Supplementary Material for: Identification of Interstitial Cajal-Like Cells in the Human Thoracic Duct

Interstitial Cajal-like cells (ICLCs) are speculated to be pacemakers in smooth muscle tissues. While the human thoracic duct (TD) is spontaneously active, the origin of this activity is unknown. We hypothesized that ICLCs could be present in the TD and using histological techniques, immunohistochemistry and immunofluorescence we have investigated the presence of ICLCs, protein markers for ICLCs and the cellular morphology of the human TD. Transmission electron microscopy was employed to investigate ultrastructure. Methylene blue staining, calcium-dependent fluorophores and confocal microscopy were used to identify ICLCs in live tissue. Methylene blue stained cells with morphology suggestive of ICLCs in the TD. Immunoreactivity localized the ICLC protein markers c-kit, CD34 and vimentin to many cells and processes associated with smooth muscle cells (SMCs): coexpression of c-kit with vimentin or CD34 was observed in some cells. Electron microscopy analysis confirmed ICLCs as a major cell type of the human TD. Lymphatic ICLCs possess caveolae, dense bands, a patchy basal lamina, intermediate filaments and specific junctions to SMCs. ICLCs were ultrastructurally differentiable from other interstitial cells observed: fibroblasts, mast cells, macrophages and pericytes. Lymphatic ICLCs were localized to the subendothelial region of the wall as well as in intimate association with smooth muscle bundles throughout the media. ICLCs were morphologically distinct with multiple processes and also spindle shapes. Confocal imaging with calcium-dependent fluorophores corroborated cell morphology and localization observed in fixed tissues. Lymphatic ICLCs thus constitute a significant cell type of the human TD and physically interact with lymphatic SMCs.

间质Cajal样细胞（Interstitial Cajal-like cells, ICLCs）被推测为平滑肌组织中的起搏细胞。人体胸导管（thoracic duct, TD）可发生自发活动，但其活动起源至今尚未明确。本研究提出如下假说：ICLCs可能存在于胸导管中，并采用组织学技术、免疫组织化学及免疫荧光法，对人体胸导管中ICLCs的存在情况、ICLCs的蛋白标志物以及细胞形态特征进行了探究；此外通过透射电子显微镜观察其超微结构。为鉴定活组织中的ICLCs，本研究还运用了亚甲蓝染色、钙依赖性荧光探针及共聚焦显微镜成像技术。亚甲蓝染色可见胸导管内存在形态符合ICLCs特征的阳性染色细胞。免疫组化结果显示，ICLCs的蛋白标志物c-kit、CD34及波形蛋白（vimentin）定位于大量与平滑肌细胞（smooth muscle cells, SMCs）相关的细胞及细胞突起；部分细胞同时存在c-kit与波形蛋白，或c-kit与CD34的共表达。电子显微镜分析证实，ICLCs是人体胸导管的主要细胞类型之一。淋巴源性ICLCs具有胞膜小凹（caveolae）、致密带、不连续基膜、中间丝，以及与平滑肌细胞形成的特异性连接结构。通过超微结构特征，可将淋巴ICLCs与成纤维细胞、肥大细胞、巨噬细胞及周细胞等其他间质细胞区分开来。淋巴ICLCs定位于血管壁的内皮下区域，同时广泛分布于中膜层并与平滑肌束紧密毗邻。ICLCs形态独特，具有多个细胞突起，呈梭形外观。钙依赖性荧光探针共聚焦成像结果进一步验证了固定组织中观察到的细胞形态与定位特征。综上，淋巴ICLCs是人体胸导管的重要细胞类型，并可与淋巴平滑肌细胞发生物理性相互作用。