Low cells in absence or presence of IGF1 at 100 and 200 ng/ml at 10 days after plating (scale bar, 100 µm). (Lower) Quantitative data are expressed as means ± sem of fold change in numbers of enteroids formed from Sox9-EGFP^Low cells grown in presence of IGF1 relative to numbers of enteroids formed from untreated Sox9-EGFP^Low cells normalized to initial numbers of cells plated per well. Quantification of enteroid numbers demonstrated that IGF1 at 100 and 200 ng/ml did not significantly affect numbers of enteroids formed from Sox9-EGFP^Low cells vs. untreated controls at 4 and 10 days after plating. B) Representative photographs of enteroids grown from Sox9-EGFP^High cells in absence or presence of IGF1 at 100 and 200 ng/ml at 10 days after plating. In the absence of IGF1, Sox9-EGFP^High cells yielded no enteroids. Addition of exogenous IGF1 resulted in the ability of Sox9-EGFP^High cells to yield enteroids. IGF1 impact on enteroid formation from Sox9-EGFP^High cells was similar at all doses of IGF1 tested.

Figure 8.

Cellular and molecular mechanisms involved in IGF1-induced crypt expansion. The schematic summarizes the cellular and molecular changes associated with crypt expansion induced by IGF1 therapy in uninjured intestine or during radiation-induced crypt regeneration.