Aging effects on intestinal homeostasis associated with expansion and dysfunction of intestinal epithelial stem cells

Abstract

Intestinal epithelial stem cells (IESCs) are critical to maintain intestinal epithelial function and homeostasis. We tested the hypothesis that aging promotes IESC dysfunction using old (18-22 months) and young (2-4 month) Sox9-EGFP IESC reporter mice. Different levels of Sox9-EGFP permit analyses of active IESC (Sox9-EGFP^Low), activatable reserve IESC and enteroendocrine cells (Sox9-EGFP^High), Sox9-EGFP^Sublow progenitors, and Sox9-EGFP^Negative differentiated lineages. Crypt-villus morphology, cellular composition and apoptosis were measured by histology. IESC function was assessed by crypt culture, and proliferation by flow cytometry and histology. Main findings were confirmed in Lgr5-EGFP and Lgr5-LacZ mice. Aging-associated gene expression changes were analyzed by Fluidigm mRNA profiling. Crypts culture from old mice yielded fewer and less complex enteroids. Histology revealed increased villus height and Paneth cells per crypt in old mice. Old mice showed increased numbers and hyperproliferation of Sox9-EGFP^Low IESC and Sox9-EGFP^High cells. Cleaved caspase-3 staining demonstrated increased apoptotic cells in crypts and villi of old mice. Gene expression profiling revealed aging-associated changes in mRNAs associated with cell cycle, oxidative stress and apoptosis specifically in IESC. These findings provide new, direct evidence for aging associated IESC dysfunction, and define potential biomarkers and targets for translational studies to assess and maintain IESC function during aging.

Keywords: Sox9; aging; intestinal epithelial stem cells; organoid.

Figure 1. Decreased enteroid forming efficiency and budding of crypts in enteroids from old compared to young animals

(A) Representative images of enterospheres and enteroids formed from crypts isolated from young and old mice and cultured in matrigel. Enterospheres are indicated by the black arrows. Buds are indicated by black triangles. Magnification : 10x, Scale bar : 100μm. (B) Quantification of enterospheres counted at day 1 that are able to grow into enteroids in matrigel culture. n=3 animals per group and 4-5 wells per animal, *p<0.05 Young vs Old, unpaired t test. (C) Quantification of enterosphere and enteroid complexity. n=3 animals per group and 4-5 wells per animal, *p<0.05 Young vs Old, unpaired t test.

Figure 2. Increased jejunal villus height and number of Paneth cells per crypt with age

(A) Representative hematoxylin and eosin stained sections of jejunal cross-sections from young and old mice showing crypt and villus architecture. Images were taken at 10x magnification. Scale bar, 50μm. (B) Representative images of crypt sections stained for the Paneth cell marker Lysozyme and nuclear marker DAPI. Magnification: 40x, Scale bar : 20μm. n=6 animals per group. (C) Representative images of crypt sections stained for the Phloxine Tartrazine and the Goblet cell marker Alcian Blue. Magnification: 40x, Scale bar : 20μm. n=5 young and 6 old animals.

Figure 3. Increased IESC in old mice

(A) Representative flow cytometry data of Sox9-EGFP expressing cells in young and old Sox9-EGFP reporter mice. Gate: R3=Sox9-EGFP^High, R4=Sox9-EGFP^Low, R5=Sox9-EGFP^Sublow. (B) Relative abundance of different Sox9-EGFP expressing cells measured by flow cytometry. n=19 animals per group, *p<0.05 Young vs. Old, unpaired t test. (C) Representative images of crypt sections from young and old Sox9-EGFP reporter mice stained with EGFP and the nuclear marker DAPI. Sox9-EGFP^Low IESC marked by closed triangles. Sox9-EGFP^High EEC marked by open triangles. Magnification : 40x, Scale bar : 20μm. (D) Quantification of the number of Sox9-EGFP^Low IESC counted per crypt section. n=8 young and 9 old animals, *p<0.05 Young vs. Old, unpaired t test. (E) Quantification of the number of Sox9-EGFP^High cells counted per crypt section. n=6 young and 7 old animals. (F) Representative images of Xgal stained crypt sections from young and old Lgr5-LacZ reporter mice. Magnification : 40x, Scale bar : 20μm. (G) Quantification of the number of Xgal stained Lgr5-LacZ IESC counted per crypt section. n=4, p<0.05 Young vs. Old, unpaired t test.

Figure 4. Old mice exhibit increased proportion of Sox9-EGFP^Low IESC and Sox9-EGFP^High cells in S-phase as assessed by flow cytometry and histology

(A and B) Relative abundance of Sox9-EGFP expressing populations that have incorporated EdU was measured by flow cytometry. n=6 animals per group, *p<0.05 Young vs. Old, unpaired t test. (C) Representative images of crypt sections from young and old Sox9-EGFP mice stained with EGFP, S-phase marker EdU and nuclear marker DAPI. Closed arrows indicate Sox9-EGFP^Low IESC in S-phase. (D) Number of total crypt cells incorporating EdU per Sox9-EGFP crypt section. n=6 animals per group. (E) Number of Sox9-EGFP^Low IESC incorporating EdU per crypt section. n=6 animals per group, *p<0.05 Young vs. Old, unpaired t test.

Figure 5. Histology to demonstrate increased proportion of Lgr5-EGFP IESC in S-phase in old mice

(A) Representative images of crypt sections from young and old Lgr5-EGFP mice stained with EGFP, S-phase marker EdU and nuclear marker DAPI. Closed arrows indicated Lgr5-EGFP IESC in S-phase. (B) Number of total crypt cells incorporating EdU per crypt section. n=5 young and 6 old animals. (C). Number of Lgr5-EGFP IESC incorporating EdU per crypt section. n=5 young and 6 old animals, *p<0.05 Young vs. Old, unpaired t test. Magnification: 40x, Scale bar : 20μm for all images

Figure 6. Increased proportion of small intestinal epithelial cell apoptosis with age

(A) Representative image of crypt and villus sections stained for cleaved caspase-3. Magnification : 10x, Scale bar : 100μm. (B) Quantification of the number of cleaved caspase-3 positive cells per villus section (n=7 animals per group) and per crypt section (n=10 young and 9 old animals). *p<0.05 Young vs. Old, unpaired t test. (C) Representative image of crypt sections stained for cleaved caspase-3. Magnification: 40x, Scale bar: 20μm. (D) Quantification of the location of cleaved caspase-3 positive cells per 40 crypts per animal by position. Inlay shows method of identifying cell position within crypt. n=10 young and 9 old animals, *p<0.05 Young vs. Old, unpaired t test.

Figure 7. Isolated Sox9-EGFP cell populations from young and old mice are enriched for known biomarkers associated with specific population

Sox9-EGFP cell populations isolated using FACS underwent high throughput qRT-PCR for the genes (A) Sox9, (B) ChgA, (C) Lgr5, (D) Hopx. n≥3 animals per group, #p<0.05 compared to all other Sox9-EGFP populations, 1-way ANOVA, Bonferroni. n=4 per group.

Figure 8. Model outlining the effects of aging on the small intestine

This model suggests that IESC specific changes in proliferation and cell cycle regulation with age are the result of oxidative stress. Cell cycle is accelerated in IESC resulting in IESC hyperproliferation and an increased IESC pool. The observed increase in Paneth cells may be required to support the increased number of IESC per crypt in aged animals. Increased IESC proliferation with age may lead to, increased DNA damage, resulting in p53 activated IESC apoptosis and decreased IESC function.