title: Activation of Wnt Signaling Increases Numbers of Enteric Neurons Derived From Neonatal Mouse and Human Progenitor Cells.
abstract: Neural stem and progenitor cells from the enteric nervous system (ENS) might serve as a source of cells for treatment of neurogastrointestinal disorders. Before we can use these cells, we must increase our understanding of the signaling mechanisms that regulate proliferation and differentiation. We systematically evaluated the effects of canonical Wnt signaling on proliferation and differentiation of cultured ENS progenitor cells from neonatal mice and humans.
  
  
  
  We isolated ENS progenitors from tunica muscularis of the small intestine of newborn (postnatal day 0) wild-type C57BL/6 mice as well as from Wnt1-Cre2 reporter mice. We also obtained intestinal tissue samples from infants (2 and 7 months old) undergoing surgery for imperforate anus or focal intestinal perforation and isolated ENS cells. ENS cells were cultured under proliferation conditions leading to formation of 3-dimensional spheres, which we activated with Wnt3a and SB216763 in order to activate the β-catenin-dependent canonical Wnt pathway. We used immunoblot and quantitative polymerase chain reaction to evaluate the molecular response to Wnt stimuli and immunohistochemistry, proliferation, and cell death assays to identify new neurons.
  
  
  
  In proliferating enterospheres derived from ENS progenitor cells, we verified the expression of Wnt receptors frizzled 1-10 and the co-receptors low-density lipoprotein receptor-related proteins 5 and 6. Pharmacologic stimulation with Wnt agonists led to intracellular accumulation of Wnt-dependent β-catenin and up-regulated expression of known Wnt target genes axin2, lef1, and lgr5. Activation of the canonical Wnt pathway promoted growth of ENS cell spheres during cell expansion and increased the number of newborn neurons derived from mouse and human progenitor cells.
  
  
  
  In studies of human and mouse ENS progenitors, we found activation of the Wnt signaling pathway to promote neurogenesis of the ENS in vitro. The neurogenic effect of Wnt agonists on ENS progenitors supports their use in generation of cell pools for autologous cell replacement therapies.
authors.affiliation: Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
authors.forename: Ying
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
authors.forename: Karin
authors.initials: K
authors.lastname: Seid
authors.affiliation: Department of Pediatric Surgery, University Children's Hospital Tübingen, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Obermayr
authors.affiliation: Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
authors.forename: Lothar
authors.initials: L
authors.lastname: Just
authors.affiliation: Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany. Electronic address: peter.neckel@uni-tuebingen.de.
authors.forename: Peter H
authors.initials: PH
authors.lastname: Neckel
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D060466:Axin Protein / Q000235:genetics; D002452:Cell Count; D016923:Cell Death; D002454:Cell Differentiation; D049109:Cell Proliferation / Q000187:drug effects; D017615:Enteric Nervous System / Q000166:cytology*; D005260:Female; D051157:Frizzled Receptors / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007223:Infant; D060492:Low Density Lipoprotein Receptor-Related Protein-5 / Q000235:genetics / Q000378:metabolism; D060488:Low Density Lipoprotein Receptor-Related Protein-6 / Q000235:genetics / Q000378:metabolism; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000235:genetics; D008297:Male; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009474:Neurons; D012333:RNA, Messenger / Q000032:analysis*; D043562:Receptors, G-Protein-Coupled / Q000235:genetics; D018874:Spheroids, Cellular / Q000378:metabolism; D013234:Stem Cells; D015854:Up-Regulation; D060449:Wnt Signaling Pathway / Q000187:drug effects; D060509:Wnt3A Protein / Q000494:pharmacology; D051176:beta Catenin / Q000378:metabolism
chemical_list: C491761:AXIN2 protein, human; D060466:Axin Protein; C111729:Axin2 protein, mouse; D051157:Frizzled Receptors; D007211:Indoles; C116884:LGR5 protein, human; C493597:Lgr5 protein, mouse; D060492:Low Density Lipoprotein Receptor-Related Protein-5; D060488:Low Density Lipoprotein Receptor-Related Protein-6; D051821:Lymphoid Enhancer-Binding Factor 1; D008301:Maleimides; D012333:RNA, Messenger; D043562:Receptors, G-Protein-Coupled; C417521:SB 216763; D060509:Wnt3A Protein; D051176:beta Catenin
keywords: Differentiation; Neural Progenitor; Neurons; Proliferation