Here we show the murine ISC number in vivo and functional activity ex vivo decline due to aging. In striking contrast, the number of Paneth cells in mice and their function in supporting ISCs are not impaired by aging. NAD + supplementation by the NAD + precursor NR can rescue these ISC defects and restore a youthful number and function of ISCs. Thus our findings suggest a translational path of NAD + replenishment for rejuvenating the aged gut.

Calorie restriction (CR) has been shown to trigger the release of cyclic ADP ribose from the ISC niche, the Paneth cells, (Yilmaz et al., 2012 ), to drive a pathway of signaling in LgR5‐expressing ISCs to promote their expansion in cell number. This pathway involves SIRT1 activation due to increases in the NAD + biosynthetic enzyme, nicotinamide phosphoribosyl transferase (Nampt), SIRT1 deacetylation of S6 kinase 1 (S6K1), and consequent phosphorylation of deacetylated S6K1 by mTORC1 (Igarashi & Guarente, 2016 ). Notably, mTORC1 activation by CR in ISCs is opposite to its observed repression during CR in many differentiated cells, including Paneth cells. As predicted by the model, the mTORC1 inhibitor rapamycin and genetic ablation of SIRT1 suppress the effect of CR on ISC expansion (Igarashi & Guarente, 2016 ).

The rapid turnover of the intestinal epithelium is sustained by ISCs. Previous studies of aging of ISCs mainly come from studies on the intestinal epithelium of Drosophila (Biteau, Hochmuth, & Jasper, 2011 ). During aging, the number and activity of cells that express stem cells marker in Drosophila midgut increase due to an environmental challenge or tissue injury (Biteau, Hochmuth, & Jasper, 2008 ; Choi, Kim, Yang, Kim, & Yoo, 2008 ; Hochmuth et al., 2011 ). In mammals, Lgr5‐expressing cells in the base of the crypt constitute the majority of ISCs under normal conditions (Barker, Tan, & Clevers, 2013 ; Barker et al., 2007 ). Recently, it was reported that aging results in a decline in ISCs function in mammals and wnt signaling ameliorated the impaired of function of aged ISCs (Mihaylova et al., 2018 ; Nalapareddy et al., 2017 ).

Aging is one of major risk factors of adult‐onset disease such as cancer, diabetes, Alzheimer's and Parkinson's disease, and cardiovascular disease (Niccoli & Partridge, 2012 ). Adult tissue homeostasis is controlled by adult stem cells, which are continuously proliferative and maintain the tissue (hematopoietic and intestinal stem cells [ISCs]) or quiescent and induced by tissue injury (muscle, liver, and neural stem cells) (Chandel, Jasper, Ho, & Passegué, 2016 ). Previous studies have described the aging‐related deterioration of adult stem cell function. In certain cases, this decline could be attributed to a loss in the activity of one of the sirtuins, which are nicotinamide adenine dinucleotide (NAD + )‐dependent deacylases that regulate aging and age‐related diseases (Guarente, 2013 ). For example, in hematopoietic stem cells, a reduction in SIRT3 or SIRT7 activity compromises the regenerative capacity of HSCs in aging mice (Brown et al., 2013 ; Mohrin et al., 2015 ). In muscle stem cells (MuSCs), reduced amounts of NAD + and the associated decline in activity of SIRT1 are determinants of aging‐related decline. Notably, treatment with NAD + precursor nicotinamide riboside (NR) (Cantó et al., 2012 ) induces the rejuvenation of MuSCs in aged mice and extends the lifespan of the animals (Zhang et al., 2016 ).

2 RESULTS

2.1 The ISC pool decreases due to aging To assess the effect of aging on gut homeostasis, we compared the histology of small intestine in young (3–5 months old) and old (more than 24 months old) C57BL/6 mice. Morphologically, aging induced an increase in villus length and crypts showed a trend to a smaller size, which did not reach significance (Figure 1a). In agreement with the increase in villus size, aging increased the numbers of differentiated cells of the gut: absorptive enterocytes, goblet cells, and chromogranin A+ enteroendocrine cells (Supporting Information Figure S1a–c). Figure 1 Open in figure viewer PowerPoint Aging reduces ISC number in vivo. (a) H & E staining images and the quantification of the crypt size and villus size in the intestine of young (3 months old) and old (more than 24 months old) C57BL/6 mice (six mice per group, approximately 50 crypt/villus units per mouse). All histological images in this and subsequent figures are typical of numerous tissue samples analyzed. (b) BrdU staining images and the quantification of BrdU‐positive crypt base columnar (CBC) cells (arrowheads) adjacent to Paneth cells at the bottom of crypts as assessed 2 hr after the injection of BrdU (three mice per group, 50 intact well‐orientated crypts per mouse). (c) In situ hybridization images of Olfm4 mRNA and the quantification of Olfm4+ ISCs (arrowheads) (three mice per group, approximately 25 intact well‐orientated crypts per mouse). (d) Lysozyme staining image (Red: lysozyme, Blue:DAPI) and the quantification of lysozyme‐positive Paneth cells (three mice per group, 50 intact well‐orientated crypts per mouse). Original magnifications: ×100 (a); ×400 (b); and ×200 (c and d). Scale bar: 100 µm (a); 25 µm (b); and 50 µm (c and d). Values represent the mean ± SEM. *p < 0.05; **p < 0.01; t test. ISC, intestinal stem cell Next, to test whether aging influences the population of proliferative cells in young versus old mice, 2‐hr BrdU labeling was performed in crypts, which marks ISCs and their immediate descendants, transit amplifying (TA) cells (Figure 1b and Supporting Information Figure S1d). There was no difference between young and old mice in the incorporation of BrdU into total crypt cells (ISCs and TA cells) (Supporting Information Figure S1d). However, there was a decrease BrdU in incorporation into the crypt base columnar (CBC) cells, which are stem cells wedged between Paneth cells (Figure 1b) (Barker, van Oudenaarden, & Clevers, 2012), indicating that proliferation of ISCs decreased in old mice compared to young mice. Next to investigate the number of ISCs in young versus old mice, we performed in situ hybridization (ISH) for Olfactomedin‐4 (Olfm4), which is a marker of ISCs (van der Flier, Haegebarth, Stange, van de Wetering, & Clevers, 2009; Flier et al., 2009). The number of Olfm4+ positive ISCs in the crypts of old mice decreased compared with young mice from about five cells per crypt to about three cells per crypt (Figure 1c). A similar finding was noted using FACS analysis of mice expressing Lgr5‐GFP+cells to count ISCs (Supporting Information Figure S1e). Paneth cells are niche cells which are adjacent to and support the proliferation of ISCs (Sato et al., 2011). Particularly, ISC proliferation in CR depends on the cyclic ADP ribose secreted from Paneth cells (Igarashi & Guarente, 2016; Yilmaz et al., 2012). However, we did not find any difference in the number of Paneth cells (lysozyme positive) between young versus and old mice (Figure 1d). These results all indicate that the number of ISCs in old mice decreases and the villi length increases, suggesting that self‐renewal of ISCs is reduced and differentiation of ISCs is consequently increased in old mice.

2.2 Aging degrades the formation of intestinal organoids from ISCs To further investigate the effects of aging on the proliferation of ISCs, we isolated crypts from young and old mice, dispersed the cells, and seeded them on Matrigel to obtain organoid colonies (Igarashi & Guarente, 2016; Sato & Clevers, 2013). Each organoid colony is seeded by a single ISC and gives rise to all the differentiated cells of the gut in the colony. This assay thus enumerates the functional ISCs ex vivo. Crypts from old mice formed fewer organoid colonies and showed a decreased number of buds，which is another indicator of stem cell function, compared to those from young mice, consistent with the in vivo data (Figure 2a and Supporting Information Figure S4a). Next to assess organoid‐forming ISCs more accurately and address how the ISCs and Paneth cells interact functionally, we isolated Lgr5‐positive ISCs and Paneth cells each to >95% purity from young and old Lgr5‐EGFP‐IRES‐CreERT2 mice, as described previously (Igarashi & Guarente, 2016). A total of 2,000 cells were plated of each cell type and in the standard media containing glycogen synthase kinase 3β (GSK3β) inhibitor CHIR99021 (CHIR), which is known to induce β‐catenin and thus stimulate organoid formation (Igarashi & Guarente, 2016; Yin et al., 2014). Lgr5‐positive ISCs isolated from old mice formed fewer and smaller colonies on day 5 of culture and fewer organoid colonies with a decreased number of buds on day 9, compared with those from young mice (Figure 2b and Supporting Information Figure S2a and S5b,c). By combining young or old ISCs and young or old Paneth cells, we determined that the Paneth cells from old mice were as functional as young Paneth cells in stimulating organoid colony formation in co‐culture with young ISCs and with or without CHIR (Figures 2c, Supporting Information Figures S2b and S5d). Old ISCs were similarly defective in organoid formation with either young or old Paneth cells. Thus, this functional ex vivo assay for ISC function and niche–stem cell interaction demonstrates a defect in old ISCs but not old Paneth cells. Figure 2 Open in figure viewer PowerPoint Aging reduces the formation of intestinal organoids from ISCs ex vivo. (a) Crypts from young and old mice were cultured in Matrigel to allow ISCs to form organoid colonies. The number of colonies was assessed at day 5 (7–8 wells/group (the sum of two different experiments). The picture shows colonies cultured from young crypts and old crypts. (b) ISCs were isolated from young and old Lgr5‐EGFP‐IRES‐CreERT2 mice (>95% pure), and 2 × 103 cells were cultured in the absence of Paneth cells in culture medium containing 10 µM CHIR. The number of colonies was assessed at day 5 (11–15 wells/group [the sum of three different experiments]). The picture shows colonies from young ISCs and old ISCs culture in the absence of Paneth cells. (c) ISCs and Paneth cells were isolated from young and old Lgr5‐EGFP‐IRES‐CreERT2 mice, and 2 × 103 cells each were co‐cultured in the medium containing 10 µM CHIR. The number of colonies was assessed at day 5 (3–4 wells/group). Original magnifications: ×100 (a and b). Scale bar: 100 µm (a and b). Values represent the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; t test. ISC, intestinal stem cell To gain insights into the mechanisms driving loss of organoid formation in old ISCs, we focused our analysis on gene networks previously associated with ISC stemness (Igarashi & Guarente, 2016) and found a significant reduction in SIRT1 protein in old crypts and ISCs (Supporting Information Figure S2c,d). Also, we observed a trend to a reduction in S6 phosphorylation in old crypts and old ISCs, indicative of a lowering in SIRT1/mTORC1 activity (Figure 4c,d). Notably, RNAseq analysis of young and old ISCs did not reveal significant differences in gene expression, suggesting that relevant changes occur post‐transcriptionally (Supporting Information Figure S3).

2.3 Nicotinamide riboside restores the colony formation in aged mice In order to probe the role of the SIRT1/mTORC1 axis ISC aging, we tested whether NAD+ supplementation affects the function of old ISCs ex vivo. Thus, we treated crypts from young and old mice with NR, a NAD+ precursor, which increases intracellular NAD+ levels and activates SIRT1 (Cantó et al., 2012). NR treatment completely rescued the diminishment in colony formation efficiency and number per organoid of differentiated buds in old crypt‐derived organoids, while having no significant effect in the colonies from young crypts (Figure 3a and Supporting Information Figure S4a). To address in more functional detail the role of the SIRT1/mTORC1 pathway in the NR‐rejuvenated ISCs, old crypts were incubated with NR plus the mTORC1 inhibitor rapamycin or the specific SIRT1 inhibitor EX527. Rapamycin or EX527 treatment of old crypts blocked the rescue of organoid formation by NR treatment (Figure 3b,c), while having a minimal effect on NR‐treated young crypts (Supporting Information Figure S4b,c). Next to be certain these effects occurred specifically in the ISCs, we purified Lgr5+ stem cells and carried out parallel experiments to the studies using crypts. We found with the pure ISCs that NR again increased organoid colony formation by old ISCs, and that this increase was blocked by rapamycin or EX527 treatment (Figure 3d,e), while having a minimal effect on NR‐treated young ISCs (Supporting Information Figure S4d,e). These studies show that ISC functional decline with aging can be rescued by NAD+ replenishment via NR and that the effect is intermediated by SIRT1/mTORC1 signaling pathway. Figure 3 Open in figure viewer PowerPoint Nicotinamide riboside (NR) restores the colony formation in aged mice. (a) Isolated young or old crypts were cultured in medium with or without 1 mM NR as indicated (5–6 wells/group). Representative images of the formed organoids at day 5 and the quantification of organoids number. Original magnifications: ×50. Scale bar: 100 µm. (b) Isolated old crypts were cultured in medium with or without 1 mM NR and 1 mM rapamycin as indicated (five wells/group). (c) Isolated old crypts were cultured in medium with or without 1 mM NR and 1 µM EX527 as indicated (4–5 wells/group). (d) Isolated old intestinal stem cells (ISCs) were cultured in medium with or without 1 mM NR and 1 mM rapamycin as indicated (four wells/group). (e) Isolated old ISCs were cultured in medium with or without 1 mM NR and 1 µM EX527 as indicated (three wells/group). Values represent the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; t test

2.4 NR treatment restores ISC number in aged mice in vivo We wished to test whether the decline in ISC number in old mice could be reversed by NAD+ replenishment. Thus, we tested whether NR treatment affects the number of ISCs in vivo in young or old mice with a 6 weeks treatment of drinking water containing NR (500 mg/kg body weight) or vehicle (Figure 4). First, we tested whether NAD+ supplementation affects the function of ISCs derived from old mice, but not Paneth cells in vivo. In vivo, NR treatment completely rescued the diminishment in colony formation efficiency in old crypts and old ISCs (Supporting Information Figure S5a–c). On the other hand, by combining ISCs and Paneth cells purified from young or old mice, we determined that NR treatment affects ISCs rather than Paneth cells (Supporting Information Figure S5d). Next, we observed that the elongation of villi induced during aging was abrogated by NR treatment (Supporting Information Figure S6a). To investigate how NR treatment influenced the activity and number of ISCs in young and old mice, BrdU labeling and ISH for the ISC marker Olfm4 were performed. NR treatment completely rescued the decrease of BrdU uptake into CBC cells (Figure 4a) and, more importantly, the decrease of Olfm4+‐positive cells (Figure 4b). Again, NR treatment had minimal effects on the expansion of ISCs in young mice (Figure 4a,b). Moreover, we found a significant increase in SIRT1 protein and S6 phosphorylation in NR‐treated old crypts and old ISCs, confirming that ISC functional improvement by in vivo NAD+ replenishment activates the SIRT1/mTORC1 signaling pathway (Figure 4c,d and Supporting Information Figure S6b). Figure 4 Open in figure viewer PowerPoint NR treatment restores ISC number in aged mice in vivo. (a) BrdU staining images and the quantification of BrdU‐positive CBC cells (arrowheads) in young or old mice administered with vehicle or NR (500 mg/kg) in drinking water for 6 weeks mice as assessed 2 hr after the injection of BrdU (3–5 mice per group, 50 intact well‐orientated crypts per mouse). (b) In situ hybridization images of Olfm4 mRNA and the quantification of Olfm4+ ISCs (arrowheads) in young or old mice administered with vehicle or NR (500 mg/kg) in drinking water for 6 weeks (eight mice per group, 50 intact well‐orientated crypts per mouse). (c) In situ phospho S6 (pS6) staining images (green, pS6; blue, DAPI) in the intestine of young or old mice administered with vehicle or NR (500 mg/kg) in drinking water for 6 weeks and the quantification of relative signal intensities per area in crypt (five mice per group). At least four different image fields per each sample were quantified by Image J. Y:Young, O:Old. Original magnifications: ×400 (a); ×200 (b); and ×100 (c). Scale bar: 25 µm (a); 50 µm (b); and 100 µm (c). (d) Immunoblotting of pS6 or S6 in ISCs isolated from young or old Lgr5-EGFP-IRES-CreERT2 mice administered with vehicle or NR. Values represent the mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; t test, CBC: crypt base columnar; ISCs: intestinal stem cells; NR: nicotinamide riboside