Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells
Open accessAbstract
Although intestinal homeostasis is maintained by intestinal stem cells (ISCs), regeneration is impaired upon aging. Here, we first uncover changes in intestinal architecture, cell number, and cell composition upon aging. Second, we identify a decline in the regenerative capacity of ISCs upon aging because of a decline in canonical Wnt signaling in ISCs. Changes in expression of Wnts are found in stem cells themselves and in their niche, including Paneth cells and mesenchyme. Third, reactivating canonical Wnt signaling enhances the function of both murine and human ISCs and, thus, ameliorates aging-associated phenotypes of ISCs in an organoid assay. Our data demonstrate a role for impaired Wnt signaling in physiological aging of ISCs and further identify potential therapeutic avenues to improve ISC regenerative potential upon aging.
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TL;DR
Aging impairs intestinal stem cell (ISC) function and regenerative capacity without reducing ISC number, largely due to declining canonical Wnt signaling. Reactivating canonical Wnt signaling with Wnt3a restores a more youthful organoid-forming phenotype in both murine and human aged ISCs.
Key findings
- Aged mouse small intestine (20–22 months vs. 2–3 months) showed decreased crypt number but increased crypt length/width, villus length, and cells per crypt; mitotic index declined, BrdU-labeled cells traveled less far into villi at 72 hr, and TUNEL-positive apoptotic crypt cells increased.
- ISC numbers were unchanged by Lgr5-EGFP and Olfm4 analyses, but ISC function declined: lineage tracing was impaired, and aged organoids had reduced formation after the third passage and severe decline after the fourth split, whereas young organoids formed through the eighth replating.
- Canonical Wnt signaling declined in aged ISCs: Wnt3 was reduced in ISCs, Paneth cells, and mesenchyme; expression of β-catenin, Ascl2, Lgr5, Myc, Ephb2, and CD44 declined in ISCs, and Ascl2/nuclear β-catenin protein was reduced. Notch1 decreased and Atoh1 increased, with increased Paneth and goblet cell numbers.
- Wnt3a treatment increased organoid number and lobes/buds from aged mouse crypts almost to young levels and ameliorated the age-related decline in human organoid formation from young (12–16 years) vs. aged (62–77 years) subjects. After 10+10 Gy irradiation, ~50% of aged mice died by day 5 vs. 12% of young mice.
Methods
- Young (2–4 months) and aged (18–22 months) C57BL/6 mice, plus Lgr5eGFPCreERT2 Rosa26YFP mice aged up to 2 years, were used for histology, immunofluorescence, TUNEL, BrdU tracing, in situ hybridization, FACS of Lgr5eGFPhi ISCs and CD24hi Paneth cells, RNA-seq, qRT-PCR, western blot, irradiation recovery, and organoid assays.
- Organoid cultures were established from mouse proximal small intestine and human tissue, with Wnt3a (100 ng/mL) added only in rescue experiments; organoid number and lobes/buds were scored on day 6 after plating or passaging.
- Statistical significance used Student’s t test or Wilcoxon/Mann-Whitney test; error bars indicate SD.
Limitations
- Stated: the precise molecular mechanisms causing reduced canonical Wnt expression/signaling upon aging, the detailed Notch–Wnt interplay, contributions of extracellular matrix/niche changes, and whether Smad/PPAR signaling is linked to Wnt changes all require further investigation; more detailed clonality experiments comparing labeling protocols are also needed.
- Stated/evident: human organoid data are limited to young (12–16 years) vs. aged (62–77 years; methods say 62–72 years) subjects, and sample sizes are not reported in the text.
- Evident: in the main mouse experiments, young mice were male and female C57BL/6 from Charles River, while aged mice were female C57BL/6 from NIA, so sex and source differences may confound aging comparisons; the authors do not discuss this.
Model-written summary. Check it against the paper before relying on it.
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- Findingown data
The average distance traveled by BrdU-positive cells from the crypt base into the villi 72 hr after BrdU administration is larger in young mice than in aged mice.
“Our data revealed that the average distance of BrdU-positive cells that had traveled from the crypt base into the villi 72 hr post BrdU administration was larger in young mice compared to aged mice (Figures 1K and 1L)”
Quote in the paperSubstantial claim - Findingown data
Wnt3 expression is reduced in aged ISCs.
“Quantitative real-time RT-PCR analyses for expression of Wnt1, 2, 2b, 3, 3a, 8a, 8b, 10a, and 10b (Farin et al., 2012) in ISCs demonstrated reduced Wnt3 (Figure 3C) levels in aged ISCs.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Farin et al., 2012 source
Not checked in the free run.
- Findingown data
RNA-seq analysis revealed changes in the gene expression profile in both Lgr5 intestinal stem cells and CD24 hi Paneth cells upon aging.
“RNA-seq analysis revealed changes in the gene expression profile in both Lgr5 intestinal stem cells as well as in CD24 hi Paneth cells (Figures S3C and S3D).”
Quote in the paperSubstantial claim - Findingown data
Notch1 expression declines in aged ISCs.
“We also observed a decline in the expression of Notch1 (Figure 3H) (expression of Notch2, 3, and 4 was not detected in ISCs; data not shown) and an increase in Atonal homolog 1 (Atoh1) (Figure 3I) gene expression.”
Quote in the paperSubstantial claim - Findingown data
In aged mice compared with young mice, crypt width is increased in the proximal and distal small intestine.
“Histological H&E analysis of intestinal tissue from young (2–3 months old) and aged mice (20–22 months old) showed a decrease in crypt number accompanied by an increase in crypt length and width in aged compared to young intestine in both the proximal and distal regions (Figures 1A–1H).”
Quote in the paperNot a substantial claim
- Findingown data
Expression of Lrig1 is reduced in aged intestinal crypts.
“Expression analyses of published +4 quiescent ISC markers (Lrig1, Hopx, Sox9, Tert, and Bmi1) revealed that the expression of Lrig1 and Tert were reduced (Figure 2F), whereas the expression levels of Hopx, Sox9, and Bmi1 (Figure 2F) did not change upon aging.”
Quote in the paperNot a substantial claim - Findingown data
In aged mice (20-22 months) compared with young mice (2-3 months), crypt number is decreased in the proximal and distal small intestine.
“Histological H&E analysis of intestinal tissue from young (2–3 months old) and aged mice (20–22 months old) showed a decrease in crypt number accompanied by an increase in crypt length and width in aged compared to young intestine in both the proximal and distal regions (Figures 1A–1H).”
Quote in the paperSubstantial claim - Findingown data
Villus length is elevated in aged mice.
“Interestingly, the length of villi and the number of cells per crypt were also elevated in aged mice (Figures S1A–S1D).”
Quote in the paperNot a substantial claim - Findingown data
Expression of CDKN1C (p57) is reduced in aged intestinal crypts.
“Consistent with a reduced rate in mitotic progression, expression of CDKN1C (p57) was reduced in aged intestinal crypts, whereas other cell cycle regulators like CDKN1A (p21), CDKN1B (p27), and CDKN2A (p16), and Cyclin D1 showed no significant change upon aging (Figure S1E)”
Quote in the paperNot a substantial claim - Findingown data
Atoh1 gene expression increases in aged ISCs.
“We also observed a decline in the expression of Notch1 (Figure 3H) (expression of Notch2, 3, and 4 was not detected in ISCs; data not shown) and an increase in Atonal homolog 1 (Atoh1) (Figure 3I) gene expression.”
Quote in the paperNot a substantial claim - Findingown data
Olfm4 RNA expression levels are similar in young and aged intestinal crypts.
“Olfm4 RNA expression levels were also similar in young and aged intestinal crypts (Figures 2D and 2E).”
Quote in the paperNot a substantial claim - Findingown data
In aged mice compared with young mice, crypt length is increased in the proximal and distal small intestine.
“Histological H&E analysis of intestinal tissue from young (2–3 months old) and aged mice (20–22 months old) showed a decrease in crypt number accompanied by an increase in crypt length and width in aged compared to young intestine in both the proximal and distal regions (Figures 1A–1H).”
Quote in the paperNot a substantial claim - Findingown data
Expression of Tert is reduced in aged intestinal crypts.
“Expression analyses of published +4 quiescent ISC markers (Lrig1, Hopx, Sox9, Tert, and Bmi1) revealed that the expression of Lrig1 and Tert were reduced (Figure 2F), whereas the expression levels of Hopx, Sox9, and Bmi1 (Figure 2F) did not change upon aging.”
Quote in the paperSubstantial claim - Findingown data
Four weeks after YFP activation in ISCs, aged mice show impaired lineage tracing similar to the one-week time point.
“Analyses 4 weeks after YFP activation in ISCs showed similar results compared to that of the 1-week time point (Figures 2G and 2H), indicating impaired ISC function upon aging.”
Quote in the paperSubstantial claim - Findingown data
Wnt3 expression is reduced in aged Paneth cells.
“Paneth (niche) cells also presented with reduced Wnt3 expression levels (Figure 3D).”
Quote in the paperSubstantial claim - Findingown data
TUNEL-positive apoptotic cells are increased in aged mouse intestinal crypts.
“We also observed an increase in terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive (apoptotic) cells in aged mouse intestinal crypts (Figures S1F and S1G).”
Quote in the paperSubstantial claim - Findingown data
The number of mitotic cells is reduced in aged compared with young intestinal crypts.
“Analyses of the mitotic index by phospho-histone H3 staining, which marks cells undergoing mitosis, revealed a decline in the number of mitotic cells in aged compared to young crypts (Figures 1I and 1J).”
Quote in the paperSubstantial claim - Findingown data
Wnt2b and Wnt2 expression do not decline in aged mesenchyme.
“Quantitative analyses on expression of canonical Wnts in mesenchyme (in the absence of crypt epithelium) from young and aged mouse small intestine also revealed a decline in Wnt3 but, for example, not for Wnt2b and Wnt2 (Figures 3E and 3F; Figure S4B)”
Quote in the paperSubstantial claim - Findingown data
Wnt3 expression declines in aged mesenchyme.
“Quantitative analyses on expression of canonical Wnts in mesenchyme (in the absence of crypt epithelium) from young and aged mouse small intestine also revealed a decline in Wnt3 but, for example, not for Wnt2b and Wnt2 (Figures 3E and 3F; Figure S4B)”
Quote in the paperSubstantial claim - Findingown data
Expression levels of Hopx, Sox9, and Bmi1 do not change in aged intestinal crypts.
“Expression analyses of published +4 quiescent ISC markers (Lrig1, Hopx, Sox9, Tert, and Bmi1) revealed that the expression of Lrig1 and Tert were reduced (Figure 2F), whereas the expression levels of Hopx, Sox9, and Bmi1 (Figure 2F) did not change upon aging.”
Quote in the paperNot a substantial claim - Findingown data
Expression of CyclinD1, Axin2, and Olfm4 does not decline in aged ISCs.
“Finally, canonical Wnt signaling target genes and genes regulating ISC function, like β-catenin, Ascl2, Lgr5, Myc, Ephb2, and CD44 (van der Flier et al., 2009b) presented with a decline in the level of expression in ISCs but, interestingly, not CyclinD1, Axin2, or Olmf4 (Figure 3G).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites van der Flier et al., 2009b
Not checked in the free run.
- Findingown data
Expression of CDKN1A (p21), CDKN1B (p27), CDKN2A (p16), and Cyclin D1 shows no significant change in aged intestinal crypts.
“Consistent with a reduced rate in mitotic progression, expression of CDKN1C (p57) was reduced in aged intestinal crypts, whereas other cell cycle regulators like CDKN1A (p21), CDKN1B (p27), and CDKN2A (p16), and Cyclin D1 showed no significant change upon aging (Figure S1E)”
Quote in the paperNot a substantial claim - Findingown data
Expression of canonical Wnt target genes and ISC function genes β-catenin, Ascl2, Lgr5, Myc, Ephb2, and CD44 declines in aged ISCs.
“Finally, canonical Wnt signaling target genes and genes regulating ISC function, like β-catenin, Ascl2, Lgr5, Myc, Ephb2, and CD44 (van der Flier et al., 2009b) presented with a decline in the level of expression in ISCs but, interestingly, not CyclinD1, Axin2, or Olmf4 (Figure 3G).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites van der Flier et al., 2009b
Not checked in the free run.
- Findingown data
The number of cells per crypt is elevated in aged mice.
“Interestingly, the length of villi and the number of cells per crypt were also elevated in aged mice (Figures S1A–S1D).”
Quote in the paperNot a substantial claim - Findingown data
Addition of Wnt3a to aged organoid cultures increases the number of lobes/buds compared with non-treated aged controls.
“Addition of Wnt3a, an inducer of canonical Wnt signaling, to aged organoid cultures resulted in an increase in the number of organoids and increase in the number of lobes/buds in organoids derived from aged animals compared to non-treated aged control organoids (Figures 6D–6F) almost to the level seen in cultures of young organoids.”
Quote in the paperSubstantial claim - Findingown data
Addition of Wnt3a to aged organoid cultures increases the number of organoids compared with non-treated aged controls.
“Addition of Wnt3a, an inducer of canonical Wnt signaling, to aged organoid cultures resulted in an increase in the number of organoids and increase in the number of lobes/buds in organoids derived from aged animals compared to non-treated aged control organoids (Figures 6D–6F) almost to the level seen in cultures of young organoids.”
Quote in the paperSubstantial claim - Findingown data
Five days after 10 Gy irradiation, aged mouse intestine has a higher number of Ki67-negative non-proliferative crypts than young intestine.
“Analysis of the mouse small intestine 5 days after 10 Gy of irradiation (Figure 5A) showed a higher number of Ki67-negative, or non-proliferative, crypts (Figures 5B and 5C) in aged compared to young intestine.”
Quote in the paperSubstantial claim - Findingown data
On day 5 after irradiation, young but not aged intestines show a marked increase in crypt depth.
“We also detected a marked increase in crypt depth and crypt fission in young but not in aged intestines on day 5 after irradiation (Figures 5E–5H).”
Quote in the paperSubstantial claim - Findingown data
The number of lobes or buds per crypt is lower in replated organoids from aged intestine.
“In addition, the number of lobes or buds per crypt, another indicator of stem cell function, was lower in replated organoids from aged intestine (Figure 6C).”
Quote in the paperSubstantial claim - Findingown data
About 50% of aged mice died by day 5 after 10+10 Gy irradiation compared with 12% of young animals.
“In addition, ~50% of aged mice died by day 5 in response to 10+10 Gy compared to only 12% of young animals (data not shown).”
Quote in the paperSubstantial claim - Findingown data
Human organoid cultures from aged subjects (62-77 years) show a decline in frequency of organoid formation upon aging compared with young subjects (12-16 years).
“Organoid cultures from young (12–16 years old) and aged (62–77 years old) human subjects showed, similar to the mouse, a decline in the frequency of organoid formation upon aging (Figures 6G and 6H) that was ameliorated by addition of Wnt3a (Figures 6G and 6H).”
Quote in the paperSubstantial claim - Findingown data
The number of goblet cells is increased in aged compared with young mouse intestine.
“The number of goblet cells (determined by Alcian blue staining), a secretory cell type, was also increased in aged compared to young mouse intestine (Figures 4C and 4D).”
Quote in the paperSubstantial claim - Findingown data
The number of Paneth cells per crypt is increased in aged mouse proximal and distal intestine.
“The number of Paneth cells per crypt, as determined by lysozyme or MMP7 staining, in both the proximal and distal mouse intestine was increased upon aging (Figures 4A and 4B; Figures S4G and S4H).”
Quote in the paperNot a substantial claim - Findingown data
There is no difference in the number of viable crypts between young and aged mouse intestines on day 5 after irradiation.
“There was no difference in the number of viable crypts between young and aged mouse intestines on day 5 after irradiation (Figures S5C and S5D).”
Quote in the paperSubstantial claim - Findingown data
Three days after 10+10 Gy irradiation, young intestinal epithelium shows a strong decline in the number of viable crypts compared with non-irradiated control.
“We observed a strong decline 3 days after 10+10 Gy irradiation in the number of viable crypts in the young intestinal epithelium compared to the non-irradiated control (Figures S5C and S5D).”
Quote in the paperNot a substantial claim - Findingown data
Organoids from aged mice have a reduced rate of organoid formation after the third passage.
“Organoids from aged mice, though, had a reduced rate of organoid formation after the third passage (Figures 6A and 6B).”
Quote in the paperSubstantial claim - Findingown data
Addition of Wnt3a ameliorates the aging-associated decline in human organoid formation.
“Organoid cultures from young (12–16 years old) and aged (62–77 years old) human subjects showed, similar to the mouse, a decline in the frequency of organoid formation upon aging (Figures 6G and 6H) that was ameliorated by addition of Wnt3a (Figures 6G and 6H).”
Quote in the paperSubstantial claim - Findingown data
Organoids derived from young and aged intestinal epithelium initially form with equal efficiency.
“Organoids derived from both young and aged intestinal epithelium were initially formed with equal efficiency (Figure S6A).”
Quote in the paperSubstantial claim - Findingown data
On day 5 after irradiation, young but not aged intestines show a marked increase in crypt fission.
“We also detected a marked increase in crypt depth and crypt fission in young but not in aged intestines on day 5 after irradiation (Figures 5E–5H).”
Quote in the paperSubstantial claim - Findingown data
Addition of Wnt3a to aged organoid cultures elevates expression of canonical Wnt target genes Axin2 and Ascl2.
“Addition of Wnt3a to aged organoid cultures resulted, as expected, in elevated levels of expression of the canonical Wnt target genes Axin2 and Ascl2 in aged organoids (Figure S6B).”
Quote in the paperSubstantial claim - Findingown data
The mitotic index is reduced in aged compared with young mouse intestinal crypt cells.
“Phospho-histone 3 staining demonstrates that the number of cells ultimately entering mitosis (mitotic index) is reduced in aged compared with young mouse intestinal crypt cells.”
Quote in the paperSubstantial claim - Findingown data
Aging of intestinal stem cells is associated with a lower level of Wnt signaling.
“Our finding of a lower level of Wnt signaling upon ISC aging is distinct from reports of aging in the muscle stem cell compartment (Brack et al., 2007), in which elevated levels of Wnts were reported to cause aging of muscle stem cells.”
Quote in the paperSubstantial claim - Findingreasoning
Aged intestinal stem cells show an increase in the number of secretory lineage cells, namely Paneth and goblet cells.
“The increase in the number of secretory lineage cells, namely Paneth and goblet cells, also implies deregulation of stem cell differentiation pathways in aged ISCs, most likely as a response to deregulated Notch signaling, like elevated levels of Atoh1 expression (Kim et al., 2014; Koch et al., 2013; Tian et al., 2015).”
Quote in the paperNot a substantial claim - Findingown data
Aging causes a decline in Ascl2 expression at both the RNA and protein levels in both crypts and intestinal stem cells.
“Our experiments reveal a critical role of a decline in Ascl2 expression at both the RNA and protein levels upon aging in both crypts and ISCs.”
Quote in the paperSubstantial claim - Findingnone given
In aged intestinal stem cells, changes in canonical Wnt signaling components such as β-catenin, Myc, and Ascl2 show little or no correlation with Axin2 expression.
“Thus it is also a surprising finding that canonical changes in canonical Wnt signaling like β catenin, Myc, and Ascl2 present with little or no correlation to Axin2 expression.”
Quote in the paperNot a substantial claim - Findingown data
Restoration of a more youthful phenotype of aged intestinal stem cell function is achieved by reactivation of canonical Wnt signaling in both murine and human intestinal organoid cultures.
“Restoration of a more youthful phenotype of aged ISC function is achieved by reactivation of canonical Wnt signaling in both murine and human intestinal organoid cultures.”
Quote in the paperSubstantial claim - Findingown data
A youthful level of organoid formation can be achieved by re-activating canonical Wnt signaling in aged intestinal stem cells.
“our data provide evidence that changes in Wnt signaling upon aging are causative for aging of intestinal ISCs because a youthful level of organoid formation can be achieved by re-activating canonical Wnt signaling in aged ISCs.”
Quote in the paperSubstantial claim - Interpretationown data
Rather than the number, the function of ISCs might be altered upon aging.
“In aggregation, these data imply that, rather than the number, the function of ISCs might be altered upon aging.”
Quote in the paperSubstantial claim - Interpretationown data
ISC function is impaired upon aging.
“indicating impaired ISC function upon aging.”
Quote in the paperSubstantial claim - Interpretationreasoning
Aging alters ISC differentiation.
“Therefore, these data also suggest altered ISC differentiation upon aging.”
Quote in the paperSubstantial claim - Interpretationown data
Aging results in changes in the architecture of the small intestine.
“Aging thus results in changes in the architecture of the small intestine.”
Quote in the paperSubstantial claim - Interpretationown data
Ascl2, not primarily Axin2, seems to be the aging target gene of canonical Wnt signaling in ISCs.
“It is interesting to note that Ascl2, and not primarily Axin2, seems to be the “aging” target gene of canonical Wnt signaling in ISCs because changes in the expression of Ascl2 are closely correlated to the aging and rejuvenation phenotype reported here.”
Quote in the paperSubstantial claim - Premisecitation
The ability to form organoids depends primarily on ISC function.
“The ability to form organoids depends primarily on ISC function (Barker et al., 2007; Sato et al., 2009).”
Quote in the paperSubstantial claim0 of 2 sources checked - Interpretationown data
Reactivation of canonical Wnt signaling in aged crypts re-establishes a more youthful regenerative potential in aged ISCs.
“Re-activation of canonical Wnt signaling in aged crypts thus re-established a more youthful regenerative potential in aged ISCs.”
Quote in the paperSubstantial claim - Interpretationown data
ISC lineage tracing experiments further substantiate a decline in stem cell function and turnover upon aging.
“Ultimately, ISC lineage tracing experiments with young and aged Lgr5-EGFP-IRES-CreERT2: Rosa26YFP animals further substantiate a decline in stem cell function and turnover upon aging.”
Quote in the paperSubstantial claim - Interpretationown data
Reduced canonical Wnt signaling in aged ISCs in mice and humans is tightly linked to reduced regenerative potential upon aging.
“These data demonstrate an important role of reduced canonical Wnt signaling in aged ISCs in mice and humans that is tightly linked to reduced regenerative potential upon aging.”
Quote in the paperSubstantial claim - Premisecitation
The organoid system is an accepted ex vivo assay that is reflective of stem cell function in vivo.
“The organoid system is an accepted ex vivo assay that is reflective of stem cell function in vivo (Boj et al., 2015).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Boj et al., 2015 source
Not checked in the free run.
- Interpretationcitation
The most likely cellular source of Wnts influencing Wnt signaling upon aging is mostly Paneth cells and in part mesenchyme.
“Because it was recently reported that Wnt3 transfer requires direct cell contact and has only a very limited range (Farin et al., 2016), the most likely cellular source of Wnts that influence Wnt signaling upon aging might thus be mostly Paneth cells and, in part, mesenchyme.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Farin et al., 2016 source
Not checked in the free run.
- Interpretationown data
The data support a likely contribution of an ISC-intrinsic mechanism of changes in Wnt expression to reduced canonical signaling in ISCs upon aging.
“Our data also support a likely contribution of an ISC-intrinsic mechanism of changes in Wnt expression to reduced canonical signaling in ISCs upon aging.”
Quote in the paperSubstantial claim - Interpretationown data
In this study, Axin2 is not the gene linked to changes in Wnt signaling in ISCs upon aging.
“Although Axin2 is a strong prototype target gene of canonical Wnt signaling, as demonstrated in multiple publications, upon aging, in our study, Axin2 is not the gene linked to changes in Wnt Signaling in ISCs.”
Quote in the paperSubstantial claim - Interpretationown data
Primarily reduced canonical Wnt signaling in ISCs causes impaired ISC function upon aging.
“Our data imply that primarily reduced canonical Wnt signaling in ISCs causes impaired ISC function upon aging.”
Quote in the paperSubstantial claim - Interpretationown data
Young ISCs exhibit greater regenerative potential than aged ISCs.
“These data support that young ISCs exhibit a greater regenerative potential compared with aged ISCs.”
Quote in the paperSubstantial claim - Interpretationreasoning
The increase in differentiated secretory cells upon aging is consistent with reduced Notch signaling and increased Atoh1.
“Our finding of an increase in the number of differentiated secretory cells upon aging is consistent with reduced Notch signaling along with an increase in the expression levels of Atoh1, which all favor ISC differentiation.”
Quote in the paperSubstantial claim - Interpretationown data
Aged ISCs have a decline in regenerative potential.
“These data demonstrate a decline in the regenerative potential of aged ISCs and are reminiscent of the loss of repopulating activity of aged haematopoietic stem cells (HSCs) upon serial transplantation (Kamminga et al., 2005).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Kamminga et al., 2005 source
Not checked in the free run.
- Interpretationown data
Impaired intestinal stem cell function in aged intestinal epithelium is because of a decline in canonical Wnt signaling.
“In summary, we demonstrate impaired ISC function in aged intestinal epithelium because of a decline in canonical Wnt signaling.”
Quote in the paperSubstantial claim - Interpretationown data
Reactivation of canonical Wnt signaling to a youthful level is a potential therapeutic approach to restore youthfulness of intestinal stem cell function and increase the regenerative capacity of aged intestinal epithelium.
“These data suggest reactivation of canonical Wnt signaling to a youthful level as a potential therapeutic approach to restore youthfulness of ISC function to increase the regenerative capacity of aged intestinal epithelium.”
Quote in the paperSubstantial claim - Premisecitation
Wnt signaling plays a prominent role in intestinal stem cell maintenance in young animals.
“Wnt signaling plays a prominent role in ISC maintenance in young animals (van der Flier et al., 2009b), and our data provide evidence that changes in Wnt signaling upon aging are causative for aging of intestinal ISCs because a youthful level of organoid formation can be achieved by re-activating canonical Wnt signaling in aged ISCs.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites van der Flier et al., 2009b
Not checked in the free run.
- Interpretationown data
Changes in Wnt signaling upon aging are causative for aging of intestinal stem cells.
“our data provide evidence that changes in Wnt signaling upon aging are causative for aging of intestinal ISCs because a youthful level of organoid formation can be achieved by re-activating canonical Wnt signaling in aged ISCs.”
Quote in the paperSubstantial claim - Interpretationcitation
The findings on intestinal stem cell aging are in line with mechanisms of aging in the hematopoietic stem cell compartment, where aging is associated with low canonical Wnt signaling.
“Our findings are in line with mechanisms of aging in the HSC compartment (Florian et al., 2013), in which aging of HSCs is also associated with low canonical Wnt signaling (Reya et al., 2003).”
Quote in the paperNot a substantial claim0 of 2 sources checked
- Classified as probably not a substantial claim. 13
- May combine two claims. 23
- The quote only partly states this claim. 1
Probably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataExpression of other Wnts (Wnt1, 2, 2b, 3a, 8a, 8b, 10a, and 10b) in ISCs and Paneth cells is below the threshold level.
“However, expression of other Wnts (like Wnt1, 2, 3a, 8a, 8b, 10a, and 10b) in both ISCs and Paneth cells was below our threshold level (data not shown).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataOne week after YFP activation, Lgr5 EGFP-positive cells from young crypts show YFP tracing into the villus, whereas the YFP-marked villus is subtle in aged mice.
“One week after yellow fluorescent protein (YFP) activation, Lgr5 EGFP-positive cells from crypts of young animals presented with YFP tracing into the villus, whereas the YFP-marked villus was subtle in aged mice (Figures S2B–S2D).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataGO terms downregulated in aged ISCs include cell proliferation, extracellular matrix, PPAR and SMAD signaling, and Wnt signaling pathways.
“Molecular processes identified by Gene Ontology (GO) terminology that were downregulated in aged ISCs included, as anticipated, cell proliferation but also extracellular matrix, PPAR and SMAD signaling, and Wnt signaling pathways (Figure 3A; Figure S4A).”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingown dataSimilar lineage tracing results were obtained with 3 days of YFP activation followed by analysis 1 week after YFP activation.
“Similar results were obtained with 3 days of YFP activation followed by analysis 1 week after YFP activation (Figures S2E–S2G).”
Quote in the paperNot a substantial claimMay combine two claims.
Findingown dataAging does not significantly change the number of Lgr5-EGFP-positive crypts, the percentage of Lgr5 EGFPhi cells, or the total number of Lgr5EGFPhi ISCs in mice.
“we did not observe a significant change in the number of crypts positive for Lgr5-EGFP (Figures 2A and 2B) as well as in the percentage of Lgr5 EGFPhi cells (as determined by flow cytometric analyses) (Figure S2A) as well as for the total number of Lgr5EGFPhi ISCs (Figure 2C) at the crypt base at positions 0 to +4 (Barker et al., 2007) in aged compared to young animals.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Barker et al., 2007 source
Not checked in the free run.
May combine two claims.
Findingown dataIn whole crypts, aging reduces expression of Wnt3, β-catenin, Ascl2, and Lgr5, and also Axin2.
“Similar data were obtained when young and aged crypts were analyzed for levels of gene expression, like a reduction in the expression of Wnt3, β-catenin, Ascl2, and Lgr5, upon aging, except for Axin2, which was also down in whole-crypt analysis. (Figures S4C and S4D).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataAscl2 and nuclear β-catenin protein levels are reduced in aged intestinal crypts.
“At the protein level, Ascl2 and nuclear β catenin were reduced (Figures S4E and S4F) in aged intestinal crypts.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataYoung crypt organoids form through the eighth replating, whereas aged organoids show a severe decline in replating efficiency after the fourth split.
“Finally, organoids derived from crypts of young mice were able to form organoids through the termination of the assay at the eighth replating, whereas organoids from aged mice showed a severe decline in replating efficiency after the fourth split.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataThere is a decline in canonical Wnt signaling in ISCs and reduced canonical Wnt expression (primarily Wnt3) in both Paneth cells and mesenchyme.
“We report here a decline in canonical Wnt signaling in ISCs and reduced canonical Wnt expression (primarily Wnt3) in both Paneth cells and mesenchyme, which might also contribute to the reduced activity of canonical Wnt/Wnt signaling in aged ISCs.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataBesides a decline in Wnt signaling in aged ISCs, there is a decline in genes regulating extracellular matrix proteins, genes altering cell proliferation, and changes in PPAR and SMAD signaling.
“Besides a decline in Wnt signaling in aged ISCs, we also observed a decline in genes regulating extra cellular matrix proteins and genes that alter cell proliferation and changes in PPAR and SMAD signaling (Figure 3).”
Quote in the paperNot a substantial claimMay combine two claims.
Interpretationown dataAging does not change the absolute number of ISCs, though some intestinal stem cell markers (Lrig1 and Tert) decrease in expression.
“Together, these data imply that aging does not result in a change in the absolute number of ISCs, though some intestinal stem cell markers (Lrig1 and Tert) present with a decrease in expression.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataAging alters crypt and villus architecture, and aged crypts exhibit reduced cell divisions and reduced survival.
“Taken together, these results indicate that aging alters the crypt and villus architecture and that aged crypts exhibit reduced cell divisions and reduced survival.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe BrdU tracing result is consistent with fewer mitotic events of ISCs and/or reduced transit-amplifying cell proliferation rates upon aging.
“consistent with fewer mitotic events of ISCs and/or reduced transit-amplifying cell proliferation rates upon aging.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataCanonical Wnt signaling declines in ISCs upon aging, linked to a decline in canonical Wnts like Wnt3 in Paneth cells, mesenchyme, and ISCs themselves.
“Together, these data imply a decline of canonical Wnt signaling in ISCs upon aging, which is linked to a decline in expression of canonical Wnts like Wnt3 in both Paneth cells and the mesenchyme as well as in ISCs themselves.”
Quote in the paperSubstantial claimMay combine two claims.
InterpretationreasoningAging results in an increase in secretory lineage cells including Paneth and goblet cells, most likely driven by changes in the differentiation potential of aged ISCs.
“Aging thus also results in an increase in the number of secretary lineage cells, including Paneth cells and goblet cells, most likely driven by changes in the differentiation potential of aged ISCs.”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataAlthough the number of ISCs is not reduced upon aging, aged ISCs show reduced regeneration upon serial radiation exposure (10+10 Gy) and a decline in organoid formation.
“Although the number of ISCs was not reduced upon aging, aged ISCs showed reduced regeneration upon serial radiation exposure (10+10 Gy) and a decline in organoid formation.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim. May combine two claims.
PremisecitationIrradiation in the intestinal epithelium is accompanied by crypt shrinking because of apoptosis, followed by a burst of proliferative response predominantly from existing/surviving ISCs.
“Irradiation in the intestinal epithelium is accompanied by crypt shrinking because of apoptosis, followed by a burst of proliferative response predominantly from existing/surviving ISCs (Metcalfe et al., 2014), usually leading to an increase in crypt depth followed by crypt fission.”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Metcalfe et al., 2014 source
Not checked in the free run.
May combine two claims.
InterpretationreasoningThe increase in Paneth cell number does not compensate for the lower Wnt3 expression because overall Wnt signaling is reduced in aged ISCs.
“This increase in Paneth cell number, though, does not compensate for the overall lower level of expression of Wnt3 upon aging because overall Wnt signaling is reduced in aged ISCs.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationLow levels of p57 have been associated with stem cell hibernation.
“low levels of p57 have been associated with stem cell hibernation (Yamazaki et al., 2006)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Yamazaki et al., 2006 source
Not checked in the free run.
Classified as probably not a substantial claim.
PremisecitationIt takes approximately 4-5 days for a progenitor cell derived from an ISC division at the crypt base to reach the tip of the villus.
“It takes approximately 4–5 days for a progenitor cell derived from an ISC division at the crypt base to reach the tip of the villus (Barker et al., 2008).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Barker et al., 2008 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationTissue damage upon aging affects the extracellular matrix, which further alters stem cell niches regulating stem cell-regenerative functions.
“Tissue damage upon aging affects the extracellular matrix, which further alters stem cell niches regulating stem cell-regenerative functions (Blau et al., 2015)”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Blau et al., 2015 source
Not checked in the free run.
Classified as probably not a substantial claim. May combine two claims.
PremisecitationWnt3 transfer requires direct cell contact and has only a very limited range.
“Because it was recently reported that Wnt3 transfer requires direct cell contact and has only a very limited range (Farin et al., 2016)”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Farin et al., 2016 source
Not checked in the free run.
May combine two claims.
Interpretationown dataUpon aging, there is decreased ISC function and regenerative potential.
“Our studies further substantiate but also significantly extend these findings by demonstrating that, upon aging, there is decreased ISC function and regenerative potential.”
Quote in the paperSubstantial claimClassified as probably not a substantial claim.
PremisecitationWnt signaling in the intestinal epithelium is critical for tissue homeostasis in young mice.
“Wnt signaling in the intestinal epithelium is well studied and critical for tissue homeostasis in young mice (Pinto et al., 2003; van der Flier et al., 2009b).”
Quote in the paperSubstantial claim0 of 2 sources checked- cites Pinto et al., 2003 source
Not checked in the free run.
- cites van der Flier et al., 2009b
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAscl2 is a central Wnt-responsive transcription factor.
“This finding is also in line with recent reports suggesting Ascl2 as a central Wnt-responsive transcription factor (Schuijers et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Schuijers et al., 2015 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAging of hematopoietic stem cells is associated with low canonical Wnt signaling.
“Our findings are in line with mechanisms of aging in the HSC compartment (Florian et al., 2013), in which aging of HSCs is also associated with low canonical Wnt signaling (Reya et al., 2003).”
Quote in the paperNot a substantial claim0 of 2 sources checkedProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationPrevious reports indicated that the number of ISCs and the number of stem cells replaced are age-independent.
“Previous reports (Kozar et al., 2013) indicated that the number of ISCs and the number of stem cells replaced are age-independent by using a specific labeling technique of intestinal epithelial cells at a young age with a follow-up to 2 years.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Kozar et al., 2013 source
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationAscl2 has a central role in determining intestinal stem cell fate.
“A contribution of changes in Ascl2 expression to aging is consistent with a report demonstrating a central role for Ascl2 in determining ISC fate (van der Flier et al., 2009b).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites van der Flier et al., 2009b
Not checked in the free run.
May combine two claims.
InterpretationcitationThe increase in secretory lineage cells in aged intestinal stem cells implies deregulation of stem cell differentiation pathways in aged ISCs, most likely as a response to deregulated Notch signaling (e.g., elevated Atoh1).
“The increase in the number of secretory lineage cells, namely Paneth and goblet cells, also implies deregulation of stem cell differentiation pathways in aged ISCs, most likely as a response to deregulated Notch signaling, like elevated levels of Atoh1 expression (Kim et al., 2014; Koch et al., 2013; Tian et al., 2015).”
Quote in the paperNot a substantial claim0 of 3 sources checkedMay combine two claims.
InterpretationcitationChanges in Ascl2 expression contribute to aging of intestinal stem cells, consistent with the central role of Ascl2 in ISC fate.
“A contribution of changes in Ascl2 expression to aging is consistent with a report demonstrating a central role for Ascl2 in determining ISC fate (van der Flier et al., 2009b).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites van der Flier et al., 2009b
Not checked in the free run.
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationLow levels of p57 upon aging were also detected in aged hematopoietic stem cells.
“low levels of p57 upon aging were also detected in aged hematopoietic stem cells (Florian et al., 2012).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Florian et al., 2012 source
Not checked in the free run.
May combine two claims.
InterpretationcitationThe finding of lower Wnt signaling upon intestinal stem cell aging is distinct from aging in the muscle stem cell compartment, where elevated Wnts cause aging.
“Our finding of a lower level of Wnt signaling upon ISC aging is distinct from reports of aging in the muscle stem cell compartment (Brack et al., 2007), in which elevated levels of Wnts were reported to cause aging of muscle stem cells.”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Brack et al., 2007 source
Not checked in the free run.
The quote only partly states this claim.
InterpretationcitationThe decline in Ascl2 expression upon aging is in line with Ascl2 being a central Wnt-responsive transcription factor.
“This finding is also in line with recent reports suggesting Ascl2 as a central Wnt-responsive transcription factor (Schuijers et al., 2015).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Schuijers et al., 2015 source
Not checked in the free run.
The paper relies on this without support.
Before this study, it had not been determined whether changes in Wnt signaling pathways contribute to changes in ISC function upon aging.
“Whether changes in Wnt signaling pathways contribute to changes in ISC function upon aging has so far not been determined.”
The paper relies on this without support.
The extent to which ISC function alters during physiological aging is still a matter of debate.
“However, the extent to which ISC function alters during physiological aging is still a matter of debate.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
In the aged GI tract, compromised epithelial and mucosal cell turnover, integrity, and function can lead to increased epithelial barrier permeability.
“In the aged GI tract, compromised epithelial and mucosal cell turnover, integrity, and function [32, 36, 76, 83] can lead to increased epithelial barrier permeability [19, 75, 118, 120]”
From Fecal microbiota transfer between young and aged mice reverses hallmarks of the aging gut, eye, and brain (2022) · cites this paper as [83]
Self-check
Percentages, sums and reported differences re-computed from the paper’s own numbers.
Each contradiction between two passages listed here was confirmed by an independent check.
Conflicting age range for older human donors
MinorThe results define the older human subjects as 62-77 years old, while the experimental procedures define them as 62-72 years old.
“Organoid cultures from young (12–16 years old) and aged (62–77 years old) human subjects”
“Young refers to 12–16 years of age, and aged refers to 62–72 years of age.”
Figures 1K and 1L are assigned to two different experiments
MinorThe results cite Figures 1K and 1L for BrdU cell migration, then cite the same figures for an ISC lineage-tracing experiment.
“Our data revealed that the average distance of BrdU-positive cells that had traveled from the crypt base into the villi 72 hr post BrdU administration was larger in young mice compared to aged mice (Figures 1K and 1L)”
“we performed short-term lineage tracing experiment on young (2–3 months old) and aged (22–24 months old) Lgr5-eGFP-IRES-CreERT2: Rosa26YFP mice (Figures 1K and 1L).”
The citation lists haven’t been fetched yet
References 38 · Cited by 280
- Authors
- Nalapareddy, K., Nattamai, K. J., Kumar, R. S., Karns, R., Wikenheiser-Brokamp, K. A., Sampson, L. L., Mahe, M. M., Sundaram, N., Yacyshyn, M.-B., Yacyshyn, B., Helmrath, M. A., Zheng, Y., & Geiger, H.
- Affiliations
- Cincinnati Children's Hospital Medical Center (US)
- University of Cincinnati (US)
- Corresponding author
- Geiger, Hartmut
- ORCID
- Venue
- Cell Reports
- Publisher
- Elsevier BV
- Published
- 2017-03
- Volume
- 18
- Issue
- 11
- Pages
- 2608-2621
- ISSN
- 2211-1247 (ISSN-L)
- PMC
- PMC5987258
- OpenAlex
- W2596902967
- Semantic Scholar
- a133094c4391e54800251feb21c623d9cdec8780
- Cited by
- 280
- References
- 38
- Citations by year
- 2026: 35 · 2025: 43 · 2024: 35 · 2023: 31 · 2022: 39 · 2021: 26 · 2020: 29 · 2019: 17 · 2018: 18 · 2017: 6
- FWCI
- 7.78
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Licence
- Locations
- Cell Reports (publishedVersion, cc-by-nc-nd)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion, cc-by-nc-nd)
- PubMed Central (acceptedVersion, cc-by-nc-nd)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- OPen Access Repositorium der Universität Ulm (OPARU) (Ulm University) (submittedVersion, cc-by-nc-nd)
- Universität Ulm, Kommunikations- und Informationszentrum (kiz)
- Funders
- NIH (R01DK104814, AG040118, DK078392)
- Digestive Health Center (DHC)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Aging › Genetics, Aging, and Longevity in Model Organisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Digestive system and related health
- Keywords
- intestinal stem cells, aging, Wnt/β-catenin signaling, intestinal homeostasis, intestinal regeneration, Paneth cells, mesenchyme, stem cell niche, organoids, regenerative capacity
- MeSH terms
- Animals, Cell Count, Female, Intestine, Small, Organoids, Regeneration, Stem Cells, Biomarkers, Cellular Senescence, Cell Proliferation, Mice, Stem Cell Niche, Wnt Signaling Pathway
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