Department of Surgery, Chang Gung Children's Hospital, College of Medicine, Chang Gung University, 5, Fu-Shin Street, Kweishan, Taoyuan, 333, Taiwan.
Graduate Institute of Clinical Medicine, College of Medicine, National Taiwan University, Taipei, 100, Taiwan.
Stem Cell Res Ther. 2023 Oct 5;14(1):290. doi: 10.1186/s13287-023-03517-y.
Neurosphere medium (NSM) and self-renewal medium (SRM) were widely used to isolate enteric neural stem cells (ENSCs) in the form of neurospheres. ENSCs or their neurosphere forms were neurogenic and gliogenic, but the compelling evidence for their capacity of assembling enteric neural networks remained lacking, raising the question of their aptitude for rebuilding the enteric nervous system (ENS) in ENSC therapeutics. It prompted us to explore an effective culture protocol or strategy for assembling ENS networks, which might also be employed as an in vitro model to simplify the biological complexity of ENS embedded in gut walls.
NSM and SRM were examined for their capacity to generate neurospheres in mass culture of dispersed murine fetal enterocytes at serially diluted doses and assemble enteric neural networks in two- and three-dimensional cell culture systems and ex vivo on gut explants. Time-lapse microphotography was employed to capture cell activities of assembled neural networks. Neurosphere transplantation was performed via rectal submucosal injection.
In mass culture of dispersed enterocytes, NSM generated discrete units of neurospheres, whereas SRM promoted neural network assembly with neurospheres akin to enteric ganglia. Both were highly affected by seeding cell doses. SRM had similar ENSC mitosis-driving capacity to NSM, but was superior in driving ENSC differentiation in company with heightened ENSC apoptosis. Enteric neurospheres were motile, capable of merging together. It argued against their clonal entities. When nurtured in SRM, enteric neurospheres proved competent to assemble neural networks on two-dimensional coverslips, in three-dimensional hydrogels and on gut explants. In the course of neural network assembly from enteric neurospheres, neurite extension was preceded by migratory expansion of gliocytes. Assembled neural networks contained motile ganglia and gliocytes that constantly underwent shapeshift. Neurospheres transplanted into rectal submucosa might reconstitute myenteric plexuses of recipients' rectum.
Enteric neurospheres mass-produced in NSM might assemble neural networks in SRM-immersed two- or three-dimensional environments and on gut explants, and reconstitute myenteric plexuses of the colon after rectal submucosal transplantation. Our results also shed first light on the dynamic entity of ENS and open the experimental avenues to explore cellular activities of ENS and facilitate ENS demystification.
神经球培养基 (NSM) 和自我更新培养基 (SRM) 广泛用于以神经球的形式分离肠神经干细胞 (ENSCs)。ENSCs 或其神经球形式具有神经发生和神经胶质发生能力，但缺乏其组装肠神经网络能力的确凿证据，这引发了关于它们在 ENSC 治疗中重建肠神经系统 (ENS) 的能力的问题。这促使我们探索一种有效的培养方案或策略来组装 ENS 网络，该方案也可以用作体外模型来简化肠壁中嵌入的 ENS 的生物学复杂性。
在连续稀释剂量的分散鼠胎肠上皮细胞的大规模培养中，检查 NSM 和 SRM 生成神经球的能力，并在二维和三维细胞培养系统以及肠外植体上组装肠神经网络。延时显微摄影用于捕获组装神经网络的细胞活动。通过直肠黏膜下注射进行神经球移植。
在分散肠上皮细胞的大规模培养中，NSM 生成离散的神经球单位，而 SRM 则促进具有类似于肠神经节的神经球的神经网络组装。两者都受到接种细胞剂量的高度影响。SRM 具有与 NSM 相似的 ENSC 有丝分裂驱动能力，但在驱动 ENSC 分化方面优于 NSM，同时伴有 ENSC 凋亡增加。肠神经球是运动的，能够融合在一起。这表明它们不是克隆实体。当在 SRM 中培养时，肠神经球能够在二维盖玻片、三维水凝胶和肠外植体上组装神经网络。在从肠神经球组装神经网络的过程中，神经突延伸之前是神经胶质细胞的迁移扩张。组装的神经网络包含运动性神经节和不断改变形状的神经胶质细胞。移植到直肠黏膜下的神经球可能会重建接受者直肠的肌间神经丛。
在 NSM 中大规模生产的肠神经球可以在 SRM 浸泡的二维或三维环境以及肠外植体上组装神经网络，并在直肠黏膜下移植后重建结肠的肌间神经丛。我们的结果还首次揭示了 ENS 的动态实体，并为探索 ENS 的细胞活动和促进 ENS 解密开辟了实验途径。
