RESUMO
BACKGROUND & AIMS: 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. METHODS: 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 ß-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. RESULTS: 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 ß-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. CONCLUSIONS: 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.
Assuntos
Diferenciação Celular , Proliferação de Células , Sistema Nervoso Entérico/citologia , Neurônios , RNA Mensageiro/análise , Via de Sinalização Wnt , Animais , Animais Recém-Nascidos , Proteína Axina/genética , Contagem de Células , Morte Celular , Proliferação de Células/efeitos dos fármacos , Feminino , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Expressão Gênica/efeitos dos fármacos , Humanos , Indóis/farmacologia , Lactente , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/genética , Masculino , Maleimidas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Acoplados a Proteínas G/genética , Esferoides Celulares/metabolismo , Células-Tronco , Regulação para Cima , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt3A/farmacologia , beta Catenina/metabolismoRESUMO
The proliferation, migration and differentiation of dentate gyrus stem and precursor cells have aroused keen interest. Neogenin and RGMb are expressed in non-overlapping compartments of the developing dentate gyrus. While Neogenin is expressed in migrating and proliferating dentate precursors, RGMb is localized in structures bordering the developing dentate, such as cornus ammonis cells and Cajal-Retzius cells in the marginal zone including the hippocampal fissure. Co-immunoprecipitation and binding assays indicate a strong physical interaction. In vitro and in vivo migration of dentate neuroepithelial cells is abolished by RGMb, and cell adhesion is reduced when cells expressing Neogenin comes into contact with cells expressing RGMb. Ectopic expression of RGMb in organotypic slice cultures and after in utero electroporation in the hippocampus modifies precursor cell migration. Our results imply that Neogenin-RGMb interaction might be involved in neuronal migration in the dentate gyrus.
Assuntos
Movimento Celular/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Animais , Moléculas de Adesão Celular Neuronais , Linhagem Celular Transformada , Eletroporação/métodos , Embrião de Mamíferos , Fluoresceínas , Proteínas Ligadas por GPI , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Proteínas de Fluorescência Verde/genética , Hipocampo/citologia , Proteínas de Homeodomínio/metabolismo , Humanos , Imunoprecipitação , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/genética , Técnicas de Cultura de Órgãos , Células-Tronco/fisiologia , Transfecção/métodos , Proteínas Supressoras de Tumor/metabolismoRESUMO
Identification and isolation of neural progenitor cells from the human enteric nervous system (ENS) is currently hampered by the lack of reliable, specific markers. Here, we define the Wnt-receptor frizzled-4 as a marker for the isolation of enteric neural progenitor cells derived from paediatric gut samples. We show that the Wnt-receptor frizzled-4 is expressed in the human colon and in Tunica muscularis-derived enterospheres. To obtain a purified culture, we carried out fluorescence-activated cell sorting (FACS) using PE-conjugated frizzled-4 antibodies. Frizzled-4positive cells gave rise to neurosphere-like bodies and ultimately differentiated into neurons as revealed by BrdU-proliferation assays and immunocytochemistry, whereas in frizzled-4negative cultures we did not detect any neuronal and glial cells. By using a patch-clamp approach, we also demonstrated the expression of functional sodium and potassium channels in frizzled-4positive cell cultures after differentiation in vitro.