Letter
https://doi.org/10.1038/s41586-019-1383-0
Notum produced by Paneth cells attenuates
regeneration of aged intestinal epithelium
Nalle Pentinmikko^1 , Sharif Iqbal1,14, Miyeko Mana2,14, Simon Andersson^1 , Armand B. Cognetta III^3 , radu M. Suciu^3 , Jatin roper^4 ,
Kalle Luopajärvi^1 , eino Markelin^1 , Swetha Gopalakrishnan^1 , Olli-Pekka Smolander^1 , Santiago Naranjo^2 , tuure Saarinen5,6,
Anne Juuti^6 , Kirsi Pietiläinen^5 , Petri Auvinen^1 , Ari ristimäki^7 , Nitin Gupta^8 , tuomas tammela^9 , tyler Jacks2,10,
David M. Sabatini2,10,11, Benjamin F. Cravatt^3 , Ömer H. Yilmaz^2 & Pekka Katajisto1,12,13*
A decline in stem cell function impairs tissue regeneration during
ageing, but the role of the stem-cell-supporting niche in ageing is
not well understood. The small intestine is maintained by actively
cycling intestinal stem cells that are regulated by the Paneth cell
niche^1 ,^2. Here we show that the regenerative potential of human
and mouse intestinal epithelium diminishes with age owing to
defects in both stem cells and their niche. The functional decline
was caused by a decrease in stemness-maintaining Wnt signalling
due to production of Notum, an extracellular Wnt inhibitor, in
aged Paneth cells. Mechanistically, high activity of mammalian
target of rapamycin complex 1 (mTORC1) in aged Paneth cells
inhibits activity of peroxisome proliferator activated receptor
α (PPAR-α)^3 , and lowered PPAR-α activity increased Notum
expression. Genetic targeting of Notum or Wnt supplementation
restored function of aged intestinal organoids. Moreover,
pharmacological inhibition of Notum in mice enhanced the
regenerative capacity of aged stem cells and promoted recovery from
chemotherapy-induced damage. Our results reveal a role of the stem
cell niche in ageing and demonstrate that targeting of Notum can
promote regeneration of aged tissues.
Tissue turnover and regenerative capacity decrease with ageing in
many tissue types^4 –^6. The intestinal epithelium is one of the fastest
renewing tissues in the human body and has previously been reported
to regenerate without loss of self-renewal in long-term in vitro organoid
culture^7. However, complications in the gastrointestinal system increase
with age^8 –^10 , and intestines of old mice regenerate more slowly after
radiation-induced damage^11 , which suggests reduced stem cell activity.
To assess possible ageing-induced changes in the human
intestinal epithelium, we used the capacity of intestinal-stem-cell
(ISC)-containing epithelial crypts to form clonogenic organoids^7 as
an in vitro assay of intestinal regenerative potential. We observed a
significant age-induced reduction in the organoid-forming capacity
of colonic crypts derived from biopsies from healthy human donors
(Fig. 1a). As the heterogeneous human colon material does not allow
downstream analysis of stem-cell-intrinsic and -extrinsic effects, we
next analysed the effects of age on mouse small-intestinal epithelium.
Crypts from old (more than 24-month-old) mice formed significantly
fewer organoids than those isolated from young (3-to-9-month-old)
mice (Extended Data Fig. 1a). Notably, regenerative growth of de novo
crypts was also diminished in the organoids formed by the old crypts
(Fig. 1b, Extended Data Fig. 1b, c), which indicates a reduction in stem
cell function. Furthermore, the reduced crypt formation observed
during serial passage of secondary crypt domains demonstrated that
the decline in epithelial regeneration was due to alterations intrinsic to
the epithelium (Extended Data Fig. 1d, e).
Intestinal tissue renewal is largely maintained by the LGR5-
expressing ISCs, which are located between Paneth cells at the crypt
base. ISCs divide regularly and produce transit-amplifying progenitor
cells that divide several additional times and gradually differentiate.
Paneth cells produce antimicrobial peptides and multiple signalling
factors, such as epidermal growth factor (EGF), Wnt3, Delta-like
ligands and cyclic ADP ribose (cADPR)^2 ,^12 , which regulate stemness
and function of the neighbouring ISCs. To more specifically address
the separate roles of stem cells and their niche in age-associated intes-
tinal decline, we used Lgr5-eGFP-IRES-creERT2 reporter mice^1 , which
enable identification and isolation of Paneth cells, Lgr5–eGFPhi ISCs,
and transit-amplifying cells that can be further divided into immediate
eGFPmed and late eGFPlo progenitors.
The aged mouse crypts did not present gross histological altera-
tions, and the fraction of ISCs and transit-amplifying cells that were 5-
ethynyl-2′-deoxyuridine (EdU) + or Ki67+ was unchanged in old mouse
and human samples (Extended Data Fig. 1f–h). However, flow cytome-
try of crypts from old mice revealed a significant drop in frequency of
Lgr5hi ISCs (Fig. 1c), whereas Paneth cell frequency was significantly
increased in old mice and humans (Fig. 1c, Extended Data Fig. 1i, j).
As the Lgr5-eGFP-IRES-creERT2 mouse model exhibits mosaic expres-
sion of the eGFP-containing construct^1 , the alterations in cellular
frequencies were also validated by immunohistochemical analyses
of olfactomedin 4 and lysozyme as markers of ISCs and Paneth cells,
respectively (Extended Data Fig. 1k). The reduction in ISC number,
together with the unchanged EdU+ frequency suggested that old crypts
may have a lower output of cells, which possibly contributes to villus
blunting and slower intestinal turnover during ageing^13. As Paneth
cells positively regulate the number and function of Lgr5hi stem cells
in young mice^2 ,^12 , the decoupling of the Lgr5hi:Paneth cell ratio in old
mice (Extended Data Fig. 1l) raised the possibility that interactions
between these two cell types change during ageing. To address this, we
investigated the organoid-forming capacity of co-cultured Lgr5hi and
Paneth cells isolated from young and old mice (Fig. 1d). Old Paneth
and Lgr5hi cells both showed cell-type-specific age-induced effects, and
initiated organoids with reduced efficiency. Consistent with previous
work, neither cell type formed organoids efficiently by themselves^2 ,^12
but, when co-cultured with Paneth cells, Lgr5hi cells from young mice
formed organoids at a higher rate than old Lgr5hi cells. Of note, the
age-induced stem cell defect was partially rescued by co-culture with
young Paneth cells, whereas old Paneth cells did not fully support(^1) Institute of Biotechnology, HiLIFE, University of Helsinki, Helsinki, Finland. (^2) The David H. Koch Institute for Integrative Cancer Research at MIT, Department of Biology, MIT, Cambridge, MA, USA.
(^3) The Skaggs Institute for Chemical Biology, Department of Chemical Physiology, The Scripps Research Institute, La Jolla, CA, USA. (^4) Department of Medicine, Division of Gastroenterology,
Duke University, Durham, NC, USA.^5 Obesity Research Unit, Research Programs Unit, Diabetes and Obesity, University of Helsinki, Helsinki, Finland.^6 Abdominal Center, Department of
Gastrointestinal Surgery, Helsinki University Hospital, Helsinki, Finland.^7 Department of Pathology, Research Programs Unit and HUSLAB, University of Helsinki and Helsinki University Hospital,
Helsinki, Finland.^8 Atlanta Gastroenterology Associates, Atlanta, GA, USA.^9 Cancer Biology and Genetics, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, NY, USA.
(^10) Howard Hughes Medical Institute, MIT, Cambridge, MA, USA. (^11) Whitehead Institute for Biomedical Research, Howard Hughes Medical Institute, Department of Biology, MIT, Cambridge, MA, USA.
(^12) Molecular and Integrative Bioscience Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki, Helsinki, Finland. (^13) Department of Biosciences and Nutrition,
Karolinska Institutet, Stockholm, Sweden.^14 These authors contributed equally: Sharif Iqbal, Miyeko Mana. *e-mail: [email protected]
398 | NAtUre | VOL 571 | 18 JULY 2019