Div. of Gastroenterology and Hepatology, Stanford Univ. School of Medicine, CA 94305, USA.
Am J Physiol Gastrointest Liver Physiol. 2011 Oct;301(4):G644-55. doi: 10.1152/ajpgi.00123.2011. Epub 2011 Aug 4.
Recent studies have explored the potential of central nervous system-derived neural stem cells (CNS-NSC) to repopulate the enteric nervous system. However, the exact phenotypic fate of gut-transplanted CNS-NSC has not been characterized. The aim of this study was to investigate the effect of the gut microenvironment on phenotypic fate of CNS-NSC in vitro. With the use of Transwell culture, differentiation of mouse embryonic CNS-NSC was studied when cocultured without direct contact with mouse intestinal longitudinal muscle-myenteric plexus preparations (LM-MP) compared with control noncocultured cells, in a differentiating medium. Differentiated cells were analyzed by immunocytochemistry and quantitative RT-PCR to assess the expression of specific markers and by whole cell patch-clamp studies for functional characterization of their phenotype. We found that LM-MP cocultured cells had a significant increase in the numbers of cells that were immune reactive against the panneuronal marker β-tubulin, neurotransmitters neuronal nitric oxide synthase (nNOS), choline acetyltransferase (ChAT), and neuropeptide vasoactive intestinal peptide (VIP) and showed an increase in expression of these genes, compared with control cells. Whole cell patch-clamp analysis showed that coculture with LM-MP decreases cell excitability and reduces voltage-gated Na(+) currents but significantly enhances A-current and late afterhyperpolarization (AHP) and increases the expression of the four AHP-generating Ca(2+)-dependent K(+) channel genes (KCNN), compared with control cells. In a separate experiment, differentiation of LM-MP cocultured CNS-NSC produced a significant increase in the numbers of cells that were immune reactive against the neurotransmitters nNOS, ChAT, and the neuropeptide VIP compared with CNS-NSC differentiated similarly in the presence of neonatal brain tissue. Our results show that the gut microenvironment induces CNS-NSC to produce neurons that share some of the characteristics of classical enteric neurons, further supporting the therapeutic use of these cells for gastrointestinal disorders.
最近的研究探索了中枢神经系统来源的神经干细胞（CNS-NSC）在肠道神经系统再殖中的潜力。然而，肠道移植的 CNS-NSC 的确切表型命运尚未得到描述。本研究旨在探讨肠道微环境对 CNS-NSC 体外表型命运的影响。通过 Transwell 培养，研究了在分化培养基中，与对照组非共培养细胞相比，无直接接触的情况下，鼠胚胎 CNS-NSC 与鼠肠纵行肌-肌间神经丛（LM-MP）共培养时的分化情况。通过免疫细胞化学和定量 RT-PCR 分析分化细胞，以评估特定标志物的表达，并通过全细胞膜片钳研究对其表型进行功能特征分析。我们发现，与对照组细胞相比，LM-MP 共培养细胞中对神经元标志物β-微管蛋白、神经递质神经元型一氧化氮合酶（nNOS）、胆碱乙酰转移酶（ChAT）和神经肽血管活性肠肽（VIP）免疫反应性的细胞数量显著增加，并且这些基因的表达也增加。全细胞膜片钳分析表明，与 LM-MP 共培养会降低细胞兴奋性，减少电压门控 Na(+) 电流，但显著增强 A 电流和晚期后超极化（AHP），并增加四个 AHP 生成的 Ca(2+) 依赖性 K(+) 通道基因（KCNN）的表达，与对照组细胞相比。在另一个实验中，与在新生脑组织存在下相似地分化的 CNS-NSC 相比，LM-MP 共培养的 CNS-NSC 分化产生了更多对神经递质 nNOS、ChAT 和神经肽 VIP 免疫反应性的细胞。我们的结果表明，肠道微环境诱导 CNS-NSC 产生具有某些经典肠神经元特征的神经元，进一步支持将这些细胞用于胃肠道疾病的治疗。
