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LETTER https://doi.org/10.1038/s41586-019-1212-5
Tracing the origin of adult intestinal stem cells
Jordi Guiu1,14, Edouard Hannezo2,3,14, Shiro Yui1,13, Samuel Demharter1, Svetlana Ulyanchenko1, Martti Maimets1,
Anne Jørgensen4, Signe Perlman5, Lene Lundvall5, Linn Salto Mamsen6, Agnete Larsen7, Rasmus H. Olesen7,
Claus Yding Andersen6, Lea Langhoff Thuesen8, Kristine Juul Hare8, Tune H. Pers9, Konstantin Khodosevich1,
Benjamin D. Simons2,10,11 & Kim B. Jensen1,12*
Adult intestinal stem cells are located at the bottom of crypts of
Lieberkühn, where they express markers such as LGR5
1,2
and fuel
the constant replenishment of the intestinal epithelium
1
. Although
fetal LGR5-expressing cells can give rise to adult intestinal stem
cells
3,4
, it remains unclear whether this population in the patterned
epithelium represents unique intestinal stem-cell precursors.
Herewe show, using unbiased quantitative lineage-tracing
approaches, biophysical modelling and intestinal transplantation,
that all cells of the mouse intestinal epithelium—irrespective of
their location and pattern of LGR5 expression in the fetal gut tube—
contribute actively to the adult intestinal stem cell pool. Using 3D
imaging, we find that during fetal development the villus undergoes
gross remodelling and fission. This brings epithelial cells from the
non-proliferative villus into the proliferative intervillus region,
whichenables them to contribute to the adult stem-cell niche.
Our results demonstrate that large-scale remodelling of the
intestinal wall and cell-fate specification are closely linked.
Moreover, these findings provide a direct link between the observed
plasticity and cellular reprogramming of differentiating cells in
adult tissues following damage5–9, revealing that stem-cell identity
is an induced rather than a hardwired property.
The intestine forms from the pseudostratified gut tube, which
becomes patterned during late fetal development into villi and a
continuous intervillus region formed by LGR5− and LGR5+ cells,
respectively10 (Fig.1a, Extended Data Fig.1a–c). The continuous
intervillus region is the major site of proliferation in the developing
intestine (Extended Data Fig.1d–f), and crypts subsequently form
from this region postnatally
11
. Despite the apparent transcriptional
similarity between fetal and adult LGR5+ cells4, it remains unclear
how the fetal immature intestine transitions into the mature structure
and how this is orchestrated at the cellular level. In particular, it is
not known whether a specialized subset of fetal cells becomes adult
intestinal stem cells or whether stem-cell identity is an induced
property.
To investigate the role of fetal LGR5+ cells in the establishment of
the adult intestinal stem cell population, we performed lineage trac-
ing on this population from embryonic day (E)16.5. Focusing on
the proximal part of the small intestine, we observed that, consistent
with previous reports3,4,12, progeny of the LGR5-expressing popula-
tion were maintained into adulthood and thereby contributed to the
adult intestinal stem-cell compartment (Fig.1b). Most of the clones
observed at postnatal day (P)0 were, as expected, located in the inter-
villus regions (Extended Data Fig.2a). Moreover, it was not until P11
that clones extended as ribbons from the base of crypts to the tips of
villi (Supplementary Video1).
The quantitative contribution from LGR5+ progeny, labelled at
E16.5, was slightly greater than the overall degree of tissue expansion
(Fig.1c, Extended Data Fig.2b–e). This confirmed that LGR5
+
cells
were an important source of tissue growth. However, given that LGR5
+
cells constituted only a small fraction of the cells (fraction of LGR5
+
cells/total cells, f=7.0%±0.9%, mean±s.e.m.) in the proximal part of
the small intestine at the time of labelling (Extended data Fig.2f–h), we
reasoned thatif LGR5+ cells were the main source of adult epithelium
(Fig.1d) they would have to expand by a ratio 1/f greater than overall
tissue to fuel growth and replace cells outside the intervillus regions.
Thus, LGR5
+
clones should expand 130-fold from P5 to adulthood,
nearly an order of magnitude higher than the measured value (Fig.1e).
Expansion of LGR5
+
progeny was thus insufficient to explain tissue
growth.
To resolve the cellular diversity in the epithelium at E16.5, we per-
formed single-cell RNA sequencing (scRNA-seq) analysis. Consistent
with our characterization of LGR5–eGFP, Lgr5 was detected in 7% of
the 3,509 cells analysed and—despite detecting only goblet cells by
immunostaining—we identified other differentiated cell types, includ-
ing Paneth cells (Lyz1), entero-endocrine cells (Chga) and enterocytes
(Alpi) (Extended Data Fig.3a, b). In the adult epithelium, the differenti-
ated villi compartment can be separated into at least five transcription-
ally distinct populations
13
. In the fetal intestine, these largely collapse
into two populations, and a gene signature for crypt proliferation was
detected beyond the LGR5
+
compartment, including cells expressing
differentiation markers
14
(Extended Data Fig.3c–e). This strongly sup-
ported the conclusion that cells in the fetal intestinal epithelium were
distinct from their adult counterparts, and that cells expressing differ-
entiation markers had not completed their differentiation program.
To test experimentally how cells outside the intervillus region con-
tributed to tissue growth, we performed fate mapping using a ubiq-
uitously expressed keratin 19 (Krt19)-driven Cre model (Fig.2a;
Extended Data Fig.3f–h). Although the scRNA-seq data revealed that
49% of Krt19-expressing cells at E16.5 score positive for the prolifer-
ation signature, the expansion of clones closely mirrored the overall
growth of the tissue (Fig.2b; Extended Data Fig.3i), whichconfirms
that Krt19-expressing cells were representative of the tissue. However,
we found that boththe long-term persistence (defined as the fraction
of surviving clones) and size of KRT19-labelled clones were very sim-
ilar to their LGR5-labelled counterparts (Fig.2c, d, Supplementary
Information, ‘Supporting clonal data’). Several independent measure-
ments confirmed that KRT19 marked a population of cells distributed
randomly along the villus–intervillus axis (Extended Data Fig.3j, k,
Supplementary Video2). Moreover, apoptotic cells at the tips of villi
appeared only from P7; this means that KRT19+ clones cannot be lost
1Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark. 2The Wellcome Trust–Cancer Research UK Gurdon Institute, University of Cambridge,
Cambridge, UK. 3Institute of Science and Technology Austria, Klosterneuburg, Austria. 4Department of Growth and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark.
5Department of Gynecology, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark. 6Laboratory of Reproductive Biology, Section 5712, The Juliane Marie Centre for Women, Children
and Reproduction, University Hospital of Copenhagen, University of Copenhagen, Copenhagen, Denmark. 7Department of Biomedicine–Pharmacology, Aarhus University, Aarhus, Denmark.
8Department of Obstetrics and Gynaecology, Hvidovre University Hospital, Hvidovre, Denmark. 9The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical
Sciences, University of Copenhagen, Copenhagen, Denmark. 10Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge, UK. 11The Wellcome Trust–Medical Research
Council Stem Cell Institute, University of Cambridge, Cambridge, UK. 12Novo Nordisk Foundation Center for Stem Cell Research, Faculty of Health and Medical Sciences, University of Copenhagen,
Copenhagen, Denmark. 13Present address: Center for Stem Cell and Regenerative Medicine, Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University (TMDU), Tokyo,
Japan. 14These authors contributed equally: Jordi Guiu, Edouard Hannezo. *e-mail: kim.jensen@bric.ku.dk
6 JUNE 2019 | VOL 570 | NATURE | 107
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