R spondin 1

Manufactured by Thermo Fisher Scientific

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R-spondin-1 is a recombinant protein that serves as a positive regulator of the Wnt signaling pathway. It is a member of the R-spondin family of secreted proteins known to enhance Wnt/β-catenin signaling.

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R-spondin (34 citations) Recombinant human R-spondin 1 (17 citations) Human R-spondin-1 (8 citations) Murine R-spondin-1 (3 citations)

52 protocols using r spondin 1

Optimizing 3D Culture of Single Cells

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Freshly sorted cell populations were plated on NuncTM MicroWellTM 96-Well Optical-Bottom plates in Matrigel (9,500 cells per 9.5 μl) and overlaid with defined basal culture medium, supplemented with Nrg1 (100 ng ml⁻¹), Noggin (100 ng ml⁻¹) and R-Spondin 1 (2.6 ng ml⁻¹; Peprotech). Lower concentrations of recombinant R-Spondin 1 from Peprotech were used compared with R-Spondin 1 from R&D (100 ng ml⁻¹) due to its superior activity. Cultures were additionally treated with Rho kinase inhibitor (Y-27632, 10 μM, Tocris) for the first 5 days. As previously observed for other cell types13 (link)54 , Y-27632 in our hands increased the colony-forming efficiency of single cells.

Jardé T., Lloyd-Lewis B., Thomas M., Kendrick H., Melchor L., Bougaret L., Watson P.D., Ewan K., Smalley M.J, & Dale T.C. (2016). Wnt and Neuregulin1/ErbB signalling extends 3D culture of hormone responsive mammary organoids. Nature Communications, 7, 13207.

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Genotyping and Phenotyping of Enteroids from Polr3b-Mutant Mice

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Small intestine was harvested from P1 Polr3b-mutant and control mice. After a 30-minute EDTA (5 mM) incubation, the intestinal crypts were filtered through a 70 μm cell strainer. Crypts were plated suspended in Matrigel (BD Biosciences, 356231) and cultured in Advanced DMEM/F-12 (Thermo Fisher Scientific; Waltham, MA; 12634010) containing the Wnt agonist R-spondin1 (Protein Expression Facility, The Wistar Institute; Philadelphia, PA), murine epidermal growth factor (mEGF) (Life Technologies; Carlsbad, CA; PMG8043), murine Noggin (mNoggin) (Peprotech; Rocky Hill, NJ; 250-38), and the Gsk3 inhibitor CHIR99021 (BioVision; Milpitas, CA; 1677-5) as reported previously^{24 (link),25 (link)}. On days 2, 4, and 6, pooled enteroids were genotyped by qPCR for the presence of the deleted allele using primers Polr3bF and Polr3bR1, as described above, and the undeleted allele using primers Polr3bF2: 5′-GCTTGATATCGAATTCCGAAG-3′ and Polr3bR: 5′-CCTCTCTGGAACTCCAACCAA-3′. The quantity of genomic DNA present in qPCR reactions was normalized to genomic β-actin. In addition, proliferation and apoptosis were assessed in developing enteroids by immunofluorescent analysis of Ki67 and cleaved caspase-3, respectively, as described above.

Kieckhaefer J.E., Lukovac S., Ye D.Z., Lee D., Beetler D.J., Pack M, & Kaestner K.H. (2016). The RNA Polymerase III Subunit Polr3b Is Required for the Maintenance of Small Intestinal Crypts in Mice. Cellular and Molecular Gastroenterology and Hepatology, 2(6), 783-795.

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Polr3b-Mutant Mouse Enteroid Assay

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Small intestine was harvested from P1 Polr3b-mutant and control mice. After a 30-minute EDTA (5 mmol/L) incubation, the intestinal crypts were filtered through a 70-μm cell strainer. Crypts were plated suspended in Matrigel (356231; BD Biosciences) and cultured in advanced Dulbecco's modified Eagle medium/F-12 (12634010; Thermo Fisher Scientific, Waltham, MA) containing the Wnt agonist R-spondin1 (Protein Expression Facility, The Wistar Institute, Philadelphia, PA), murine epidermal growth factor (PMG8043; Life Technologies), murine Noggin (250-38; Peprotech, Rocky Hill, NJ), and the Gsk3 inhibitor CHIR99021 (1677-5; BioVision, Milpitas, CA) as reported previously.24 (link), 25 (link) On days 2, 4, and 6, pooled enteroids were genotyped by quantitative PCR (qPCR) for the presence of the deleted allele using primers Polr3bF and Polr3bR1, as described earlier, and the undeleted allele using primers Polr3bF2: 5’-GCTTGATATCGAATTCCGAAG-3’ and Polr3bR: 5’-CCTCTCTGGAACTCCAACCAA-3’. The quantity of genomic DNA present in qPCR reactions was normalized to genomic β-actin. In addition, proliferation and apoptosis were assessed in developing enteroids by immunofluorescent analysis of Ki67 and cleaved caspase-3, respectively, as described earlier.

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Intestine-specific PPAR-Alpha Knockout Mice

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Villin-cre mice were obtained from Deborah L. Gumucio, University of Michigan.^{16 (link)}Ppara^fl/fl mice, on the C57BL/6N genetic background, were generated as previously described.^{17 (link)}Ppara^fl/fl mice were crossed with Villin-cre mice to produce intestine-specific Ppara null mice (Ppara^ΔIE). Human PPARA transgenic mice, on the Sv129 background, with the complete human PPARA transgene and the mouse Ppara-null allele, were described previously.^{18 (link)} Mice were maintained under a standard 12-h light/12-h dark cycle with water and chow provided ad libitum. All animal experiments were carried out in accordance with Institute of Laboratory Animal Resources guidelines and protocols approved by the National Cancer Institute Animal Care and Use Committee. AOM was purchased from Syncon (Anaheim, CA). 5-Aza-2’-deoxycytidine (5-Aza) and PD0332991 were purchased from Sigma-Aldrich (St. Louis, MO) and DSS (molecular mass 36,000–50,000 Da) was from MP Biochemicals (Solon, OH). EPZ020411 (EPZ) was purchase from Cayman Chemical (Ann Arbor, MI). EGF, R-Spondin 1, and Noggin were purchase from PeproTech (Rocky Hill, NJ).

Luo Y., Xie C., Brocker C.N., Fan J., Wu X., Feng L., Wang Q., Zhao J., Lu D., Tandon M., Cam M., Krausz K.W., Liu W, & Gonzalez F.J. (2019). Intestinal PPARα Protects Against Colon Carcinogenesis via Regulation of Methyltransferases DNMT1 and PRMT6. Gastroenterology, 157(3), 744-759.e4.

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Culture and Transduction of CRC Organoids

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Human CRC organoid was cultured as described previously^{60 (link),61 (link)}. In brief, fresh CRC tumor tissues were washed with cold PBS containing penicillin-streptomycin, and cut into 3–5 mm fragments. Pieces were digested with EDTA (5 mM) on ice for 60 min with mixing. After being digested into clumps of cells, the sample was mixed with Matrigel and seeded into a 24-well plate. After Matrigel polymerization (10 min at 37 °C), 500 μL/well advanced DMEM/F12 medium containing 10 mM HEPES, 100 U/mL penicillin/streptomycin, 2 mM GlutaMAX, 1× B27, 1× N2 (Life Technologies), 10 nM gastrin I (Biogems), 500 ng/mL R-spondin1 (Peprotech), 10 μM SB202190 (Sigma), 10 μM Y-27632 (Abmole), 50 ng/mL recombinant EGF, 500 nM A83-01 (Biogems), 100 ng/mL recombinant Noggin (Peprotech), 10 mM nicotinamide (Sigma), 1 mM N-acetylcysteine (Sigma) was added to each well containing organoids. On the second day, the organoids were transduced with shCTL or shCHD6 lentivirus using polybrene (10 μg/mL) (Millipore, TR-1003-G).

Zhang B., Liu Q., Wen W., Gao H., Wei W., Tang A., Qin B., Lyu H., Meng X., Li K., Jin H., Yu F., Pan Q., Lin J, & Lee M.H. (2022). The chromatin remodeler CHD6 promotes colorectal cancer development by regulating TMEM65-mediated mitochondrial dynamics via EGF and Wnt signaling. Cell Discovery, 8, 130.

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Establishment of Pancreatic Cancer Organoids

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PDAC organoids were established from KPC PCCs, which were established using the outgrowth method as described [21 ]. PCCs were embedded in growth factor-reduced Matrigel (Cat#356231; BD Bioscience, CA, USA), and cultured in human complete medium at 37 C° for 14 days [21 , 25 (link)]. Human complete medium was AdDMEM/F12 (Cat#12634–010; Invitrogen, CA, USA), medium supplemented with 1 M HEPES (Invitrogen), GlutaMax (Cat#35050–061; Invitrogen), penicillin/streptomycin (Cat#15140122; Invitrogen), B27 (Cat#17504044; Invitrogen), N-acetyl-l-cysteine (Cat#A9165; Sigma-Aldrich Co.), Wnt-3a (Cat#5036-WN-010; R&D Systems, MN, USA), R-Spondin 1 (Cat#120–38; Peprotech, NJ, USA), Noggin (Cat#120-10C; Invitrogen), epidermal growth factor (EGF, Cat#AF-100-15; Peprotech), fibroblast growth factor (FGF, Cat#100–26; Peprotech), nicotinamide (Cat#N0636; Sigma-Aldrich Co.), Y-27263 (Cat# Y0503; Sigma-Aldrich Co.) and A83–01 (Cat#2939/10; R&D Systems).

Yan Z., Ohuchida K., Fei S., Zheng B., Guan W., Feng H., Kibe S., Ando Y., Koikawa K., Abe T., Iwamoto C., Shindo K., Moriyama T., Nakata K., Miyasaka Y., Ohtsuka T., Mizumoto K., Hashizume M, & Nakamura M. (2019). Inhibition of ERK1/2 in cancer-associated pancreatic stellate cells suppresses cancer–stromal interaction and metastasis. Journal of Experimental & Clinical Cancer Research : CR, 38, 221.

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Induction of Stem Cell Differentiation

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Cell culture medium (RPMI 1640, advanced DMEM/F12 medium), defined fetal bovine serum (dFBS), and B27 were acquired from Thermo Fisher (Waltham, MA, USA). Activin A, fibroblast growth factor 4 (FGF4), WNT3A, epidermal growth factor (EGF), Noggin, and recombinant human IL-2 were purchased from R&D Systems (Minneapolis, MN, USA). R-Spondin 1 was purchased from PeproTech Inc (Rocky Hill, NJ, USA). Specific inhibitors of STAT3, including S3I-201 or Stattic, phorbol myristate acetate (PMA), and calcium ionophore A23187 were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Jung K.B., Kwon O., Lee M.O., Lee H., Son Y.S., Habib O., Oh J.H., Cho H.S., Jung C.R., Kim J, & Son M.Y. (2019). Blockade of STAT3 Causes Severe In Vitro and In Vivo Maturation Defects in Intestinal Organoids Derived from Human Embryonic Stem Cells. Journal of Clinical Medicine, 8(7), 976.

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Gastric Cancer Organoid Establishment

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Gastric cancer tissue was obtained from The Peking University Cancer Hospital, and the study was approved by Peking University Cancer Hospital Review Board (protocol number: 2019KT111) in accordance with the Declaration of Helsinki. Gastric cancer organoids were established and cultured as previously reported (22 (link)). Briefly, established organoids were embedded in Matrigel (Corning; catalog no.: 356231) with addition of organoid culture medium, including advanced Dulbecco's modified Eagle's medium (DMEM)/F12, 1% penicillin/streptomycin (Invitrogen), 1× Gluta Max (Invitrogen), 1× Hepes (Invitrogen), 1× B27 (Invitrogen), 10 mM N-acetylcystenine (Sigma), 50 mg/ml nicotinamide (Sigma), 500 ng/ml FGF10 (PeproTech, Inc), 500 ng/ml Noggin (PeproTech, Inc), 500 ng/ml R-spondin-1 (PeproTech, Inc), 2 μM A-8301 (Tocris), 100 ng/ml Wnt3a (R&D Systems), 10 μM Y-27632 (Sigma), epidermal growth factor (Invitrogen), gastrin (Tocris), and primocin (0.1 mg/ml; Invivogen). Fresh medium was changed every 2 to 3 days. Organoids were split every 5 to 7 days in a 1:3 ratio using mechanical dissociation and plated in fresh Matrigel.

Wang M., Yu H., Zhang T., Cao L., Du Y., Xie Y., Ji J, & Wu J. (2021). In-Depth Comparison of Matrigel Dissolving Methods on Proteomic Profiling of Organoids. Molecular & Cellular Proteomics : MCP, 21(1), 100181.

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Prostate Organoid Culture from Pten Mice

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Prostate organoid cultures were established from Pten^L2/L2 and Pten^(i)pe−/− mice at 3 months AGI following previously described protocols (52 (link), 53 (link)). Briefly, prostates were dissociated into single cells, which were embedded in Matrigel (Corning, 356231) and seeded in 24-well plates (40 μl of Matrigel drop per well). Organoid medium was prepared by adding B27 (50× diluted; Gibco, 17504044), N-acetyl cysteine (1.25 mM; Sigma-Aldrich, A9165-25G), Hepes (10 mM; Gibco, 15630080), GlutaMAX (100× diluted; Gibco, 35050061), and P/S [1% (v/v); Gibco, 15140122] to advanced DMEM/F-12 (Gibco, 12634010). Complete medium was prepared by adding EGF (50 ng/ml; PeproTech, 315-09), Noggin (100 ng/ml; PeproTech, 120-10C), R-spondin 1 (500 ng/ml; PeproTech, 120-38), dihydrotestosterone (1 nM; Sigma-Aldrich, A8380-1G), Y-27632 (10 μM; Tocris, 1254), and A83-01 (200 nM; PeproTech, 9094360) to the organoid medium. Upon solidification of the Matrigel domes, 500 μl of complete medium was added to each well.

Abu el Maaty M.A., Terzic J., Keime C., Rovito D., Lutzing R., Yanushko D., Parisotto M., Grelet E., Namer I.J., Lindner V., Laverny G, & Metzger D. (2022). Hypoxia-mediated stabilization of HIF1A in prostatic intraepithelial neoplasia promotes cell plasticity and malignant progression. Science Advances, 8(29), eabo2295.

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Hepatic Differentiation of DFAT Cells

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DFAT cells were cultured under a humidified atmosphere of 5% CO₂ and 95% air at 37°C on collagen type I‐C (Nitta Gelatin)‐coated tissue 40‐mm culture dishes (TPP) containing Dulbecco's modified Eagle's medium (DMEM) (Nissui Pharmaceutical) supplemented with 10% fetal bovine serum (FBS) (Moregate BioTech). DFAT cells were grown to semiconfluence for differentiation. Hepatic differentiation was induced by changing the medium to SHM + YAC (Chen et al., 2012), namely DMEM/F12 (Gibco) containing 2.4 g/L NaHCO₃ and l‐glutamine, which was supplemented with 5 mM HEPES (Sigma‐Aldrich), 30 mg/L l‐proline (Sigma‐Aldrich), 0.05% BSA (Sigma‐Aldrich), 10 ng/ml epidermal growth factor (PeproTech), insulin–transferrin–serine (ITS)‐X (Gibco), 10^–7 M Dexamethasone (Dex)(Sigma‐Aldrich), 10 mM nicotinamide (Wako), 1 mM ascorbic acid‐2 phosphate (Sigma‐Aldrich), 10 mM Y‐27632 (Wako), 0.5 mMA83‐01 (Wako) and 3 mM CHIR99021 (Axon Medchem). After 2 days, the culture medium was replaced with SHM + YAC, which was supplemented with 20 ng/ml oncostatin M (OsM) (Wako), 10⁻⁶ M Dex and 500 ng/ml R‐spondin1 (PeproTech). The cells were then cultured for 12 days, with fresh medium provided every 2 days.

Hagiwara R., Oki Y., Matsumaru T., Ibayashi S, & Kano K. (2020). Generation of metabolically functional hepatocyte‐like cells from dedifferentiated fat cells by Foxa2, Hnf4a and Sall1 transduction. Genes to Cells, 25(12), 811-824.

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