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Tracing the origin of adult intestinal stem cells

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Adult intestinal stem cells are located at the bottom of crypts of Lieberkühn, where they express markers such as LGR51,2 and fuel the constant replenishment of the intestinal epithelium1. Although fetal LGR5-expressing cells can give rise to adult intestinal stem cells3,4, it remains unclear whether this population in the patterned epithelium represents unique intestinal stem-cell precursors. Here we 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, which enables 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 damage5,6,7,8,9, revealing that stem-cell identity is an induced rather than a hardwired property.
LGR5-derived clones are located in intervillus regions and qualitative and quantitative morphological analysis of the intestine from fetal to adult stages a, Quantification of localization of labelled clones at P0 following labelling at E16.5 in Rosa26-lsl-Confetti;Lgr5-eGFP-ires-creERT2 mice. Villi containing clones were divided into three equal regions (T, top; M, middle; B, bottom) based on the z projections in 3D to determine the clone localization at P0 (n = 24 clones). b, Detection of E-cadherin (red) in whole mounts at the indicated time points. Scale bars, 100 μm. A minimum of three mice were analysed at each time point and representative images are shown (E16.5 n = 3, P0 n = 9, P5 n = 9, P11 n = 9, adult n = 9 mice). c, Measurements of the total epithelial volume per unit area based on detection of E-cadherin relative to the area of the intestine (samples from b). Dots represent independent biological samples; data are mean ± s.e.m. d, Length of the small intestine at E16.5 (n = 12), P0 (n = 3), P5 (n = 3), P11 (n = 8) and adult (n = 4). Dots represent individual mice and lines represent the mean ± s.e.m. e, Luminal perimeter of the small intestine at E16.5, P0, P5, P11 and adult. Dots represent individual mice (n = 3) and lines represent the mean ± s.e.m. f–h, Quantification of GFP as a proxy for LGR5 in proximal and distal small intestine at E13.5 (n = 3 mice both proximal and distal) and E16.5 (n = 7 mice proximal, n = 5 mice distal) small intestines (f). g, Fluorescence minus one (GFP) controls used to establish the positive gates. h, Representative FACS dot plot illustrating the gating strategy to quantify the size of the LGR5–DTR–eGFP⁺ population (gate, DAPI⁻CD31⁻CD45⁻EpCAM⁺). Dots represent measurement in individual mice; data are mean ± s.e.m. Source data
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Characterization of the fetal small intestinal epithelium a, t-distributed stochastic neighbor embedding (t-SNE) plots from scRNA-seq of epithelial cells from the proximal small intestine showing expression of intestinal stem-cell (Lgr5) and differentiation markers (Muc2, Lyz1, ChgA and Alpi). Darker colour indicates higher normalized gene expression. Each dot represents a cell; a total of 3,509 cells is shown. b, Detection of differentiation markers in E16.5 and adult small intestine. Tissue is counterstained with haematoxylin and eosin. Scale bar, 250 μm. Samples from n = 3 mice were analysed at each time point and representative images are shown. c, Cartoon depicting that adult villi are transcriptionally zonated in five regions numbered from bottom to top (z, zone). d, t-SNE plots showing enrichment of villi clusters in the scRNA-seq from E16.5 small intestine. e, t-SNE plot showing the enrichment of a proliferation signature in specific cell populations (left) and fraction of cells in each subpopulation scoring positive for the proliferation gene signature (right). A darker colour in the t-SNE plot indicates higher expression levels of the proliferation gene signature. f, t-SNE plot showing expression of keratin 19 (Krt19). Darker colour indicates higher normalized gene expression levels. g, Detection of KRT19 (red) and GFP (Lgr5-DTR-eGFP) at E16.5 in proximal small intestine. Tissue is counterstained with DAPI (cyan). Scale bars, 50 μm. Samples from three mice were analysed and a representative image is shown. h, Detection of KRT19 (red) at different time points in tissue from the small intestine. Tissue is counterstained with DAPI (cyan). Scale bars, 50 μm. Samples from three mice were analysed per time point and representative images are shown. i, Relative volume (projected) of KRT19 clones and epithelium based on E-cadherin. E-cadherin is also shown in Fig. 1c and KRT19 clones are also shown in Fig. 2. KRT19 clones, number of biologically independent samples: P0 n = 1; P5 n = 3, P11 n = 6, adult n = 3; E-cadherin, number of biologically independent samples: P0 n = 9, P5 n = 9, P11 n = 9, adult n = 9. j, Quantification of the localization of labelled clones at P0 following administration of 4-hydroxytamoxifen at E16.5 in Rosa26-lsl-Confetti;Krt19-creERT mice. Villi containing clones were divided in three equal regions (T, top; M, middle; B, bottom) based on the z projection in 3D to determine where clones were located at P0 (n = 27). k, Detection of GFP (green) and RFP (red) in whole mounts from the proximal part of the small intestine isolated from mT/mG Krt19-creERT mice at P0 following induction at E16.5. A representative image of n = 3 biologically independent samples is shown. Scale bars, 25 μm. l, Apoptotic cells were detected by cleaved caspase-3. Arrowheads demarcate positive cells in inserts. Samples from three mice were analysed per time point and representative pictures are shown. Scale bars, 250 μm. Source data
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Villi formation and parameter description of villi fissions a, Total number of villi (projected) in the proximal half of the small intestine based on equal density along the length. Samples from three mice were analysed per time point. Each dot represents a mouse; data are mean ± s.e.m. b, The fold change in villi numbers between the indicated time points based on three samples analysed per time point. Each dot represents an independent sample and lines represent the mean ± s.e.m. c, Villus height at the different time points. The demarcated red lines indicate the interval containing villi with sharing stroma. The length was assessed in 25 villi per mouse and in 3 mice per time point. Dots represent independent measurements and lines represent the mean. d, Quantification of the number of villi sharing stroma in three mice per indicated time point (E16.5 n = 233, P0 n = 412, P5 n = 406, P11 n = 412, adult n = 129 villi were counted). Dots represent the percentage of villi sharing stroma in each independent mouse and the line represents the mean. e, Detection of E-cadherin (red) and PDGFRA (yellow) in whole mounts indicating villi with sharing stroma (arrowhead). Samples from three mice were analysed per time point and representative images are shown. Scale bars, 100 μm. f, Detection of E-cadherin (magenta) and GFP (Lgr5-DTR-eGFP) in E16.5 intestinal whole mount. Boxed area (1) indicates a villus undergoing fission, which is shown at higher magnification (middle). Transverse sections (2 and 3) illustrating villi surrounded by LGR5-expressing cells (2) and villus with shared mesenchyme (3). The arrowhead indicates that pockets formed in a fissioning villus are LGR5-negative, the dashed line outlines the epithelium. Samples from three mice were analysed and a representative image is shown. g, Detection of EdU incorporation (green) in the epithelium (E-cadherin, red) following a 1-h chase in E16.5 intestinal whole mounts. Arrowheads indicate proliferative cells at the edge of putative villi undergoing fission. These are detected in 11 out of 16 structures. Representative pictures from three mice analysed are shown. Scale bars, 50 μm. h, Quantification of EdU intensity in the fissioning areas compared to the surrounding villi at the same height quantified as depicted in the cartoon on the basis of thresholded intensity in the demarcated boxes. n = 16 independent villi sharing stroma were quantified. Box plots show the median, box edges represent the first and third quartiles, and the whiskers extend to minimum and maximum values. Dots represent fluorescent ratio of the independent villi sharing stroma. Paired t-test. i, Height of the proliferative fissioning areas compared to the surrounding intervillus regions were quantified as depicted in the cartoon. n = 11 independent villi sharing stroma were quantified. j, Images showing the start and end points from live imaging of villi undergoing fission (Supplementary Video 6). Image from mT/mG;Villin-cre mice, where the epithelium is shown Green (mG) and remaining cells in red (mT). A representative fission event from five analysed mice is shown. Scale bar, 50 μm. Source data
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Outline of the model and typical outputs from simulations a, Schematics of the model for the renewal of intervillus LGR5⁺ cells. On the basis of proliferation data, we assume that the classical model of symmetrically dividing and competing LGR5⁺ cells holds embryonically, with a division rate once a day. The ‘losing’ cell is expelled into the transit-amplifying (TA) compartment, displacing all cells above it by one position. b, Schematics of the model for the dynamics of differentiated cells on the villus. The model dynamics are separated into two phases. The first phase occurs from E16.5 to P5: LGR5⁺ cells are the only proliferative cells, and villi fission occurs as a stochastic event, resulting in the duplication of a villus subregion into a whole new intervillus–villus grid, and a resulting shift of cells along the existing villus. A second phase occurs after P5: the dynamics are similar to adulthood, with rapidly dividing transit-amplifying cells (occupying 1/5th of the villus at the bottom) and cell loss at the top. Proliferation of transit-amplifying cells is again exclusively along the top–bottom axis, resulting in unidirectional displacement of all cells above the dividing cell. c, Three snapshots from a numerical simulation of the epithelium as a growing elastic sheet on a growing elastic medium. The growth is assumed to be quasi-static, so that the epithelium maintains a deformation at a given wavelength, minimizing the elastic energy of the sheet and substrate. Where the system grows (top to bottom panels), this results in de novo villi formation from local deformations of the epithelial sheet, resulting in villus and intervillus regions shifting places via tissue bending (the dashed lines serve as a guide to represent how a cell in a given position x can change height z). d, e, Two sets of snapshots from two numerical simulations of an E16.5 LGR5 tracing, according to the rules outlined in a, b. Each black box represents an intervillus–villus grid, with the number increasing in time owing to random villi fission. Red squares indicate the labelled cells at E16.5 (initially in the bottom-most layer of LGR5⁺ cells). An example of a clone that becomes lost in time, despite having participated in villi fission between E18.5 and P1 is shown in d. An example of a clone that becomes fixed within one of the villi having formed de novo during the simulation (while the labelled cell in the original intervillus–villus region of induction was shifted away from the intervillus by a villi fission event between E16.5 and E17.5) is shown in e.
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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.
Herewe 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,
whichenables 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 Video1).
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 thatif 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), whichconfirms
that Krt19-expressing cells were representative of the tissue. However,
we found that boththe 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 Video2). 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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