The Clevers group studies the molecular mechanisms of tissue development and cancer of various organs using organoids made from adult Lgr5 stem cells.

Tcf as Wnt effector

In 1991, we reported the cloning of a T cell specific transcription factor that we termed TCF1 (1). Related genes exist in genomes throughout the animal kingdom.We have shown in frogs (4), flies (7) and worms (11) that the TCF proteins constitute the effectors of the canonical Wnt pathway. Upon Wnt signaling, ß-catenin binds and activates nuclear TCFs by providing a trans-activation domain. For these studies, we designed the widely used pTOPFLASH Wnt reporters. In the absence of Wnt signaling, we found that Tcf factors associate with proteins of the Groucho family of transcriptional repressors to repress target gene transcription (9).

Wnt signaling in cancer

The tumor suppressor protein APC forms the core of a cytoplasmic complex which binds ß-catenin and targets it for degradation in the proteasome. In APC-deficient colon carcinoma cells, we demonstrated that ß-catenin accumulates and is constitutively complexed with the TCF family member TCF4, providing a molecular explanation for the initiation of colon cancer (5).

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 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, 21).

A GFP knock-in into the Lgr5 locus visualizes the stem cells of the small intestine of mice at the base of crypts (23)

Lgr5 as adult stem cell marker

Amongst the Wnt target genes, we found the Lgr5 gene to be unique in that it marks small cycling cells at crypt bottoms. These cells represent the epithelial stem cells of the small intestine and colon (23), the hair follicle (24), the stomach (28) and -probably- all other epithelial stem cell types of the mammalian body. They also represent the cells-of-origin of adenomas in the gut (25) and within adenomas Lgr5 stem cells act as adenoma stem cells (36). The related Lgr6 marks multipotent skin stem cells (29).

Lgr5 stem cell biology

Lgr5 crypt stem cells behave in unanticipated ways: Against common belief, they divide constantly. Stem cells numbers remain fixed because stem cells compete ‘neutrally’  for niche space. Thus, they do not divide asymmetrically (32), a phenomenon that was confirmed by in vivo imaging (43). Daughters of the small intestinal stem cells, the Paneth cells, serve as crypt niche cells by providing Wnt, Notch and EGF signals (30). The Wnt target gene encoding the transcription factor Achaete scute-like 2 controls the fate of the intestinal stem cell (26).

Lgr5 is the R-spondin receptor

Lgr5 resides in Wnt receptor complexes and mediates signaling of the R-spondin Wnt agonists (33), explaining the unique dependence of Lgr5 stem cells of various epithelia on 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. They serve in a negative feedback loop to control the size of the stem cell zone (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

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 mini-guts (27, 31), mini-stomachs (28), colon cancer organoids (31, 48) liver organoids (39, 46), prostate organoids (45), breast cancer organoids (53) and organoids representing multiple other adult tissues. 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 (46). The direct cloning of multiple individual cells from primary tumors allows molecular and functional analysis of tumor heterogeneity with an unprecedented resolution (54). Human organoids are readily amenable to CRISP-mediated genome modification to model for instance malignant transformation (49) and mutagenesis upon faulty DNA repair (52).

As proof-of-concept, the CFTR locus was repaired in single gut stem cells from two Cystic Fibrosis patients, using CRISPR/Cas9 technology in conjunction with homologous recombination. Repaired stem cells were clonally expanded into mini-guts and shown -in a swelling assay- to contain a functional CFTR channel (43). The organoid-based swelling assay has meanwhile become clinical practice in the Netherlands to identify patient with rare mutations that respond to the Vertex drugs. To this end, we founded the non-for-profit HUB foundation which currently builds a biobank of all 1500 Dutch CF patients funded by our national insurance companies. The HUB also maintains large biobanks of colon-, breast-, lung- and pancreas cancer organoids, accessible by academia and industry.

Key Publications

1)              van de Wetering, M., Oosterwegel, M., Dooijes, D., and Clevers, H.C. 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.C. An HMG box containing T-cell factor required for thymocyte differentiation.
Nature 374: 70-74 (1995)

3)              Schilham, M., Oosterwegel, M., Moerer, P., Jing Ya, de Boer, P., van de Wetering, M., Verbeek, S., 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. Review
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., 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., 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., 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., Clevers, H.C. 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., 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., Clevers, H., Wnt signalling in stem cells and cancer. Review.
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., 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., 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., 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., 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., 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., Clevers H.  Lgr6 marks stem cells in the hair follicle that generate all cell lineages of the skin.
Science 327: 1385-1389 (2010)

30)           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., Clevers, H. Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.
Nature 469: 415-418 (2011)

31)           Sato T, Stange DE, Ferrante M, Vries RG, Van Es JH, Van den Brink S, Van Houdt WJ, Pronk A, Van Gorp J, Siersema PD, Clevers H. Long-term expansion of epithelial organoids from human colon, adenoma, adenocarcinoma, and Barrett’s epithelium.
Gastroenterology 141: 1762-1772 (2011).

32)           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)

33)           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., Clevers, H. Lgr5 homologues associate with Wnt receptors and mediate R-spondin signalling.
Nature 476: 293-297 (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 Hans Clevers. 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., 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., 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., 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., Clevers, H. In vitro expansion of single Lgr5+ liver stem cells induced by Wnt-driven regeneration.
Nature 494: 247-250 (2013)

40)           Sato, T., 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., 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., 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., 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., Clevers, H.C. 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., Clevers, H. Long-term culture of genome-stable bipotent stem cells from adult human liver.
Cell 160: 299-312 (2015)

47)           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)

48)           van de Wetering, M., Francies, H.E., Francis, J.M., Bounova, G., Iorio, 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., Clevers, H. Prospective derivation of a ‘Living Organoid Biobank’ of colorectal cancer patients.
Cell 161: 933-945 (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., 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 O, Gogola E, Bounova G, Weeber F, Balgobind AV, Wind K, Gracanin A, Begthel H, Korving J, van Boxtel R, Duarte AA, Lelieveld D, van Hoeck A, Ernst RF, Blokzijl F, Nijman IJ, Hoogstraat M, van de Ven M, Egan DA, Zinzalla V, Moll J, Boj SF, Voest EE, Wessels L, van Diest PJ, Rottenberg S, Vries RGJ, Cuppen E, Clevers H. A Living Biobank of Breast Cancer Organoids Captures Disease Heterogeneity.
Cell 172:373-386 (2018).

54)           Roerink, S.F., Sasaki, N., …, Stratton, MR, and Clevers H. Intra-tumour diversification in colorectal cancer at the single-cell level.
Nature 556, 437-462 (2018).