title: Lipid-mediated Wnt protein stabilization enables serum-free culture of human organ stem cells.
abstract: Wnt signalling proteins are essential for culture of human organ stem cells in organoids, but most Wnt protein formulations are poorly active in serum-free media. Here we show that purified Wnt3a protein is ineffective because it rapidly loses activity in culture media due to its hydrophobic nature, and its solubilization requires a detergent, CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), that interferes with stem cell self-renewal. By stabilizing the Wnt3a protein using phospholipids and cholesterol as carriers, we address both problems: Wnt activity remains stable in serum-free media, while non-toxic carriers allow the use of high Wnt concentrations. Stabilized Wnt3a supports strongly increased self-renewal of organ and embryonic stem cells and the serum-free establishment of human organoids from healthy and diseased intestine and liver. Moreover, the lipophilicity of Wnt3a protein greatly facilitates its purification. Our findings remove a major obstacle impeding clinical applications of adult stem cells and offer advantages for all cell culture uses of Wnt3a protein.
authors.affiliation: Department of Cell Biology, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Nesrin
authors.initials: N
authors.lastname: Tüysüz
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, Utrecht 3584 CG, The Netherlands.
authors.forename: Louis
authors.initials: L
authors.lastname: van Bloois
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Cancer Genomics.nl and University Medical Center Utrecht, Uppsalalaan 8, Utrecht 3584 CT, The Netherlands.|Foundation Hubrecht Organoid Technology (HUB), Utrecht 3584 CT, The Netherlands.
authors.forename: Stieneke
authors.initials: S
authors.lastname: van den Brink
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Cancer Genomics.nl and University Medical Center Utrecht, Uppsalalaan 8, Utrecht 3584 CT, The Netherlands.|Foundation Hubrecht Organoid Technology (HUB), Utrecht 3584 CT, The Netherlands.
authors.forename: Harry
authors.initials: H
authors.lastname: Begthel
authors.affiliation: Department of Surgery, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Monique M A
authors.initials: MM
authors.lastname: Verstegen
authors.affiliation: Experimental Molecular Imaging, Department of Radiology, Leiden University Medical Center, Albinusdreef 2, Leiden 2333 ZA, The Netherlands.
authors.forename: Luis J
authors.initials: LJ
authors.lastname: Cruz
authors.affiliation: Shanghai Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Lijian
authors.initials: L
authors.lastname: Hui
authors.affiliation: Department of Surgery, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Luc J W
authors.initials: LJ
authors.lastname: van der Laan
authors.affiliation: Department of Surgery, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Jeroen
authors.initials: J
authors.lastname: de Jonge
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Cancer Genomics.nl and University Medical Center Utrecht, Uppsalalaan 8, Utrecht 3584 CT, The Netherlands.|Foundation Hubrecht Organoid Technology (HUB), Utrecht 3584 CT, The Netherlands.
authors.forename: Robert
authors.initials: R
authors.lastname: Vries
authors.affiliation: Department of Hematology, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Eric
authors.initials: E
authors.lastname: Braakman
authors.affiliation: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, Utrecht 3584 CG, The Netherlands.
authors.forename: Enrico
authors.initials: E
authors.lastname: Mastrobattista
authors.affiliation: Department of Hematology, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Jan J
authors.initials: JJ
authors.lastname: Cornelissen
authors.affiliation: Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Cancer Genomics.nl and University Medical Center Utrecht, Uppsalalaan 8, Utrecht 3584 CT, The Netherlands.|Foundation Hubrecht Organoid Technology (HUB), Utrecht 3584 CT, The Netherlands.
authors.forename: Hans
authors.initials: H
authors.lastname: Clevers
authors.affiliation: Department of Cell Biology, Erasmus University Medical Center, PO Box 2040, Rotterdam 3000 CA, The Netherlands.
authors.forename: Derk
authors.initials: D
authors.lastname: Ten Berge
mesh_terms: D053687:Adult Stem Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D001706:Biopsy; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002784:Cholesterol / Q000737:chemistry*; D017077:Culture Media, Conditioned / Q000494:pharmacology*; D058625:End Stage Liver Disease / Q000473:pathology; D006526:Hepatitis C / Q000473:pathology; D006527:Hepatolenticular Degeneration / Q000473:pathology; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D007583:Jejunum / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008081:Liposomes / Q000008:administration & dosage / Q000737:chemistry; D008099:Liver / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D008113:Liver Neoplasms / Q000473:pathology; D009940:Organoids / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D010743:Phospholipids / Q000737:chemistry*; D046509:Tissue Culture Techniques; D060509:Wnt3A Protein / Q000494:pharmacology*
chemical_list: D017077:Culture Media, Conditioned; D008081:Liposomes; D010743:Phospholipids; C555510:WNT3A protein, human; D060509:Wnt3A Protein; D002784:Cholesterol