Yang Li-Ju, Ma Ying, Li Yuan, Dang Qing-Ya, Cheng Jun, Yang Yan, Li Peng-Yun
Key Laboratory of Medical Electrophysiology, Ministry of Education & Medical Electrophysiological Key Laboratory of Sichuan Province, Collaborative Innovation Center for Prevention and Treatment of Cardiovascular Disease of Sichuan Province, Institute of Cardiovascular Research, Southwest Medical University, Luzhou 646000, China.
Department of Cardiology, the Affiliated Hospital of Southwest Medical University, Luzhou 646000, China.
Sheng Li Xue Bao. 2023 Apr 25;75(2):205-215.
Vascular wall-resident stem cells (VW-SCs) play a critical role in maintaining normal vascular function and regulating vascular repair. Understanding the basic functional characteristics of the VW-SCs will facilitate the study of their regulation and potential therapeutic applications. The aim of this study was to establish a stable method for the isolation, culture, and validation of the CD34 VW-SCs from mice, and to provide abundant and reliable cell sources for further study of the mechanisms involved in proliferation, migration and differentiation of the VW-SCs under various physiological and pathological conditions. The vascular wall cells of mouse aortic adventitia and mesenteric artery were obtained by the method of tissue block attachment and purified by magnetic microbead sorting and flow cytometry to obtain the CD34 VW-SCs. Cell immunofluorescence staining was performed to detect the stem cell markers (CD34, Flk-1, c-kit, Sca-1), smooth muscle markers (SM22, SM MHC), endothelial marker (CD31), and intranuclear division proliferation-related protein (Ki-67). To verify the multipotency of the isolated CD34 VW-SCs, endothelial differentiation medium EBM-2 and fibroblast differentiation medium FM-2 were used. After culture for 7 days and 3 days respectively, endothelial cell markers and fibroblast markers of the differentiated cells were evaluated by immunofluorescence staining and q-PCR. Furthermore, the intracellular Ca release and extracellular Ca entry signaling were evaluated by TILLvisION system in Fura-2/AM loaded cells. The results showed that: (1) High purity (more than 90%) CD34 VW-SCs from aortic adventitia and mesenteric artery of mice were harvested by means of tissue block attachment method and magnetic microbead sorting; (2) CD34 VW-SCs were able to differentiate into endothelial cells and fibroblasts in vitro; (3) Caffeine and ATP significantly activated intracellular Ca release from endoplasmic reticulum of CD34 VW-SCs. Store-operated Ca entry (SOCE) was activated by using thapsigargin (TG) applied in Ca-free/Ca reintroduction protocol. This study successfully established a stable and efficient method for isolation, culture and validation of the CD34 VW-SCs from mice, which provides an ideal VW-SCs sources for the further study of cardiovascular diseases.
血管壁驻留干细胞（VW-SCs）在维持正常血管功能和调节血管修复中起关键作用。了解VW-SCs的基本功能特性将有助于对其调控及潜在治疗应用的研究。本研究的目的是建立一种稳定的方法，用于从小鼠中分离、培养和鉴定CD34 VW-SCs，并为进一步研究VW-SCs在各种生理和病理条件下的增殖、迁移和分化机制提供丰富且可靠的细胞来源。通过组织块贴壁法获取小鼠主动脉外膜和肠系膜动脉的血管壁细胞，并通过磁性微珠分选和流式细胞术进行纯化，以获得CD34 VW-SCs。进行细胞免疫荧光染色以检测干细胞标志物（CD34、Flk-1、c-kit、Sca-1）、平滑肌标志物（SM22、SM MHC）、内皮标志物（CD31）以及核内分裂增殖相关蛋白（Ki-67）。为验证分离出的CD34 VW-SCs的多能性，使用了内皮分化培养基EBM-2和成纤维细胞分化培养基FM-2。分别培养7天和3天后，通过免疫荧光染色和q-PCR评估分化细胞的内皮细胞标志物和成纤维细胞标志物。此外，在加载Fura-2/AM的细胞中，通过TILLvisION系统评估细胞内钙释放和细胞外钙内流信号。结果表明：（1）通过组织块贴壁法和磁性微珠分选，从小鼠主动脉外膜和肠系膜动脉中收获了高纯度（超过90%）的CD34 VW-SCs；（2）CD34 VW-SCs在体外能够分化为内皮细胞和成纤维细胞；（3）咖啡因和ATP显著激活了CD34 VW-SCs内质网的细胞内钙释放。通过在无钙/再引入钙方案中应用毒胡萝卜素（TG）激活了储存式钙内流（SOCE）。本研究成功建立了一种稳定且高效的方法，用于从小鼠中分离、培养和鉴定CD34 VW-SCs，为心血管疾病的进一步研究提供了理想的VW-SCs来源。
