Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche
Abstract
The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.
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Tissues including small intestine, colon, stomach, esophagus, tongue, liver, lung, pancreas, heart, ear, and skin have been obtained from iPSCs, adult or fetal cells, either stem/progenitor cells or differentiated cells.
“Under defined physicochemical conditions, tissues such as small intestine7, colon30,31, stomach32,33, esophagus30, tongue34, liver35–38, lung15, pancreas39–41, heart42, ear43, and skin44 have been obtained from iPSCs, adult or fetal cells, either stem/progenitor cells or differentiated cells.”
From Organoids (2022) · cites this paper as [7]
Adult intestinal stem cells placed in Matrigel with a tissue-specific growth factor cocktail self-organize into three-dimensional crypt-villus structures.
“adult intestinal stem cells embedded in Matrigel and in the presence of tissue-specific cocktail of growth factors were also shown to self-organize into 3D crypt-villus structures7”
From Organoids (2022) · cites this paper as [7]
Intestinal organoids were the first tissue-derived organoid type to be established.
“as this was the first tissue-derived-organoid type established7.”
From Organoids (2022) · cites this paper as [7]
Organoids can arise either from round colonies generated by single cells or from initial multicellular structures such as intestinal crypts, cell aggregates, or micropatterned cells.
“Organoids can arise either from round colonies generated by single cells7,35 or, from initial multicellular structures such as intestinal crypts30,63, cell aggregates24 or micropatterned cells26.”
From Organoids (2022) · cites this paper as [7]
Intestinal organoids need to be fragmented into smaller cell clusters and reseeded regularly because of inadequate nutrient and waste diffusion.
“This is the reason why intestinal organoids need to be fragmented into smaller cell clusters and reseeded regularly7.”
From Organoids (2022) · cites this paper as [7]
Intestinal organoids were one of the earliest organoid types to be successfully cultured in vitro.
“Intestinal organoids are among the first organoid types successfully established in vitro7,30.”
From Organoids (2022) · cites this paper as [7]
Most intestinal stem cells could survive, proliferate, and form organoids when Matrigel mimicked the laminin-enriched crypt environment with exogenous growth factors.
“Most intestinal stem cells could survive, proliferate, and form organoids7.”
From Organoids (2022) · cites this paper as [7]
Organoids represent robust in vitro cultures that preserve tissue architecture, stem cell activity, multilineage differentiation, genomic alterations, histology, and pathology of primary tissues.
“Organoids represent robust in vitro cultures that preserve tissue architecture, stem cell activity, multilineage differentiation, genomic alterations, histology, and pathology of primary tissues1,2.”
From Large-scale CRISPR screening in primary human 3D gastric organoids enables comprehensive dissection of gene-drug interactions (2025) · cites this paper as [1]
Multiplexed CRISPR base-editing is versatile and flexible for cancer modeling across several epithelial tissues by mutating liver, endometrium, and organoids.
“We further demonstrate the versatility and flexibility of multiplexed CRISPR base-editing for cancer modeling across several epithelial tissues by mutating liver, endometrium, and organoids32–34.”
From One-step generation of tumor models by base editor multiplexing in adult stem cell-derived organoids (2023) · cites this paper as [34]
RhoA signalling might potentially contribute to cell death, because it can promote apoptosis in low-adhesion environments by increasing cell contractility.
“RhoA signalling might also potentially contribute to cell death, because it can promote apoptosis in low-adhesion environments by increasing cell contractility, explaining why ROCK inhibitors are a standard component of media for stem cells and organoid isolation and maintenance in culture 77-81 .”
From Taxane-induced conformational changes in the microtubule lattice activate GEF-H1-dependent RhoA signalling (2026) · cites this paper as [78]
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).”
From Canonical Wnt Signaling Ameliorates Aging of Intestinal Stem Cells (2017) · cites this paper as Sato et al., 2009
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Self-citation
- Authors
- Sato, T., Vries, R. G., Snippert, H. J., van de Wetering, M., Barker, N., Stange, D. E., van Es, J. H., Abo, A., Kujala, P., Peters, P. J., & Clevers, H.
- Affiliations
- University Medical Center Utrecht (NL)
- The Netherlands Cancer Institute (NL)
- Corresponding author
- Clevers, Hans
- ORCID
- Venue
- Nature
- Published
- 2009-03-29
- Volume
- 459
- Issue
- 7244
- Pages
- 262-265
- ISSN
- 0028-0836 (ISSN-L)
- 1476-4687
- OpenAlex
- W1965157206
- Cited by
- 7,204
- References
- 33
- Citations by year
- 2026: 537 · 2025: 663 · 2024: 658 · 2023: 663 · 2022: 640 · 2021: 680 · 2020: 636 · 2019: 433 · 2018: 450 · 2017: 397 · 2016: 341 · 2015: 260 · 2014: 223 · 2013: 204 · 2012: 159
- FWCI
- 69.23
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Nature (publishedVersion)
- PubMed (publishedVersion)
- Data Archiving and Networked Services (DANS) (submittedVersion)
- RePEc: Research Papers in Economics (submittedVersion)
- KNAW Research Portal (The Royal Netherlands Academy of Arts and Sciences) (submittedVersion)
- Topics
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Wnt/β-catenin signaling in development and cancer
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Digestive system and related health
- Keywords
- Lgr5, intestinal stem cells, intestinal crypts, self-organizing structures, mesenchymal niche, intestinal epithelium, stem cell hierarchy, crypt fission, cell tracing
- MeSH terms
- Animals, Cell Separation, Intestinal Mucosa, Intestines, Mesoderm, Organoids, Regeneration, Stem Cells, Gene Expression Regulation, Developmental, Cell Culture Techniques, Cell Lineage, Paneth Cells, Multipotent Stem Cells, Receptors, G-Protein-Coupled, Mice, Receptors, Notch, Stem Cell Niche
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