Institute of Clinical Anatomy and Cell Analysis, University of Tübingen, Tübingen, Germany.
Department of Pediatric Surgery, University Children's Hospital Tübingen, Germany.
Gastroenterology. 2017 Jul;153(1):154-165.e9. doi: 10.1053/j.gastro.2017.03.019. Epub 2017 Mar 27.
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.
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.
肠神经系统（ENS）中的神经干细胞和祖细胞可能作为治疗神经胃肠疾病的细胞来源。在我们能够使用这些细胞之前，我们必须增加对调节增殖和分化的信号机制的理解。我们系统地评估了经典 Wnt 信号对培养的新生小鼠和人类 ENS 祖细胞增殖和分化的影响。
我们从小鼠（新生后第 0 天）和 Wnt1-Cre2 报告小鼠的小肠黏膜肌层中分离出 ENS 祖细胞。我们还从因肛门闭锁或局部肠穿孔而接受手术的婴儿（2 个月和 7 个月大）的肠道组织样本中获得了 ENS 细胞。ENS 细胞在增殖条件下培养，形成 3 维球体，我们用 Wnt3a 和 SB216763 激活这些球体，以激活依赖β-连环蛋白的经典 Wnt 通路。我们使用免疫印迹和定量聚合酶链反应来评估对 Wnt 刺激的分子反应，并使用免疫组织化学、增殖和细胞死亡测定来鉴定新的神经元。
在 ENS 祖细胞来源的增殖性 enterospheres 中，我们验证了 Wnt 受体 frizzled 1-10 和共受体低密度脂蛋白受体相关蛋白 5 和 6 的表达。Wnt 激动剂的药理刺激导致 Wnt 依赖性β-连环蛋白的细胞内积累，并上调已知的 Wnt 靶基因 axin2、lef1 和 lgr5 的表达。经典 Wnt 通路的激活促进了 ENS 细胞球体在细胞扩增期间的生长，并增加了源自小鼠和人类祖细胞的新生神经元的数量。
在对人类和小鼠 ENS 祖细胞的研究中，我们发现激活 Wnt 信号通路可促进 ENS 在体外的神经发生。Wnt 激动剂对 ENS 祖细胞的神经发生作用支持它们用于生成用于自体细胞替代治疗的细胞池。
