Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis
2013; Springer Nature; Volume: 32; Issue: 20 Linguagem: Inglês
10.1038/emboj.2013.204
ISSN1460-2075
AutoresMeritxell Huch, Paola Bonfanti, Sylvia F. Boj, Toshiro Sato, Cindy J.M. Loomans, Marc van de Wetering, Mozhdeh Sojoodi, Vivian Li, Jurian Schuijers, Ana Gudelj Gračanin, Femke Ringnalda, Harry Begthel, Karien M. Hamer, Jan Mulder, Johan H. van Es, Eelco J.P. de Koning, Robert G. Vries, Harry Heimberg, Hans Clevers,
Tópico(s)Pancreatic function and diabetes
ResumoArticle17 September 2013Open Access Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis Meritxell Huch Meritxell Huch Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Paola Bonfanti Paola Bonfanti Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Search for more papers by this author Sylvia F Boj Sylvia F Boj Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Toshiro Sato Toshiro Sato Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The NetherlandsPresent address: Department of Gastroenterology, School of Medicine, Keio University, 35 Shinanomachi, Shinnjukuku, Tokyo 160-8582, Japan Search for more papers by this author Cindy J M Loomans Cindy J M Loomans Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Search for more papers by this author Marc van de Wetering Marc van de Wetering Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Mozhdeh Sojoodi Mozhdeh Sojoodi Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Search for more papers by this author Vivian S W Li Vivian S W Li Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The NetherlandsPresent address: Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Search for more papers by this author Jurian Schuijers Jurian Schuijers Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Ana Gracanin Ana Gracanin Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Femke Ringnalda Femke Ringnalda Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Search for more papers by this author Harry Begthel Harry Begthel Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Karien Hamer Karien Hamer Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Joyce Mulder Joyce Mulder Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Johan H van Es Johan H van Es Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Eelco de Koning Eelco de Koning Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Search for more papers by this author Robert G J Vries Robert G J Vries Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Harry Heimberg Corresponding Author Harry Heimberg Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Search for more papers by this author Hans Clevers Corresponding Author Hans Clevers Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Meritxell Huch Meritxell Huch Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Paola Bonfanti Paola Bonfanti Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Search for more papers by this author Sylvia F Boj Sylvia F Boj Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Toshiro Sato Toshiro Sato Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The NetherlandsPresent address: Department of Gastroenterology, School of Medicine, Keio University, 35 Shinanomachi, Shinnjukuku, Tokyo 160-8582, Japan Search for more papers by this author Cindy J M Loomans Cindy J M Loomans Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Search for more papers by this author Marc van de Wetering Marc van de Wetering Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Mozhdeh Sojoodi Mozhdeh Sojoodi Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Search for more papers by this author Vivian S W Li Vivian S W Li Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The NetherlandsPresent address: Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK Search for more papers by this author Jurian Schuijers Jurian Schuijers Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Ana Gracanin Ana Gracanin Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Femke Ringnalda Femke Ringnalda Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Search for more papers by this author Harry Begthel Harry Begthel Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Karien Hamer Karien Hamer Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Joyce Mulder Joyce Mulder Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Johan H van Es Johan H van Es Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Eelco de Koning Eelco de Koning Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands Search for more papers by this author Robert G J Vries Robert G J Vries Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Harry Heimberg Corresponding Author Harry Heimberg Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium Search for more papers by this author Hans Clevers Corresponding Author Hans Clevers Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands Search for more papers by this author Author Information Meritxell Huch1,‡, Paola Bonfanti2,‡, Sylvia F Boj1,‡, Toshiro Sato1,‡, Cindy J M Loomans1,3, Marc van de Wetering1, Mozhdeh Sojoodi2, Vivian S W Li1, Jurian Schuijers1, Ana Gracanin1, Femke Ringnalda1,3, Harry Begthel1, Karien Hamer1, Joyce Mulder1, Johan H van Es1, Eelco de Koning1,3, Robert G J Vries1, Harry Heimberg 2,‡ and Hans Clevers 1,‡ 1Hubrecht Institute for Developmental Biology and Stem Cell Research, University Medical Centre Utrecht, Utrecht, The Netherlands 2Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium 3Department of Nephrology, Leiden University Medical Center, Albinusdreef 2, Leiden, The Netherlands ‡These authors contributed equally to this work. *Corresponding authors. Hubrecht Institute for Developmental Biology and Stem Cell Research, Uppsalalaan 8, 3584CT Utrecht & University Medical Centre Utrecht, Utrecht, The Netherlands. Tel.:+31 30 212 1800; Fax:+31 30 251 6464; E-mail: [email protected] Research Center, Vrije Universiteit Brussel, Laarbeeklaan 103, D2, B1090 Brussel, Belgium. Tel.:+3224774477; Fax:+3224774472; E-mail: [email protected] The EMBO Journal (2013)32:2708-2721https://doi.org/10.1038/emboj.2013.204 Present address: Department of Gastroenterology, School of Medicine, Keio University, 35 Shinanomachi, Shinnjukuku, Tokyo 160-8582, Japan PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality. Introduction As first demonstrated for intestinal crypts (Korinek et al, 1998), Wnt signalling plays a crucial role in the regulation of multiple types of adult stem cells and progenitors (Clevers and Nusse, 2012). The Wnt target gene Lgr5 marks actively dividing stem cells in Wnt-driven, continuously self-renewing tissues such as small intestine and colon (Barker et al, 2007), stomach (Barker et al, 2010) and hair follicles (Jaks et al, 2008). However, expression of Lgr5 is not observed in endodermal organs with a low rate of spontaneous self-renewal, such as liver or pancreas. In the liver, we have recently described that Wnt signalling is highly activated during the regenerative response following liver damage. Lgr5 marks an injury-induced population of liver progenitor cells capable of regenerating the tissue after injury (Huch et al, 2013). In the adult pancreas, Wnt signalling is inactive (Pasca di Magliano et al, 2007), yet it is essential for its development during embryogenesis (Murtaugh et al, 2005; Heiser et al, 2006). The embryonic pancreas harbours multipotent progenitor cells that can give rise to all pancreatic lineages (acinar, duct and endocrine) (Zaret and Grompe, 2008). Injury to the pancreas can reactivate the formation of new pancreatic islets, called islet neogenesis, by mechanisms still not entirely understood but that resemble development of the embryonic pancreas (Bouwens, 1998; Gu et al, 2003). Lineage tracing studies have demonstrated that these ‘de novo beta cells’ can be derived from pre-existing beta cells (Dor et al, 2004), or by conversion of alpha cells, after almost 90% beta-cell ablation (Thorel et al, 2010). Also, severe damage to the pancreas, by means of partial duct ligation (PDL) or acinar ablation, can stimulate non-endocrine precursors, such as duct cells, to proliferate and differentiate towards acinar (Criscimanna et al, 2011; Furuyama et al, 2011), duct (Criscimanna et al, 2011; Furuyama et al, 2011; Kopp et al, 2011) and also endocrine lineages (including beta cells) (Xu et al, 2008; Criscimanna et al, 2011; Pan et al, 2013; Van de Casteele et al, 2013), suggesting the existence of a pancreas progenitor pool within the ductal tree of the adult pancreas. The development of a primary culture system based on the adult, non-transformed progenitor pancreas cells would represent an essential step in the study of the relationships between pancreas progenitor cells, their descendants and the signals required to instruct them into a particular lineage fate. Also, the production of an unlimited supply of adult pancreas cells would facilitate the development of efficient cell replacement therapies. Most of the available pancreas adult stem cell-based culture protocols yield cell populations that undergo senescence over time unless the cells become transformed. It is fair to say that no robust, long-term culture system exists today that is capable of maintaining potent, clonal expansion of adult non-transformed pancreas progenitors over long periods of time under defined conditions. Recently, endoderm progenitors derived from embryonic stem cells (ESCs) (Cheng et al, 2012; Sneddon et al, 2012) or induced pluriportent stem cells (iPSCs) (Cheng et al, 2012) were serially expanded, in co-culture with pancreas mesenchyme or MEFs, respectively, and gave rise to glucose-responsive beta cells in vitro (Cheng et al, 2012) and glucose-sensing and insulin-secreting cells, when transplanted, in vivo (Sneddon et al, 2012). We have recently described a 3D culture system that allows long-term expansion of adult small intestine, stomach and liver cells without the need of a mesenchymal niche, while preserving the characteristics of the original adult epithelium (Sato et al, 2009; Barker et al, 2010; Huch et al, 2013). A crucial component of this culture medium is the Wnt agonist RSPO1 (Kim et al, 2005; Blaydon et al, 2006), the recently reported ligand of Lgr5 and its homologues (Carmon et al, 2011; de Lau et al, 2011). Here, we describe that Wnt signalling and Lgr5 are strongly upregulated in remodelling duct-like structures upon injury by PDL. We exploit the Wnt-Lgr5-Rspo signalling axis to generate culture conditions that allow long-term expansion of adult pancreatic duct cells, which maintain the ability to differentiate towards both duct and endocrine lineages when provided the proper signals. Results Wnt signalling and Lgr5 expression are upregulated during pancreas regeneration following PDL We first sought to document Wnt pathway activation in normal adult pancreas and following acute damage. We used the Axin2-LacZ allele as a general reporter for Wnt signalling (Leung et al, 2002; Lustig et al, 2002; Yu et al, 2005). In the head of a pancreas injured by PDL, where there is still healthy tissue, the reporter was inactive (Figure 1A), in agreement with the previous observations made with the TOPGAL Wnt reporter mice (DasGupta and Fuchs, 1999; Pasca di Magliano et al, 2007). However, after controlled injury by PDL (Watanabe et al, 1995; Xu et al, 2008), the Axin2LacZ reporter was highly activated along the ductal tree of the ligated part of the pancreas (Figure 1B). Axin2 activation in the pancreas was already detectable at day 3 post injury, as assessed by qPCR (Figure 1C). Co-labelling with duct (pancytokeratin, CK) and endocrine (insulin, INS) markers revealed that the Axin2 upregulation was restricted to the duct compartment (Figure 1D). Thus, pancreas injury by PDL led to activation of Wnt target genes in the proliferative duct cell compartment (Scoggins et al, 2000) during the regenerative response. Figure 1.Induction of Axin2 and Lgr5 expression upon damage on adult pancreas. (A, B) Axin2-LacZ induction in newly formed pancreatic ducts upon PDL. Axin2-LacZ mice (n=6) underwent PDL as explained in Materials and methods. Mice were sacrificed at the indicated time points and the non-ligated pancreatic tissue (Head-PDL) was separated from the ligated part (Tail-PDL). (A) Head-PDL and (B) Tail-PDL portion 13 days after injury. Arrows indicate XGAL-specific staining exclusively detected in the pancreatic ducts of the ligated pancreas. Scale bars 200 μm (A, B, left panels) and 50 μm (A, B, right panels). (C) qPCR analysis of Axin2 and Lgr5 mRNA in adult pancreas following PDL. Results are represented as mean±s.e.m. of at least three independent experiments. The Hprt housekeeping gene was used to normalize for differences in RNA input. Non-parametric Mann–Whitney test was used. ***P 2 months (∼passage 8) (Supplementary Figure S2A). The cultures maintained exponential growth with cell doubling times essentially unchanged during the culturing period (Figure 2C). Using these culture conditions, we have been able to expand the cultures by passaging at a 1:4–1:5 ratio weekly for over 10 months (Figure 2B). These culture conditions allowed the recovery of the cells after freezing and thawing. Of note, when transplanted into immunocompromised mice, the cultures did give rise only to ductal structures, and no tumour formation was detected in any of the mice analysed (n=5), confirming the non-transformed origin of the cultured cells (Supplementary Figure S2C and D). Also, the karyotype analysis revealed that chromosome numbers were essentially normal, even after >5 months in culture (Supplementary Figure S2B). Figure 2.Establishment of the pancreas organoids from adult pancreatic ducts. (A) Scheme representing the isolation method of the pancreatic ducts and the establishment of the pancreatic organoid culture. The pancreatic ducts were isolated from adult mouse pancreas after digestion, handpicked manually and embedded in matrigel. Twenty-four hours after, the pancreatic ducts closed and generated cystic structures. After several days in culture, the cystic structures started folding and budding. (B) Representative serial DIC images of a pancreatic organoid culture growing at the indicated time points. Magnifications: × 10 (days 0, 2, 4, 6, and 8) and × 4 (day 10 onwards). (C) Growth curves of pancreas cultures originated from isolated pancreatic ducts cultured as described in Materials and methods. Note that the cultures followed an exponential growth curve within each time window analysed. Graphs illustrate the number of cells counted per well at each passage from passages P1–P3 (left), P5–P7 (middle) and P10–P12 (right). The doubling time (hours) is indicated in each graph. Data represent mean±s.e.m., n=2. (D) Representative DIC images of XGAL staining in WT (left), Axin2-LacZ (middle) and Lgr5-LacZ (right) derived pancreas organoids. Download figure Download PowerPoint Organoids generated from Axin2LacZ and Lgr5LacZ knock-in mice allowed localization of the Axin2- and Lgr5-positive cells. We observed XGAL staining in Axin2LacZ pancreas organoids throughout the cysts, whereas XGAL staining in the Lgr5LacZ-derived pancreas organoids was mainly restricted to small budding structures (Figure 2D). These results resembled the in vivo situation after pancreas injury by PDL, where only the ductal buds were Lgr5+, whereas the Axin2 reporter showed a broader expression pattern (compare Figure 1B versus Figure 1F). Prospectively isolated single pancreatic duct cells but not endocrine or acinar cells self-renew long term in vitro We then prospectively isolated the different pancreatic epithelial cells (duct, acinar and endocrine lineages) and cultured the different populations in our defined 3D culture system. A prospective isolation procedure that allows isolation of single cells of the different pancreatic epithelial cell types and maintenance of their viability in culture has not been established yet. The epithelial cell-surface marker EpCAM and the high concentration of Zn2+ in secretory granules of endocrine cells, that allows binding of the fluorescent chelator TSQ (6-methoxy-8-p-toluenesulfonamido-quilone), were used as a basis for cell isolation. Pancreas tissue from both WT or transgenic mice that constitutively and ubiquitously express eGFP (Okabe et al, 1997) was dissociated into single cells. After depletion of non-epithelial (EpCAM−) and haematopoietic cells (CD45+, CD31+), the cell suspension was FACS sorted in order to separate the granulated endocrine fraction (EpCAM+TSQ+) from the non-endocrine component (EpCAM+TSQ-) with high purity (>99.6%) (Figure 3A–D; Supplementary Figure S3A and B). To rule out the possibility that endocrine cells might de-granulate during the isolation procedure and thus contaminate the non-endocrine fraction, we repeated the protocol on pancreas cells obtained from mouse insulin promoter (Mip)-RFP mice and found no RFP+ cells in the non-endocrine fraction (Supplementary Figure S4A). The separated fractions were then tested for their ability to survive, proliferate and give rise to organoids under the above-defined conditions. Only the EpCAM+TSQ− exocrine cells were able to generate duct-like structures that gave rise to larger organoids (1–1.5% organoid formation efficiency) and had to be split once a week (Figure 3E). As expected, the growth pattern of the single sorted cells followed an exponential curve (Supplementary Figure S3D). The duct-derived cell cultures were maintained for >5 months (Figure 3E). The EpCAM+TSQ+ endocrine cells did not proliferate, but survived for at least 30 days in culture (Figure 3F). Figure 3.Isolation and in vitro expansion of single, endocrine-depleted pancreatic epithelial cells. (A) Representative fluorescence-activated cell sorting (FACS) plot illustrating the distribution of EpCAM+ and EpCAM− cells from dissociated adult mouse pancreas, following epithelial cell enrichment by magnetic beads as described in Materials and methods. (B) Representative FACS plot showing the distribution of EpCAM+ non-granulated TSQ− epithelial cells and EpCAM+ granulated TSQ+ endocrine cells. (C) EpCAM+TSQ+ and EpCAM+TSQ− sorted fractions were cytocentrifuged and immunostained (red) for Synaptophysin (SYP), Amylase (AMY) and pancytokeratin (CK); nuclei were counterstained with Hoechst 33342 (blue). Magnification: × 40. (D) Representative FACS analysis purity of sorted EpCAM+TSQ− cells indicating that this population is isolated with high purity (>99.6%). (E) EpCAM+TSQ−-sorted single cells were assessed for their growth potential in 3D expansion culture conditions: this population gave rise to organoids that could be expanded for many passages (>5 months). (F) EpCAM+TSQ+-sorted single cells were assessed for their growth potential under the same conditions: endocrine TSQ+ cells survived in culture but did not proliferate. Scale bars: 30 μm. Download figure Download PowerPoint Acino-ductal metaplasia can happen under conditions of stress or following injury (Means et al, 2005; Blaine et al, 2010). To confirm that duct rather than acinar cells are the long-term expanded cells isolated from the EpCAM+TSQ− fraction, we traced the progeny of isolated duct (Sox9+) or acinar (Ptf1a+) cells in vitro. Transgenic mice with a Ptf1aCreER allele, that is exclusively expressed in the acinar compartment (Kopp et al, 2012; Pan et al, 2013), or mice carrying the Sox9CreER allele, that is expressed predominantly (but is not absolutely restricted to) the duct cell compartment (Furuyama, et al, 2011; Kopp et al, 2012) were crossed with Rosa26RYFP mice and subcutaneously injected with tamoxifen as described in Supplementary Figure S5A. After the washout period, the pancreas was dissociated and single Sox9YFP+ or Ptf1aYFP+ cells were FACS sorted and cultured in our defined pancreas culture medium (Supplementary Figure S5B–D). Only Sox9YFP+ cells grew into budding organoids that expanded long term in culture, even when starting from a single cell (Supplementary Figure S5D, top panel). By contrast, the cultures derived from Ptf1aYFP+ cells gave rise to smaller duct-like structures that were able to proliferate only for 3–4 passages, after which they arrested proliferation (Supplementary Figure S5D, bottom panel). In conclusion, these data indicated that the long-term expanding pancreas organoid cultures derive from duct cells. Lgr5 cells sustain the growth of pancreas organoids that have a duct cell phenotype To test whether the Lgr5-expressing cells maintained the growth potential of the pancreas organoids, we sorted single Lgr5LacZ+ cells from in vitro expanded organoids derived from Lgr5-LacZ knock-in mice (Barker et al, 2007). Indeed, the isolated Lgr5+ cells grew and formed organoids (Figure 4A–E) that were subsequently expanded for >4 months in culture by splitting the cultures weekly at a 1:6–1:8 ratio. The colony formation efficiency was ∼16%, similar to the colony formation of Lgr5 cells of small intestine and stomach (Barker et al, 2010; Sato et al, 2011) (Figure 4C). Of note, 1.6% of the Lgr5neg-sorted population also grew into organoids (Figure 4C; Supplementary Figure S6A–D). These Lgr5neg-derived clones rapidly re-expressed Lgr5 (Supplementary
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