Differential effects of 2- and 3-series E-prostaglandins on in vitro expansion of Lgr5 + colonic stem cells

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1 Carcinogenesis vol.35 no.3 pp , 2014 doi: /carcin/bgt412 Advance Access publication December 11, 2013 Differential effects of 2- and 3-series E-prostaglandins on in vitro expansion of Lgr5 + colonic stem cells Yang-Yi Fan 1,2, Laurie A.Davidson 1,2,3, Evelyn S.Callaway 1,2, Jennifer S.Goldsby 1,2 and Robert S.Chapkin 1,2,3,4, * 1 Program in Integrative Nutrition and Complex Diseases, 2 Department of Nutrition and Food Science, 3 Center for Translational Environmental Health Research and 4 Department of Biochemistry and Biophysics, Texas A&M University, College Station, TX 77843, USA *To whom correspondence should be addressed. Tel: ; Fax: ; r-chapkin@tamu.edu Arachidonic acid (20:4 5,8,11,14, AA)-derived prostaglandin E 2 (PGE 2 ) promotes colon cancer development. In contrast, chemoprotective n-3 polyunsaturated fatty acids supplant AA, thereby decreasing PGE 2 biosynthesis in colonocytes, with eicosapentaenoic acid (20:5 5,8,11,14,17, EPA) in particular being metabolized to a novel 3-series E-prostaglandin (PGE 3 ), a putative anti-tumorigenic-cyclooxygenase metabolite. Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5)-enhanced green fluorescent protein-internal ribosome entry site (IRES)-creER T2 knock-in mouse model to isolate and culture colonic organoids, in order to document ex vivo responses to exogenous PGE 2 and PGE 3. Colonic crypts were isolated from transgenic mice and cultured in a Matrigel-based three-dimensional platform. Organoids were treated with exogenous PGE 2, PGE 3 or dimethyl sulfoxide (vehicle control) for 5 days and the number of viable organoids was recorded daily. Subsequently, samples were processed for immunohistochemistry, flow cytometry and real-time PCR analyses. PGE 2 promoted optimal organoid growth and induced significantly higher levels of cell proliferation (P < 0.05) compared with PGE 3 and control. In contrast, the Lgr5-green fluorescent protein-positive stem cell number was uniquely elevated by >2-fold in PGE 2 -treated cultures compared with PGE 3 and control. This coincided with the upregulation of stem-cell-related Sox9, Axin2 and Cd44 messenger RNAs. Our results demonstrate that relative to AA-derived PGE 2, a known promoter of colon tumorigenesis, EPA-derived PGE 3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids. Introduction The epithelial layer of the human colon is made up of a single sheet of columnar epithelial cells, which form finger-like invaginations into the underlying connective tissue of the lamina propria to form the basic functional unit of the intestine, the crypt. Intestinal crypts house tissue-specific, multipotential stem cells, which are located in the niche at the base of the intestinal crypt and are capable of regenerating all intestinal cell types. However, it has been a challenge to isolate and culture these cells, or induce them to differentiate and form structures that are similar to those observed in vivo. Recently, Sato et al. (1,2) and Ootani et al. (3) have successfully cultured intestinal crypts without a mesenchymal niche in vitro. This unique three-dimensional culture system allows the crypt expansion to create organoids, comprising Abbreviations: AA, arachidonic acid; DMSO, dimethyl sulfoxide; EdU, 5-ethynyl-2ʹ-deoxyuridine; EGFP, enhanced green fluorescent protein; EPA, eicosapentaenoic acid; GFP, green fluorescent protein; Lgr5, leucine-richrepeat-containing G-protein-coupled receptor 5; PBS, phosphate-buffered saline; PGE, prostaglandin E; PUFA, n-3 polyunsaturated fatty acids; SE, standard error. multiple crypt-like domains surrounding a central lumen lined by a villus-like epithelium, similar to the basic crypt villus physiology in vivo. Recent advances have been made in the identification of stem cells (approximately 6 14 per crypt), which replenish the intestinal epithelium every 3 5 days (4). It has been demonstrated that Lgr5 (leucine-rich-repeat-containing G-protein-coupled receptor 5, also known as Gpr49), a receptor for R-spondins (5), marks a long-lived pool of cycling stem cells in the small intestine and colon (4,6 9). The distribution of Lgr5 + cells within stem-cell-derived adenomas indicates that a stem cell/progenitor cell hierarchy is maintained in early neoplastic lesions. By crossing stem-cell-specific Lgr5-EGFP- IRES-creER T2 knock-in mice to Apc flox/flox mice, it was unequivocally shown that crypt stem cells are the cells of origin of intestinal cancer (7). With respect to irrefutable stem cell characterization, a single Lgr5 + intestinal stem cell can generate a continuously expanding, self-organizing epithelial structure reminiscent of normal gut (8). In addition, it has been demonstrated that intestinal crypt homeostasis results from neutral competition between symmetrically dividing Lgr5 + stem cells (10). This highly relevant assertion is supported by a recent publication indicating that chemoprevention of colon cancer by non-steroidal anti-inflammatory drugs is the result of elimination of Lgr5 + stem cells that are inappropriately activated by oncogenic events via the induction of apoptosis (11). Collectively, these data provide cogent evidence indicating that Lgr5 + is a marker of adult intestinal stem cells and that they are clearly the cells of origin for colon tumors. With respect to disease prevention, the long-chain n-3 polyunsaturated fatty acids (PUFA) present in dietary fish oil, e.g. eicosapentaenoic acid (EPA) and docosahexaenoic acid, affect diverse physiological processes in the colon, including eicosanoid [prostaglandin (PGE)] signaling (12,13). This is significant because a predominant product of cyclooxygenase activity in the intestine, PGE 2, promotes colon tumorigenesis, in part by activating Wnt signaling (14 18). One major consequence of fish oil feeding is the decreased arachidonic acid (AA, 20:4 5,8,11,14 ) to EPA (20:5 5,8,11,14,17 ) ratio in colonic mucosa (19,20), which increases production of the novel PGE 3 relative to PGE 2 (21,22). In contrast to PGE 2, PGE 3 appears to exhibit attenuated bioactivity (12,23). This difference could underpin the ability of n-3 PUFA to suppress colon tumorigenesis. Therefore, we further characterized the putative chemoprotective properties of PGE 3 with respect to directed differentiation of pluripotent stem cells in epithelial organoids isolated from mouse colon. Materials and methods Animals Lgr5-EGFP-IRES-creER T2 transgenic mice were originally generated by H.Clevers at the Hubrecht Institute University Medical Center, Utrecht, The Netherlands (6). A total of 10 male (8 13 weeks of age) and 10 female mice (9 14 weeks of age) were randomized equally to the different experimental groups in all experiments to minimize the potential effect of gender and age on colonic stem cells. All procedures adhered to the guidelines approved by Public Health Service and the Institutional Animal Care and Use Committee at Texas A&M University. Animals were fed a commercial 10% safflower oil diet (Research Diets, New Brunswick, NJ) for at least 2 weeks prior to the experiments. Colonic crypt isolation and three-dimensional culture Colonic crypts were isolated using the method of Sato et al. (1) with modification. Briefly, intact colons were everted on a disposable mouse gavage needle (Instech Laboratories, Plymouth Meeting, PA) and incubated with 20 mm ethylenediaminetetraacetic acid in Ca/Mg-free Hanks balanced salt solution at 37 C for 30 min. Colonic crypts were released by mechanical disruption (vigorous vortexing) and purified by a series of phosphate-buffered saline (PBS) The Author Published by Oxford University Press. All rights reserved. For Permissions, please journals.permissions@oup.com 606

2 PGE2 promotes in vitro expansion of colonic stem cells washes and centrifugation steps as described previously (24). Purified crypts were kept on ice and resuspended in cold growth factor reduced, phenol-redfree Matrigel (BD Biosciences, San Jose, CA) at a density of 15 crypts/µl. Crypts from the same animal were mixed well, evenly distributed to multiple wells for different PGE incubations to ensure the equal distribution of Lgr5+ cells among the treatment groups. A total of 750 crypts/50 µl Matrigel were plated onto the center of wells in a 24-well plate and incubated at 37 C for 5 10 min. After polymerization, 500 µl of complete medium containing advanced Dulbecco s modified Eagle s medium/f12 (ADF; Life Technologies, Grand Island, NY), epidermal growth factor (50 ng/ml; Life Technologies), Noggin (100 ng/ml; Peprotech, Rocky Hill, NJ), R-Spondin (500 ng/ml; Sino Biological, Beijing, China), N2 supplement (1 ; Invitrogen), B27 supplement (1X; Life Technologies), N-acetylcysteine (1 µm; Sigma, St Louis, MO) and Wnt-conditioned medium as described previously by Barker et al. (25) (1:1 dilution) was added. The culture was then incubated at 37 C. Exogenous PGE2, PGE3 (Cayman Chemical, Ann Arbor, MI) and dimethyl sulfoxide (DMSO; control vehicle) were added to cultures. Live organoid evaluation The growth of organoids in three-dimensional culture before and after exogenous PGE treatment was evaluated by directly enumerating live organoids in culture wells on a daily basis. Twenty-four hours after initial plating, all live organoids were counted in the wells. Organoid viability was assessed under bright field imaging, with live organoids exhibiting a translucent halo. In contrast, non-viable organoids appeared opaque in nature. Organoid harvesting After 5 days in culture, Matrigel embedded colonic organoid cultures were washed with cold PBS, followed by addition of ADF medium to the well. Organoids were released by mechanical pipetting. The dissociated organoids were transferred to a pre-chilled 15 ml conical tube, followed by cold PBS washes and centrifugation to remove residual Matrigel and proteins from the medium. For samples used for immunohistochemistry analysis, organoids were pelleted and fixed in 4% paraformaldehyde at room temperature for 10 min, followed by dilution in 0.1 M glycine in PBS to terminate the fixation process. Samples were centrifuged and washed once with PBS, followed by a series of ethanol dehydration washes, then embedded in paraffin. For reverse transcription PCR analyses, organoid pellets were resuspended in lysis buffer in order to isolate RNA using an RNAqueous 4-PCR kit (Ambion, Austin, TX) as per the manufacturer s protocol. Immunohistochemistry Colonic cell proliferation in organoids was measured using the Click-IT EdU kit (Life Technologies). Organoid cultures were pulsed with 5-ethynyl2ʹ-deoxyuridine (EdU, 10 µm) 4 h prior to harvesting. Apoptosis was assessed with a terminal deoxynucleotidyl transferase labeling kit (Trevigen, Gaithersburg, MD) as described previously (26,27). Goblet cells were detected based on mucin enrichment (28). Mucin staining was obtained by sequential incubation with 0.5% periodic acid (Sigma), 50% Schiff s reagent (Sigma) and Mayer s hematoxylin (Sigma). Sections were then dehydrated, cleared and mounted in Permount mounting medium (Sigma). Neuroendocrine cells were detected based on chromogranin A expression (29). Briefly, antigen retrieval was performed by sub-boiling in 10 mm Tris base/1 mm ethylenediaminetetraacetic acid (ph 9.0), after which goat anti-chromogranin A (Santa Cruz) primary antibody was applied at 1:400 overnight at 4 C. An Alexa-488 rabbit anti-goat secondary antibody (Life Technologies) was used at a dilution of 1:1000 at room temperature for 1 h. For enumeration of immunohistochemical staining, the average number of positive cells from a minimum of 35 organoids (typically >70) was assessed from three experiments per treatment. Flow cytometry In select studies, after organoids were harvested from Matrigel, samples were further incubated with trypsin/dnase to produce a single cell suspension for flow cytometry of green fluorescent protein (GFP)-positive cells using an Accuri C6 flow cytometer (BD Biosciences) as described previously (24). Fig. 1. Dose-dependent growth of colonic organoid cultures. Lgr5-EGFP-IRES-creERT2 mouse colonic crypts were isolated and cultured in Matrigel containing advanced Dulbecco s modified Eagle s/f12 medium, supplemented with epidermal growth factor, Noggin, R-Spondin, N2, B27 supplements, N-acetylcysteine and Wnt-conditioned medium. Exogenous PGE2, PGE3 and DMSO (control vehicle) were added the following day (Day 0). Fresh complete media containing 0.1, 1 or 10 µm of PGE2, PGE3 or DMSO was changed daily. (A) Representative micrograph of isolated colonic crypts. Note Lgr5-GFP-positive stem cells at the base of the crypts. (B) Representative Day 5 colonic organoids. (C) All live organoids (~35 50 organoids per well) were counted daily. Data are presented as the percentage of live organoids per well at Day 0 for PGE2- and PGE3-treated groups, mean ± standard error (SE), n = 3 separate experiments per treatment ( 10 magnification, bar represents 50 µm). 607

3 Y-Y.Fan et al. Real-time PCR Isolated organoid RNA was reverse transcribed to complementary DNA using random hexamers and oligo(deoxythymidine) primers with Superscript II RT (Life Technologies). Gene expression was quantified by predeveloped TaqMan assays using an ABI 7900HT (Applied Biosystems). Gene expression was quantified for the following genes: Lgr5, Axin2, Sox9, Cd44, EphB3, β-catenin, Hopx and 18S (for normalization). Statistics Statistical analyses were carried out using the SuperANOVA software. Data were analyzed using the one-way analysis of variance and the results from treatments showing significant overall changes were subjected to post-hoc Tukey s test with significance for P < Results PGE2 maintains primary Lgr5 colonic organoid culture viability and rates of cell proliferation In initial studies, the effects of PGE2/PGE3 dose (0 10 µm) and duration (1 5 days) on crypt viability were evaluated (Figure 1). Crypt viability was optimally maintained using 10 µm of PGE2 after 5 days. This concentration was consistent with previously reported primary culture studies (30,31) and comparable with human rectal mucosa cellular levels (4 120 µm) (32,33). Therefore, the remaining studies utilized complete media containing 10 µm of PGE2, PGE3 or DMSO changed out every other day. After 5 days in culture, ~2% of the total collected cells from DMSO-treated colonic organoids were classified as GFP-positive stem cells. This compares favorably to freshly isolated colonic crypts, where ~2% of crypt cells were GFP-positive. Representative colonic crypts are shown at plating and 5 days in culture (Figure 1A and B). Without PGE supplementation, the growth and viability of organoids decreased by 40% after 1 day in culture (Figure 1C). PGE2 dose-dependently enhanced the growth of organoids, with a dose of 10 µm exhibiting the greatest effect compared with PGE3 and vehicle control groups over the 5 day culture period (Figure 1C). Subsequently, PGE2 (10 µm) supplementation for 5 days significantly (P < 0.05) increased (by 28%) the percentage of proliferative cells in organoid culture compared with the control group (Figure 2). Although PGE3 also increased organoid cell proliferation, the effect was moderated. Interestingly, both PGE2 and PGE3 had a nominal effect on apoptosis relative to the control group (Figure 2). Exogenous PGE2 modulates intestinal cell differentiation The percentage of goblet cells in colonic crypts was increased by 37% after 5 days with PGE2 treatment compared with control (Figure 2). In comparison, the effect of PGE3 was slightly moderated. Specifically, relative to control, the percentage of neuroendocrine cells was decreased by 41% in PGE2-treated cultures and by 36% in the PGE3 treatment group (Figure 2). PGE2 promotes GFP-positive stem cell expansion Following PGE treatment for 5 days, GFP-positive stem cells from isolated organoid cultures were quantified by flow cytometry. Interestingly, the percentage of stem cells was significantly elevated (~180%) by PGE2 treatment relative to control and PGE3-treated organoids (Figure 3). Because the effect of n-3 PUFA and its metabolites on mediators of the stem cell niche, e.g. Wnt signature, remain largely unexplored, we assessed Wnt-signaling-related gene expression following PGE treatment. Consistent with effects on stem cell numbers in harvested organoids, messenger RNA levels of stem cell markers (Lgr5, Hopx, Sox9, EphB3 and Cd44) and Wnt signaling (Axin2 and β-catenin) were maximally increased by PGE2 (Figure 4). Discussion Monoclonal intestinal crypts are maintained by rapidly cycling crypt base columnar Lgr5+ stem cells (34). These stem cells are capable of giving rise to all cell lineages resulting in the establishment of a cellular hierarchy in tissues and in tumors (1,4). Emerging evidence indicates that both quiescent +4 cells and crypt base columnar cells have cooperative functional roles, exhibiting bi-directional lineage relationships in the small intestine and to some degree in the colon Fig. 2. Effect of PGEs on colonic cell proliferation, apoptosis and differentiation in organoids. Colonic crypts were isolated from Lgr5-EGFP-IRES-creERT2 mice and cultured in Matrigel with enriched medium. Organoids were treated with 10 µm of PGE2, PGE3 or control vehicle (DMSO) for 5 days. Subsequently, organoids were harvested, fixed in 4% PFA and paraffin embedded. Levels of cell proliferation and apoptosis in organoids were measured by the EdU Click-It and terminal deoxynucleotidyl transferase assays, respectively. In addition, goblet cells were detected with periodic acid-schiff stain and neuroendocrine cells were measured by immunohistochemistry. (A) Representative micrographs for EdU Alexa-647-stained (red) proliferative cells ( 10); terminal deoxynucleotidyl transferase diaminobenzidine- (brown) stained apoptotic cells ( 10); periodic acid-schiff-stained (magenta) goblet cells ( 20) and chromogranin A Alexa488-stained (green) neuroendocrine cells ( 10). (B) Data are expressed as percentage of EdU-labeled, apoptotic, goblet or neuroendocrine cells relative to the total number of cells per organoid. (mean ± SE, n = ). Treatments with different letters are significantly different (P < 0.05). 608

4 PGE 2 promotes in vitro expansion of colonic stem cells Fig. 3. Percentage of Lgr5-GFP-positive stem cells in organoids following PGE treatment. Organoids were treated with 10 µm of PGE 2, PGE 3 or control vehicle (DMSO) for 5 days. On Day 5, organoids were harvested, single cells prepared and processed by flow cytometry to quantify the percentage of Lgr5-GFP-positive stem cells in the cell population. (A) Representative flow cytometry profiles from wild-type mice and the gate setting for GFP-positive cell population. (B) Representative flow cytometry profiles from DMSO- (control), PGE 2 - and PGE 3 -treated Lgr5-EGFP-IRES-creER T2 mice. (C) Data are expressed as percentage of GFP-positive stem cells (mean ± SE, n = 9). Treatments with different letters are significantly different (P < 0.05). See Figure 2 for legend details. (7,11,24,25,35 37). Hence, it is now possible for the first time to visualize stem cells and examine their behavior in the context of tissue remodeling and cancer chemoprevention. Using a recently described three-dimensional culture system (1), we were able to monitor the growth of mouse colonic crypts, stem cell self-renewal and differentiation in vitro. This in vitro organoid culture model fully recapitulates the Wnt- and Notch-dependent intestinal stem cell niche (3). It is well known that diet plays an important role in colon cancer development (38,39). The high incidence of colon cancer in Westernized countries has been partly attributed to the Western diet, which contains a very high n-6 to n-3 PUFA ratio. We have previously demonstrated that mice fed an n-6-pufa-enriched diet had significantly higher levels of PGE 2 relative to PGE 3 in colonic mucosa, compared with mice fed an n-3-pufa-enriched diet (22). These findings were corroborated in fat-1 transgenic mice, which synthesize long-chain n-3 PUFA de novo (21). Because transformation of adult stem cells is an extremely important route toward initiating intestinal cancer, we utilized the Lgr5-EGFP-IRES-creER T2 knock-in mouse model to isolate and culture adult intestinal stem cells, in order to document ex vivo responses to exogenous PGE 2 and PGE 3. Our findings indicate that exogenous pro-tumorigenic AA-derived PGE 2 promotes mouse colonic organoid viability, cell proliferation and Lgr5 + stem cell expansion. The ability of PGE 2 to support epithelial stem cell expansion in sphere-like organoids is consistent with reports 609

5 Y-Y.Fan et al. Fig. 4. Wnt-signaling-related gene expression levels in organoid cultures. Organoids were treated with 10 µm of PGE 2, PGE 3 or control vehicle (DMSO) for 5 days. On Day 5, RNA was extracted and the expression level of selected genes was measured by quantitative reverse transcription PCR. Data are expressed as percentage of control group (mean ± SE, n = 8 11 samples per treatment). Treatments with different letters are significantly different (P < 0.05). 610

6 PGE 2 promotes in vitro expansion of colonic stem cells in other model systems (30,31,40). In contrast, the novel PGE 3, a putative chemoprotective-epa-derived metabolite, had little or no effect on organoid viability or stem cell number. Because colonic stem cells differentiate into enterocytes (absorbing water and nutrients), goblet cells (secreting mucus) and neuroendocrine cells (secreting hormones), renewing the colonic epithelium every 4 5 days, we also compared the effects of PGE 2 and PGE3 on cell-lineage-directed differentiation. Our data show that PGE 2 increased the percentage of goblet cells and decreased the percentage of neuroendocrine cells in colonic organoids after 5 days in culture, whereas PGE 3 only decreased neuroendocrine cell percentage. Interestingly, with respect to lineage plasticity, it has been reported that Bak-Null mice exhibit a similar phenotype, exhibiting colonic goblet cell hyperplasia and endocrine cell hypoplasia, and have higher number of mitotic cells in colonic crypts (41). This suggests that PGE 2 can modulate stem cell differentiation ex vivo and promote a proliferative phenotype, which is consistent with in vivo pro-tumorigenesis properties of this AA-derived eicosanoid. Lgr5 serves as a receptor for R-spondins and is a member of the Wnt signaling pathway (5,34). The Wnt family of secreted proteins is critical for cell fate specification in adult stem cell niches. For example, Hirata et al. (42) have documented the dose-dependent effects of canonical Wnt signaling in de novo crypt formation and cell cycle regulation in the colonic epithelium. These data complement earlier findings in which manipulation of Wnt signaling was shown to influence the self-renewal process of the intestinal epithelium, including cell proliferation (34,43,44). With respect to targeting Wnt signaling in colon cancer, AA-derived PGE 2 has been shown to promote colon tumorigenesis, in part by activating Wnt signaling where it may promote stem cell function (14 18). Mechanistically, PGE 2 acts to prevent β-catenin degradation by inhibiting both GSK-3β and Axin2 function, thereby activating Wnt signaling (30). For these reasons, we further examined the effect of E-series PGE on Wnt-targeted stem/progenitor cell marker genes in the context of in vitro expansion of organoids. With regard to genes controlling intestinal cell fate, Al-Kharusi et al. (45) recently demonstrated that PGE 2 promotes adenoma cell survival, in part, by increasing the expression of Lgr5. These data are consistent with findings in our study, where we demonstrate that PGE 2 promoted Lgr5 expression and stem cell number in mouse colonic organoids in the absence of a mesenchymal niche. Additionally, PGE 2 as compared with control, significantly elevated the expression of Sox9, a Wntactivated progenitor/stem cell marker (46); β-catenin, an intracellular signal transducer in the Wnt signaling pathway (47); Axin2, which regulates stability of β-catenin (48); EphB3, a protein tyrosine kinase capable of regulating cell sorting in the mature epithelium (49); Hopx, a putative slow cycling stem cell marker (50) and Cd44, a cell adhesion molecule and cancer stem cell surface marker (51). These results indicate the important role of PGE 2 in upregulating stem-cell-related, Wnt-signaling-dependent, gene expression. In contrast, EPA-derived PGE 3 only exhibited a moderate or had no effect on genes related to Wnt signaling and stemness. Overall, our observations are consistent with chemoprevention studies, where enhanced PGE 2 substrate levels were associated with the upregulation of several Wnt-targeted genes in colonic stem cells (24). The consonant gene expression profiles in our current in vitro study and previous in vivo studies further support the utility of the three-dimensional colonic organoid culture model in studying the impact of diet on colonic stem cell biology. Importantly, although the changes in gene expression were modest (but statistically significant), the fact that downstream signaling amplification of gene regulation would impact multiple pathways, suggest that the effects of PGE 2 /PGE 3 on Wnt signaling are biologically relevant. The fact that single Lgr5 stem cells can build crypt structures in vitro without a mesenchymal niche is a tremendous advantage for probing the effect of putative non-epithelial-derived mediators. With respect to the in vitro modulation of PGE 2 and PGE 3 on colonic organoid stem cell numbers, the presented organoid data are consistent with our in vivo dietary (n-6 versus n-3 PUFA) observations with respect to the modulation of genes, which regulate colonic crypt stem cell numbers (24). These initial findings suggest that the organoid system has utility for screening chemopreventative agents, specifically with respect to the regulation of intestinal stem cell self-renewal and differentiation. In conclusion, we provide evidence that chemoprotective EPAderived PGE 3 has diminished ability to support colonic stem cell expansion in mouse colonic organoids as compared with AA-derived PGE 2. This in part is consistent with a reduced ability of PGE 3 to upregulate messenger RNA expression profiles of stem cell mediators. Although we did not directly determine whether PGE 3 is a competitive inhibitor of PGE 2, it is well known that n-3 fatty acids competitively inhibit AA, thereby reducing PGE 2 levels (21,34). Future studies will focus on the combinatorial effects of PGE 2 /PGE 3 on stem cell biology. We propose that the enablement of primary intestinal organoid culture will have widespread application for elucidating the molecular mechanisms of nutrient action on gut biology. Funding National Institutes of Health (CA and CA129444); Cancer Prevention and Research Institute of Texas (RP100473). Acknowledgements The authors would like to thank T.Hou for formatting assistance. Conflict of Interest Statement: None declared. References 1. Sato,T. et al. (2009) Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature, 459, Sato,T. et al. (2013) Primary mouse small intestinal epithelial cell cultures. Methods Mol. Biol., 945, Ootani,A. et al. 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