Clevers Group: Adult stem cell-based organoids

Wnt signaling in adult stem cells
In mammals, physiological Wnt signaling is intimately involved with the biology of adult stem cells and self-renewing tissues (18,19). We were the first to link Wnt signaling with adult stem cell biology, when we showed that TCF4 gene disruption leads to the abolition of stem cell activity in crypts of the small intestine (8), and that TCF1 gene knockout severely disables the stem cell compartment of the thymus (2). The Tcf4-driven target gene program in colorectal cancer cells is the malignant counterpart of a physiological gene program in selfrenewing crypts (13, 14).

Lgr5 stem cell biology
The Wnt target gene encoding the transcription factor Achaete scute-like 2 controls the intestinal stem cell state (26). Lgr5 crypt stem cells behave in unanticipated ways: Against common belief, they divide constantly and in a symmetric fashion. Stem cell numbers remain fixed because stem cells compete ‘neutrally’ for niche space (30). This phenomenon was confirmed by in vivo imaging (44). Daughters of the small intestinal stem cells, the Paneth cells, serve as crypt niche cells by providing Wnt, Notch and EGF signals (33). In a very recent study, we describe how intestinal epithelial cells die at the end of their one week lifespan, on the tips of villi (80).

Lgr5 is the R-spondin receptor
Lgr5 resides in Wnt receptor complexes and mediates signaling of the Wnt-agonistic R-spondins (31), explaining the unique dependence of Lgr5 stem cells on secreted R-spondins in vivo and in vitro. Two other Wnt target genes, RNF43 and ZNRF3, encode stem cell-specific E3 ligases that downregulate Wnt receptors in a negative feedback loop (35). Independent work by the Feng Cong lab has first shown that R-spondin, when bound to Lgr5, captures and inactivates RNF43/ZNRF3.

Long-term clonal culturing of organoids from Lgr5 stem cells. Modeling of infectious, hereditary disease and cancer in organoids (reviewed in 51)
Wnt signaling intimately interacts with the BMP and Notch cascades to drive proliferation and inhibit differentiation in intestinal crypts and adenomas (17, 20). Based on these combined insights, we have established Lgr5/R-spondin-based culture systems that allow the outgrowth of single mouse or human Lgr5 stem cells into ever-expanding organoids. Some examples are mini-guts (27, 32), mini-stomachs (28), colon cancer organoids (32, 47), liver organoids (39, 46, 55), prostate organoids (45), breast cancer organoids (53), ovarian cancer organoids (59), pancreas cancer organoids (48), and even snake venom gland organoids (61). These epithelial organoid cultures are genetically and phenotypically extremely stable, allowing transplantation of the cultured offspring of a single stem cell, as well as disease modeling by growing organoids directly from diseased patient tissues (32, 47, 53). The direct cloning of multiple individual cells from primary tumors allows molecular and functional analysis of tumor heterogeneity with unprecedented resolution (54).
Organoids are readily amenable to CRISPR-mediated genome modification to model for instance malignant transformation (49) and mutagenesis upon faulty DNA repair (52), or to rapidly create knock-in alleles of genes of interest (62, 66). Human rectal organoids model the hereditary disease Cystic Fibrosis, are now routinely used to predict drug response in CF patients. In 2013, we have provided the first proof-of-concept for CRISPR-mediated repair of a hereditary mutation in patient stem cells (43, 64). Human organoids also model infectious disease, as demonstrated for instance for Cryptosporidium (55), a mutagenic E. coli strain (63) and for SARS-CoV-2 (65).

Characterization of rare epithelial cell types using (human) intestinal organoids.

Human organoids can be cultured under conditions designed to generate rare epithelial cell types.
Enteroendocrine cells (EECs): The transcriptional hierarchy of the six EEC lineages, including the incretin Glp1-producing L cells, was mapped in mouse and man (58, 66). Using an unbiased transcription factor CRISPR screen, ZNF800 was identified as master repressor of EEC differentiation (72), while single cell deep mRNA sequencing of human organoid EEC followed by targeted CRISPR mutation and pharmacologic activation of hormone secretion delineated roles of individual human EEC sensors in hormone secretion. These receptors represent potential pharmacological targets to influence appetite, bowel movement, insulin sensitivity and mucosal immunity (76).

Tuft cells: An Il4/Il13-driven organoid protocol allows the generation of large numbers of Tuft cells, which was used to demonstrate that these cells act as regenerative (reserve) stem cells in the human intestine (75).

BEST4+ cells: These cells, unique to man, were previously only known from sc-mRNA sequencing datasets. A specific organoid culturing protocol allowed the generation of these cells in large numbers. We demonstrated that BEST4 cells are induced by g-interferon, control water and electrolyte balance across the gut epithelium and are responsible for bacterially induced diarrea (79).

M cells: As well as facilitating luminal antigen transport to Peyer’s path immune cells, we show that human M cells also directly present antigens (i.e gluten) via their class II major histocompatibility complex (MHC-II) (81).

In sum, stem cell-derived organoids (as first described by Sasai for pluripotent stem cells and by us for adult stem cells) are rapidly gaining ground as research tools in a wide range of scientific disciplines, including basic developmental and cell biology, infectiology, toxicology and research on hereditary diseases and cancer.

Cancer Modeling Meets Human Organoid Technology

In this video, Hans Clevers summarizes the use of organoids in cancer research.

Selected papers (out of ~900 peer-reviewed papers with ~223,000 citations in Scopus; h-index 223) 

1. van de Wetering, M., Oosterwegel, M., Dooijes, D. and Clevers, H., Identification and cloning of TCF-1, a T cell-specific transcription factor containing a sequence-specific HMG box. EMBO J. 10:123-132 (1991)
2. Verbeek, J.S., Ison, D., Hofhuis, F., Robanus-Maandag, E., te Riele, H., van de Wetering, M., Oosterwegel, M., Wilson, A., MacDonald, H.R. and Clevers, H. An HMG box containing T-cell factor required for thymocyte differentiation. Nature 374:70-74 (1995)
3. Schilham, M., Oosterwegel, M., Moerer, P., Jing, Y., de Boer, P., van de Wetering, M., Verbeek, S., Lamers, W., Kruisbeek, A., Cumano, A. and Clevers, H. Sox-4 gene is required for cardiac outflow tract formation and pro-B lymphocyte expansion. Nature 380:711-714 (1996)
4. Molenaar, M., van de Wetering, M., Oosterwegel, M., Peterson-Maduro, J., Godsave, S., Korinek, V., Roose, J., Destrée, O. and Clevers, H. Xtcf-3 Transcription factor mediates beta-catenin-induced axis formation in xenopus embryos. Cell 86:391-399 (1996)
5. Korinek, V, Barker, N., Morin, P.J., van Wichen, D., de Weger, R., Kinzler, K.W., Vogelstein, B. and Clevers, H. Constitutive Transcriptional Activation by a beta-catenin-Tcf complex in APC-/- Colon Carcinoma. Science 275:1784-1787 (1997)
6. Morin, P.J., Sparks, A., Korinek, V., Barker, N., Clevers, H., Vogelstein, B. and Kinzler, K., Activation of beta-catenin-Tcf signaling in colon cancer by mutations in beta-catenin or APC. Science 275:1787-1790 (1997)
7. van de Wetering, M., Cavallo, R., Dooijes, D., van Beest, M., van Es, J., Loureiro, J., Ypma, A., Hursh, D., Jones, T., Bejsovec, A., Peifer, M., Mortin, M. and Clevers, H. Armadillo co-activates transcription driven by the product of the Drosophila segment polarity gene dTCF. Cell 88:789-799 (1997)
8. Korinek, V., Barker, N., Moerer, P., van Donselaar, E., Huls, G., Peters, P.J. and Clevers, H. Depletion of epithelial stem cell compartments in the small intestine of mice lacking Tcf 4. Nat Genet 19:379-383 (1998)
9. Roose, J., Molenaar, M., Peterson, J., Hurenkamp, J., Brantjes, H., Moerer, P., van de Wetering, M., Destree, O. and Clevers, H. The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors. Nature 395:608-612 (1998)
10. Roose, J., Huls, G., van Beest, M., Moerer, P., van der Horn, K., Goldschmeding, R., Logtenberg, T. and Clevers, H. Synergie between tumor suppressor APC and the beta-catenin/Tcf4 target gene Tcf1. Science 285:1923-1926 (1999)
11. Korswagen, R., Herman, M. and Clevers, H. Separate beta-catenins mediate Wnt signaling and cadherin adhesion in C. elegans. Nature 406:527-532 (2000)
12. Bienz, M. and Clevers, H. Linking colorectal cancer to Wnt signaling. Cell 103:311-320 (2000)
13. van de Wetering, M., Sancho, E., Verweij, C., de Lau, W., Oving, I., Hurlstone, A., van der Horn, K., Batlle, E., Coudreuse, D., Haramis, A-P., Tjon-Pon-Fong, M., Moerer, P., van den Born, M., Soete, G., Pals, S., Eilers, M., Medema, R. and Clevers, H. The beta catenin/TCF4 complex imposes a crypt progenitor phenotype on colorectal cancer cells. Cell 111:241-250 (2002)
14. Batlle, E., Henderson, J.T., Beghtel, H., van den Born, M., Sancho, E., Huls, G., Meeldijk, J., Robertson, J., van de Wetering, M., Pawson, T. and Clevers, H. Beta- catenin and TCF mediate cell positioning in the intestinal epithelium by controlling the expression of EphB/ephrinB. Cell 111:251-263 (2002)
15. Hurlstone, A.F., Haramis, A.P., Wienholds, E., Begthel, H., Korving, J., van Eeden, F., Cuppen, E., Zivkovic, D., Plasterk, R.H. and Clevers, H. The Wnt/beta-catenin pathway regulates cardiac valve formation. Nature 425:633-637 (2003)
16. Baas, A.F., Kuipers, J., van der Wel, N.N., Batlle, E., Koerten, H.K., Peters, P.J. and Clevers, H. Complete polarization of single intestinal epithelial cells upon activation of LKB1 by STRAD. Cell 116:457-466 (2004)
17. Haramis, A.P., Begthel, H., van den Born, M., van Es, J., Jonkheer, S., Offerhaus, G.J. and Clevers, H. De novo crypt formation and Juvenile Polyposis upon BMP inhibition. Science 303:1684-1686 (2004)
18. Radtke, F. and Clevers, H. Self-renewal and cancer of the gut: Two sides of a coin. Review Science 307:1904-1909 (2005)
19. Reya, T. and Clevers, H. Wnt signalling in stem cells and cancer. Nature 434:843-850 (2005)
20. van Es, J.H., Van Gijn, M.E., Riccio, O., van den Born, M., Vooijs, M., Begthel, H., Cozijnsen, M., Robine, S., Winton, D.J., Radtke, F. and Clevers H. Notch pathway/γ-secretase inhibition turns proliferative cells in intestinal crypts and neoplasia into Goblet cells. Nature 435:959-963 (2005)
21. Batlle, E., Bacani, J., Begthel, H., Jonkheer, S., Gregorieff, A., van de Born, M., Malats, N., Sancho, E., Boon, E., Pawson, T., Gallinger, S., Pals, S. and Clevers, H. EphB activity suppresses colorectal cancer progression. Nature 435:1126-1130 (2005)
22. Clevers, H. Wnt/β-catenin signaling in development and disease, Review Cell 127:469-480 (2006)
23. Barker, N., Van Es, J.H., Kuipers, J., Kujala, P., Van den Born, M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J. and Clevers, H. Identification of stem cells in small intestine and colon by the marker gene LGR5. Nature 449:1003-1007 (2007)
24. Jaks, V., Barker, N, Kasper, M., van Es, J.H., Snippert, H.J., Clevers, H., Toftgård, R. Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Nat Genet. 40:1291-1299 (2008)
25. Barker, N., Ridgway, R.A., van Es, J.H., van de Wetering, M., Begthel, H., van den Born, M., Danenberg, E., Clarke, A.R., Sansom, O.J. and Clevers, H. Crypt Stem Cells as the Cells-of-Origin of Intestinal Cancer. Nature 457:608-611 (2009)
26. van der Flier, L.G., van Gijn, M.E., Hatzis, P., Kujala, P., Haegebarth, A., Stange, D.E., Begthel, H., van den Born, M., Guryev, V., Oving, I., van Es, J.H., Barker, N., Peters, P.J., van de Wetering, M. and Clevers, H. Transcription Factor Achaete Scute-Like 2 Controls Intestinal Stem Cell Fate. Cell 136:903-912 (2009)
27. Sato, T., Vries, R., Snippert, H., van de Wetering, M., Barker, N., Stange, D., van Es, J., Abo, A., Kujala, P., Peters, P. and Clevers, H. Single lgr5 gut stem cells build crypt-villus structures in vitro without a stromal niche. Nature 459:262-265 (2009)
28. Barker, N., Huch, M., Kujala, P., van de Wetering, M., Snippert, H.J., van Es, J.H., Sato, T., Stange, D.E., Begthel, H., van den Born, M., Danenberg, E., van den Brink, S., Korving, J., Abo, A., Peters, P.J., Wright, N., Poulsom, R. and Clevers, H. Lgr5(+ve) stem cells drive self-renewal in the stomach and build long-lived gastric units in vitro. Cell Stem Cell 6:25-36 (2010)
29. Snippert, H.J., Haegebarth, A., Kasper, M., Jaks, V., van Es, J.H., Barker, N., van de Wetering, M., van den Born, M., Begthel, H., Vries, R.G., Stange, D.E., Toftgård, R. and Clevers H. Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin. Science 327:1385-1389 (2010)
30. Snippert, J., van der Flier, L.G., Sato, T., van Es, J.H., van den Born, M., Kroon-Veenboer, C., Barker, N., Klein, A.M., van Rheenen, J. Benjamin D. Simons, B.D. and Clevers, H. Intestinal Crypt Homeostasis results from Neutral Competition between Symmetrically Dividing Lgr5 Stem Cells. Cell 143:134-44 (2010)
31. de Lau, W., Barker, N., Low, T.Y., Koo, B.K., Li, V.S., Teunissen, H., Kujala, P., Haegebarth, A., Peters, P.J., van de Wetering, M., Stange, D.E., van Es, J., Guardavaccaro, D., Schasfoort, R.B., Mohri, Y., Nishimori, K., Mohammed,S., Heck, A.J. and Clevers, H. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling. Nature 476:293-297 (2011)
32. Sato T., Stange, D.E., Ferrante, M., Vries R.G., Van Es, J.H., van den Brink, S., van Houdt, W.J., Pronk, A, van Gorp, J., Siersema, P.D. and Clevers, H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium. Gastroenterology 141: 1762-1772 (2011)
33. Sato, T., van Es, J.H., Snippert, H.J., Stange, D.E., Vries, R.G., van den Born, M., Barker, N., Shroyer, N.F., van de Wetering, M. and Clevers, H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts. Nature 469:415-418 (2011)
34. Li, V.S., Ng, S.S., Boersema, P.J., Low, T.Y., Karthaus, W.R., Gerlach, J.P., Mohammed, S., Heck, A.J., Maurice, M.M., Mahmoudi, T. and Clevers, H. Wnt signaling inhibits proteasomal β-catenin degradation within a compositionally intact Axin1 complex. Cell 149:1245-1256 (2012)
35. Koo, B-K., Spit, M. Jordens, I., Low, T.Y., Stange, D.E., van de Wetering, M., van Es, J.H., Mohammed, S., Heck, A.J.R., Maurice, M.M. and Clevers, H. Tumour suppressor RNF43 is a stem cell E3 ligase that induces endocytosis of Wnt receptors. Nature 488:665-669 (2012)
36. Schepers, A.G., Snippert, H.J., Stange, D.E., van den Born, M., van Es, J.H., van de Wetering, M. and Clevers, H. Lineage Tracing Reveals Lgr5+ Stem Cell Activity in Mouse Intestinal Adenomas. Science 337:730-735 (2012)
37. van Es, J.H., Sato, T., van de Wetering, M., Lyubimova, A., Yee Nee, A.N., Gregorieff, A., Sasaki, N., Zeinstra, L., van den Born, M., Korving, J., Martens, A.C., Barker, N., van Oudenaarden, A. and Clevers, H. Dll1(+) secretory progenitor cells revert to stem cells upon crypt damage. Nat Cell Biol. 14:1099-1104 (2012)
38. Boj, S,F., van Es, J.H., Huch. M., Li, V.S., Jose, A., Hatzis, P., Mokry, M., Haegebarth, A., van den Born, M., Chambon, P., Voshol, P., Dor, Y., Cuppenm E., Fillat, C. and Clevers, H. Diabetes risk gene and Wnt effector Tcf7l2/TCF4 controls hepatic response to perinatal and adult metabolic demand. Cell 151:1595-1607 (2012)
39. Huch, M., Dorrell, C., Boj, S.F., van Es, J.H., van de Wetering, M., Li, V.S.W., Hamer, K., Sasaki, N., Finegold, M.J., Haft, A., Grompe, M. and Clevers, H. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration. Nature 494:247-250 (2013)
40. Sato, T. and Clevers, H. Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Review Science 340:1190-1194 (2013)
41. Clevers, H. The intestinal crypt, a prototype stem cell compartment. Cell 154:274-284 (2013)
42. Stange, D.E., Koo, B.K., Huch, M., Sibbel, G., Basak, O., Lyubimova, A., Kujalla, P., Bartfeld, S., Koster, J., Geahlen, J.H., Peters, P.J., van Es, J., van de Wetering, M., Mills, J.C. and Clevers, H. Differentiated Troy+ chief cells act as ‘reserve’ stem cells to generate all lineages of the stomach epithelium. Cell 155:357-368 (2013)
43. Schwank, G., Koo, B.K., Sasselli, V., Dekkers, J.F., Heo, I., Demircan, T., Sasaki, N., Boymans, S., Cuppen, E., van der Ent, C.K., Nieuwenhuis, E.E., Beekman, J.M. and Clevers, H. Functional Repair of CFTR by CRISPR/Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients. Cell Stem Cell 13:653-658 (2013)
44. Ritsma, L., Ellenbroek, S.I., Zomer, A., Snippert, H.J., de Sauvage, F.J., Simons, B.D., Clevers, H. and van Rheenen, J. Intestinal crypt homeostasis revealed at single-stem-cell level by in vivo live imaging. Nature 507:362-365 (2014)
45. Karthaus, W.R., Iaquinta, P.J., Drost, J., Gracanin, A., van Boxtel, R., Wongvipat, J., Dowling, C.M., Gao, D., Begthel, H., Sachs, N., Vries, R.G., Cuppen, E., Chen, Y., Sawyers, C.L. and Clevers, H. Identification of Multipotent Luminal Progenitor Cells in Human Prostate Organoid Cultures. Cell 159:163-175 (2014)
46. Huch, M., Gehart, H., van Boxtel, R., Hamer, K., Blokzijl, F., Verstegen, M., Ellis, E., van Wenum, M., Fuchs, S., de Ligt, J., van de Wetering, M., Sasaki, N., Boers, S., Kemperman, H., de Jonge, J. IJzermans, J., Niewenhuis, E., Hoekstra, R., Strom, S., Vries, R., van der Laan, L., Cuppen, E. and Clevers, H. Long-term culture of genome-stable bipotent stem cells from adult human liver. Cell 160:299-312 (2015)
47. van de Wetering, M., Francies, H.E., Francis, J.M., Bounova, G., Lorio, F., Pronk, A., van Houdt, W., van Gorp, J., Taylor-Weiner, A., Kester, L., McLaren-Douglas, A., Blokker, J., Jaksani, S., Bartfeld, S., Volckman, R., van Sluis, P., Li, V.S.W., Seepo, S., Sekhar Pedamallu, C., Cibulskis, C., Carter, S.L., McKenna, A., Lawrence, M.S., Lichtenstein, L., Stewart, C., Koster, J., Versteeg, R., van Oudenaarden, A., Saez-Rodriguez, J., Vries, R.G.J., Getz, G., Wessels, L., Stratton, M.R., McDermott, U., Meyerson, M., Garnett, M.J and Clevers, H. Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients. Cell 161:933-945 (2015)
48. Boj, S.F., Hwang, C.I., Baker, L.A., Chio, I.I., Engle, D.D., Corbo, V., Jager, M., Ponz-Sarvise, M., Tiriac, H., Spector, M.S., Gracanin, A., Oni, T., Yu, K.H., van Boxtel, R., Huch, M., Rivera, K.D., Wilson, J.P., Feigin, M.E., Öhlund, D., Handly-Santana, A., Ardito-Abraham, C.M., Ludwig, M., Elyada, E., Alagesan, B., Biffi, G., Yordanov, G.N., Delcuze, B., Creighton, B., Wright, K., Park, Y., Morsink, F.H., Molenaar, I.Q., Borel Rinkes, I.H., Cuppen, E., Hao, Y., Jin, Y., Nijman, I.J., Iacobuzio-Donahue, C., Leach, S.D., Pappin, D.J., Hammell, M., Klimstra, D.S., Basturk, O., Hruban RH, Offerhaus GJ, Vries RG, Clevers H, Tuveson DA. Organoid models of human and mouse ductal pancreatic cancer. Cell 160: 324-338 (2015)
49. Drost, J., van Jaarsveld, R.H., Ponsioen, B., Zimberlin, C., van Boxtel, R., Buijs, A., Sachs, N., Overmeer, R.M., Offerhaus, G.J., Begthel, H. Korving, J., van de Wetering, M., Schwank, G. Logtenberg, M., Cuppen, E., Snippert, H.J., Medema, J.P., Kops, G.J.P.L. and Clevers, H. Sequential cancer mutations in cultured human intestinal stem cells. Nature 521:43-47 (2015)
50. Farin, H.F., Jordens, I., Mosa, M.H., Basak, O., Korving, J., Tauriello, D.V.F., de Punder, K., Angers, S., Peters, P.J. Maurice, M.M. and Clevers, H. Visualization of the short-range Wnt gradient in the intestinal stem cell niche. Nature 530:340-343 (2016)
51. Clevers, H. Modeling development and disease with organoids. Cell 165:1586-1597 (2016)
52. Drost, J., van Boxtel, R., Blokzijl, F., Mizutani, T., Sasaki, N., Sasselli, V., de Ligt, J., Behjati, S., Grolleman, J.E., van Wezel, T., Nik-Zainal, S., Kuiper, R.P., Cuppen, E. and Clevers, H. Use of CRISPR-modified human stem cell organoids to study the origin of mutational signatures in cancer. Science 358:234-238 (2017)
53. Sachs, N., de Ligt, J., Kopper, , Gogola E., Bounova G., Weeber, F., Balgobind, A.V., Wind, K., Gracanin, A., Begthel, H., Korving, J., van Boxtel, R., Duarte, A.A., Lelieveld, D., van Hoeck, A., Ernst, R.F., Blokzijl, F., Nijman, I.J., Hoogstraat, M., van de Ven, M., Egan, D.A., Zinzalla, V., Moll, J., Boj, S.F., Voest, E.E., Wessels, L., van Diest, P.J., Rottenberg, S., Vries, R.G.J., Cuppen, E. and Clevers, H. A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity. Cell 172:373-386 (2018)
54. Roerink, S., Sasaki, N., Lee-Six, H., Young, M., Alexandrov, L., Behjati, S., Mitchell, T., Grossmann, S., Lightfoot, H., Egan, D., Pronk, A., Smakman, N., van Gorp, J., Anderson, E., Gamble, S., Alder, C., van de Wetering, M., Campbell, P., Stratton, M. and Clevers, H. Intra-tumour diversification in colorectal cancer at the single-cell level. Nature 556:457-462 (2018)
55. Heo, I., Dutta,, Schaefer, D.A., Lakobachvili, N., Artegiani, B., Sachs, N., Boonekamp, K.E., Bowden, G., Hendrickx, A.P.A., Willems, R.J.L., Peters, P.J., Riggs, M.W., O’Connor, R. and Clevers, H. Modelling Cryptosporidium infection in human small intestinal and lung organoids. Nat Microbiol. 3:814-823 (2018)
56. Hu, H., Gehart, H., Artegiani, B., Löpez-Iglesias, C., Dekkers, F., Basak, O., van Es, J., Chuva de Sousa Lopes, S.M., Begthel, H., Korving, J., van den Born, M., Zou, C., Quirk, C., Chiriboga, L., Rice, C.M., Ma, S., Rios, A., Peters, P.J., de Jong, Y.P. and Clevers, Long-Term Expansion of Functional Mouse and Human Hepatocytes as 3D Organoids. Cell 175:1591-1606 (2018)
57. Tuveson, D. and Clevers, Cancer modeling meets human organoid technology. Science 364:952-955 (2019)
58. Gehart, H., van Es, J.H., Hamer, K., Beumer, J., Kretzschmar, K., Dekkers, J.F., Rios, A. and Clevers, Identification of Enteroendocrine Regulators by Real-Time Single-Cell Differentiation Mapping. Cell 176:1158-1173 (2019)
59. Kopper , O., de Witte, C.J., Lõhmussaar, K., Valle-Inclan, J.E., Hami, N., Kester, L., Balgobind, A.V., Korving, J., Proost, N., Begthel, H., van Wijk, L.M., Revilla, S.A., Theeuwsen, R., van de Ven, M., van Roosmalen, M.J., Ponsioen, B., Ho, V.W.H., Neel, B.G., Bosse, T., Gaarenstroom, K.N., Vrieling, H., Vreeswijk, M.P.G., van Diest, P.J., Witteveen, P.O., Jonges, T., Bos, J.L., van Oudenaarden, A., Zweemer, R.P., Snippert, H.J.G., Kloosterman, W.P. and Clevers, An organoid platform for ovarian cancer captures intra- and interpatient heterogeneity. Nat Med. 25:838-849 (2019)
60. Tuveson, D. and Clevers, H. Cancer modeling meets human organoid technology. Science 364:952-955 (2019)
61. Post, Y., Puschhof, J., Beumer, J., Kerkkamp, H.M., de Bakker, M.A.G., Slagboom, J., de Barbanson, B., Wevers, N.R., Spijkers, X.M., Olivier, T., Kazandjian, T.D., Ainsworth, S., Iglesias, C.L., van de Wetering, W.J., Heinz, M.C., van Ineveld, R.L., van Kleef, R.G.D.M., Begthel, H., Korving, J., Bar-Ephraim, Y.E., Getreuer, W., Rios, A.C., Westerink, R.H.S., Snippert, H.J.G., van Oudenaarden, A., Peters, P.J., Vonk, F.J., Kool, J., Richardson, M.K., Casewell, N.R. and Clevers, Snake Venom Gland Organoids. Cell 180:233-247 (2020)
62. Artegiani, B., Hendriks, D., Beumer, J., Kok, R., Zheng, X., Joore, I., Chuva de Sousa Lopes, S., van Zon, J., Tans, S. and Clevers, H. Fast and efficient generation of knock-in human organoids using homology-independent CRISPR-Cas9 precision genome editing. Nat Cell Biol. 22:321-331 (2020)
63. Pleguezuelos-Manzano, C., Puschhof, J., Huber, A.R., van Hoeck, A., Wood, H.M., Nomburg, J., Gurjao, C., Manders, F., Dalmasso, G., Stege, P.B., Paganelli, F.L., Geurts, M.H., Beumer, J., Mizutani, T., van der Linden, R., van Elst, S.; Genomics England Research Consortium, Top J., Willems, R.J.L., Giannakis, M., Bonnet, R., Quirke, P., Meyerson, M., Cuppen, E., van Boxtel, R. and Clevers, H. Mutational signature in colorectal cancer caused by genotoxic pks₊ coli. Nature 580:269-273 (2020)
64. Geurts, M.H., de Poel, E., Amatngalim, G.D., Oka, R., Meijers, F.M., Kruisselbrink, E., van Mourik, P., Berkers, G., de Winter-de Groot, K.M., Michel, S., Muilwijk, D., Aalbers, B.L., Mullenders, J., Boj, S.F., Suen, S.W.F., Brunsveld, J.E., Janssens, H.M., Mall, M.A., Graeber, S.Y., van Boxtel, R., van der Ent, C.K., Beekman, J.M. and Clevers, H. CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank. Cell Stem Cell 26:503-510 (2020)
65. Lamers, M.M., Beumer, J., van der Vaart, J., Knoops, K., Puschhof, J., Breugem, T.I., Ravelli, R.B.G., van Schayck, J.P., Mykytyn, A.Z., Duimel, H.Q., van Donselaar, E., Riesebosch, S., Kuijpers, H.J.H., Schipper, D., van de Wetering, W.J., de Graaf, M., Koopmans, M., Cuppen, E., Peters, P.J., Haagmans, B.L. and Clevers, H. SARS-CoV-2 productively Infects Human Gut Enterocytes. Science 369:50-54 (2020)
66. Beumer, J., Puschhof, J., Bauzá-Martinez, J., Martínez-Silgado, A., Elmentaite, R., James, K.R., Ross, A., Hendriks, D., Artegiani, B., Busslinger, G., Ponsioen, B., Andersson-Rolf, A., Saftien, A., Boot, C., Kretzschmar, K., Geurts, M.H., Bar-Ephraim, Y.E., Pleguezuelos Manzano, C., Post, Y., Begthel, H., van der Linden, F., Lopez-Iglesias, C., van de Wetering, W.J., van der Linden, R., Peters, P.J., Heck, A.J.R., Goedhart, J., Snippert, H., Zilbauer, M., Teichmann, S.A., Wu, W. and Clevers, H. High Resolution mRNA and Secretome Atlas of Human Enteroendocrine Cells. Cell 181:1291-1306 (2020)
67. Lohmussaar K., Oka R., Espejo Valle-Inclan J., Veersema S., […] van Boxtel R., Clevers H. Patient-derived organoids model cervical tissue dynamics and viral oncogenesis in cervical cancer.
    Cell Stem Cell 28:1-17 (2021)
68. Bannier-Hélaouët M., Post Y., Korving J. […] Imhoff S., Clevers H. Exploring the human lacrimal gland using organoids and single-cell sequencing. Cell Stem Cell 28:1-12 (2021)
69. He GW, Lin L, DeMartino J, Zheng X, […] Holstege F, Clevers H. Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation. Cell Stem Cell 29:1333-1345 (2022)
70. Hendriks D, Brouwers JF, […] Artegiani B, Clevers H. Engineered human hepatocyte organoids enable CRISPR-based target discovery and drug screening for steatosis.
    Biotechnology 41(11): 1567-1581 (2023)
71. Dayton T, Alcala N, Moonen L, Den Hartigh L […] Fernández-Cuesta L, Clevers H. Druggable Growth Dependencies and Tumor Evolution Analysis in Patient-Derived Organoids of Neuroendocrine Neoplasms from Multiple Body Sites. Cancer Cell, 41(12):2083-2099.e9. (2023).
72. Lin L, DeMartino J, Wang D, Van Son JF,  […] Van Es J, Clevers, H. Unbiased transcription factor CRISPR screen identifies ZNF800 as master repressor of enteroendocrine differentiation. Science, 27, 382: 451-458 (2023)
73. Hendriks D, Pagliaro A, Andreatta F, Ma Z, […] Clevers H*, Artegiani B* (*: equal contribution). Human fetal brain self-organizes into long-term expanding organoids. Cell 178: 712-732 (2024)
74. Bannier-Hélaouët M, Korving J, Ma Z, Begthel H, […] Wu W, Clevers H.  Human conjunctiva organoids to study ocular surface homeostasis and diseases. Cell Stem Cell 31:227-243 (2024).
75. Huang L, Bernink JH, Giladi A, Krueger D […] Peters PJ, Clevers HTuft cells act as regenerative stem cells in the human intestine. Nature 634(8035):929-935 (2024)
76. Beumer, J, Geurts, MH, Geurts, V, Andersson-Rolf, A, Akkerman, A, […] Van Es, J, Clevers, H. Description and functional validation of human enteroendocrine cell sensors, Science 18;386(6719):341-348 (2024)
77. Mizutani T, Boretto M, Lim S, Drost J, […] van Boxtel R, Clevers H. Recapitulating the adenoma-carcinoma sequence by selection of four spontaneous oncogenic mutations in mismatch-repair-deficient human colon organoids, Nature Cancer (2024)
78. Andersson-Rolf, A, Groot, K, Korving, J, Begthel, H, […] van Es, J, Clevers, H. Long-term in vitro expansion of a human fetal pancreas stem cell that generates all three pancreatic cell lineages,
    Cell 187, 7394–7413 (2024)
79. Wang D, Spoelstra W, Lin L, Akkerman N, […] van Es J, Clevers H. Interferon-responsive intestinal BEST4/CA7+ cells are targets of bacterial diarrheal toxins. Cell Stem Cell 32, 1-15 (2025)
80. Krueger D, Spoelstra WK, Mastebroek JW, Rutger Kok, NU, […] Tans, SJ, Clevers, H. Epithelial tension controls intestinal cell extrusion. Science 389(6764): eadr8753 (2025).
81. Wang, D, Lim, S., … and Clevers, H. Human gut M cells resemble dendritic cells and present gluten antigen. Nature 650, pages251–260 (2026)
82. Clevers, H. Shifting Paradigms in Tissue Stem Cell Biology: Insights from the Intestine (review). Cell 189: 706-724 (2026).