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

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-EGFPLow), activatable reserve IESC and enteroendocrine cells (Sox9-EGFPHigh), Sox9-EGFPSublow progenitors, and Sox9-EGFPNegative 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-EGFPLow IESC and Sox9-EGFPHigh 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.


INTRODUCTION
Aging is a complex process resulting in decreased tissue function, and cellular and molecular damage. Adult stem cells that self-renew and maintain progenitors that appropriately differentiate into functional cell types are essential to maintain tissue homeostasis and repair throughout lifespan [1]. Aging-associated exhaustion and dysfunction have been reported in highly prolife-rative hematopoietic stem cells (HSC) [2], hair follicle stem cells (HFSC) [3], muscle stem cells [4] and adipose-derived mesenchymal stem cells [5]. Reduced stem cell function contributes to aging associated degenerative diseases [6], and is associated with molecular changes including accumulation of reactive oxygen species (ROS) [7], defects in cell cycle regulation [8] and cellular senescence [5].
AGING Rapid renewal of the intestinal epithelium is driven by resident intestinal epithelial stem cells (IESC). IESC divide to self-renew and generate progenitor cells that undergo rapid proliferation followed by terminal differentiation into cell types including enterocytes, Paneth cells, goblet cells and enteroendocrine cells (EEC) [9]. Differentiated cells perform absorptive, barrier, antimicrobial and secretory functions, and endocrine and metabolic regulatory functions essential to health. In Drosophila, aging is associated with increased IESC number and proliferation, and IESC dysfunction, including activation of stress response pathways such as JNK [10] and p38-MAPK [11]. In mammals, it has been demonstrated that aging is associated with decreased intestinal barrier function [12], impaired nutrient absorption [13], small bowel bacterial overgrowth [14] and increased risk of gastrointestinal (GI) cancers [15,16]. Previous reports in mouse models of accelerating aging indicate phenotypic changes in the intestinal epithelium including defective regeneration, cell cycle dysregulation [17] and altered Wnt signaling [18], indicating that aging may inhibit IESC/progenitor cell maintenance or function.
Prior studies in rodent models suggest that aging may be associated expansion of IESC or progenitor cells, but with reduced capacity for tissue repair following injury [19][20][21][22]. However, the direct effects of aging on IESC could not be examined in these studies due to a lack of IESC specific biomarkers to permit direct IESC visualization, isolation and functional analyses. The identification of specific biomarkers of IESC such as Lgr5 [23] and the development of fluorescent IESC reporter mice allow direct IESC evaluation in vivo and IESC isolation for ex vivo studies and molecular analyses [9]. Sox9-EGFP reporter mice provide a useful model because distinct levels of enhanced green fluorescent protein (EGFP) expression mark different intestinal epithelial cell (IEC) populations [24,25]. Sox9-EGFP High cells are hormone-producing EEC and cells with functional characteristics of activatable, reserve IESC [25,26]. Sox9-EGFP Low cells are active IESC also termed crypt based columnar cells (CBC) [23,27]. Sox9-EGFP Low cells have been functionally validated as IESC based on the ability of single Sox9-EGFP Low cells to form enteroids in vitro when isolated from uninjured small intestine [24,25,28]. Sox9-EGFP Low cells are enriched for Lgr5 mRNA and many other mRNAs expressed at high levels in Lgr5-expressing IESC-CBC [25,29]. Sox9-EGFP Sublow cells are rapidly dividing progenitor cells that give rise to differentiated progeny [24,25]. Sox9-EGFPN egative cells contain differentiated IEC, particularly absorptive enterocytes, Paneth cells and goblet cells [25], based on histologic analysis and high level expression of biomarkers of these cell types.
This study used Sox9-EGFP, Lgr5-EGFP and Lgr5-LacZ reporter models to test the hypothesis that aging leads to IESC dysfunction and altered intestinal epithelial homeostasis. Results reveal new and direct evidence for IESC dysfunction in old mice associated with IESC hyperproliferation and expansion but increased apoptosis. These functional changes in IESC were associated with altered expression of mRNAs encoding proteins and pathways that stimulate cell cycle progression, DNA damage and apoptosis specifically in IESC, providing new biomarkers and potential mechanisms of IESC aging.

RESULTS
Aging-associated decreases in enterosphere formation and enteroid development Crypt culture in a 3D matrigel system provides a useful and widely used system to assess IESC function [30]. Isolated intestinal crypts form round enterospheres that expand and differentiate to form more complex enteroids with bud structures after several days in culture [31]. Crypt culture revealed that intestinal crypts isolated from both young and old animals formed enterospheres in vitro on day 1 after plating ( Figure  1A). Efficiency of crypt culture was calculated by dividing the number of enterospheres and enteroids present at day 4 or 8 by the number of enterospheres present on day 1 in each well. This provides a measurement of how many enterospheres initially plated were able to survive and grow in crypt culture conditions. Efficiency measurements revealed a trend for decreased enteroid survival in old vs. young at day 4 post plating and a significant decrease in enteroid survival in old animals at day 8 post plating ( Figure  1B). Crypt-derived enteroids typically begin to show bud structures by 3-7 days post plating [31]. Each bud represents a crypt structure that containing stem and progenitor cells and the number of buds provide a surrogate for IESC function [32]. The numbers of buds per enteroid were counted at days 4 and 8 post plating, categorizing enteroids with 2 buds or fewer as less complex, and enteroids containing 3 or more buds as more complex. Following 4 days in culture there was no difference in the enteroid complexity between young and old ( Figure 1C). By day 8 post plating, enteroids from old mice showed a decrease in complexity compared to those from young mice as significantly fewer enteroids from old animals contained 3 or more buds ( Figure 1C). At 15 days post plating very complex enteroids had formed from the crypts derived from young mice, while the enteroids formed from old mice were much less complex ( Figure 1A).

Increased villus height and Paneth cell number in small intestine of old mice
Jejunal morphology and morphometry and the presence of differentiated cell types were assessed by histology. Results revealed no significant difference in crypt depth, crypt or villus density, total number of cells per crypt, or mucosal circumference between young and old mice, but demonstrated a significant increase in villus height in old vs. young mice (Table 1 and Figure 2A). Immunofluorescence for lysozyme, a Paneth cell biomarker, revealed a small but significant increase in the number of Paneth cells per crypt in old vs. young (Table  1 and Figure 2B). Alcian Blue positive goblet cells were quantified and revealed no change in the number of mucus producing goblet cells between the young and old mice (Table 1 and Figure 2C).

Old mice exhibit increased IESC number and enhanced IESC proliferation
Flow cytometry on IEC isolated from Sox9-EGFP mice revealed significantly more IESC (Sox9-EGFP Low ) and progenitor cells (Sox9-EGFP Sublow ) in old vs. to young ( Figure 3A and 3B). However, the Sox9-EGFP High cell population that contains enteroendocrine cells (EECs) and a reserve stem cell population activatable upon injury and regeneration [25] did not differ between young and old mice ( Figure 3A and 3B). Results were confirmed by EGFP immunofluorescence, which revealed no change in the number of Sox9-EGFP High cells per crypt in old vs. young ( Figure 3C and 3E) but demonstrated a significant increase in the number of Sox9-EGFP Low IESC per crypt in old animals ( Figure  3C and 3D). This was further confirmed in the Lgr5-LacZ IESC reporter mouse model [23] where expansion of Lgr5-LacZ IESC was observed in old vs. young ( Figure 3F and 3G). Of note, using histology it is not possible to reliably quantify Sox9-EGFP Sublow progenitor cells by EGFP immunofluorescence because the very low EGFP expression cannot be readily distinguished from background.
Flow cytometry of dissociated jejunal IEC from young and old Sox9-EGFP reporter mice detected incorporation of the S-phase marker EdU in 3 distinct EGFP-expressing cell populations ( Figure 4B). As expected, rapidly dividing Sox9-EGFP Sublow progenitor cell population showed the most EdU positive cells compared to other Sox9-EGFP populations ( Figure 4). AGING Based on percentage of cells incorporating EdU, there was no detectable difference in proliferation of Sox9-EGFP Sublow progenitors between young (33.7±2.7%) and old (37.6±5.0%) ( Figure 4). However, there was a significant increase in the percentage of EdU-positive Sox9-EGFP Low IESC in old (39.5±4.3%) vs. young (26.3±3.8%), indicating hyperproliferation of IESC in old mice. The percentage of EdU-labeled Sox9-EGFP High cells was significantly higher in old (7.8±1.6%) vs. young (2.4±0.5%) ( Figure 4). This provides evidence that old mice have a greater proportion of "activatable reserve" IESC in S phase.
Immunofluorescence for EdU revealed no difference in the total number of EdU positive cells per crypt in old vs. young ( Figure 4C, 4D). However, co-immunofluorescence for EdU and GFP revealed a significant increase in the number of EdU positive Sox9-EGFP Low IESC located at the crypt base in old mice ( Figure 4C, 4D). Results were confirmed in an independent IESC reporter mouse model, Lgr5-EGFP [23] ( Figure 5A-C), which revealed significantly more Lgr5-EGFP IESC co-labeled with EdU in old (2.3±0.2) vs. young (1.5±0.2) ( Figure 5C). Together these data provide strong evidence that aging is associated with a selective increase in proliferation and/or altered cell cycle dynamics in IESC of old mice.

Old mice exhibit increased apoptosis of the small intestinal epithelial cells
Given the age-associated IESC dysfunction indicated by our results in the ex vivo enteroid culture system, we tested whether apoptosis was increased in the small intestinal crypt of old vs. young mice. Cleaved caspase-3 immunohistochemistry revealed a significant increase in the number of apoptotic cells per crypt section in old vs. young ( Figure 6A, 6B-right and 6C). The location of cleaved caspase-3 positive cells within the crypt was assessed by dividing the crypt in half lengthwise and counting the position of apoptotic cells from the base of the crypt ( Figure 6D). Most apoptotic cells in old animals were at the base of the crypt in positions 1-4 ( Figure 6C and 6D), indicating preferential increases in apoptosis within the IESC zone. Old mice showed a significant increase in cleaved caspase-3 positive cells in the villi ( Figure 6A and 6B-left), that were strikingly located along the villus epithelium rather than the villus tip.

Aging-associated changes in gene expression
To gain insight into molecular mechanisms or biomarkers of age associated functional changes in IESC and other cell populations between old and young mice, we used FACS (Fluorescence Activated Cell Sorting) coupled with Fluidigm mRNA profiling. First we validated that profiles of Sox9 mRNA and biomar- AGING kers of specific IEC subtypes exhibited predicted profiles in isolated cell populations from young and old Sox9-EGFP mice ( Figure 7A-D). Our data verified that the expression profiles of Sox9 mRNA in FACSisolated cells matched the profiles of Sox9-EGFP used to FACS isolate cell populations ( Figure 7A). Furthermore, Sox9 mRNA profiles across Sox9 negative, sublow, low and high populations were consistent between young and old. The IESC specific marker Lgr5 was enriched in the Sox9-EGFP Low population from both young and old, consistent with phenotypic similarities between Sox9-EGFP Low and Lgr5-EGFP-expressing IESC ( Figure 7B). Sox9-EGFP High populations from young and old were enriched for EEC specific mRNA ChgA ( Figure 7C) and Hopx, a putative biomarker of activatable reserve IESC ( Figure 7D). To gain insight into the mechanisms driving aging-associated IESC expansion and hyperproliferation, we examined expression of mRNAs in-volved in cell cycle, apoptosis and maintenance of homeostasis in cell populations isolated from Sox9-EGFP mice. Consistent with histology and flow cytometry data suggesting increased proportion of active Lgr5-expressing and activatable reserve IESC in S-phase in old mice, we observed a significant increase in Ccnd1 and Cdk6 mRNAs in Sox9-EGFP Low cells and a dramatic increase in Ccnd1 mRNA in Sox9-EGFP High cells from old vs. young (Table 2A, 2B). Ccnd1 and Cdk6 are required for cell cycle progression and activate G1 to S phase transition [33,34]. Additional mRNAs involved in regulating the cell cycle and proliferation were significantly different in cell populations from old vs. young including Max in Sox9-EGFP High and p21 in Sox9-EGFP Low (p21) ( Table 2A,  AGING EGFP Low IESC from old mice (Table 2B). We found a significant increase in the mRNA encoding Nrf2, a marker of oxidative stress, in Sox9-EGFP Low IESC from old mice (Table 2B). Significantly decreased levels of mRNAs associated with proliferation, apoptosis and homeostasis were observed in the old Sox9-EGFP Sublow progenitor cell population, including Pten, Yap1, Ephb2, Notch2 and Bbc3 (Table 2C). H2afx mRNA levels were increased specifically in Sox9-EGFP Negative cells from old mice, consistent with and providing a potential biomarker of age-associated increases in apoptosis of differentiated cells on the villus. Decreased levels of mRNAs known to be associated with tissue homeostasis with aging in Sox9-EGFP Negative cells from old mice are consistent with increases in villus height, a sign of altered homeostasis.

DISCUSSION
Until 2007 the study of IESC and the effect of aging on IESC was hampered by a lack of specific biomarkers. Our findings in the Sox9-EGFP reporter mouse, confirmed in Lgr5-EGFP and Lgr5-LacZ reporter mice models, provide new and direct evidence that aging is associated with increases in IESC number, proliferation, and increases in IESC apoptosis in vivo and functional impairment of isolated IESC in vitro. Our findings of increased villus height and expansion of Paneth cells in old mice indicate altered intestinal homeostasis with aging.
Consistent with our findings, aging-associated changes in small intestinal morphology including increased in villus height have been reported in mice [35], rats [36] and humans [37]. Previous findings that old rats exhibit decreased absorption of fatty acids and glucose indicate aging-associated functional impairment in nutrient absorption [38]. Aging-associated increases in villus height may therefore represent a compensatory mechanism to reduce risk of malnutrition with aging.
Our results showing increased cleaved caspase-3 positive cells on villi of old mice provide additional support for aging-associated functional impairment of differentiated enterocytes. Our findings also support a model where increased villus height in old animals is maintained by an expanded pool of hyperproliferative IESC that is needed to generate additional differentiated enterocytes to enhance nutrient absorption. AGING Table 2. mRNAs significantly different in specific cell populations isolated from old versus young Sox9-EGFP mice.

AGING
Paneth cells release trophic factors in the IESC niche, contribute to maintained IESC function [39], and secrete antimicrobial peptides to provide host defense against microbes [40]. Age-associated increases in bacterial load have been reported in the intestinal lumen of Drosophila and were associated with increased IESC proliferation [41]. Furthermore, previous studies in mice and humans reported changes in intestinal microbiome with age including changes in proportion of major microbes and decreased overall microbiota diversity [42,43]. Our observed increases in Paneth cell number in old mice therefore suggest an adaptive response that may promote IESC expansion and could reflect ageassociated changes in microbiota.
Our findings in the Sox9-EGFP mouse model and confirmative data in Lgr5-EGFP and Lgr5-LacZ IESC provide, to our knowledge, the first direct evidence for age-associated expansion and hyperproliferation of mammalian IESC. Our findings that crypts from old animals have reduced enteroid forming ability and yield enteroids with reduced complexity compared with enteroids formed from young crypts provide new evidence for age-associated functional impairment of IESC.
The Sox9-EGFP mouse model offers the opportunity to study and isolate progenitor cells (Sox9-EGFP Sublow ) and Sox9-EGFP High cells that contain EEC cells and an activatable reserve stem cell population [25]. A recent study suggested that subsets of secretory EEC cells can act as reserve stem cells in humans [45]. Interestingly, we observed an increased proportion of Sox9-EGFP High cells in S phase of the cell cycle in old mice, yet no expansion in cell number. This provides novel evidence that aging is associated with activation and proliferation of reserve stem cells as a mechanism to maintain epithelial renewal during aging.
Based on in vivo findings of aging-associated IESC hyperproliferation and increased apoptosis in the stem cell zone of the crypt, we assessed levels of mRNAs encoding proteins linked to cell cycle regulation, oxidative stress, DNA damage and apoptosis in the different Sox9-EGFP cell populations isolated from young and old mice (Probes for mRNAs showing significant differences between cells from young and old mice are described in Supplementary Table 1). Sox9-EGFP High cells and Sox9-EGFP Low IESC from old mice exhibited increased levels of mRNAs encoding proteins AGING that accelerate G1 to S phase transition including Ccnd1, Cdk6, Max and Mlx [46]. IESC hyperproliferation may increase the probability of genomic instability, triggering a DNA damage response and apoptosis [46]. Elevation of Nrf2 mRNA in Sox9-EGFP Low IESC from old mice adds support to a model of aging-associated cellular and molecular damage in IESC [48,49]. Nrf2 promotes accumulation of reactive oxygen species (ROS), which causes DNA damage, and has been linked to aging in both drosophila and mammals [47]. Elevation of mRNAs encoding proteins known to stimulate cell apoptosis including p53, Sirt7, Max, Bak1 and Bax in actively cycling Sox9-EGFP Low IESC provide new evidence for IESC-specific mediators linked to IESC dysfunction. p53 is induced in response to DNA damage or cellular stress and activates cell cycle arrest, apoptosis or senescence pathways [50,51]. Our observed induction of both p53 mRNA and the downstream mediator, Perp [52,53] in old IESC support a model of aging-linked oxidative stress and DNA damage-induced apoptosis. Overall, mRNA profiling data on young and old IESC and differentiated lineages provide new evidence that aging is associated with altered expression of multiple genes involved in regulation of cell cycle, proliferation, apoptosis, DNA damage and repair.
Together our findings support a model where dysregulated cell cycle progression and evasion of cell cycle checkpoints promotes age-related hyperproliferation and expansion of actively cycling IESC, increasing cellular stress, DNA damage, and leading to increased apoptosis and impaired IESC function ( Figure  8). This model is consistent with aging-associated dysfunction and loss of stem cell populations in other organs [54]. Observed increases in proliferation of Sox9-EGFP High cells, indicate that reserve, activatable IESC are mobilized to compensate for hyperproliferation but impaired function of actively cycling IESC. Age-associated IESC hyperproliferation may be necessary to support increased villus height, compensate for increased villus apoptosis and maintain functions of the intestinal epithelium with aging. Our identification of mediators specifically altered in IESC from old mice provides a basis for translational studies of mediators and pathways of IESC aging in humans and to identify potential targets for new approaches or therapies that may ameliorate aging-associated dysfunction or disease of the AGING intestine. Importantly, our IESC-reporter mice provide the models for preclinical studies of the effect of potential interventions specifically on IESC aging.

Mouse models
Sox9-EGFP mice on an outbred CD-1 background were maintained as heterozygotes and genotyped as previously described [24,28]. Lgr5-EGFP-IRES-CreERT2 (Lgr5-EGFP) mice on a C57BL/6 background were ordered from The Jackson Laboratory (Bar Harbor, ME) and maintained as heterozygotes. Original Lgr5-LacZ mice on a mixed background were generously provided by Lexicon Pharmaceuticals (The Woodlands, TX). Lgr5-LacZ mice on C57BL/6 background that were used in this study were generated after 10 generations of backcrossing the original Lgr5-LacZ mice with C57BL/6 wild type mice. Young mice studied were 2 to 4 months old. Old mice were 18 to 24 months old. Young and old female and male mice were studied. For the old study groups, females were 20-24 months old and males 18-20 months old. These ages were selected based on pilot studies demonstrating no overt signs of disease in female or male mice at these ages.

Crypt isolation and culture
Jejunal crypts were isolated and in vitro crypt culture was performed as previously described [32].

Intestinal epithelial cell isolation, flow cytometry and FACS
Intestinal epithelial cells were isolated from the jejunum of young and old Sox9-EGFP mice for flow cytometry as previously described [25].  [56]. β-actin was used as the reference gene to normalize mRNA input and allow accurate comparisons of results between samples. An unsorted, pooled RNA sample was set as the control sample to which all other samples were compared. GraphPad Prism 5 was used to perform a 1-way ANOVA with Bonferroni post-hoc test to test for differences in levels of mRNA across different cell populations and between cells from young and old mice.

Statistical analysis
Data are expressed as mean ± SEM. For histology morphometry, histology, flow cytometry and in vitro culture experiments, unpaired Student's t-tests were performed to compare data from young and old mice.

AGING
For high throughput qRT-PCR experiments one-way ANOVA with Bonferroni post hoc test was performed for comparisons of multiple groups. A P value of less than 0.05 was considered statistically significant.